Synthesis and properties of DNA containing a spore photoproduct analog

Article abstract of DOI:10.1039/b810008j

The spore photoproduct is a unique photolesion, formed in spores upon irradiation with UV light; to investigate the properties of spore photoproduct containing DNA we have synthesized 5S and 5R lesion analogs and incorporated them into DNA. The Royal Soci

Full text of DOI:10.1039/b810008j

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Synthesis and properties of DNA containing a spore photoproduct
analogw
Eva Burckstummer and Thomas Carell*
¨ ¨
Received (in Cambridge, UK) 13th June 2008, Accepted 18th July 2008
First published as an Advance Article on the web 30th July 2008
DOI: 10.1039/b810008j
The spore photoproduct is a unique photolesion, formed in spores
upon irradiation with UV light; to investigate the properties of
spore photoproduct containing DNA we have synthesized 5S and
5R lesion analogs and incorporated them into DNA.
Bacteria of the Clostridium and Bacillus species form meta-
bolically dormant endospores in response to nutrient deple-
tion.1 The DNA of these dormant spores is extremely well
protected against damage imposed by heat, desiccation, var-
ious chemicals, UV- and g-irradiation.2 Consequently, spores
can store genetic material over long periods.3 They are in every
Fig. 1 The two diastereomeric phosphoramidites 1a and 1b prepared
respect the most long lasting information storage systems on
earth. Factors which contribute to the amazing DNA stability
inside spores are the small acid soluble proteins (SASPs),
which complex the DNA together with dipicolinic acid under
low water conditions.4 Although the DNA is rather stable
even in the presence of UV light, UV irradiation triggers the
formation of a unique DNA lesion in spore DNA, which needs
to be repaired. This lesion is the spore photoproduct (SP)
formed between two adjacent thymidines.5–7 Repair of the
lesion is achieved by the spore photoproduct lyase, a radical
SAM enzyme active during germination.8–13 To date, a defined
synthetic system which allows monitoring of the repair process
in vitro does not exist and the exact configuration of the lesion
at C5 is not known. We were, however, recently able to show
with dinucleotide lesion analogs that only one of the two
possible SP-lesion diastereomers, the 5S isomer, is efficiently
repaired by the SP-lyase.14 We now report the incorporation
of the two lesion analogs 1a and 1b (Fig. 1), which possess
either 5S or 5R configuration at C5, into DNA in order to
probe how they influence the duplex stability.15 We show that
both affect the duplex stability by different amounts with the
5S isomer causing the smaller destabilization. The synthesized
lesion analogs only lack the central phosphodiester linkage
between the two lesion substructures, which is however
important to allow the needed enzymatic investigations. Stu-
dies with cyclobutane pyrimidine dimers lacking the same
phosphate established that these analogs can serve as superb
substrates. Since repair requires chemistry at the heterocycles
for the incorporation of the spore analogs into DNA. DMT =
dimethoxytrityl, TBDPS = tert-butyldiphenylsilyl, SEM = 2-(tri-
methylsilyl)ethoxymethyl.
of the lesion the backbone modification might influence the
lesion recognition step but has likely only a limited influence
on the repair process.
Our synthetic strategy is summarized in Scheme 1.16 The key
step of the synthesis is the formation of the diastereomeric
dinucleotide spore photoproducts 2a and 2b by coupling of the
dihydrothymidine 3 with the allyl bromide 4 followed by
selective manipulations of the various protecting groups.
Protected dihydrothymidine 3 and thymidine 5 were synthe-
sized using standard procedures. Allyl bromide 4 was prepared
by Wohl–Ziegler bromination of thymidine 5. Reaction of 4
with the enolate of 3 provided the coupled but still protected
dinucleotides, which were selectively TES- and TBDMS-de-
protected to give 2a/b with pTsOH.17 Separation of the
diastereoisomers 2a and 2b was achieved by np-HPLC
(120 A, 7 mm). The configuration at C5 of 2a and 2b was
assigned using NOESY data. As indicated in the supplemen-
tary information,w for the 5S isomer 2a we measured strong
NOEs between Hb and the methyl group protons and of the Hc
with Ha. For the 5R isomer 2b, in contrast, we determined
strong NOEs between C30-H, Ha and the methyl protons. This
assignment is in agreement with previous work in our group.16
For the synthesis of the phosphoramidites 1a and 1b, we
protected the free primary hydroxyl group of 2a and 2b with
DMTOTf to obtain 6a and 6b.18 Use of the triflate was
essential for this conversion because of the steric bulk around
the primary hydroxyl group, which did not allow any reaction
with DMTCl. Transformation of the two diastereoisomers 6a
and 6b with CEDCl furnished the needed phosphoramidites 1a
and 1b ready for incorporation into DNA.
Center for Integrative Protein Science at the Department of
Chemistry and Biochemistry, LMU Munich, Butenandtstr. 5-13,
D-81377 Munich, Germany. E-mail: Thomas.Carell@cup.uni-
muenchen.de; Fax: +49 89 2180 77756; Tel: +49 89 2180 77750
w Electronic supplementary information (ESI) available: Synthesis of
the SP-isomers 1a and 1b, HPLC chromatograms of the separated
isomers 2a and 2b, 1H-NMR spectra from 2a and 2b and NOE data
for the isomers 2a/b, DNA melting curves. See DOI: 10.1039/b810008j
Having obtained both phosphoramidites it was possible to
perform the DNA synthesis on a 1 mmol-scale. The coupling of
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Table 1 DNA sequence, name and melting temperature for the
corresponding duplex. ASA/CSA synthesized with 1a, ASB/CSB
synthesized with 1b
Melting
temperature
Name
Sequence
Tm/1C
AS0
ASA
ASB
CS0
CSA
CSB
50-ATC GGC TTC GCG CA-30
50-ATC GGC T^TC GCG CA-30
50-ATC GGC T^TC GCG CA-30
50-T ATT GCA TCA TGC-30
50-T AT^T GCA TCA TGC-30
50-T AT^T GCA TCA TGC-30
65–66
45–46
42–43
51–52
43–44
41–42
are listed in Table 1. In order to determine how much the
various stereoisomers influence the stability of the duplex we
prepared two series of oligonucleotides: one in which we
inserted the spore lesion analogs in the middle of the duplex
(AS-series) and one in which the lesion is placed close to the 50
end (CS series). These latter strands are typical substrates for
primer extension studies.
The DNA strands were hybridized with the appropriate
counter strands and the melting points of the duplexes were
measured in comparison to the unmodified DNA (CS0 and
AS0). The obtained data are compiled in Table 1. The first
result of the study is that the spore lesion dramatically reduces
the stability of the duplex. Important is a direct comparison
between the 5S (CSA and ASA) and the 5R (CSB and ASB)
isomers. Here the data show that the R isomer reduces in both
cases the duplex stability (45 1C and 43 1C) by an additional
2–3 1C, in comparison to the S isomer (42 1C and 41 1C).
The amount of destabilization is clearly detectable even if
one assumes a rather large experimental error of about 1 1C. It
is therefore clear that the melting point decrease is much
stronger for duplexes with ASB and CSB. In other words
the lesion with 5R configuration gives a less stable DNA. This
would suggest that in nature the S isomer might be formed to a
larger extent. This result together with the observation that
only the 5S isomer is repaired by spore photoproduct lyases
indicates that it is the 5S isomer which is predominantly
formed in spores. More important, however, is that we
achieved for the first time the synthesis of pure oligonucleo-
tides containing repairable, site specific spore photoproduct
lyase substrates in sufficient amounts for future biochemical
studies.
Scheme 1 Synthesis of the spore photoproduct lesion starting from
thymidine 7 and 50-TBDPS-protected dihydrothymidine 8. TBDPSCl
= tert-butyldiphenylsilyl chloride, TBDMSCl = tert-butyldimethyl-
silyl chloride, SEMCl
DIEA diisopropylethylamine, NBS
DBPO = dibenzoyl peroxide, TESCl = triethylsilyl chloride, LDA
lithium diisopropylamide, pTsOH p-toluenesulfonic acid,
=
2-(trimethylsilyl)ethoxymethyl chloride,
=
=
N-bromosuccinimide,
=
=
DMTOTf = dimethoxytrityl triflate, CEDCl = 2-cyanoethyl N,N-
diisopropylchlorophosphoramidite.
the natural nucleotides was carried out with a standard
protocol. Coupling of the dinucleotide spore photoproduct
analogs, however, required modification of the protocol. We
had to prolong the coupling time of 1a and 1b and in some
cases double coupling was necessary to achieve a sufficiently
high reaction yield on the synthesizer as needed particularly
for the synthesis of longer oligonucleotides. Complicated,
however, was the cleavage of the SEM-groups of 1a and 1b
after full DNA assembly. All the standard methods based e.g.
on treatment of the compounds with fluorides,19–21 bases22 or
acids23,24 failed. However, we were finally able to cleave the
SEM groups in the presence of DNA directly on the solid
support with a 1 M SnCl4-solution in CH2Cl2, which was
slowly rinsed through the cartridge containing the solid phase
material with the DNA attached.25 After subsequent cleavage
of the DNA from the solid phase with NH3–ethanol we finally
cleaved the TBDPS-groups with TBAF.
This work was supported by the Deutsche Forschungsge-
meinschaft (DFG: SFB 749 and Excellence cluster CiPSM) and
the Fonds der Chemischen Industrie (pre-doctoral fellowship
to E.B.). Support by Novartis is gratefully acknowledged.
Notes and references
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