Photocleavage of peptides and oligodeoxynucleotides carrying 2-nitrobenzyl groups

Article abstract of DOI:10.1002/hlca.200800361

Peptides and oligodeoxynucleotides containing photolabile 2-nitrobenzyl groups as mid-sequences were prepared. Photocleavage of aqueous solutions of these compounds neared completion within 30 min to a few hours depending on the photolabile group used. A

Full text of DOI:10.1002/hlca.200800361

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Photocleavage of Peptides and Oligodeoxynucleotides Carrying 2-Nitrobenzyl
Groups
by Roger Ramosa), Brendan Manninga), Anna Avin˜o´ a), Raimundo Gargallob), and Ramon Eritja*a)
a) Institute for Research in Biomedicine, IQAC-CSIC, CIBER-BBN Networking Center on
Bioengineering, Biomaterials, and Nanomedicine, Edifici Helix, Baldiri Reixac 15, E-08028 Barcelona
(phone: þ 34-934039942; fax: þ 34-932045904; e-mail: recgma@cid.csic.es)
b) Department of Analytical Chemistry, University of Barcelona, Diagonal 647, E-08028 Barcelona
Peptides and oligodeoxynucleotides containing photolabile 2-nitrobenzyl groups as mid-sequences
were prepared. Photocleavage of aqueous solutions of these compounds neared completion within
30 min to a few hours depending on the photolabile group used. A photolabile group was introduced in
the loop of an intramolecular oligodeoxynucleotide hairpin. Melting curves of the hairpin with and
without the complementary oligodeoxynucleotide showed a preference for the intramolecular hairpin
form, but an intermolecular duplex was observed after photolysis. These results open the possibility of
using photolabile DNA hairpins for the fabrication of patterned surfaces.
Introduction. – Photolysis has been used extensively in organic chemistry and
biochemistry. Several molecular groups are light-sensitive, and this property has been
exploited to mask groups that are to be liberated at a particular time. 2-Nitrobenzyl
groups are some of the most widely studied photolabile groups [1 – 2]. In the photolysis
of 2-nitrobenzyl derivatives, the NO2 group is reduced to NO, and the benzyl CH2
group is oxidized to C¼O (Scheme 1) [3 – 4]. Several derivatives of the 2-nitrobenzyl
group have been used for the protection of amines, alcohols, carboxylic acids,
imidazoles, phenols, and phosphates [2][5]. In some cases, low yields attributable to the
reaction of the nitroso aldehyde with the amino groups have been reported [5]. The
introduction of a Me group in the benzylic position accelerates photolysis and
minimizes secondary reactions due to the formation of a methyl ketone that is less
reactive than the aldehyde group [2][5]. The 2-nitrobenzyl group has also been used as
a photolabile link between peptide and the solid support in solid-phase synthesis [6 – 8].
The photolytic cleavage of the peptide supports allows the isolation of protected
Scheme 1. Synthesis of a Photolabile Linker Used for the Introduction of 2-Nitrobenzyl Groups in
Peptides
ꢀ 2009 Verlag Helvetica Chimica Acta AG, Zꢁrich

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peptide segments for use in the synthesis of longer peptides [7][9]. Moreover,
photolabile groups are also used to analyze drug – protein interactions. In this case,
masked or caged drug derivatives are synthesized and delivered to cells. Fast
photoremoval of the masking groups allows the study of the in vivo behavior of the
drug [10]. Finally, photolabile groups are used for the protection of the 5’-end of
nucleoside derivatives in the preparation of DNA chips. This technique allows the
parallel synthesis in a single chip of thousands of 20 – 30mer fragments corresponding
to a complete chromosome [11].
Here, we report the synthesis and photolysis of synthetic biomolecules (peptides
and oligodeoxynucleotides) carrying a photolabile link in the middle of their sequence.
The ultimate aim of this work is to link these photolabile molecules onto surfaces to
obtain functionalized surfaces. Photolithography of these surfaces will result in the
cleavage of the biomolecules in the illuminated area, while, in the masked area, the
biomolecules will remain intact. Utilizing the self-assembling properties of these
photolabile biomolecules will enable selective binding of various molecules to the
cleaved or uncleaved biomolecules in the photolyzed and unphotolyzed areas,
respectively.
Results and Discussion. – 1. Synthesis of Photolabile Linkers. First, an amino acid
derivative carrying 2-nitrobenzyl was prepared (Scheme 2). We selected a derivative of
4-(aminomethyl)-2-nitrobenzoic acid described in [8][12][13]. The synthesis of this
compound was achieved according to the literature [12][13] with small changes. First,
the NH2 group of 4-(aminomethyl)benzoic acid (1) was protected by the acid-stable
trifluoroacetyl (TFA) group using ethyl trifluoroacetate (Scheme 2). Nitration of the
resulting compound led to excellent yields of the TFA derivative, 3, of 4-(amino-
methyl)-2-nitrobenzoic acid, which was subsequently hydrolyzed with NH3 to give
compound 4. Finally, the (9H-fluorenyl-9-methoxycarbonyl (Fmoc) derivative, 5, of 4-
(aminomethyl)-2-nitrobenzoic acid was prepared. This compound is suitable for the
incorporation of 2-nitrobenzyl groups in peptides using solid-phase peptide protocols.
Scheme 2. Synthesis of the Photolabile Linker Needed for the Incorporation of 2-Nitrobenzyl Linkers in
Peptides
2. Synthesis and Photolysis of Peptides Carrying Photolabile Linkers. The peptide
sequence TyrAla-6-PheLysGly (Scheme 3) was prepared. Standard Fmoc chemistry
and solid-phase protocols were applied. The desired peptides were purified by
reversed-phase (RP) HPLC and characterized by mass spectrometry.

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Scheme 3. Structure of Photolabile Linkers 6 and 7 and Hydrolysis Product Found During the
Purification of the DMT-Oligonucleotide Hairpin
Aqueous solutions of the peptide sequence were irradiated with a Black Eye lamp
(340 nm). The progress of the reaction was followed by HPLC/MS analysis of aliquots
taken at different times. Photolysis of the peptide was nearly complete after 2 h of
irradiation (Fig. 1). The N-terminal fragment without the 2-nitrosobenzaldehyde group
was identified at the beginning of the chromatogram, but the tripeptide carrying the 2-
nitrosobenzaldehyde group was not observed. Instead, several small peaks appeared
along the chromatogram. It has been reported that the 2-nitrosobenzaldehyde group
may further react with amino groups [5][14]. This side reaction prevents the isolation
of the moiety carrying the 2-nitrosobenzaldehyde group.
3. Synthesis and Photolysis of a Model Oligodeoxynucleotide Carrying a Photolabile
Linker. Next, an oligodeoxynucleotide carrying a photolabile group mid-sequence was
prepared. Oligodeoxynucleotide sequence A, 5’-d(CCCCC-6-TTTT), was prepared on
a 1-mmol scale. The phosphoramidite 8 shown in Fig. 2 was used to introduce the
photolabile 2-nitrobenzyl group at the midpoint of the sequence. This phosphoramidite
has a Me group at the benzyl position, which increases the reaction rate and prevents
side reactions, as the methyl ketone group resulting from photolysis is less reactive than
the aldehyde group. This compound 8 was obtained from commercial sources. After
ammonia deprotection, the resulting oligodeoxynucleotide was purified by RP-HPLC.
Oligodeoxynucleotide A eluted at 10.2 min (Fig. 3) as a broad double peak. The double
peak was attributed to the presence of two diastereoisomers on the phosphoramidite
used. The purified compound was characterized by mass spectrometry (MALDI). In
addition to the expected mass, we observed two peaks corresponding to the
fragmentation of the oligodeoxynucleotide (T4 and C5-photolabile) that occurred
during the acquisition of the spectra.
Oligodeoxynucleotide A was photolyzed with a Black Eye lamp (340 nm) for
30 min at room temperature. Fig. 3 shows the HPLC profile of samples taken at the
start of the photolysis and after 30 min of photolysis. Photolysis was almost completed

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Fig. 1. Photolysis of peptide H-NTyr-Ala-6-Phe-Lys-GlyC-OH. HPLC Profiles of samples at the start (t ¼
0) and after 120 min of photolysis.
Fig. 2. Phosphoramidite derivative 8 used for the incorporation of 2-nitrobenzyl linkers in oligonucleo-
tides

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Fig. 3. Photolysis of oligonucleotide 5’-CCCCC-7-TTTT-3’ (Sequence A). HPLC Profiles of samples at
the start (t ¼ 0) and after 30 min of photolysis.
after 30 min, as judged by the disappearance of the starting oligodeoxynucleotide. As
the peak corresponding to sequence A disappeared, a new peak at 9 min was observed,
which was identified as T4 by HPLC analysis of the enzyme digestion, giving thymidine
as a single compound. The product containing five cytosines and the 2-nitrosophenyl
ketone group was not isolated. We speculate that the photolabile C5-moiety undergoes
further reactions, yielding several compounds. The presence of the Me group at the
benzyl position accelerated the photolysis, but it did not prevent the formation of side-
products in the fragment carrying the 2-nitrosophenyl ketone group.
4. Synthesis and Photolysis of an Oligodeoxynucleotide Hairpin Carrying a
Photolabile Linker at the Apex of the Loop. Hairpin oligodeoxynucleotide sequence
B, 5’-d(CTCAATGACTCGTT-7-TTCGAGTCATTGAGTCATTTTT)-3’, was pre-
pared. The oligonucleotide has a self-complementary sequence of 15 base pairs linked
by a tetrathymidine loop. In the middle of the loop, there is the photolabile 2-
nitrobenzyl group (cf. 7). This hairpin was designed to be immobilized to SiO2 surfaces

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in forthcoming experiments. For this reason, there are five thymidines at the 3’-end that
will be used as a spacer to separate the hairpin from the surface.
After the assembly of the sequence and ammonia deprotection, the resulting
oligonucleotide with the dimetoxytrityl (DMT) group was purified by RP-HPLC and
characterized by mass spectrometry. During HPLC purification, a minor peak (20% of
the desired product) was observed corresponding to an extra oligonucleotide sequence
with the DMT group. Mass spectrometry showed that the minor DMT-oligonucleotide
sequence was a hydrolysis product of the desired compound near the photolabile site
(Fig. 2). This indicates that the photolabile linker is not totally stable under ammonia-
deprotection conditions, but the desired full-length compound is easily separated from
the hydrolysis compound. Photolysis was performed as described for the short
oligonucleotide sequence, leading to similar results (Fig. 4). Most of the starting
material disappeared after 30 min. This time, a small peak was observed after the
starting hairpin that was assigned to the 5’-fragment with the rest of the photolabile
group. Denaturing gel electrophoresis confirmed the fragmentation of the hairpin and
the formation of two bands of 14 and 24 bases.
Fig. 4. Photolysis of oligonucleotide hairpin B. HPLC Profiles of samples at the start (t ¼ 0) and after
30 min of photolysis.
Melting curves of the hairpin oligodeoxynucleotide (sequence B) with and without
the presence of its complementary sequence (sequence C) were performed before and
after photolysis. The hairpin shows a single duplex-to-random-coil transition with a
melting temperature at 758 (Fig. 5,a). The transition at 758 was also the major

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Fig. 5. Melting curves of oligonucleotide hairpin B. a) Hairpin alone before and after photolysis; b)
hairpin in the presence of complementary oligodeoxynucleotide (sequence C) before and after
photolysis. Conditions: 0.15m NaCl, 1 mm MgCl2, and 10 mm Na3PO4 pH 7.13.
transition of the hairpin in the presence of an equimolar amount of its complementary
sequence (Fig. 5,b).
After 30 min of photolysis, the melting curve was repeated. This time, the transition
was observed at a lower melting temperature (Tm ¼ 508, Fig. 5,a and b), indicating that
the intramolecular duplex was more stable than the intermolecular duplex. We are now
examining the use of this difference in stability to fabricate patterns on surfaces. To this
end, we plan to immobilize oligodeoxynucleotide hairpins carrying photolabile groups
on surfaces and create patterns of double-stranded and single-stranded DNA
sequences by photolysis. Hybridization with complementary oligodeoxynucleotide
conjugates is expected to allow selective deposition of nanomaterials in the areas of
single-stranded DNA resulting from the photolysis.

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Conclusions. – We have demonstrated that photolabile 2-nitrobenzyl groups can be
introduced into the middle of peptide and oligodeoxynucleotide derivatives, leading to
stable derivatives that can be purified and characterized according to standard solid-
phase synthesis protocols. Photocleavage of these compounds in aqueous solutions is
achieved in minutes or in hours depending on the presence or absence of a Me group in
the benzylic position. After photolysis, the moiety of the molecule carrying the
resulting NO derivative is not easily found, probably due to further reactions of the
resulting NO derivative [5][14]. In contrast, the moiety of the molecule without the
NO derivative is clearly observed and identified.
An oligodeoxynucleotide hairpin carrying a photolabile group in the loop has been
prepared. The modified hairpin has a very stable double-stranded form, as evidenced
by melting experiments. In the presence of the complementary strand, the modified
hairpin maintains the entropy-favored intramolecular form, as judged by the analysis of
the melting curve. Photolysis of the hairpin generates two single-stranded molecules
that have a lower melting temperature than the hairpin.
These results set the basis for the use of photolabile DNA hairpins in the
manufacture of patterned surfaces by photolithography. Further work in this direction
is in progress.
´
´
We thank Dr. Sonia Varon and Dr. Miriam Royo from the Combinatorial Chemistry Unit at PCB for
their help in the HPLC-MS experiments. This study was supported by the Institute for Research in
Biomedicine (IRB Barcelona), the Spanish Ministry of Education (BFU2007-63287), Generalitat de
Catalunya (2005/SGR/00693), the Instituto de Salud Carlos III (CIBER-BNN, CB06_01_0019), and the
European Community (NANO-3D NMP4-CT2005-014006).
Experimental Part
General. Phosphoramidites and ancillary reagents used during oligodeoxynucleotide synthesis were
from Applied Biosystems (PE Biosystems Hispania S.A., Spain). The photolabile phosphoramidite 8 was
from Link Technologies (Link Technologies Ltd., Scotland). The Fmoc-amino acids were from
Novabiochem (Novabiochem GmbH, Switzerland). The rest of the chemicals were purchased from
Aldrich, Sigma, or Fluka (Sigma-Aldrich Quꢀmica S.A., Spain).
4-{[(Trifluoroacetyl)amino]methyl}benzoic Acid (2). 4-(Aminomethyl)benzoic acid (1; 3 g,
20 mmol) was reacted with CF3COOEt (4.2 ml) in 20 ml of EtOH containing 4.2 ml of Et3N at r.t.
overnight [13]. The mixture was concentrated to dryness, and the resulting residue was dissolved in hot
H2O (70 ml). The product precipitated on cooling, and a few drops of CF3COOH (TFA) were added,
which induced more abundant precipitation. The desired product was isolated by filtration to yield 4.37 g
(18 mmol, 90%) of 2. White solid. Physical and spectroscopic properties were as described in [12][13].
2-Nitro-{[(trifluoroacetyl)amino]methyl}benzoic Acid (3). Compound 2 (18 mmol) was dissolved in
95% conc. H2SO4 (60 ml), and the soln. was cooled on ice. To the cooled soln., a mixture of conc. H2SO4
(2 ml) and 68% HNO3 (1.6 ml) was added dropwise. After the addition, the mixture was allowed to warm
to r.t. The resulting mixture was poured over 500 g of crushed ice. The resulting precipitate was filtered to
yield 5.3 g (18 mmol) of 3 as a solid that was used without further purification. Physical and spectroscopic
properties were as described in [12][13].
4-[({[(9H-Fluoren-9-yl)methoxy]carbonyl}amino)methyl]-2-nitrobenzoic Acid (5). Compound 3
(0.2 g, 0.68 mmol) was dissolved in dioxane/30% aq. NH3 1:1 (10 ml), and the resulting soln. was stirred
overnight at r.t. The resulting soln. was concentrated to dryness to yield a brown residue, 4, that was used
without further purification.
The residue was dissolved in dioxane/10% aq. Na2CO3 1:1 (20 ml). The resulting mixture was cooled
in an ice bath, and 100 mg of N-hydroxysuccinimyl (9H-fluoren-9-yl)methoxycarbonate (Fmoc-OSu)

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dissolved in dioxane (10 ml) was added. The mixture was stirred at 48 for 45 min and allowed to warm to
r.t. After 1 h of magnetic stirring, H2O (30 ml) was added, and the mixture was treated with Et2O (3 ꢀ
20 ml). The aq. layer was concentrated to half of its volume, and the resulting soln. was cooled in an ice
bath. The cooled soln. was acidified to pH 2 with 32% aq. HCl soln. A white precipitate is formed that
was collected by filtration to yield 184 mg (0.44 mmol, 65%) of a white solid. Physical and spectroscopic
properties were as described in [12].
Peptide Synthesis. The peptide sequence H-NTyr-Ala-6-Phe-Lys-GlyC-OH was prepared using solid-
phase methodology and Fmoc-amino acids. Tyrosine was protected with the t-Bu group and lysine with
the (tert-butoxy)carbonyl group. The peptide was assembled on polystyrene support functionalized with
Fmoc-Gly from commercial sources (Fmoc-Gly-Wang-polystyrene [15]; 0.51 mmol/g, 50 mg, 25 mmol).
The peptide chain was elongated in DMF (0.5 ml) using fivefold excess of the Fmoc-amino acid and
fivefold excess of (benzotriazol-1-yl)-N-oxytris(pyrrolidino)phosphonium hexafluorophosphate (Py-
BOP) and tenfold excess of EtN(i-Pr)2 for 1 h. After the assembly of the sequences, the support was
treated with a soln. of TFA/H2O 95 :5 (2 ml) for 3 h. The support was removed by filtration, and the
resulting soln. was treated with Et2O (50 ml). A white precipitate was isolated by filtration. The resulting
product was purified by RP-HPLC, and the desired product was obtained as the major component (Mr
763.4; calc. 762.9).
Oligodeoxynucleotide Synthesis. Oligodeoxynucleotide sequences A (5’-d(CCCCC-7-TTTT)-3’), B
(5’-d(CTCAATGACTCGTT-7-TTCGAGTCATTGAGTCATTTTT)-3’), and C (5’-d(TGACTCAAT-
GACTCG)-3’) were prepared using solid-phase methodology and 2-cyanoethyl phosphoramidites as
monomers. The photolabile phosphoramidite (PC spacer-CE phosphoramidite, Link Technologies) was
used to introduce the photolabile function. The syntheses were performed on an Applied Biosystems
Model 3400 DNA synthesizer. After the assembly of sequences, ammonia deprotection was performed
either for 2 h at r.t. (sequence A) or overnight at 558 (sequence B). Oligonucleotides were purified by
RP-HPLC. HPLC Solns. were as follows: solvent A: 5% MeCN in 100 mm Et3N · OAc (pH 6.5) and
solvent B: 70% MeCN in 100 mm Et3N · OAc (pH 6.5); columns: Nucleosil 120C18 (10 mm, 200 ꢀ
10 mm); flow rate: 3 ml/min. Conditions A (DMT on): 20 min linear gradient from 15 – 80% B.
Conditions B (DMT off): 20 min linear gradient from 0 – 50% B.
MS: Sequence A (MALDI): found 2942.8 ([M ꢁ H]ꢁ); anal. calc. for C98H130N25O63P9: 2943.5). In
addition to the expected mass, we observed two peaks corresponding to the fragmentation of the
oligodeoxynucleotide (T4; found 1233.5; anal. calc. for C40H49N8O29P4: 1229.2 and C5-photolabile group,
found 1710.4; anal. calc. for C58H71N17O34P5: 1704.3). Yield: 7 O.D. Units at 260 nm.
MS: Sequence B (MALDI): found 11336.7 ([M ꢁ H]ꢁ); anal. calc. for C366H465N120O230P36: 11334.9.
In addition to the expected mass, we observed two peaks corresponding to the fragmentation of the
oligodeoxynucleotide (3’-fragment, found 6804.7; anal. calc. for C217H277N71O140P22: 6798.1 and 5’-
fragment-photolabile group, found 4545.85; anal. calc. for C149H189N49O90P14: 4537.8). Yield: 33 O.D. units
at 260 nm.
In addition to the expected product, a minor product corresponding to an oligodeoxynucleotide
sequence with a DMT group was also observed. This was identified as the product of the hydrolysis or
ammonolysis of the amide present in the photolabile compound (Mr 4385).
Photolysis of Peptide and Oligodeoxynucleotides. Aq. solns. (2 ml) of peptide (0.9 mM) or
oligodeoxynucleotide (0.07 mm) were photolyzed with a Black Eye lamp (340 nm) at r.t. Aliquots of
0.2 ml were taken at different time points (10, 30, 60, and 120 min, resp.) and analyzed by HPLC. The
peptide solns. were analyzed by HPLC/MS. HPLC Solns. were as follows. Solvent A: 0.1% HCOOH in
H2O and solvent B: 0.07% HCOOH in MeCN; columns: Spherisorb C18 (5 mm, 100 ꢀ 5 mm); flow rate:
1 ml/min. Conditions A: 10 min linear gradient from 0 – 50% B. Fig. 1 shows the HPLC profile of samples
taken at the start and after 2 h. The starting material (tR 4.6 min, Mr 763.4) disappeared, and the fragment
TyrAla was found at tR 2.4 min (Mr 272.1 ([M þ Naþ ꢁ Hþ]); calc. 251.2). The oligodeoxynucleotide
solns. were analyzed by HPLC as described in the purification of oligodeoxynucleotides (conditions B).
Figs. 3 and 4 show the HPLC profile of samples taken at the start of the photolysis and after 30 min. In the
photolysis of sequence A, a new peak at 9 min was observed and identified as T4 by HPLC analysis of the
enzyme digestion of the product (snake venom phosphodiesterase and alkaline phosphatase [16]) that
gave thymidine as single compound. The product containing five cytosines and the photolabile group was

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not isolated. In the photolysis of oligodeoxynucleotide sequence B, a small peak was observed eluting
after the hairpin, which was assigned to the fragment at 5’ with the rest of the photolabile groups.
Denaturing gel electrophoresis confirmed the fragmentation of the hairpin, and the formation of two
bands of 14 and 24 bases.
Melting Studies. Oligodeoxynucleotide hairpin B with or without an equimolar amount of its
complementary sequence (sequence C) was dissolved in an aq. buffer containing 0.15m NaCl, 1 mm
MgCl2, and 10 mm Na3PO4, pH 7.13, and placed in a spectrophotometer equipped with temp. controller.
Melting experiments were performed in the temp. range from 15 to 808 with a linear temp. ramp of 0.58/
min. Absorbance spectra were recorded every 18. Each sample was allowed to equilibrate at the initial
temp. for 30 min before the melting experiment was started.
HPLC Analysis of the soln. of the hairpin alone obtained after the melting experiment showed the
presence of 15% of the photolyzed hairpin. This partial photolytic cleavage is due to the UV light used
during the melting experiment. A second melting experiment with the same sample showed a small
transition at ca. 508 due to the presence of the photolyzed hairpin produced during the first melting
experiment.
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Received September 19, 2008