4-Aminometyl-3-nitrobenzoic acid - A photocleavable linker for oligonucleotides containing combinatorial libraries

Article abstract of DOI:10.1080/15257770500230509

We detail the design, synthesis, and characterization of an o-nitrobenzyl - based photolabile linker containing amine and carboxyl anchor groups. A model nucleoside monomer modified with an imidazole residue and a precursor unit linked to a heterocyclic b

Full text of DOI:10.1080/15257770500230509

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4-Aminometyl-3-Nitrobenzoic Acid—A Photocleavable
Linker for Oligonucleotides Containing Combinatorial
Libraries
Tatiana V. Abramova a & Vladimir N. Silnikov a
a The Institute of Chemical Biology and Fundamental Medicine, Novosibirsk, Russia
Published online: 31 Aug 2006.
To cite this article: Tatiana V. Abramova & Vladimir N. Silnikov (2005) 4-Aminometyl-3-Nitrobenzoic Acid—A Photocleavable
Linker for Oligonucleotides Containing Combinatorial Libraries, Nucleosides, Nucleotides and Nucleic Acids, 24:9, 1333-1343,
DOI: 10.1080/15257770500230509
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Nucleosides, Nucleotides, and Nucleic Acids, 24 (9):1333–1343, (2005)
Copyright D Taylor & Francis, Inc.
ISSN: 1525-7770 print/ 1532-2335 online
DOI: 10.1080/15257770500230509
4-AMINOMETYL-3-NITROBENZOIC ACID—A PHOTOCLEAVABLE
LINKER FOR OLIGONUCLEOTIDES CONTAINING
COMBINATORIAL LIBRARIES
5
Tatiana V. Abramova and Vladimir N. Silnikov
The Institute of Chemical Biology
and Fundamental Medicine, Novosibirsk, Russia
5
We detail the design, synthesis, and characterization of an o-nitrobenzyl --based photolabile linker
containing amine and carboxyl anchor groups. A model nucleoside monomer modified with an imidazole
residue and a precursor unit linked to a heterocyclic base through a photolabile tether is constructed.
Upon UV irradiation (313 --365 nm), the imidazole containing part of this molecule is released.
Keywords Modified Nucleosides, Photolabile Linker, Combinatorial Libraries
INTRODUCTION
The design and synthesis of a small synthetic molecule that mimics the active
center of a natural ribonuclease and that is capable of cleaving RNA molecules
is an interesting research subject.[1] The conjugation of such artificial ribo-
nucleases to a nucleic acid would potentially result in a ribonuclease capable of
cleaving its target mRNA in a sequence-specific manner. Our study on artificial
ribonucleases has resulted in the synthesis of imidazole-derived constructs in
combination with or without additional amino, hydroxy, or guanidinium
groups.[2–4] Different procedures for derivatization of oligonucleotides have been
reviewed recently.[5] However, the procedure of derivatization of polyfunctional
molecules is a difficult task.
Recently, we have described a series of monomers for oligonucleotide
conjugate synthesis[6,7] using a novel precursor strategy.[8] Using this technique we
have prepared a series of oligonucleotide based artificial ribonucleases bearing
diimidazole catalytic group.[9] We believe that this strategy in combination with
Accepted 2 May 2005.
This work was supported by the Russian Fund for Fundamental Research, Grants No 02-04-48664a, 03-04-
48562a, 04-04-48566a, and 04-03-32490a. Authors are grateful to Dr. D. Kolpashchikov (Columbia University, New
York) for fruitful discussion and Eric Green (Columbia University, New York) for careful reading of the manuscript.
Address correspondence to Tatiana V. Abramova, The Institute of Chemical Biology and Fundamental
Medicine, Lavrentev Ave. 8, Novosibirsk 630090, Russia; E-mail: abramova@niboch.nsc.ru
1333
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1334
T. V. Abramova and V. N. Silnikov
FIGURE 1 General scheme of using a cleavable linker and precursor subunit in optimization of catalytic group
structure in oligonucleotide based RNAse mimic library.
elements of combinatorial chemistry may be useful for optimization of catalytic
group structure (Figure 1).
For realization of this idea we have combined a photocleavable linker and
active precursor group in one molecule. We demonstrate the possibility of such
approach by the synthesis and preliminary photolytic study and establishment of
the minimal RNAse mimic model structure (Figure 2).
RESULTS AND DISCUSSION
Design of the Photocleavable Linker
In the last decade, special attention has been devoted to linkers potentially
suitable for the purposes of combinatorial chemistry: stable under reaction
conditions and easily cleavable without damaging biopolymers.[10–13] Well-known
o-nitrobenzyl and related compounds seem to be the most promising due to their
FIGURE 2 RNAse mimic model.

A Photocleavable Oligonucleotide Linker
1335
FIGURE 3 Photolabile linker based on the o-nitrobenzyl fragment.
ability to release a linked (or ‘‘caged’’) residue upon UV irradiation, with minimal
or no modifications.[14]
Some constructs based on the o-nitrobenzyl fragment have a chiral carbon atom
in their structure (Figure 3, when R1
6
¼R2). This is not problematic for the purpose of
creating an oligomer library, since the diastereomeric linker is absent from the target
compounds.[13,15–17] In some cases, the authors have assumed that a racemic
mixture of linker did not effect the functioning of the final compounds they
obtained.[18,19] Since our target compounds will include the photocleavable linker,
as well as a library, we must assume that different isomers of the linker could affect
the RNA cleaving effectiveness of the RNAse mimic. Thus, the stereochemistry of
the conjugate (Figure 2) is very important, and racemic mixtures in any part of the
molecule have to be avoided. Hence, R1 must be equal R2 (Figure 3); both may be a
hydrogen atom for simplicity and diminution of any rotational hindrance at this site.
Since most biopolymers do not absorb light in the range 300–400 nm, long
wavelength UV light is less damaging. According to the literature o-nitroveratryl
(when R3 = R4 = OCH3, Figure 3) and related compounds are more sensitive
to UV light at wavelengths greater than 300 nm (e360 5000 Mꢀ1cmꢀ1) than
o-nitrobenzyl compounds (e350 500 Mꢀ1cmꢀ1)[20] and display faster photolytic
kinetics.[21] To combine the advantages of a long wavelength UV absorbance band
and the utility of a carboxyl functional group, a number of studies synthesized
and used o-nitroveratrole based compounds, where R3 is –OCH3 and R4
is –O(CH2)nCOOH.[12,13,21–24] While the o-nitroveratryl derivatives have a larger
absorbance than o-nitrobenzyl within the range of 300–400 nm, quantum yield of the
photolytic cleavage in this case is less, resulting in a less than tenfold improvement in
sensitivity for long wavelength UV light.[20,25] As shown previously, the rate of
photolysis depends on the nature of the bond broken and the reaction media.[14]
Also, the introduction of additional elements such as methoxy and alkoxy groups in
the linker structure complicates the synthesis. Keeping in mind that the possible
damaging of the oligonucleotide addressing part of the target molecule (Figures 1, 2)
during UV irradiation is not important for our purposes (analysis of the catalytic
subunits of RNAse mimics libraries), we chose to construct a linker (Figure 3) where
R1 = R2 = R3 = H and R4 = COOH.
Synthesis of the RNAse Mimic Model
In order to verify the applicability of the designed 4-(aminomethyl)-3-
nitrobenzoic acid as a photocleavable linker for analysis of oligonucleotide RNAse

1336
T. V. Abramova and V. N. Silnikov
SCHEME 1 Synthesis of the model RNAse mimic. i) Dicyclohexylcarbodiimide, 1-hydroxybenzotriazole,
compound 2, acetone; ii) NH3/H2O; iii) dimethyl oxalate, TEA, MeOH; iv) histamine.2HCl, TEA, MeOH;
v) HOAc/H2O.
mimics, we synthesized a model compound consisting of addressing, photo-
cleavable, precursor and catalytic subunits (Figure 2). First, the protected
photocleavable linker was synthesized. Previously, linkers based on 4-(amino-
methyl)-3-nitrobenzoic acid were used for solid-phase peptide synthesis.[17,26] In
both cases nitration was performed on 4-(bromomethyl)benzoic acid. We decided
to use commercially available 4-(aminomethyl)benzoic acid as a starting material.
An amine group of this acid was blocked with trifluoroacetyl residue. The nitration
of 4-(trifluoroacetamidomethyl)benzoic acid 1 with 68% nitric acid in sulfuric acid
solution at 0°C afforded 3-nitro-4-(trifluoroacetamidomethyl)benzoic acid 2 with a
75% yield. The protected amino acid 2 was converted to a 1-hydroxybenzotriazole
ester and coupled to the 4-N-(6-aminohexyl)-5’-O-(4,4’-dimetoxytrityl)-2’-deoxycyti-
dine 3 (Scheme 1) to give the aminoacylated derivative 4 (Scheme 2). Compound 3
was prepared similarly to a 2’-deoxycytidine derivative with a three methylene
group linker.[6] Compound 4 was deblocked by ammonia treatment and reacted
with dimethyl oxalate to give 5 (Scheme 1). At this stage it would be possible to
convert the 5’-DMTr-2’-deoxycytidine derivative 5 to a 3’-phosphoramidite by a
standard procedure;[6] the resulting monomer could be used in oligodeoxyribo-
nucleotide synthesis to obtain oligonucleotide containing libraries with a photo-
cleavable tether using the precursor strategy (Figure 1). This is a work in progress,
SCHEME 2 Synthesis of the amidooxalhistamide 7. i) Histamine 2HCl, TEA, MeOH; ii) NH3/MeOH.

A Photocleavable Oligonucleotide Linker
1337
SCHEME 3 Products of the photolytic cleavage of the RNAse mimic model 6.
and in this article we decide to limit an addressing part of the RNAse mimic model
to monomer (Figure 2); so we do not include results concerning the oligonucleotide
synthesis. Imidazole containing constructs are widely used as small RNAse
mimics.[27] It has been shown previously that the simplest artificial RNAse includes
histamine residue.[28] Therefore, we treated compound 5 with histamine to
introduce an imidazole containing residue as a model for the catalytic group.
Finally, we treated the crude product with acetic acid to deblock the final conjugate
(Scheme 1). Compound 6 was purified by reverse-phase chromatography, and
characterized by NMR and mass spectroscopy.
Photolysis of the Model RNAse Mimic
When 6 was subjected to long wavelength UV light (313–365 nm), the starting
compound disappeared with a t1/2 of 5.0 min. The photofragmentation reaction
was monitored using HPLC (data not shown). These data are in agreement with the
half-life for photolabile linker published earlier.[18] Based on the well-known
mechanism of photolytic cleavage of o-nitrobenzyl and related systems,[14] we
expected the formation of the amidooxalhistamide 7 (Scheme 2) as an imidazole
containing photolysis product. In order to confirm this we synthesized compound 7
separately (Scheme 3) to use as a standard in the search for product 7. The molar
absorption coefficient e260 of compound 7 was found to be rather low (334
Mꢀ1cmꢀ1). Using multiwave detection and calculations of the spectral ratios we
succeeded in identifying the amidooxalhistamide in HPLC profiles of irradiated
samples of 6, using authentic sample of 7 as a reference substance. In a separate
experiment compound 7 was proved to be stable during irradiation (data not
shown). The liberated product was separated from photolysis reaction mixture by
HPLC and was subjected to NMR, UV, and mass spectra analysis. The spectral
data were found to be identical to amidooxalhistamide 7.
EXPERIMENTAL SECTION
General
The following reagents and equipment were used: N4-benzoyl-5’-O-(4,4’-
dimethoxytrityl)-2’-deoxycytidine (ChemGenes Corporation, Wilmington, MA,
USA), 4-(Aminomethyl)benzoic acid and 1,5,7-triazabicyclo[4.4.0]dec-5-en (Fluka

1338
T. V. Abramova and V. N. Silnikov
Chemica, Buchs, Switzerland), histamine dihydrochloride (Sigma, St. Louis, MO,
USA). All other reagents were from Aldrich, WI, USA. Organic solvents were dried
and purified by standard procedures. NMR spectra were acquired on Brucker AM-
400 and AC-200 spectrometers in approapriate deuterated solvents at 30°C.
Chemical shifts (d) are reported in ppm relative to TMS signals (internal standard, in
the case of 19F C6F6 was used as an external standard). Coupling constants J are
reported in Hertz. Mass spectra were run on a Finnigan-MAT-8200 (EI, 70 eV)
(Thermo Electron Corp., Somerset, NJ, USA) and Reflex III (MALDI) (Brucker,
Bremen, Germany). Dihydroxybenzoic acid was used as a matrix in MALDI
spectra. Melting points were measured on a Kofler hot stage (VEB Analytik). HPLC
was performed on a Milichrom-4 (Econova, Novosibirsk, Russia). A 2 ꢁ 60 mm
column packed with Nucleosil C18 (5 mm), having a gradient elution of acetonitrile in
0.03 M LiClO4 (0 to 50%) over 25 min, with an elution rate 0.1 mL per min, was used
for the analysis. Thin layer chromatography was performed using Alufolien
Kieselgel 60 F254 plates (Merck, Darmstadt, Germany) in appropriate solvent
mixtures and visualized by UV irradiation, ninhydrin (amine groups), cystein/
sulfuric acid (nucleoside) or p-diazobezenesulfonic acid (imidazole residue).
4-(Trifluoroacetamidomethyl)benzoic acid (1). 4-(Aminome-
thyl)benzoic acid (3 g, 20 mmol) was suspended in a mixture of methanol (20
mL) and triethylamine (4.2 mL, 30 mmol). Ethyl trifluoroacetate (4.2 mL, 36
mmol) was added dropwise to the suspension and the reaction mixture was stirred
overnight. After the reaction was complete the clear solution was evaporated. The
residue was treated with boiling water (70 mL). The clear solution was chilled to
room temperature and acidified to pH 3 with trifluoroacetic acid. The acidified
solution was cooled in an ice bath, and afforded 4-(trifluoroacetamidomethyl)-
benzoic acid 1 as a white powder (4.23 g, 84%) after filtration and drying in a
vacuum. Rf 0.44 (CH2Cl2:MeOH 9:1); m.p. 206–208°C; 1H NMR (200 MHz, [D6]-
DMSO): d 4.46 (2H, d, J 6.0, –CH2NH–), 7.39 (2H, d, J 8.0, m-CH–), 7.93 (2H,
d, J 8.0, o-CH–), 10.03 (1H, br t, J 6.0, –NH–), 12.67 (1H, br s, –OH); 13C NMR
(50 MHz, [D6]-DMSO): d 42.30, 115.89 (q, JC,F 287), 127.27, 129.44, 129.83, 142.28,
156.43 (q, JC,F 37), 166.91; 19F NMR (188 MHz, [D6]-DMSO): d 90.52; EI-MS: m/z
(%) 247 (91) [M+], 202 (100) [M+–COOH]; elemental analysis calcd for
C10H8F3NO3 (247.17): C 48.59, H 3.26, F 23.06, N 5.67; found, C 48.86, H 3.15,
F 22.92, N 5.46.
3-Nitro-4-(trifluoroacetamidomethyl)benzoic acid (2). (Tri-
fluoroacetamidomethyl)benzoic acid 1 (4 g, 16 mmol) was dissolved in sulfuric
acid (60 mL) and the solution was chilled in an ice bath. A mixture of nitric acid
(68%, 1.6 mL) and sulfuric acid (98%, 1.92 mL) was added to the solution
dropwise with stirring over a period of half an hour. The ice was removed and
the reaction mixture was left to warm to room temperature. After 2 h the solution
was poured onto 500 g of ice. The resultant slurry was filtered to give a white

A Photocleavable Oligonucleotide Linker
1339
powder. The solid was washed several times with water and recystallized from
ethanol/water to afford the nitrated trifluoroacetamide 2 (4.0 g, 86%). Rf 0.28
1
(CH2Cl2:MeOH 9:1); m.p. 196–198°C; H NMR (400 MHz, [D6]-DMSO): d 4.78
(1H, d, J 5.6, –CH2NH–), 7.66 (1H, d, J 8.0, –CH5–), 8.25 (1H, dd, J 8.0, 1.6,
–CH6–), 8.48 (1H, d, J 1.6,–CH2–), 9.87 (1H, br t, J 5.6, –NHCO–); 13C
NMR (100 MHz, [D6]- DMSO): d 40.13, 115.54 (q, JC,F 286), 124.86, 130.01,
131.46, 133.45, 135.71, 147.86, 156.51 (q, JC,F 37), 164.81; 19F NMR (376 MHz,
[D6]- DMSO): d 89.64; MS (MALDI) m/z 291.29 [M-H], 246.14 [M-NO2], 178.82
[C8H5NO4] (3-nitroso-4-formylbenzoic acid, the product of the photolysis of the
compound 2); elemental analysis calcd for C10H7F3N2O5 (292.17): C 41.11, H
2.41, F 19.51, N 9.59; found, C 40.95, H 2.32, F 19.43, N 9.33.
4-N-(6-Aminohexyl)-5’-O-(4,4’-dimethoxytrityl)-2’-deoxycyti-
dine (3). 2’-Deoxycytine derivative 3 (Scheme 1) was prepared from 5’-O-
DMTr-2’-dCBz following a published procedure[6] using 1,6-diaminohexane instead
1
of 1,3-diaminopropane. Yield was 40%. Rf 0.36 (CH2CL2:MeOH:TEA 7:2:1); H
NMR (200 MHz, [D6]-acetone plus several drops of [D5]-pyridine) d 1.30–1.43 (4H,
m, 2x-CH2–), 1.48–1.63 (4H, m, 2x-CH2–), 2.11–2.22 (1H, m, H2’), 2.29–2.40 (1H,
m, H2@), 3.14 (2H, t, J 6.5, –CH2NH2), 3.28–3.42 (4H, m, H5’,5@,–NHCH2–), 3.75
(6H, s, 2xCH3O–), 4.01–4.10 (1H, m, H4’), 4.46–4.59 (1H, m, H3’), 5.63 (1H, d, J
7.1, H5), 6.35 (1H, app. t, J 6.1, H1’), 6.85 (4H, d, J 8.6, DMTr), 7.15–7.50 (9H, m,
DMTr), 7.73 (1H, d, J 7.1, H6).
4-N-{6-[3-Nitro-4-(trifluoroacetamidomethyl)]benzamido-
hexyl}-5’-O-(4,4’-dimethoxytrityl)-2’-deoxycytidine (4). 3-Nitro-4-
(trifluoroacetamidomethyl)benzoic acid 2 (Scheme 1) (93 mg, 0.3 mmol),
dicyclohexylcarbodiimide (80 mg, 0.4 mmol), and 1-hydroxybenzotriazole (47
mg, 0.35 mmol) were dissolved in acetone (2 mL) and the reaction mixture was
stirred for 1 h. Compound 3 (157 mg, 0.25 mmol) and TEA (0.07 mL, 0.5 mmol)
were then added. After 2 h of stirring, the reaction mixture was evaporated, and
the residue was suspended in methylene chloride and filtered. The filtrate was
applied to a column packed with silica gel (50 mL). Elution was performed with
a linear gradient of methanol in methylene chloride (0–10%) containing 0.1% of
pyridine. Appropriate fractions were pooled and evaporated to give 140 mg
(0.155 mmol, 62%) of derivative 4 as white foam. Rf 0.27 (CH2Cl2:MeOH 9:1);
1H NMR (200 MHz, [D6]-acetone): d 1.28–1.49 (4H, m, 2x-CH2–), 1.49–1.72
(4H, m, 2x-CH2–), 2.12–2.22 (1H, m, H2’), 2.29–2.40 (1H, m, H2@), 3.31–3.50
(6H, m, H5’,5@, –NHCH–, –CH2NHCO–), 3.78 (6H, s, 2xCH3O–), 3.97–4.09
(1H, m, H4’), 4.42–4.58 (1H, m, H3’), 4.89 (2H, s, Ar-CH2NH–), 5.60 (1H, d, J
7.5, H5), 6.35 (1H, app. t, J 6.0, H1’), 6.85 (4H, dt, J 8.6, 2.7, DMTr), 7.18–7.52
(9H, m, DMTr), 7.76 (1H, d, J 8.0, –CH5-(Ar)), 7.87 (1H, d, J 7.5, H6), 8.30 (1H,
dd, J 8.0, 1.6, –CH6–(Ar)), 8.52 (2H, m, 2x-NHCO–), 8.65 (1H, d, J 1.6, –
CH2–(Ar)).

1340
T. V. Abramova and V. N. Silnikov
4-N-{6-[3-Nitro-4-(methoxyoxalamidomethyl)]benzamido-
hexyl}-5’-O-(4,4’-dimethoxytrityl)-2’-deoxycytidine (5). Com-
pound 4 (Scheme 1) (140 mg, 0.155 mmol) was dissolved in 5 mL of 1,4-
dioxane. Concentrated ammonia (5 mL) was added to the solution and the reaction
mixture was stirred overnight. The solution was evaporated; the residue was
dissolved in 25 mL of methylene chloride and washed several times with water. The
organic layer was dried with Na2SO4 and concentrated to 5 mL. The nucleoside
derivative was precipitated with a tenfold volume of hexane to give 4-N-{6-[3-nitro-
4-(aminomethyl)]benzamidohexyl}-5’-O-(4,4’-dimethoxytrityl)-2’-deoxycytidine (120
1
mg, 93%) as a white solid. Rf 0.1 (CH2Cl2:MeOH 8:2); H NMR (200 MHz, [D6]-
acetone plus several drops of [D5]-pyridine): d 1.30–1.50 (4H, m, 2x-CH2–), 1.51–
1.77 (4H, m, 2x-CH2–), 2.09–2.28 (1H, m, H2’), 2.32–2.50 (1H, m, H2@), 3.28–3.51
(6H, m, H5’,5@,-NHCH–,–CH2NHCO–), 3.77 (6H, s, 2xCH3O–), 4.00–4.15 (1H,
m, H4’), 4.48–4.60 (1H, m, H3’), 4.71 (2H, s, Ar-CH2NH2–), 5.62 (1H, d, J 7.5, H5),
6.36 (1H, app. t, J 6.0, H1’), 6.85 (4H, dt, J 8.6, 2.7, DMTr), 7.18–7.52 (9H, m,
DMTr), 7.77 (1H, d, –CH5- (Ar)), 7.91 (1H, d, J 7.5, H6), 8.27 (1H, dd, J 8.0,
1.6, –CH6– (Ar)), 8.56 (1H, br t, J 5.0, –NHCO–), 8.58 (1H, d, J 1.6, –CH2–
(Ar)). The nucleoside derivative with the free amino group (120 mg, 0.145 mmol)
was dissolved in the mixture of methanol (1.5 mL) and TEA (0.15 mL) and the
solution was added to solution of dimethyl oxalate (85 mg, 0.7 mmol) in 2 mL of
methanol over a 6-h period. The reaction mixture was stirred overnight, and then
evaporated. The residue was dissolved in methylene chloride (5 mL) and the solution
was applied to a column packed with silica gel (50 mL). Elution was performed with a
linear gradient of methanol in methylene chloride (0–10%) containing 0.1% of py-
ridine. Appropriate fractions were pooled and evaporated to give derivative 5
1
(Scheme 1) (90 mg, 69%) as a white foam. Rf 0.25 (CH2Cl2:MeOH 9:1); H NMR
(200 MHz, [D6]-acetone): d 1.35–1.50 (4H, m, 2x-CH2–), 1.51–1.76 (4H, m,
2x-CH2–), 2.10–2.26 (1H, m, H2’), 2.27–2.43 (1H, m, H2@), 3.30–3.49 (6H, m,
H5’,5@, –NHCH–, –CH2NHCO–), 3.77 (6H, s, 2xCH3O– (DMTr)), 3.82 (3H, s,
–OCH3(Ox)), 3.98–4.07 (1H, m, H4’), 4.46–4.58 (1H, m, H3’), 4.84 (2H, br s, Ar-
CH2NH–), 5.61 (2H, d, J 7.5, H5), 6.29 (1H, app. t, J 6.0, H1’), 6.85 (4H, dt,
J 8.6, 2.7, DMTr), 7.18–7.52 (9H, m, DMTr), 7.71 (9H, d, J 8.0, –CH5–(Ar)),
7.77 (1H, d, J 7.5, H6), 8.28 (1H, dd, J 8.0, 1.6, –CH6–(Ar)), 8.54–8.72 (3H, m,
–CH2–(Ar), 2x-NHCO–).
4-N-{6-[3-Nitro-4-(2-(4-imidazolyl)ethylamidooxalamidome-
thyl)]benzamidohexyl}-2’-deoxycytidine (6). Histamine dihydro-
chloride (54 mg, 0.4 mmol) was dissolved in a mixture of methanol (2 mL) and
TEA (0.5 mL). The derivative 5 (Scheme 1) (90 mg, 0.1 mmol) was dissolved in
methanol (1 mL), and both solutions were combined. The reaction mixture was
stirred overnight, and then evaporated; the residue was dissolved in methylene
chloride (10 mL) and washed several times with water. The organic layer was
evaporated; its residue was dissolved in 80% aqueous acetic acid (5 mL). After the

A Photocleavable Oligonucleotide Linker
1341
deprotection was complete (1 h) the reaction mixture was evaporated several times
with water to remove acetic acid, the residue was dissolved in water (10 mL), the
solution was filtered and applied to a column (30 mL) packed with Porasil C18 (55–
105 mm, Waters, Milford, MA, USA). Elution was performed with a linear gradient
of acetonitrile in water (0–50%). Fractions containing the target product were
1
evaporated to dryness to give the title compound 6 (Scheme 1) (20 mg, 30%). H
NMR (200 M, [D6]-acetone): d 1.30–1.48 (4H, m, 2x-CH2–), 1.49–1.69 (4H, m, 2x-
CH2–), 2.10–2.22 (1H, m, H2’), 2.25–2.40 (1H, m, H2@), 2.78 (2H, t, J 7.0, -
CH2CH-Im), 3.40–3.68 (6H, m, –NHCH–, 2x-CH2NHCO–), 3.70–3.89 (2H, m,
H5’,5@), 4.21–4.41 (2H, m, H3’,H4’), 4.69–4.79 (2H, m, Ar-CH2NH–), 5.74 (1H, d,
J 7.5, H5), 6.24 (1H, app. t, J 6.0, H1’), 6.87 (1H, s, H5(Im)), 7.61 (1H, s, H2(Im)),
7.62 (1H, d, J 8.0, –CH5–(Ar)), 7.80 (1H, d, J 7.5, H6), 8.24 (1H, dd, J 8.0, 1.6, –
CH6–(Ar)), 8.56 (1H, d, J 1.6, –CH2–(Ar)), 8.72–8.86 (2H, m, 2x-NHCO–), 9.28
(2H, t, J 5.5, –NHCO); MS (MALDI) m/z 669.97 [M + H].
2-(4-Imidazolyl)ethylamidooxalamide (7). Dimethyl oxalate (236
mg, 2 mmol) was dissolved in methanol (2 mL). Histamine dihydrochloride (331
mg, 1.8 mmol) was dissolved in a mixture of methanol (2 mL) and TEA (0.6 mL),
and this solution was added to the dimethyl oxalate over period of 2 h with
continuous stirring. The reaction mixture was heated under reflux for 30 min and
left overnight at room temperature. The reaction mixture was concentrated twofold
and the precipitate was collected by filtration, washed with methanol, and dried
under a vacuum. Intermediate methoxyoxalyl-2(4-imidazolyl)ethylamide was
treated with 6.6 M NH3/MeOH (5 mL) for 10 min. The solution was evaporated,
and the residue was recrystallized from water to afford the title compound 7
(Scheme 2) (0.150 g, 46%). Rf 0.25 (CH2Cl2:MeOH 8:2); m.p. 210–212°C; 1H NMR
(400 MHz, [D6]- DMSO): d 2.69 (2H, t, J 7.0, –CH-Im), 3.36 (2H, dt, J 7.0,
6.0, –NHCH–CH2–), 6.81 (1H, s, H5 (Im)), 7.53 (1H, s, H2 (Im)), 7.78 (1H, s,
NH2–), 8.05 (1H, s, NH2–), 8.75 (1H, t, J 6.0, –NHCO–), 11.82 (1H, br s, –NH-
(Im)); 13C NMR (50 MHz, [D6]-DMSO): d 26.43, 38.88, 116.13, 116.40, 134.67,
160.05, 162.12; EI-MS m/z (%): 182 (7.5) [M+], 138 (23.3) [M+-H2NCO]; elemental
analysis calcd. for C7H10N4O2 (182.18): C 46.15, H 5.53, N 30.75; found, C 45.86, H
5.48, N 30.46.
Photolysis Conditions
Photocleavage of model RNAse mimic 6 was performed at room temperature
using irradiation in the range of 313–365 nm (filters BS and UFS-2) with a high
pressure Hg lamp (DRK-120, St. Petersburg, Russia, W 0.3 mW/cm2). The
methanolic solution of 7 (5.4 ꢁ 10ꢀ3 M, 0.05 ml) was diluted tenfold with water
and 0.1 mL aliquots were placed under the lamp at a distance of 10 cm. Aliquots
were analyzed by the reverse-phase HPLC. The degree of the photolysis was
estimated by the disappearance of the starting compound 6.

1342
T. V. Abramova and V. N. Silnikov
CONCLUSION
The design and synthesis of the photocleavable o-nitrobenzyl based linker
suitable for the synthesis and analysis of oligonucleotide containing combinatorial
libraries were successful. The simplest RNAse mimic model containing the ad-
dressing, photocleavable, precursor and catalytic subunits was designed and syn-
thesized. Upon irradiation (313–365 nm), it released the catalytic subunit combined
with the precursor residue which was identified in the course of chromato-
graphic analysis.
In the future we plan to construct a chimeric oligonucleotide that includes both
an addressing part of RNAse mimic and an RNA target. It will also use a library of
catalytically active constructs joined to the addressing part of the molecule through
the photocleavable linker described in this paper. Active in RNA cleavage chimeras
will be separated from inactive constructs by gel electrophoresis. After selecting the
most active constructs, the catalytic subunits would be cleaved from the rest of the
molecule and analyzed by chromatomass spectroscopy.
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