Photoanalogues of the initiation substrates of the RNA polymerase II, 5-azido-2-nitrobenzoyl derivatives of the ATP γ-amidophosphate: The possible photoinduced degradation of the functional group to an n-arylhydroxylamine

Article abstract of DOI:10.1081/NCN-200026041

Photoanlogues of the initiation substrates of the RNA polymerase II, N ₃Ar-NH(CH₂)_nNHpppA where N₃Ar is 5-azido-2-nitrobenzoyl group (n=2 or 4) were synthesized, allowing the preparation of photoreactive oligonu

Full text of DOI:10.1081/NCN-200026041

NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 23, Nos. 6 & 7, pp. 921–925, 2004
Photoanalogues of the Initiation Substrates of the RNA
Polymerase II, 5-Azido-2-Nitrobenzoyl Derivatives
of the ATP g-Amidophosphate: The Possible
Photoinduced Degradation of the Functional
Group to an N-Arylhydroxylamine
T. V. Popova,1 V. S. Mal’shakova,1 P. V. Alekseyev,1 N. V. Kudryashova,1
M. M. Shakirov,2 L. L. Savinkova,3 I. A. Drachkova,3
and T. S. Godovikova1,
*
1Institute of Chemical Biology and Fundamental Medicine, Novosibirsk, Russia
2Institute of Organic Chemistry, Novosibirsk, Russia
3Institute of Cytology and Genetics, Novosibirsk, Russia
ABSTRACT
Photoanlogues of the initiation substrates of the RNA polymerase II, N3Ar-
NH(CH2)nNHpppA where N3Ar is 5-azido-2-nitrobenzoyl group (n=2 or 4) were
synthesized, allowing the preparation of photoreactive oligonucleotides in situ by
RNA polymerase II for application as photolabels. Photolysis of p-nitro-substituted
aromatic azide in aqueous medium was investigated. Using the azoxy-coupling
reaction it was possible to determine whether a nitrene or p-nitrophenyl
hydroxylamine azoxy compound is the trappable intermediate that is generated at
ambient temperature in aqueous solution.
Key Words: Photoanalogues NTP; Aryl azide; Long-lived intermediate.
*
Correspondence: T. S. Godovikova, Institute of Chemical Biology and Fundamental Medicine,
8 Lavrentyeva Ave., Novosibirsk, 630090, Russia; E-mail: godov@niboch.nsu.ru.
921
DOI: 10.1081/NCN-200026041
1525-7770 (Print); 1532-2335 (Online)
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Copyright D 2004 by Marcel Dekker, Inc.

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Popova et al.
INTRODUCTION
Nucleoside 5’-triphosphate derivatives carrying nitro-substituted aryl azide residue
were proposed a few years ago as substrates of the nucleic acid polymerases for the
preparation of photoreactive oligonucleotides in situ, which can be used for studying
supramolecular structures of nucleoprotein complexes.[1,2] Despite this importance, the
photochemistry of these compounds in aqueous solution has not been studied in detail.
In this paper, we describe substrate activity of ATP photoanalogues 1 and 2 (Scheme 1)
in polymerization catalyzed by RNA polymerase II and focus on the photochemistry of
its functional group in aqueous media.
RESULTS AND DISCUSSION
We find that the ATP photoanalogues 1 and 2 have initiation substrate activity
similar to those of ATP (Fig. 1, lanes 1 and 2). The fact that synthesis of RNA is
aborted (Fig. 1, lane 4) in the presence of a-amanitin (1 mg/ml) demonstrates that
transcription is realized by RNA-polymerase II alone.
The photochemistry of p-nitro-substituted aromatic azides has been the subject of
several previous studies.[3,4] Photolysis of 4-nitrophenyl azide (4NPA) in O2-saturated
organic solution gives p-dinitrobenzene, 4-nitronitrosobenzene (4NNB) and 4,4’-
dinitroazoxybenzene (4,4’DNAB).[3] The related reaction products have been observed
from the photolysis of 4-nitro-substituted aromatic azides in aqueous solution.[4] Also,
irradiation of an aqueous deoxygenated solution of this aryl azide gives 4,4’DNAB
derivative. The formation of azoxybenzene in the absence of O2 is an intriguing
process. On the contrary, irradiation of 4NPA in deoxygenated organic solution gives
azobenzene as the single characterizable product.[3]
In the course of the photolysis giving 4,4’-DNAB, the following three-step process,
involving p-nitrophenylhydroxylamine (4NPHA) as a transient intermediate, may be
adopted on the basis of the facts described below (Scheme 2).
Under the conditions of assays, a primary arylhydroxylamine is susceptible to oxida-
tion, disproportionation and condensation.[5] On the other hand, a related products reaction
has been generated from the triplet nitrene.[3] A derivatization scheme was sought that
would convert the hydroxylamine to a stable product permitting analysis to be carried out
Scheme 1. Structural formulae of photoreactive ATP derivatives.

Initiation Substrates of the RNA Polymerase II
923
Figure 1. Autoradiogram of electrophoretically analyzed transcripts (20% polyacrylamide gel, 7 M
urea): lane 1–transcription with NTP and ATP (4 Â 10À4 M); lanes 2 and 3–transcription with NTP
and ATP photoanalogue 2 (4 Â 10À4 M and 10À5 M respectively); lane 4–transcription in the
presence of a-amanitin; lane 5–heterologous 32P-labeled 17-base oligoribonucleotide.
at a more leisurely pace with minimal decomposition. The derivatization approach that
has been exploited involves the addition of nitroso (as a trap of arylhydroxylamine) to the
aryl azide; the photolysis product being converted to a more stable azoxy compound.
We began with an experiment with 4NNB using prephotolyzed 4NPA (0.7/1
mixture) and found that a reaction proceeds to give 4,4’DNAB (68 %). Since it was
determined that O2 has a marked effect on the photochemistry of aryl azides,[3,4] all
studies were performed in deoxygenated aqueous solution. Photolysis of 4NPA in the
presence of the 4NNB resulted in an increased yield of 4,4’DNAB (Table 1). These
observations point to the operation of a route for formation of 4,4’DNAB that does not
pass through a triplet nitrene. One possibility for the non-nitrene route to 4,4’DNAB is
the reaction of the 4NNB with 4NPHA (Scheme 2).
In photoaffinity labeling experiments, the phenomenon of formation of long-lived
species upon irradiation of reagents carrying 5-azido-2-nitrobenzoyl residue has been

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Popova et al.
Scheme 2. The course of the photolysis of [p]-nitrophenylazide aqueous solution.
traced to.[6] Our study has demonstrated that 4NPHA as a reactive intermediate may be
formed from photolysis of these photoreagents in aqueous solution.
EXPERIMENTAL PROCEDURES
The plasmid pUC119 containing adenovirus major late promoter (AdML) (À39/
+20) was kindly provided by J. Kadonaga. A transcriptionally active complex was
prepared from Saccharomyces cerevisiae and the transcription reactions were
performed as previously described.[7] Our results for the synthesis of the ATP photo-
analogues 1 and 2 will be published in due course.
UV irradiation of the samples was performed as previously described.[5] Sample
solutions were prepared in quartz cells with Teflon stopcocks and purged with Ar for
20 min at 0°C prior to each experiment. A water solution of 4NNB was mixed with
either 5.3 Â 10À4 M solution of prephotolyzed 4NPA or with 4NPA (reaction mixture
I) in molar ratio according Table 1. The reaction mixture I was subjected to irradiation.
All mixtures were cooled in an ice water bath. The resulting precipitates were
collected, dried in vacuo and identified as 4,4’DNAB.[8] 1H NMR ((CD3)2CO, J, Hz, d,
Table 1. Effect of 4NNB on the 4,4’DNAB formed on photolysis of 4NPA.
4NPA/4NNB
Absence of 4NNB
15
3.6/1
31
0.7/1
73
The yield* of 4,4’DNAB, %
*Yields are described as molar equivalent estimated from one mole of the starting 4NPA.

Initiation Substrates of the RNA Polymerase II
925
p.p.m.) 8,32 (d.d., J2’,3’=J6’,5’=9.0, J2’,6’=J6’,2’=2,5 H2’, H6’, 2H), 8,44 (d.d.,
3’,2’=J5’,6’=9.0, J3’,5’=J5’,3’=2,5 H3’, H5’, 2H), 8,51 (d.d., J3,2=J5,6=9.0, J3,5=J5,3=2,5
H3, H5, 2H), 8,64 (d.d., J2,3=J6,5=9.0, J2,6=J6,2=2,5 H2, H6, 2H).
J
ACKNOWLEDGMENT
We are grateful for grant support provided by the Russian Foundation for Basic
Research (00-04-49258, 02-04-48080 and 03-04-06132).
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Received December 2, 2003
Accepted April 23, 2004