Intramolecular sensitization of photocleavage of the photolabile 2-(2-nitrophenyl)propoxycarbonyl (NPPOC) protecting group: Photoproducts and photokinetics of the release of nucleosides

Article abstract of DOI:10.1002/chem.200800613

Novel photolabile protecting groups based on the 2-(2-nitrophenyl)- propoxycarbonyl (NPPOC) group with a covalently linked thioxanthone as an intramolecular triplet sensitizer exhibit significantly enhanced light sensitivity under continuous illumination. Herein we present a detailed study of the photokinetics and photoproducts of nucleosides caged with these new protecting groups. Relative to the parent NPPOC group, the light sensitivity of the new photolabile protecting groups is enhanced by up to a factor of 21 at 366 nm and is still quite high at 405 nm, the wavelength at which the sensitivity of the parent compound is practically zero. A new pathway for de-protection of the NPPOC group pro_ceeding through a nitroso benzylalcohol intermediate has been discovered to complement the main mechanism, which involves β elimination. Under standard conditions of lithographic DNA-chip synthesis, some of the new compounds, while maintaining the same chip quality, react ten times faster than the unmodified NPPOC-protected nucleosides.

Full text of DOI:10.1002/chem.200800613

DOI: 10.1002/chem.200800613
Intramolecular Sensitization of Photocleavage of the Photolabile
2-(2-Nitrophenyl)propoxycarbonyl (NPPOC) Protecting Group:
Photoproducts and Photokinetics of the Release of Nucleosides
Dominik Wçll,*[a, b] Julia Smirnova,[a] Marina Galetskaya,[a] Tamara Prykota,[a]
Jochen Bühler,[c] Klaus-Peter Stengele,[c] Wolfgang Pfleiderer,[a] and Ulrich E. Steiner*[a]
Abstract: Novel photolabile protecting
groups based on the 2-(2-nitrophenyl)-
propoxycarbonyl (NPPOC) group with
a covalently linked thioxanthone as an
intramolecular triplet sensitizer exhibit
significantly enhanced light sensitivity
under continuous illumination. Herein
we present a detailed study of the pho-
tokinetics and photoproducts of nu-
cleosides caged with these new protect-
ing groups. Relative to the parent
NPPOC group, the light sensitivity of
the new photolabile protecting groups
is enhanced by up to a factor of 21 at
366 nm and is still quite high at
405 nm, the wavelength at which the
sensitivity of the parent compound is
practically zero. A new pathway for de-
protection of the NPPOC group pro-
ceeding through a nitroso benzylalco-
hol intermediate has been discovered
to complement the main mechanism,
which involves b elimination. Under
standard conditions of lithographic
DNA-chip synthesis, some of the new
compounds, while maintaining the
same chip quality, react ten times
faster than the unmodified NPPOC-
protected nucleosides.
Keywords: DNA chips
· energy
transfer · photochemistry · protect-
ing groups · sensitization
Introduction
age conditions. Even a “photoorthogonality” of photolabile
protecting groups based on their distinction by the wave-
length of the actinic light has been demonstrated.[5] Because
light-induced reactions are usually very fast, photocleavage
by using short light pulses can be achieved on a short time-
scale and the release of the previously “caged” compounds
can be exploited in many ways, for example, as phototrig-
gers for biokinetic studies.[6–8] Furthermore, the exposure to
light can be spatially controlled. This is a prerequisite, for
example, in the synthesis of high-density DNA chips,[9,10] in
which many oligonucleotides of different base sequences are
immobilized on a glass substrate in arrays of up to one mil-
lion spots per square centimeter. With such chips, huge
amounts of genetic information become amenable to mas-
sive parallel detection.[11–14]
Photolabile protecting groups used for DNA-chip synthe-
sis should be removable in high yields, that is, have a low
tendency for side reactions to avoid errors in the oligonu-
cleotide sequence. Furthermore, they should exhibit high
light sensitivity to reduce the probability of light-induced
damage of the chip and to keep the overall processing time
within economic limits. The light sensitivity is determined
by the product of two factors, the quantum yield (f) of the
cleavage reaction and the absorption coefficient (e) at the
Blocking of functional groups by protecting groups and
their deblocking under chemically selective conditions is a
key technique in organic, particularly in bioorganic, synthe-
sis.[1]
A
special advantage of photolabile protecting
groups[2,3] results from their specific photochemical reactivi-
ty that can be triggered by the absorption of light and
makes them “orthogonal”[4] to many other chemical cleav-
[a] Dr. D. Wçll, Dr. J. Smirnova, M. Galetskaya, Dr. T. Prykota,
Prof. Dr. W. Pfleiderer, Prof. Dr. U. E. Steiner
Fachbereich Chemie, Universität Konstanz
Universitätsstraße 10, 78457 Konstanz (Germany)
Fax : (+49)7531-88-3014
E-mail: dominik.woell@uni-konstanz.de
ulrich.steiner@uni-konstanz.de
[b] Dr. D. Wçll
Present address: Laboratory for Photochemistry and Spectroscopy
Katholieke Universiteit Leuven, Celestijnenlaan 200F
3001 Heverlee (Belgium)
[c] Dr. J. Bühler, Dr. K.-P. Stengele
NimbleGen Systems GmbH, Beuthener Str. 2
84478 Waldkraiburg (Germany)
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
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FULL PAPER
wavelength of irradiation. An overview of photoremovable
protecting groups used in DNA-chip synthesis has been
given in a review by Pirrung and Rana.[6] Clearly, com-
pounds with an o-nitrophenylalkyl function play a dominant
role. Currently, the methylnitropiperonyloxycarbonyl
(MeNPOC) group seems to be used most.[15,16] It is of the o-
nitrobenzyl-type in which cleavage occurs at the benzylic
carbon and the leaving moiety is an aromatic o-nitrosocar-
bonyl compound.[17] In photoremovable protecting groups
involving the 2-(2-nitrophenyl)propoxycarbonyl (NPPOC)
group, which was introduced by Pfleiderer and co-work-
ers,[18–20] cleavage is due to b elimination, and formation of
the reactive and problematic nitroso byproduct[21] can be
suppressed by using suitable reaction conditions
(Scheme 1).[22] This is probably the reason why chips of
higher quality result when using the NPPOC group.[23,24]
At 366 nm, the most commonly used mercury line for ir-
in the lowest photoexcited state as a consequence of the in-
creased p-electron density in the aromatic ring. In NPPOC,
the quantum yield is high, but the light sensitivity is limited
by the low value of e. To avoid any compensating effect be-
tween e and f when trying to improve the performance of
NPPOC, we chose sensitization as a way to separate the
process of light absorption from the photochemical reaction,
so that both properties could be independently optimized.
Thioxanthone turned out to be the most suitable sensitizer
for the NPPOC group. It has been shown that intermolecu-
lar sensitization works well in homogeneous solution and on
a chip surface, and that under either conditions the kinetics
can be quantitatively modeled.[25]
When using free sensitizer molecules, diffusion-controlled
encounters between the photoexcited sensitizer and the pho-
toreactive chromophore have to occur during the lifetime of
the excited sensitizer and each photoreactive chromophore
has to find an excited sensitizer. These requirements essen-
tially limit the efficiency of the process. Therefore, we
choose to covalently link the two partners such that the sen-
sitizer moiety can function as an intramolecular antenna for
the photoreactive moiety.[26] The enhancement of photolysis
of a photolabile protecting group by a covalently linked an-
tenna molecule has been reported by Papageorgiou and co-
workers[27,28] and our own
radiation, the light sensitivities ef of MeNPOC (e366nm,MeOH
=
3000mꢀ1 cmꢀ1,
fMeOH =0.03) and NPPOC (e366nm,MeOH
=
250mꢀ1 cmꢀ1, fMeOH =0.41) are very similar. However, it is of
interest to note that the large gain in e caused by the substi-
tution of the aromatic ring in MeNPOC is completely com-
pensated for by a concomitant decrease in the quantum
yield, which indicates a loss of reactivity of the nitro group
group. The syntheses of the
compounds shown in Scheme 2
are described in reference [29].
The letter/number code for
these compounds is of the gen-
eral structure TmLn-OR. The
first letter indicates the antenna
moiety (T for thioxanthone),
the number m gives the posi-
tion on the NPPOC moiety at
which the linker is attached (1–
6 for the aromatic positions, 7
for the benzylic position), the
letter L characterizes the type
of chain that links NPPOC and
the sensitizer (S: saturated
carbon chain, D: single bond
carbon chain including one
double bond, T: single bond
carbon chain including one
triple bond, E: ester bridge),
the number n is the length of
the chain connecting the aro-
matic rings of NPPOC and thio-
xanthone, and throughout this
paper, the protected moiety (R)
is a nucleoside connected to
NPPOC by a carbonate linker.
A preliminary account of the
properties of these compounds
has appeared.[30] Furthermore, a
Scheme 1. Principal cleavage pathways and products of NPPOC-protected substrates.
detailed spectroscopic study of
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D. Wçll, U. E. Steiner et al.
Results and Discussion
The compounds investigated in
this work are shown in
Scheme 2. Their synthesis and
spectroscopic properties are de-
scribed elsewhere.[29,31]
Products after continuous ir-
radiation: For the characteriza-
tion of the light sensitivity of
the new compounds and the
distribution of photoproducts,
continuous irradiation experi-
ments were carried out with
deaerated solutions in methanol
by using the lines at 366 and
405 nm, respectively, of a mer-
cury high-pressure light source.
At 405 nm, the absorption of
the photolabile NPPOC group
is practically negligible. Hence,
photoreactions observed at this
wavelength are almost exclu-
sively due to the sensitization
effect of the thioxanthone an-
tenna. The reaction progress
was followed by HPLC analyses
Scheme 2. NPPOC-conjugates investigated in this work.
after suitable irradiation peri-
ods (see the Supporting Infor-
the mechanism of sensitization that led to new insights into
the photophysics of thioxanthone has also been published.[31]
Herein, the photokinetics and photoproduct formation of
the new compounds under continuous irradiation are de-
scribed in detail.
mation). As an example, in Figure 1 we present the results
for the irradiation of T7S4-O(CO)Thy at 366 nm. As in pre-
vious experiments with NPPOC and free thioxanthone as
the sensitizer,[25] we observe the decay of the peak of the ini-
tial protected thymidine (tR =17.3min) and a concomitant
rise of the peak of free thymidine (tR =6.6 min). The peak at
Figure 1. HPLC chromatograms representing the photoproduct distribution after irradiating T7S4-O(CO)Thy (0.117 mm) in deaerated MeOH for the
times indicated at each trace. Irradiation wavelength=366 nm, I0 =1.75”10ꢀ8 einsteincmꢀ2 sꢀ1
.
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Intramolecular Sensitization of the Photolabile NPPOC Group
FULL PAPER
tR =23.8 min is assigned to the styrene derivative from the
b-elimination reaction (styrene product in Scheme 1). As
observed previously, this peak decreases at longer irradia-
tion times due to subsequent transformations of this prod-
uct.[22,25,32] The peak at tR =20.8 min is assigned to a nitroso
product that originates from the primary aci-nitro intermedi-
ate by a reaction pathway competing with b elimination
(Scheme 1).[22] This pathway primarily results in formation
of a nitroso product (nitroso product 1 in Scheme 1). As a
detailed structural analysis has shown, an intramolecular re-
arrangement induced by the electrophilic attack of the
newly formed benzylic OH group at the carbon center of
the carbonate linker gives rise to nitroso product 2 (see
Scheme 1). Whether the latter reaction occurs, and to what
extent, depends on the solvent. It could be shown that in
acetonitrile the irradiation of T7S4-O(CO)Thy results
mainly in the formation of nitroso product 1 (peak at tR =
16.4 min in Figure S4 in the Supporting Information), where-
as in methanol nitroso product 2 is formed (peak at tR =
20.8 min in Figure 1).
The five species in Scheme 1 (protected nucleoside, free
nucleoside, styrene derivative, nitroso product 1, nitroso
product 2) are sufficient to account for the essential features
of the photochemical product distribution for all compounds
investigated except for T7D4 and T7T4 in which a double
or a triple bond, respectively, is present in the linker chain;
these compounds will be discussed in further detail below.
Photokinetic analysis, yields, quantum yields, and light
sensitivity: The disappearance of the starting material upon
irradiation gives a qualitative impression of the light sensi-
tivity of the compounds investigated. Pertinent photokinetic
curves for irradiation at 366 and 405 nm are shown in
Figure 2.
In comparison with the parent NPPOC compound it is
evident that an enormous enhancement of the light sensitivi-
ty is achieved through the intramolecular attachment of a
thioxanthone antenna. Even at 405 nm, the wavelength at
which the NPPOC compounds are not photosensitive at all
(curve not shown), the new thioxanthone/NPPOC conju-
gates show high sensitivity.
Figure 2. Decay kinetics of thioxanthone/NPPOC-protected thymidine
under continuous irradiation at 366 (top) and 405 nm (bottom). For
better comparability of the kinetic curves measured at different irradia-
tion light intensities, the remaining fraction of starting material is plotted
against the dose of light t”I0. The lines shown represent kinetic fits ac-
cording to Equation (1). Within the light-dosage range shown, no signifi-
cant reaction was observed for NPPOC-Thy at 405 nm.
The photokinetic data were analyzed according to the
rate law given as Equation (1):
the starting material was also used to extrapolate the HPLC
data points for the concentration of deprotected thymidine
to infinite irradiation time. The evaluated quantum yields
(f) and nucleoside deprotection yields (YNucl) are listed in
Table 1. We also quote the values of the product ef, which
is the appropriate value of merit for comparing the light
sensitivities of the various compounds.
dc
dt
Fd ð1ꢀ10ꢀAðtÞ
Þ
ð1Þ
¼ ꢀI0
ecðtÞꢀ
V
AðtÞ
in which I0 represents the photon irradiance, F is the illumi-
nated cross section, d is the optical path length, V is the
volume of the solution, A(t) is the absorbance of the solu-
tion after irradiation time t, e is the molar absorption coeffi-
cient of the starting compound, c is its concentration, and f
is the sum of the quantum yields of all photoreactions in-
volving the NPPOC chromophore. The quantum yield (f)
was determined by fitting the result of the numerical inte-
gration of Equation (1) to the observed time dependence of
c(t) (Figure 2). The simulated curve for concentration c(t) of
By comparing the values of ef as the benchmark figures
of light sensitivity for the antenna bearing new compounds
with the values for the unsensitized reaction of NPPOC-
Thy, it is clear that a considerable enhancement is achieved
by intramolecular sensitization. For irradiation at 366 nm, a
maximum enhancement factor of 21 is achieved with T5S0-
O(CO)Thy. For irradiation at 405 nm, where the sensitivity
of the parent NPPOC compound is almost zero, somewhat
lower sensitivities than at 366 nm, but still higher than for
NPPOC at 366 nm, are achieved. This is particularly useful
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D. Wçll, U. E. Steiner et al.
Table 1. Quantum yields (f), light sensitivities (ef), and nucleoside
yields (YNucl) of protected nucleosides for irradiation in deaerated metha-
nol.
assigned to the HPLC chromatograms: the free nucleoside
at retention times of approximately 8 min, nitroso product 1
at retention times slightly earlier than the protected nucleo-
sides, nitroso product 2 at approximately 21 min, and the
styrene product at approximately 24 min. Because nitroso
product 2 and the styrene product are independent of the in-
itial protected nucleoside, their UV-visible spectra are iden-
tical in all cases. In contrast, the UV-visible spectrum of ni-
troso product 1 shows a signature of the investigated nucleo-
side.
Irradiation at 366 nm
Irradiation at 405 nm
Compound
f
ef[a]
YNucl
f
ef[a]
YNucl
NPPOC-Thy
0.41
0.18
0.21
0.21
0.08
0.42
0.09
0.26
0.29
0.36
100
690
820
800
250
1500
360
2100
2400
2900
920
0.90
0.60
0.75
0.86
0.27
0.21
0.92
0.66
0.61
0.52
0.52
0.41
0.09
0.14
0.14
0.05
0.38
0.09
0.27
0.23580
0.24
0.06
7
T7S2-O(CO)Thy
T7S3-O(CO)Thy
T7S4-O(CO)Thy
T7D4-O(CO)Thy
T7T4-O(CO)Thy
T4E2-O(CO)Thy
T7S0-O(CO)Thy
T7S0-O(CO)CytAc
T7S0-O(CO)AdeBz
160
320
320
170
1000
160
650
0.63
580
160
0.63
0.79
0.75
0.38
0.33
0.82
0.89
The light sensitivity of protected deoxyguanosine amidite
is less than half of the value of the other protected nucleo-
sides, as shown in Figure 3and Table 1. The reason for this
0.54
0.64
T7S0-O(CO)GuaAm 0.11
[a] in mꢀ1 cmꢀ1
because diode lasers at this wavelength have become com-
mercially available.
Whereas the increase in e is the determining factor in the
sensitivity gain by the introduction of an antenna molecule,
a general decrease in the photochemical quantum yield
could not be avoided, but was kept within a modest range of
1
about a factor of = relative to direct excitation of NPPOC.
2
(T7D4-OR and T7T4-OR should not be considered because
other photochemical primary reactions, which do not lead to
a release of the protected substrate, must also be taken into
account; see below). It is not yet clear, whether the reduc-
tion in quantum yield is due to a lower efficiency of forma-
tion of the aci-nitro intermediate or to an increased reversi-
bility of the formation of this species. This question must be
left open for further studies. The photochemical quantum
yields are fairly independent of the excitation wavelength
Figure 3. Decay kinetics of thioxanthone/NPPOC-protected nucleosides
under continuous irradiation at 366 nm. The lines represent kinetic fits
according to Equation (1).
except for compounds T7Sn-O(CO)Thy with short aliphatic
1
linkers (n=2 to 4), for which a drop by a factor of = is ob-
2
served between 366 and 405 nm.
is a decrease in the deprotection quantum yield due to com-
peting electron transfer from the deoxyguanosine to the
thioACHTRExUNG anthone moiety. This electron transfer is reversible and
For practical purposes, the total yield of release of depro-
tected substrate is at least as important as the light sensitivi-
ty. With the new compounds, these yields are between 52
and 92%. It should be noted, however, that under reaction
conditions for the synthesis of DNA chips, the yields can be
different and, of course, reaction conditions can be further
optimized for maximizing this quantity. For example, the
yield of thymidine release from NPPOC protected thymi-
dines has been reported to be as high as 98% on a chip,[25]
whereas under the conditions of our experiments the yield is
only 90%.
Experiments with other nucleosides: Thymidine has
served as the standard nucleoside for most of our investiga-
tions. To demonstrate the principal suitability of the new
type of antenna bearing protecting groups, the T7S0 conju-
gates were also synthesized for deoxyadenosine, deoxycyti-
dine, and deoxyguanosine, which also have a further protect-
ing group at their amino position (for structures, see
Scheme 2). The products formed after irradiation were simi-
lar for all nucleosides (see Figures S8–S11 in the Supporting
Information). The structures of the four main products are
results in a recovery of the reactants. Experiments for inter-
molecular quenching of thioxanthone by deoxyguanosine
amidite gave a bimolecular rate constant of 2.3”109 mꢀ1 sꢀ1
(see the Supporting Information).
Due to the reduced quantum efficiency for the deprotec-
tion of deoxyguanosine amidite, the gain in light sensitivity
when using the new photolabile protecting groups is less
pronounced than for the other nucleosides. However, we be-
lieve that by using a different protecting group for the gua-
nine amino function, the oxidation potential of the nucleo-
side could be reduced and thus, electron transfer would be
avoided or at least reduced in its efficiency.
Photokinetics of the T7D4-group: In the case of the
T7D4 group, energy transfer to the NPPOC chromophore
and subsequent photorelease of the substrate has to com-
pete with a very efficient photochemical trans/cis isomeriza-
tion. The situation is described in Scheme 3.
The time profiles of the species observed when irradiating
T7D4-O(CO)Thy is shown in Figure 4.
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Intramolecular Sensitization of the Photolabile NPPOC Group
FULL PAPER
figures ef of the corresponding processes, an example of
which is given in Equation (5) (see Scheme 3).
Fd
V
k0T ¼
ln 10etransꢀT ¼ 0:7675etransꢀT
ð5Þ
Scheme 3. Photoisomerization and photoreaction of trans- and cis-T7D4-
O(CO)Thy.
in which F is the illuminated area, d is the thickness of the
cuvette, V is the illuminated volume, etrans is the absorption
coefficient of the trans isomer, and fT is the quantum yield
of sensitized NPPOC triplet formation of the trans isomer.
The definitions of k0C, kT0 C, and kC0 T are analogous.
The light sensitivity figures for these processes are given
in Table 2. The values confirm that the isomerization be-
Table 2. Light sensitivity values (ef) for the processes of T7D4 shown in
Scheme 3for irradiation in MeOH at 366 or 405 nm, respectively.
etransfTC
ecisfCT
etransfT
ecisfC
[mꢀ1 cmꢀ1
]
[mꢀ1 cmꢀ1
]
[mꢀ1 cmꢀ1
]
[mꢀ1 cmꢀ1
]
366 nm
deaerated
366 nm
aerated
405 nm
630
680
420
620
760
440
130
170
110
170
150
110
deaerated
&
Figure 4. HPLC-derived concentrations of trans-T7D4-O(CO)Thy ( ),
~
*
cis-T7D4-O(CO)Thy ( ), and thymidine ( ) during irradiation at 366 nm
in deaerated MeOH.
tween the trans and cis form is more efficient than the re-
lease of thymidine. Therefore, the photostationary equilibri-
um between the trans and cis form of T7D4-O(CO)Thy is
reached before most of the substrate has been deprotected,
and the quantum yield of release is decreased by a factor of
around three relative to T7S4-O(CO)Thy, which is the cor-
responding compound with a saturated linker.
Photochemistry of the T7T4-group: In case of the T7T4-
group, the triple bond gives rise to the formation of an
isomer, T7T4!P2, of the starting compound, presumably
through the cycloaddition product, T7T4!P1, as represent-
ed in Scheme 4 (see the Supporting Information for details
of the structural assignment of compound T7T4!P2).
Chip experiments: In preliminary experiments, the new
photolabile protecting groups were also tested under condi-
tions for real chip production. For the synthesis of an oligo-
thymidine test chip (Figure 5), the protected thymidines
were converted into their corresponding phosphoramidites
by established procedures.[33] The stepwise yield for extend-
The kinetics of the observed compounds can be modeled
by differential Equations (2) to (4) :
d½transŠ
¼ ꢀk0T½transŠꢀkT0 C½transŠþkC0 T½cisŠ
ð2Þ
ð3Þ
ð4Þ
dEhn
d½cisŠ
dEhn
¼ k0TC½transŠꢀkC0 T½cisŠꢀk0C½cisŠ
d½ThymidineŠ
dEhn
¼ ðk0T½transŠþk0C½cisŠÞhThymidine
in which [trans], [cis], and [Thymidine] are the concentra-
tions of the compounds indicated, Ehn is the dose of light
[einsteincmꢀ2], and hThymidine is the efficiency of thymidine
release after population of the NPPOC triplet. The effective
rate constants are related to the pertinent light sensitivity
Scheme 4. Dominant photochemical reaction pathway for T7T4-O(CO)Thy.
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D. Wçll, U. E. Steiner et al.
Conclusion
A novel class of photoremovable protecting groups based
on the NPPOC group enhanced by a thioxanthone chromo-
phore covalently linked as an intramolecular antenna has
been presented. Although the quantum yield of the photo-
cleavage of NPPOC is somewhat smaller in the thioxanthon/
NPPOC conjugates, the light sensitivity of the new com-
plexes, as indicated by ef, is significantly higher. Enhance-
ment factors of about 20 at 366 nm and about 140 at 405 nm
have been observed. Unless there are other photoreactive
functions, such as those introduced by the linkers with
double or triple bonds, the yield of photorelease of the
caged substrate is high (52–92%). The new protecting
groups react via the intermediates known from unsensitized
NPPOC photochemistry to give nitrostyrene and/or nitroso-
benzene derivatives. As shown in this work for the first
time, however, the nitroso product can continue reacting. It
undergoes an intramolecular re-esterification, which results
in deprotection of the nucleoside. All four nucleosides show
similar chemical yields and products. However, the gain in
light sensitivity for deoxyguanosine amidite is less due to
thioxanthone triplet quenching by intramolecular reversible
electron transfer from the nucleoside.
The new protecting groups were tested under standard
conditions of photolithographic DNA-chip synthesis. Similar
chip quality as those produced with NPPOC could be ach-
ieved in 10-fold shorter irradiation times.
Figure 5. Synthesis of oligothymidines (1–12 nucleotides when going from
the top to the bottom) on a chip with the new protecting groups T7S4
(top) and T7S0 (bottom), as described in reference [15]. After final de-
protection, the free OH groups on each spot are treated with the fluores-
cence label reagent Cy3-amidite. A section of the chip produced is shown
together with the fluorescence intensity along one vertical line of spots.
The irradiation time per cycle was approximately 5 s in DMSO. T indi-
cates the number of thymidines in the oligothymidine chains produced.
Finally, it is worth noting that the photosensitivity of the
new compounds is only slightly affected by the presence of
oxygen because of the short triplet lifetime of the thioxan-
thone moiety in the proximity of the NPPOC chromophore.
For practical applications, this represents another advantage
because the reaction systems need not be operated under
air-free conditions. In conclusion, it has been shown that in-
tramolecular sensitization is an efficient strategy for improv-
ing the light sensitivity of photolabile protecting groups.
This is of particular interest in applications in which, for sci-
entific or economic reasons, short irradiation times are es-
sential.
ing the oligonucleotide chain by one thymidine was deter-
mined by using the fluorescence labeling method described
in references [15] and [25]. In summary, a series of features
composed of oligonucleotides of different length ranging
from one to twelve bases was grown by employing the cor-
responding number of illumination and reaction cycles.
After synthesis of the oligonucleotides, all of the features
were deprotected, and Cy3–amidite was coupled to the ter-
minal hydroxyl groups. The resulting fluorescence image
represents a relative measure of the remaining hydroxyl
groups.
The irradiation times are approximately ten times shorter
than those usually applied for NPPOC. In both cases, T7S4
(Figure 5, top) and T7S0 (Figure 5, bottom), a yield of ap-
proximately 90% was obtained. Conditions were not opti-
mized, but we believe that with optimization almost quanti-
tative yields are possible. This is underlined by the fact that
no significant changes in quality or yields were observed in
comparable experiments with the commercial NPPOC
group.
Experimental Section
NMR and MS spectra: NMR spectra were measured with a JEOL GX
400 FT-NMR or a Bruker DRX 600 spectrometer. EI and FAB mass
spectra were recorded with a Varian MAT 312 mass spectrometer. The
FAB ion source was from AMD.
Continuous irradiation in solution:
A high-pressure mercury lamp
(Osram HBO 200) was used. The light was passed through a heat filter
(optical length 5 cm, filled with a saturated aqueous solution of CuSO4),
a collimating lens, an electronic shutter, a 366 nm interference filter
(Schott), and a thermostated optical cell holder with a cell of 1 cm path
length (Hellma QS) equipped with a magnetic stirrer bar. The light flux
was determined by azobenzene actinometry.[34] Typical values were
around 3”10ꢀ8 einsteincmꢀ2 sꢀ1. The cell was filled with the probe solu-
tions (3mL) and sealed by a rubber septum. It was purged with nitrogen
for 15 min and irradiated for fixed intervals of time. UV/Vis absorption
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Chem. Eur. J. 2008, 14, 6490 – 6497

Intramolecular Sensitization of the Photolabile NPPOC Group
FULL PAPER
spectra were recorded before and after irradiation. Reactants and prod-
ucts were separated and quantified by HPLC.
[14] DNA Microarrays: A Molecular CloningManual (Eds.: D. Bowtell,
J. Sambrook), Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY, 2003.
HPLC: HPLC measurements were carried out on
a Merck-Hitachi
[15] G. H. McGall, A. D. Barone, M. Diggelsmann, S. P. A. Fodor, E.
Gentalen, N. Ngo, J. Am. Chem. Soc. 1997, 119, 5081–5090.
[16] 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.
[17] F. Bley, K. Schaper, H. Gçrner, Photochem. Photobiol. 2008, 84,
162–171.
HPLC system, including a LiChrospher 100 RP-18 (5 mm) column, a L-
7100 pump, a L-7200 autosampler, a L-7450A UV diode array detector,
and a L-7000 interface by using the “HSM-manager” software. A gradi-
ent from pure water to pure acetonitrile was used.
[18] 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.
[19] H. Giegrich, S. Eisele-Bühler, C. Hermann, E. Kvasyuk, R. Charu-
bala, W. Pfleiderer, Nucleosides Nucleotides 1998, 17, 1987–1996.
[20] S. Bühler, I. Lagoja, H. Giegrich, K.-P. Stengele, W. Pfleiderer, Helv.
Chim. Acta 2004, 87, 620–659.
Acknowledgements
This work was carried out within Research Unit 434 (Oligosaccharide
and DNA chips—recognition of secondary gene products) funded by the
Deutsche Forschungsgesellschaft (DFG Ste 283/7).
[21] L. Krçck, A. Heckel, Angew. Chem. 2005, 117, 475–477; Angew.
Chem. Int. Ed. 2005, 44, 471–473.
[22] S. Walbert, W. Pfleiderer, U. E. Steiner, Helv. Chim. Acta 2001, 84,
1601–1611.
[1] T. W. Greene, P. G. M. Wuts, Protective Groups in Organic Synthesis,
Wiley, New York, 1999.
[2] C. G. Bochet, J. Chem. Soc. Perkin Trans. 1 2002, 125–142.
[3] A. P. Pelliccioli, J. Wirz, Photochem. Photobiol. Sci. 2002, 1, 441–
458.
[4] R. B. Merrifield, G. Barany, W. L. Cosand, M. Engelhard, S. Mojsov,
Pept. Proc. Am. Pept. Symp. 5th 1977, 488–502.
[5] C. G. Bochet, Synlett 2004, 13, 2268–2274.
[6] M. C. Pirrung, V. S. Rana in Dynamic Studies in Biology: Phototrig-
gers, Photoswitches, and Caged Compounds (Eds.: M. Goeldner,
R. S. Givens), Wiley, New York, 2005, 341–368.
[7] J. A. McCray, D. R. Trentham, Annu. Rev. Biophys. Biophys. Chem.
1989, 18, 239–270.
[23] M. Beier, J. D. Hoheisel, Nucleic Acids Res. 2000, 28, e11, i-vi.
[24] J. D. Hoheisel, F. Diehl, M. Scheideler, N. Hauser, V. Aign, S. Maty-
siak, M. Beier in Novel Approaches in Biosensors and Rapid Diag-
nostic Assays (Eds.: Z. Liron, A. Brohmberg, M. Fisher), Kluwer
Academic/Plenum Publishers, London, 2001, 165–172.
[25] D. Wçll, 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.
[26] U. E. Steiner, D. Wçll, (Universität Konstanz), DE 10315772(A1),
WO 2004089529, 2004; [Chem. Abstr. 2004, 141, 366034]
[27] G. Papageorgiou, D. Ogden, E. T. Corrie, J. Org. Chem. 2004, 69,
7228–7233.
[8] V. Hagen, B. Dekowski, N. Kotzur, R. Lechler, B. Wiesner, B.
Briand, M. Beyermann, Chem. Eur. J. 2008, 14, 1621–1627.
[9] R. J. Lipshutz, S. P. A. Fodor, T. R. Gingeras, D. J. Lockhart, Nat.
Genet. 1999, 21, 20–24.
[28] G. Papageorgiou, M. Lukeman, P. Wan, J. E. T. Corrie, Photochem.
Photobiol. Sci. 2004, 3, 366–373.
[29] J. Smirnova, D. Wçll, W. Pfleiderer, U. E. Steiner, Helv. Chim. Acta
2005, 88, 891–904.
[10] M. C. Pirrung, Angew. Chem. 2002, 114, 1326–1341; Angew. Chem.
Int. Ed. 2002, 41, 1276–1289.
[30] D. Wçll, J. Smirnova, W. Pfleiderer, U. E. Steiner, Angew. Chem.
2006, 118, 3042–3045; Angew. Chem. Int. Ed. 2006, 45, 2975–2978.
[31] D. Wçll, S. Laimgruber, M. Galetskaya, J. Smirnova, W. Pfleiderer,
B. Heinz, P. Gilch, U. E. Steiner, J. Am. Chem. Soc. 2007, 129,
12148–12158.
[32] H. Giegrich, PhD thesis, Universität Konstanz 1996.
[33] W. Bannwarth, A. Trzeciak, Helv. Chim. Acta 1987, 70, 175–186.
[34] G. Gauglitz, J. Photochem. 1976, 5, 41–47.
[11] For an overview, see: Nat. Genet. Suppl. 1999, 21, 1–60.
[12] D. G. Wang, J.-B. Fan, C.-J. Siao, A. Berno, P. Young, R. Sapolsky,
G. Ghandour, N. Perkins, E. Winchester, J. Spencer, L. Kruglyak, L.
Stein, L. Hsie, T. Topaloglou, E. Hubbell, E. Robinson, M. Mitt-
mann, M. S. Morris, N. Shen, D. Kilburn, J. Rioux, C. Nusbaum, S.
Rozen, T. J. Hudson, R. Lipshutz, M. Chee, E. S. Lander, Science
1998, 280, 1077–1082.
Received: April 2, 2008
[13] D. J. Lockhart, E. A. Winzeler, Nature 2000, 405, 827–836.
Published online: June 9, 2008
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