Chemical synthesis, crystal structure and enzymatic evaluation of a dinucleotide spore photoproduct analogue containing a formacetal linker

Article abstract of DOI:10.1002/chem.201101821

Spore photoproduct (SP) is the exclusive DNA photodamage product found in bacterial endospores. Its photoformation and repair by a metalloenzyme spore photoproduct lyase (SPL) composes the unique SP biochemistry. Despite the fact that the SP was discovered almost 50 years ago, its crystal structure is still unknown and the lack of structural information greatly hinders the study of SP biochemistry. Employing a formacetal linker and organic synthesis, we successfully prepared a dinucleotide SP isostere 5R-CH₂SP, which contains a neutral CH₂ moiety between the two thymine residues instead of a phosphate. The neutral linker dramatically facilitates the crystallization process, allowing us to obtain the crystal structure for this intriguing thymine dimer half a century after its discovery. Further ROESY spectroscopic, DFT computational, and enzymatic studies of this 5R-CH ₂SP compound prove that it possesses similar properties with the 5R-SP species, suggesting that the revealed structure truly reflects that of SP generated in Nature.

Full text of DOI:10.1002/chem.201101821

DOI: 10.1002/chem.201101821
Chemical Synthesis, Crystal Structure and Enzymatic Evaluation of a
Dinucleotide Spore Photoproduct Analogue Containing a Formacetal Linker
Gengjie Lin,[a] Chun-Hsing Chen,[b] Maren Pink,[b] Jingzhi Pu,[a] and Lei Li*[a, c]
Abstract: Spore photoproduct (SP) is
the exclusive DNA photodamage prod-
uct found in bacterial endospores. Its
photoformation and repair by a metal-
loenzyme spore photoproduct lyase
(SPL) composes the unique SP bio-
chemistry. Despite the fact that the SP
was discovered almost 50 years ago, its
crystal structure is still unknown and
the lack of structural information
greatly hinders the study of SP bio-
linker and organic synthesis, we suc-
cessfully prepared a dinucleotide SP
isostere 5R-CH2SP, which contains a
neutral CH2 moiety between the two
thymine residues instead of a phos-
phate. The neutral linker dramatically
facilitates the crystallization process, al-
lowing us to obtain the crystal structure
for this intriguing thymine dimer half a
century after its discovery. Further
ROESY spectroscopic, DFT computa-
tional, and enzymatic studies of this
5R-CH2SP compound prove that it pos-
sesses similar properties with the 5R-
SP species, suggesting that the revealed
structure truly reflects that of SP gener-
ated in Nature.
Keywords: DNA
enzymes oligonucleotides
photochemistry · UV irradiation
damage
·
·
·
chemistry. Employing
a
formacetal
Introduction
Endospore-forming bacterial strains are responsible for a
number of human diseases such as anthrax and tetanus. Due
to the unique spore DNA photochemistry coupled to effi-
cient damage repair in germinated spores, these spores are
extremely resistant to UV radiation.[1] Contrasting to the
normal cells where the UV radiation induces thymine dime-
rization in genomic DNA to generate cyclobutane pyrimi-
dine dimers (CPD) and pyrimidine (6-4) photoproducts, the
exclusive DNA photoproduct in endospores is a special thy-
mine dimer 5-thyminyl-5,6-dihydrothymine, also called
Scheme 1.
stand its formation and repair,[1a,4] the process is not com-
pletely understood.
spore photoproduct or SP.[2] Spores contain
a special
enzyme, the spore photoproduct lyase (SPL), to effectively
reverse SP formation at the early germination phase, resum-
ing their normal life cycle (Scheme 1).[1a,2] However, despite
the fact that SP was discovered nearly half a century ago[3]
and the strong interest of the scientific community to under-
Our group is interested in the mechanistic elucidation of
both aspects of the SP biochemistry. By selectively labeling
a dinucleotide TpT with deuterium and following the deute-
rium migration during the photoreaction, we demonstrated
that one of the three methyl hydrogen atoms migrates to the
pro-S position of the C6 carbon in resulting SP.[5a] Further-
more, utilizing the deuterium-labeled SP substrate generated
via photochemistry, we proved that the hydrogen atom ab-
stracted by SPL in the initial step of the enzyme repair pro-
cess was not returned to the resulting TpT product, a pro-
tein residue to be identified instead serves as the H-atom
donor.[6] Our data suggest that the currently hypothesized
enzyme mechanism, which excludes the involvement of pro-
tein residue in catalysis, needs to be modified;[1f,7] the SPL
reaction is much more complex than currently thought.[6]
Although being discovered almost half a century ago, the
structure of SP remains unknown. In fact, among the three
thymine dimers, CPD, (6-4) photoproduct and SP, only CPD
is structurally characterized in duplex DNA[8] as well as in
dinucleotide fragment.[9] The lack of structural information
[a] Dr. G. Lin, Prof. Dr. J. Pu, Prof. Dr. L. Li
Department of Chemistry and Chemical Biology
Indiana University, Purdue University Indianapolis (IUPUI)
402 N. Blackford St., Indianapolis, IN 46202 (USA)
Fax : (+1)317-274-4701
E-mail: lilei@iupui.edu
[b] Dr. C.-H. Chen, Dr. M. Pink
Indiana University Molecular Structure Center, Chemistry
A421, Indiana University, 800 E. Kirkwood Avenue
Bloomington, IN 47405 (USA)
[c] Prof. Dr. L. Li
Department of Biochemistry and Molecular Biology
Indiana University School of Medicine, 635 Barnhill Drive
Indianapolis, IN 46202 (USA)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201101821.
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FULL PAPER
has drastically hindered our understanding of the SP bio-
chemistry. As SP contains a chiral center in C5, both R and
S isomers can be obtained should the thymine residues react
freely.[10] Such a free rotation does not happen due to the re-
striction of the double helical structure in genomic DNA,
which determines that only one of two SP isomers can possi-
bly form in Nature. There was a heated debate in the past
several years on the steric configuration of SP in bacterial
DNA.[11] Although recent NMR spectroscopic studies estab-
lished SP to adopt a 5R configuration, a crystal structure
which allows a direct observation of the atom arrangement
in SP is clearly needed.
Dinucleotide is the smallest DNA fragment to accommo-
date a SP dimer. Such a SP possesses the size of a small or-
ganic molecule; it can thus provide the precise structural de-
tails by the single-crystal X-ray diffraction methods. We ra-
tionalized that the negatively charged phosphate moiety in
natural DNA may hinder the crystallization process, as re-
flected by the fact that all the CPD dimers crystallized to
date contain either no or neutral linker between the two T
residues.[9,12] A formacetal is the simplest and smallest non-
chiral isostere for a phosphate moiety and has proved very
useful in oligonucleotide biochemical studies.[9a,13] It was
used to successfully crystallize a dinucleotide CPD analogue
formed between two uracil residues.[9a] It was included be-
tween the thymine residues of a CPD which was incorporat-
ed into the middle of a duplex DNA strand to facilitate the
crystallization of the photolyase–DNA complex as well.[14]
We therefore decided to incorporate a formacetal linker
into the SP dinucleotide to examine if the resulting com-
pound would mimic the properties of natural phosphate
containing SP. Moreover, the neutral methylene linker was
expected to facilitate the crystallization process, allowing us
to gain structural insight into this important thymine photo-
damage product.
Results and Discussion
Chemical synthesis of 5R-CH2SP: As summarized in
Scheme 2, the formacetal containing SP analog was synthe-
sized using a 15-step chemical reaction via a modified proce-
dure employed in the synthesis of phosphate containing di-
nucleotide SP adopted by Begley and co-worker.[11d] Howev-
er, our synthesis is not a simple repetition of the previous
work. For instance, SEM ([2-(trimethylsilyl)ethoxy]methyl)
was used to protect the thymine N3 in Begleyꢁs original ap-
proach; such a protecting reagent proved futile in our syn-
thesis as it is incompatible with the formacetal linkage in
the SnCl4-promoted de-protection step to obtain compound
11. Changing to other de-protection reagents such as TBAF/
DMPU, or TBAF/HMPA[15] led to a complete decomposi-
tion of SEM-protected 11 as well. We therefore chose the
benzyloxymethyl group (CH2OBn) for N3 protection, which
as a bonus increased the diastereoselectivity of the 5R over
5S isomer to 2.6:1 after the coupling step to yield compound
Scheme 2.
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L. Li et al.
5, contrasting to the non-selective 1:1 mixture obtained in
the previous work.[11d] (Note: The steric configurations of
the compounds involved were assigned in consistence to the
SP assignment in the previous NMR studies.[4]) To remove
the CH2OBn from compound 10, the traditional hydrogena-
tion reaction did not work probably due to the intoxication
of the Pd/C catalyst by the thymine nucleobase.[16] Fortu-
nately, the oxidative de-benzylation strategy using the 2,3-di-
chloro-5,6-dicyano-1,4-benzoquinone (DDQ) proved effec-
tive;[17] the 5R-CH2SP was successfully obtained after a stan-
dard desilylation reaction via TBAF (tetrabutylammonium
fluoride) treatment.
suggests that the TCH T adopts a conformation different
from that of TpT at the solid state.
2
Although the solid state is found to promote SP formation
under UV photolysis probably due to the unique thymine
stacking interaction, the exact thymine conformation under
such a state is unclear. Our research to date found that this
photoreaction can be easily disturbed by small modifications
on the nucleotide residues. For instance, disturbing the
methyl–methyl interaction between the thymine residues
could dramatically alter the outcome of SP photochemistry.
Due to the lower zero-point energy, deuterium is slightly
smaller that protium even though it is the heavier isotope.[20]
However, substituting the CH3 of 5’-T by a smaller CD3
moiety while leaving the CH3 group of the 3’-T which is in-
volved in the H-transfer process intact decreases the yield of
SP by 2.5-fold after photolysis of TpT.[5a] Moreover, the di-
nucleotide UpT which does not contain such a Me–Me inter-
action totally stopped the SP formation in the dry film reac-
tion[5b] although the mechanism suggests a SP analogue to
be produced should the UpT adopt a similar conformation
as TpT.
Chemical synthesis and photolysis of TCH T: The synthesis of
2
thymine dinucleotide with a formacetal linker was achieved
using a modified procedure of the UCH U synthesis,[9a] as
2
shown in Scheme 3. The TCH T was readily obtained after
2
the standard deprotection step via ammonium hydroxide
treatment.
The highly sensitive DNA conformation favoring SP for-
mation indicates that changing the negatively charged phos-
phate moiety to a neutral and smaller formacetal linker is
likely to alter the thymine conformation in the dry state,
making it to no longer support the SP formation upon UV
excitation. The observation suggests that to obtain the for-
macetal containing SP, the lengthy chemical synthesis shown
above becomes the only choice, which is in sharp contrast to
the cyclobutane pyrimidine dimer formation in the UCH U
2
photolysis conducted by Carell and co-workers.[9a] In their
studies, the cis–syn CPD product was isolated as the major
photoproduct with a 44% yield, making the CPD dimer
preparation much easier.
Crystal structure of the 5R-CH2SP: The resulting 5R-CH2SP
is very soluble in water due to the hydrophilic nature of the
thymidine residue. More importantly, as expected, this neu-
tral linker dramatically improved the crystallization property
of SP. Dissolving the 5R-CH2SP in water and allowing the
solvent to slowly evaporate in two days afforded a colorless
block crystal. The X-ray analysis of the crystal revealed the
structure of SP half a century after it was discovered.[3]
As shown in Figure 1, the most noteworthy feature in the
SP structure is the methylene bridge between the two C5
carbons of the thymine residues. The C5 carbon on the 5’-T
is chiral as the consequence of the methyl addition; and our
structure clearly shows that it adopts an R configuration,
confirming the assignment by the previous NMR spectro-
scopic studies.[4] (Note: in the structure shown in Figure 1,
C5 was used to label the methylene bridge between the thy-
mine bases in the PDB file; while C6 was used to label the
C5 position of thymine, which adopts an R configuration.).
The 3’-thymine ring in SP is still planar; in contrast, the 5’-
thymine is distorted because of the loss of C5=C6 bond and
the subsequent loss of the ring aromaticity.
Scheme 3.
However, UV irradiation of the resulting TCH T in either
2
dry film or ice at 77 K under the established photochemical
procedure[5,18] led to no formation of the SP analog 5R-
CH2SP, as suggested by the HPLC analysis using the synthe-
sized 5R-CH2SP as the standard. This finding is in sharp
contrast to what was observed in the TpT photolysis, which
readily produced 5R-SP in ~1% yield in the dry film reac-
tion as shown in our previous studies.[5a] As the outcome of
DNA photoreaction is determined by the DNA conforma-
tion at the instant of photoexcitation,[19] such an observation
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A Dinucleotide Spore Photoproduct Analogue
FULL PAPER
ies.[4] Comparing with the (J1’2’ + J1’2’’) value, the values of
J1’2’/(J1’2’ + J3’4’) and J3’4’/(J1’2’ + J3’4’) are more commonly used
to calculate the percentages of C2’- and C3’-endo conform-
ers in the mixture.[21–22] As shown in Table 1, the calculation
shows that the majority of the deoxyriboses at the 5’-end of
SP adopt the C3’-endo conformation in both 5R-SP and 5R-
CH2SP species. The large J1’2’ (7.1 Hz) in the deoxyriboses at
the 3’-end of 5R-SP suggests that the C2’-endo conformer
could be the dominant species as suggested by the previous
NMR studies,[4] although significant amount of C2’-endo
conformers may still exist in the 3’-end ribose of the 5R-
CH2SP as suggested by the relatively small J1’2’ (5.9 Hz)
value. However, the absolute percentages cannot be ob-
tained due to the lack of J3’4’ value for both SP species. Once
crystallized, the C3’-endo conformation prevails for both
ribose rings, suggesting such a conformation is more stable
in the solid state.
The slightly higher percentages of C3’-endo conformer for
both deoxyriboses in 5R-CH2SP than its phosphate counter-
part seem to suggest that the formacetal linker may induce
the ribose to adopt the C3’-endo conformation in solution.
However, the formacetal linker proved not to change the
C2’-endo deoxyribose conformation in regular duplex DNA
as revealed by a previous NMR spectroscopic studies.[13b]
Figure 1. X-ray structure of the 5R-CH2SP with the nitrogen and oxygen
atoms labeled. For clarity purpose, only the bridging methylene carbon
between the two thymine bases (C5), the newly formed chiral center at
the 5’-T (C6) and the formacetal carbon (C16) are labeled among the 21
carbon atoms in the structure. The structure clearly shows that the chiral
center (C6) adopts an R configuration. The distance between the H6a-pro-S
and the C5 carbon (shown in blue arrow) is found to be 2.63 ꢂ and that
between the H6a-pro-R and the C5 carbon (shown in red arrow) is found to
be 3.36 ꢂ. Details about the bond lengths and bond angles are available
in Supporting Information.
Both deoxyriboses adopt the C3’-endo conformation in
the structure, contrasting to the C2’-endo claimed in the pre-
vious NMR studies.[4] Re-examining the NMR studies re-
vealed that the C2’-endo conformation was reached as the
values of (J1’2’ + J1’2’’) for the two deoxyribose rings were
Moreover, the deoxyriboses in a UCH U CPD dimer with a
2
formacetal linker exhibit the C2’-endo conformation as
well.[9a] These results suggest that the linker alone is not re-
sponsible for the ribose conformation; such a conformation
is more likely due to the intrinsic property of the SP com-
pound.
C3’-endo is typically observed in A-form DNA while the
C2’-endo is the feature of B-form DNA.[23] It is widely ac-
cepted that thymine dimerization in B-form DNA produces
CPD as the dominant species; while DNA conformation has
to change to an A-like DNA form before SP product can be
generated upon UV radiation.[1a,24] The deoxyribose confor-
mation observed in our structure thus provides a direct evi-
found to be 11.7 and 15.1 Hz, respectively. The (J1’2’ + J1’2’’
)
value ranging from 7.1 to 16.1 Hz was suggested to be char-
acteristic for the C2’-endo conformation, which we believe is
not correct. As the C2’-endo and C3’-endo conformers are in
equilibrium in solution in most cases, a (J1’2’ + J1’2’’) value of
7.1 Hz roughly corresponds to a 100% C3’-endo conformer;
while a (J1’2’ + J1’2’’) value of 16.1 Hz roughly corresponds to
a 100% C2’-endo conformer.[21] Therefore, what was ob-
served in the previous NMR studies also indicates a mixture
of C2’- and C3’-endo conformers, and the question thus is
which conformer dominates?
À
We therefore re-visited the 1D-NMR studies of both 5R-
SP and 5R-CH2SP species and obtained the coupling con-
stants for the ribose protons. As shown in Table 1, all cou-
pling constants for the deoxyribose attached to the 5’-T can
be readily obtained; however, the J3’4’ for the deoxyribose at
the 3’-end was difficult to determine owing to the poor reso-
lution. The (J1’2’ + J1’2’’) values for the 5R-SP species were
found to be 12.1 and 14.2 Hz, respectively, which are compa-
rable to the values determined in the previous NMR stud-
dence to support this assumption. The C O bonds associat-
ed with the formacetal linker are found to be 1.412 ꢂ long,
À
which is about 0.2 ꢂ shorter than the typical P O bond in
DNA.[23] However, such a bond shortening is suggested to
be readily compensated by small changes of the backbone
torsional angles and is not projected to result in big DNA
conformational change.[9a]
As shown in our previous deuterium labeling studies, the
H6a-pro-S atom of SP originates from the methyl group of the
3’-T in dinucleotide TpT.[5a] The distance between the H6a-pro-
and the bridging methylene carbon is found to be 2.63 ꢂ
in our SP structure (distance indicated by the double-head
S
Table 1. J1’2& and J3’4& coupling constants (Hz) and population distribu-
tion of conformers in the deoxyribose in 5R-SP and 5R-CH2SP.
À
blue arrow in Figure 1), only 1 ꢂ longer than a typical C H
5R-SP
5R-CH2SP
bond. Should our structure reflect that of natural SP and a
direct H atom abstraction occur during SP formation as pro-
posed by our mechanism,[5a] only relatively small conforma-
tional change is needed from the reactive TpT to the resul-
tant SP. In contrast, the distance between the H6a-pro-R, which
is the retained H atom at 5’-T during SP formation, and the
methylene carbon, is 0.7 ꢂ longer at 3.36 ꢂ (distance indi-
5’-end
3’-end
5’-end
3’-end
J1’2’
J1’2’’
J3’4’
4.0
8.1
6.9
37
7.1
7.1
n/d
3.3
8.6
7.3
31
5.9
8.6
n/d
% of C2’-endo[a]
% of C3’-endo[b]
63
69
[a] Calculated from J1’2’/(J1’2’ + J3’4’). [b] Calculated from J3’4’/(J1’2’ + J3’4’).
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L. Li et al.
cated by the double-head red arrow in Figure 1). This obser-
vation supports our findings in previous labeling studies that
one of the methyl H atoms migrates to the H6a-pro-S position
during SP photo-formation; relatively minimal structural
changes are required as the consequence of this H atom mi-
gration even in dinucleotide TpT.
Structural comparison of 5R-SP and 5R-CH2SP via 2D
ROESY spectroscopy: To confirm that the formacetal link-
age does not alter the base interaction in SP and the 5R-
CH2SP truly reflects the conformation of 5R-SP formed in
Nature, we first compared the solution structure of these
two species using 2D-ROESY NMR spectroscopy which re-
flects the H–H correlations through space. As summarized
in Figure 2d, strong cross-peaks were observed among pro-
tons located in front of the thymine ring (H6a-pro-R, CH3, HS)
or the protons behind this ring (H6a-pro-S, H6b, HR) in ROESY
spectrum of 5R-SP.[4–5] Between the two C6 protons on the
5’-T of 5R-SP, Only the H6a-pro-S exhibits a strong interaction
with the deoxyribose protons H2’A/2’’A and H3’A. Moreover, al-
though both protons interact with the -CH3 group, the cou-
pling with H6a-pro-S is weaker than that with H6a-pro-R. All
these interactions have been observed in the ROESY spec-
trum of the 5R-CH2SP species except that the coupling be-
tween H6a-pro-S and -CH3 is also strong. In the two ROESY
spectra exhibited in Figure 2, only the coupling interactions
among the protons associated with the 5’-T ring are high-
lighted for clarity purpose. Furthermore, H–H coupling can
be typically observed for two protons 3–4 ꢂ away from each
other in the ROESY spectrum. Comparing the H–H distan-
ces predicted by the ROESY spectrum with those found in
the X-ray structure reveals that these two agree with each
other fairly well, suggesting that the SP crystal structure ob-
tained in solid state largely reflects its solution structure.
Structural comparison of 5R-SP and 5R-CH2SP via DFT
calculation: Theoretical calculations further support that the
formacetal linkage does not change the overall structure of
the SP molecule. The 5R-CH2SP and 5R-SP structures were
optimized using density functional theory at the B3LYP/6-
Figure 2. Top) Zoom-in view of 1H-ROESY NMR spectra for 5R-CH2SP
and 5R-SP, respectively in DMSO. The key H-H correlations from pro-
tons associated with the 5’-thymine ring are highlighted by the green
arrows in these spectra (Note: in the ROESY of 5R-CH2SP, the spots
pointed by two arrows are actually composed by two dots, which can be
separated in a further zoom-in view). Bottom) Key NOESY cross-peaks
determined from the NMR spectra of these SP species. The almost iden-
tical coupling patterns between these two compounds suggest them to
possess very similar steric conformation in the nucleobase structure. Full
1D- and 2D-NMR spectra for these SP species can be found in SI.
31+GACHTUNGTRENNUNG
(d,p) level and basis set (Figure 3).[25] The root-mean-
square-deviation (RMSD) of common heavy atoms between
the optimized 5R-CH2SP and the crystal structure is
0.290 ꢂ. The RMSD between the optimized 5R-CH2SP and
À
5R-SP is 0.372 ꢂ. In 5R-CH2SP, the C O bond distances as-
sociated with the formacetal linkage were calculated to be
1.413 and 1.405 ꢂ, respectively, which are comparable to
those revealed by the X-ray structure but shorter than the
À
corresponding P O bonds of 1.689 ꢂ found in 5R-SP, sug-
gesting a slightly tighter linkage between the two deoxyri-
bose rings in 5R-CH2SP. However, such a difference at the
ribose interface does not alter the overall structural features
of SP, with only negligible differences bond lengths and
bond angles obtained in other part of these two species.[25]
In addition to these essentially identical geometric param-
eters, a few changes were observed for the stacking configu-
ration between two thymine residues in these two SPs: the
dihedral angles between the two base planes are 49.9 and
50.08, in 5R-CH2SP and 5R-SP, respectively. The conserva-
tion of angular features in SPs upon PO2/CH2 replacement
may be ascribed to the geometric restraint imposed by the
methylene bridge between two rigid thymine bases, which
results a minimum structural impact of the PO2/CH2 re-
placement in the more flexible ribose part. Population anal-
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A Dinucleotide Spore Photoproduct Analogue
FULL PAPER
Figure 3. Superposition of the 5R-CH2SP and 5R-SP structures computa-
tionally optimized at B3LYP/6-31+GACTHNUGTRNEUNG(d,p) level. Carbon atoms in 5R-
CH2SP and 5R-SP are colored in dark-gray and green, respectively; the
phosphorus atom in 5R-SP is colored in orange and oxygen atoms are in
red. These two structures are found to overlap perfectly for most part of
the molecules except for the linkage section.
Figure 4. A) HPLC chromatograph of the 5R-CH2SP repair mediated by
SPL. 5’-dA was eluted at 16.8 min, 5R-CH2SP at 18.5 min and TCH T at
2
28.2 min. B) Enzyme reaction rates were calculated via plotting the
ysis of the SPs further suggests that the formacetal linkage
does not change the atomic charge distribution in the nucle-
obase, with the atomic charges in the two SPs essentially the
same.[25]
amount of TCH T or TpT isolated versus reaction time.
2
The significant repair rate exhibited by 5R-CH2SP thus dem-
onstrates that it is a SPL substrate and its structure revealed
in 5R-CH2SP must be very similar to the naturally occurring
5R-SP.
Structural comparison of 5R-SP and 5R-CH2SP via enzy-
matic reaction: In germinated spores, the SP dimer is re-
paired by a radical SAM enzyme spore photoproduct lyase
(SPL).[1d–g,6,26] SPL can tolerate a number of substrates, rang-
ing from SP containing plasmid DNA[1d,f,7] to a 6-mer oligo-
nucleotide.[1g] SPL even repairs the dinucleotide SP, the min-
imal substrate possible, to TpT.[1e,6,27] The phosphate linker
in SP proved important to maintain its conformation, ena-
bling its fitting into the enzyme binding pocket. The pres-
ence of phosphate results a 50-fold rate increase in enzyme
reaction compared with a SP substrate with no such a link-
er.[11b,c] Comparing the SPL reactions using SP substrates
within DNA strand of different length; the dinucleotide 5R-
SP results in a relatively slow repair rate due to its weak
binding affinity to SPL. Despite that, our recent work
proves that it still exhibits all the important substrate prop-
erties during SPL catalysis. If 5R-CH2SP adopts a similar
conformation as the 5R-SP, as proved above, it should sup-
port a reasonable SPL activity as well.
Conclusion
In summary, by employing a neutral formacetal linker and
synthetic chemistry, a dinucleotide SP isostere was success-
fully prepared. Such a compound can only be obtained via
this lengthy synthetic route. Photolysis of the dinucleotide
T
CH T did not result in any SP analogue formation, indicat-
2
ing that the loss of the negative charge in the phosphate
after the linker replacement and/or the smaller size of the
formacetal linker change the relative positions of the thy-
mine residues, making them to no longer support the SP
photochemistry. Such a neutral linker greatly facilitates the
crystallization process, allowing the SP to be crystallized and
its structure determined 50 years after its discovery. ROESY
spectroscopy, DFT calculations as well as enzymatic activity
studies all verify that the resulting 5R-CH2SP possesses a
similar structure to the phosphate-containing 5R-SP, suggest-
We therefore incubated the 5R-CH2SP with the SPL
enzyme using the experimental conditions described in our
previous studies.[6] The substrate concentration was kept at
~150-fold of the enzyme concentration; decreasing the con-
centration by half did not change the reaction rate. This ob-
servation suggests that the reaction rate determined very
ing that different from the TCH T residue, in SP the methyl-
2
ene bridge formed between the methyl moiety of the 3’-T
and the C5 carbon of the 5’-T in the rigid nucleobase region
dictates the overall SP structure, making the influence of
such a linker change trivial.
likely reflects the Vmax. As shown in Figure 4, the TCH T spe-
2
cies was produced as the consequence of the enzyme repair
Such a simple structure of dinucleotide SP isostere still
provides several valuable mechanistic insights regarding to
the SP photo-formation. The shorter distance between the
H6a-pro-S and the methylene bridge between the two thymine
bases supports our mechanism that both the H6a-pro-S and the
methylene bridge originate from the methyl group of the 3-
T.[5a] While the C3’-endo conformation, which is typically ob-
served in A-form DNA, for both deoxyriboses in 5R-CH2SP
reaction; plotting the amount of TCH T isolated versus the
2
reaction time leads to a Vmax of 0.17Æ0.01 minÀ1, which is
roughly half of the Vmax determined using 5R-SP as the
enzyme substrate.[6] The slightly decreased reaction rate sug-
gests that the phosphate linker in natural SP may be in-
volved in substrate recognition. Of the 5R and 5S SP iso-
mers, SPL is proved to only take one as its substrate.[11b,c]
Chem. Eur. J. 2011, 17, 9658 – 9668
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9663

L. Li et al.
2489 UV/Visible detector and an Agilent 1100 LC-MS system under both
a positive and negative ionization mode. Sonic disruption of E. coli cells
was conducted by a Fisher Scientific* Model 500 Digital Sonic Dismem-
brator. DNA sequencing was performed by the Indiana University Nucle-
ic Acid Core Facility at IU School of Medicine.
supports the assumption that the spore genomic DNA has
to change to the A-like conformation due to the binding of
the small acid soluble proteins before SP can be produced
upon UV irradiation.[24] Our data further suggests that after
SP formation, the methylene bridge between the thymine
bases becomes the dominant factor, forcing the two deoxy-
riboses to maintain the A-like conformation even though
the DNA binding proteins are absent.
The fact that 5R-CH2SP is still an enzyme substrate sug-
gests that the phosphate moiety between the two thymine
residues in SP most likely plays a structural role to hold the
two thymine residues in the right positions so that SP can fit
into the enzyme binding pocket. Such a linker may also be
recognized by some protein residues due to its negative
charge as the linker change to a neutral formacetal moiety
results a 50% rate decrease in the enzyme reaction. The for-
macetal linker has proved to be highly useful in studying the
DNA repairing enzymes. For instance, the formacetal con-
taining CPD dimer was incorporated into a duplex DNA,
enabling the co-crystallization of such a DNA sequence with
the CPD repair enzyme - DNA photolyase.[14] Using this 5R-
CH2SP to explore the reaction mechanism of SPL is current-
ly in progress.[33]
TCH T photochemical studies: The photolysis of TCH T was conducted
2
2
using the same precedure in our previous studies.[5a] Briefly, a 5 mL 1 mm
aqueous solution of TCH T was prepared by titration with 0.3m NaOH to
2
pH 7.0. After removal of water under vacuum, the resulting
TCH T
2
sodium salt was dissolved in MeOH (5 mLs). The solution was trans-
ferred to a 10 cm petri dish and methanol evaporation afforded a nice
thin film. The film was then exposed for 45 min to the UVC (254 nm)
light. The products were subsequently dissolved in water and analyzed by
reverse phase HPLC in the gradient mode using triethyl ammonium ace-
tate, pH 6.5, and acetonitrile as solvents.
In another set of experiments, the resulting TCH T sodium salt was re-dis-
2
solved in a glycerol/water solution (1:1, 5mL total) and flash freeze in
liquid N2 to obtain a transparent glass. The glass was subsequently irradi-
ated for 45 min under 254 nm UV light in liquid N2. The glass was al-
lowed to warm up to ambient temperature and the UV reaction products
in the glycerol/water solution analyzed directly by HPLC.
DFT calculation: The computational procedures are described below. To
obtain an initial geometry of 5R-SP, we started from the crystal structure
of 5R-CH2SP, where we replaced the carbon atom in the CH2 linkage by
À
a phosphorous atom. Then the two oxygen atoms in PO2 were placed at
initial positions that are 1.6 ꢂ away from the phosphorous atom along
À
the crystallographic C H bond directions. We noticed that there is a crys-
tal water molecule captured in the X-ray structure, where inclusion of
the crystal water in the models introduced additional computational cost
for optimizing the intermolecular interaction between SPs and the sol-
vent. Our preliminary calculations showed that the presence of the crys-
tal water did not change the essential features of the SPs in geometry
and charge distributions. Therefore, we omitted the crystal water from
our models in the subsequent calculations. The 5R-CH2SP crystal struc-
ture and the 5R-SP structure generated with above procedures were then
optimized with density function theory (DFT) calculations at the B3LYP/
Experimental Section
General methods: Unless otherwise stated all solvents and chemicals
used were of commercially available analytical grade. They were pur-
chased from Sigma, Fisher, or VWR and used without further purifica-
tion. All reactions were carried out using oven or flame-dried glassware
under a nitrogen atmosphere in distilled solvents. Dichloromethane and
pyridine were distilled over calcium hydride. Purification of reaction
products was carried out by flash chromatography using silica gel (Dy-
namic Adsorbents Inc, 32–63 mm). For TLC analysis, precoated plates (w/
h F254, Dynamic Adsorbents Inc, 0.25 mm thick) were used. The 1H and
13C NMR spectra were obtained on a Bruker 500 MHz NMR Fourier
transform spectrometer. NMR spectra were recorded in the following
solvents: CDCl3, with residual chloroform (d 77.2 ppm for 13C NMR) and
TMS (d 0 ppm for 1H NMR), CD3OH (d 3.31 ppm for 1H NMR and d
49.1 ppm for 13C NMR) or [D6]DMSO, with residual methyl sulfoxide (d
2.50 ppm for 1H NMR and d 39.5 ppm for 13C NMR) taken as the stan-
dard. MS analysis was obtained using Agilent 1100 series LC/MSD
6-31+GACTHNUGRTENUNG(d,p) level and basis set, by using the electronic structure pack-
age Gaussian03.[28]
The computationally optimized structures were superimposed with the
original X-ray structure based on common heavy atoms using the macro-
molecular modeling package CHARMM,[29] where for the comparison of
À
5R-CH2SP and 5R-SP, the O/P atoms involved in the CH2/PO2 replace-
ment were included in the superposition, but the two oxygen atoms in
À
PO2 were excluded. The dihedral angles between the two thymine
planes were calculated by the least square fitting plane utility (LSQP) of
the COOR module in CHARMM, where 12 atoms are included to
define the 3’-thymine ring, and 10 atoms are included to define the 5’-thy-
mine ring.
system with Electrospray Ionization (ESI). The TCH T photoreactions
2
Chemical synthesis of 5R-CH2SP: As shown in Scheme 2, the synthesis
of the 5R-CH2SP was achieved via a modified procedure used in the syn-
thesis of 5R-SP originally adopted by Begley and co-workers.[30]
under UVC light were carried out using a Spectroline germicidal UV
sterilizing lamp at 254 nm (Dual-tube, 15 W, intensity: 1550 mWcmÀ2).
All DNA-modifying enzymes and reagents were purchased from Fermen-
tas Life Sciences (Glen Burnie, MD). B. subtilis strain 168 chromosomal
DNA was purchased from the ATCC (ATCC 23857D). Oligonucleotide
primers were obtained from Integrated DNA Technologies (Coralville,
N3-Benzyloxymethyl-3’,5’-O-di(tert-butyldimethylsilyl)-5-bromomethyl-2’-
deoxyuridine (2): A mixture of 1 (10.0 g, 18.2 mmol), iPr2NEt (11.1 mL,
63.7 mmol) and BnOCH2Cl (6.35 mL, 45.5 mmol) in anhydrous CH2Cl2
(25 mL) was stirred for 1 d.[31] The reaction solution was diluted with
CH2Cl2 (200 mL), washed with saturated aqueous sodium bicarbonate
(3ꢃ20 mL) and dried over anhydrous sodium sulfate. After filtration and
concentration under vacuum, the residue was purified by flash chroma-
tography (hexane/EtOAc 6:1) to afford 2 as a yellow oil (4.40 g, 36%).
1H NMR (CDCl3): d = 0.08 (s, 3H), 0.09 (s, 3H), 0.12 (s, 3H), 0.13 (s,
3H), 0.90 (s, 9H), 0.94 (s, 9H), 1.96 (ddd, J=5.9, 7.7, 13.5 Hz, 1H), 2.33
(ddd, J=2.6, 5.8, 13.5 Hz, 1H), 3.77 (dd, J=2.6, 11.4 Hz, 1H), 3.87 (dd,
J=2.8, 11.4 Hz, 1H), 3.96–4.00 (m, 1H), 4.32 (d, J=12.0 Hz, 1H), 4.36–
4.41 (m, 1H), 4.39 (d, J=12.0 Hz, 1H), 4.71 (s, 2H), 5.50 (s, 2H), 6.29
(dd, J=5.8, 7.7 Hz, 1H), 7.24–7.28 (m, 1H), 7.29–7.35 (m, 2H), 7.35–7.39
(m, 2H), 7.82 ppm (s, 1H); 13C NMR (CDCl3): d = À5.3 (2C), À4.7,
À4.5, 18.1, 18.5, 25.8, 26.1, 39.5, 41.9, 63.1, 70.7, 72.4, 72.5, 86.3, 88.3,
IA). E. coli BL21ACHTUNGTRENNUNG(DE3) and expression vector pET-28a were purchased
from Novagen (Madison, WI). The construct containing the SPL gene
was co-expressed with plasmid pDB1282, which was a generous gift from
Professor Squire Booker at the Pennsylvania State University. 5’-deoxya-
denosine (5’-dA) and S-adenosylmethionine (SAM) was purchased from
Aldrich and used directly without further purification. 5 Prime Perfect-
pro* Nickel nitrilotriacetic acid (Ni-NTA) resin was purchased from
Fisher Scientific. All other buffers and chemicals were of the highest
grade available.
The protein purification as well as the enzyme reactions were carried out
under an inert atmosphere using a Coylab anaerobic chamber (Grass
Lake, MI) with the H2 concentration around 3%. Product analysis was
conducted using a Waters (Milford, MA) breeze HPLC system with a
9664
www.chemeurj.org
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2011, 17, 9658 – 9668

A Dinucleotide Spore Photoproduct Analogue
FULL PAPER
110.7, 127.8, 128.4, 137.9, 138.1, 150.5, 161.6 ppm; ESI-MS (positive ion):
dried over anhydrous sodium sulfate. After filtration and concentration
under vacuum, the residue was purified by flash chromatography
(hexane/EtOAc/MeOH 1:1:0.2) to afford 6 as a colorless oil (1.25 g,
78%). 1H NMR (CDCl3): d = 0.09 (s, 6H), 0.12 (s, 6H), 0.90 (s, 9H),
0.93 (s, 9H), 1.11 (s, 3H), 1.95 (ddd, J=5.9, 7.9, 13.4, Hz, 1H), 2.00 (ddd,
J=3.3, 6.4, 13.6 Hz, 1H), 2.08–2.15 (m, 1H), 2.20 (ddd, J=2.6, 5.8,
13.4 Hz, 1H), 2.68 (d, J=14.3 Hz, 1H), 2.69 (brs, 1H), 2.74 (d, J=
14.3 Hz, 1H), 2.93 (brs, 1H), 2.94 (d, J=13.1 Hz, 1H), 3.35 (d, J=
13.1 Hz, 1H), 3.43–3.65 (m, 1H), 3.58–3.65 (m, 1H), 3.73–3.82 (m, 3H),
3.92–3.97 (m, 1H), 4.26–4.31 (m, 1H), 4.39–4.43 (m, 1H), 4.58 (d, J=
12.4 Hz, 1H), 4.62 (d, J=12.4 Hz, 1H), 4.68 (d, J=11.8 Hz, 1H), 4.71 (d,
J=11.8 Hz, 1H), 5.28 (d, J=9.9 Hz, 1H), 5.35 (d, J=9.9 Hz, 1H), 5.41
(d, J=9.4 Hz, 1H), 5.52 (d, J=9.4 Hz, 1H), 6.19 (dd, J1 =J2 =6.8 Hz,
1H), 6.33 (dd, J=5.9, 7.9 Hz, 1H), 7.22–7.29 (m, 6H), 7.30–7.34 (m, 2H),
7.34–7.38 (m, 2H), 7.54 ppm (s, 1H); 13C NMR (CDCl3): d = À5.3, À5.2,
À4.7, À4.6, 18.0, 18.5, 21.0, 25.8, 26.1, 32.0, 37.5, 40.7, 42.3, 44.8, 62.5,
63.2, 70.8, 70.9, 71.9, 72.0, 72.4, 72.5, 85.5, 85.8, 86.1, 87.9, 108.7, 127.5,
127.7, 127.8, 128.1, 128.4, 128.5, 137.6, 138.5, 139.1, 150.7, 152.3, 163.7,
174.3 ppm; ESI-MS (positive ion): m/z: calcd for C48H73N4O12Si2+: 953.5
[M+H]+, found 953.5.
m/z: calcd for C30H50BrN2O6Si2+: 669.2 [M+H]+, found 669.2.
N3-Benzyloxymethyl-3’,5’-O-di(triethylsilyl)-5,6-dihydrothymidine (4): A
mixture of 3 (3.40 g, 7.19 mmol), iPr2NEt (5.66 mL, 32.5 mmol) and
BnOCH2Cl (3.00 mL, 21.7 mmol) in anhydrous CH2Cl2 (15 mL) was
stirred for 36 h.[11d] The reaction solution was diluted with CH2Cl2
(150 mL), washed with saturated aqueous sodium bicarbonate (3ꢃ
10 mL) and dried over anhydrous sodium sulfate. After filtration and
concentration under vacuum, the residue was purified by flash chroma-
tography (hexane/EtOAc 6:1) to afford 4 as a colorless oil (3.27 g, 77%).
1H NMR (CDCl3): d = 0.61 (q, J=7.9 Hz, 12H), 0.96 (t, J=7.9 Hz, 9H),
0.97 (t, J=7.9 Hz, 9H), 1.21 (d, J=7.1 Hz, 3H), 1.92–1.98 (m, 2H), 2.54
(ddq, J=5.2, 8.8, 7.1 Hz, 1H), 3.08 (dd, J=8.8, 12.9 Hz, 1H), 3.17 (dd,
J=5.2, 12.9 Hz, 1H), 3.67–3.71 (m, 2H), 3.80–3.84 (m, 1H), 4.32–4.36 (m,
1H), 4.66 (s, 2H), 5.33 (d, J=9.9 Hz, 1H), 5.36 (d, J=9.9 Hz, 1H), 6.36
(dd, J=6.3, 8.1 Hz, 1H), 7.23–7.27 (m, 1H), 7.27–7.33 (m, 2H), 7.33–
7.37 ppm (m, 2H); 13C NMR (CDCl3): d = 4.3, 4.8, 6.8, 13.4, 35.8, 38.1,
40.5, 62.9, 70.4, 71.8, 72.2, 84.6, 86.7, 127.5, 127.6, 128.3, 138.6, 153.0,
172.8 ppm; ESI-MS (positive ion): m/z: calcd for C30H53N2O6Si2+: 593.3
[M+H]+, found 593.3.
Selective protection of 6 to yield 7: A solution of 6 (1.06 g, 1.11 mmol),
tert-butyl diphenylchlorosilane (TBDPSCl) (0.31 mL, 1.20 mmol) and
imidazole (152 mg, 2.23 mmol) in DMF (5 mL) was stirred at 08C for
16 h. The reaction was allowed to warm to room temperature and stirred
for another 20 h. The reaction was quenched by water (30 mL). The mix-
ture was extracted with Et2O (5ꢃ50 mL). The collected extracts were
washed with brine (20 mL) and dried over anhydrous sodium sulfate.
After filtration and concentration under vacuum, the residue was purified
by flash chromatography (hexane/EtOAc 1:1) to afford 7 as a colorless
Synthesis of 5: LDA solution (3.20 mL, 2.00m in THF) was added to a so-
lution of dihydrothymidine 4 (2.50 g, 4.22 mmol) in anhydrous THF
(25 mL) at À788C. The reaction mixture was stirred at À788C for 3 h
before the addition of 2 (2.90 g, 4.33 mmol, 0.25m in THF). The reaction
mixture was stirred at À788C for 3 h and allowed to warm to room tem-
perature for overnight. The reaction was quenched by saturated aqueous
sodium bicarbonate (40 mL) and the aqueous phase was extracted with
CH2Cl2 (3ꢃ100 mL). The collected extracts were dried over anhydrous
sodium sulfate. After filtration and concentration under vacuum, the resi-
due was purified by flash chromatography (hexane/EtOAc 6:1) to afford
isomer 5R-5 as a pale yellow oil (1.95 g, 39%). Further purification by
flash chromatography (hexane/CH2Cl2/EtOAc 2:1:0.3) afforded isomer
5S-5 as a pale yellow oil (0.74 g, 15%). 5R-5: 1H NMR (CDCl3): d =
0.08 (s, 3H), 0.09 (s, 3H), 0.12 (s, 6H), 0.60 (q, J=7.9 Hz, 6H), 0.65 (q,
J=8.0 Hz, 6H), 0.89 (s, 9H), 0.93 (s, 9H), 0.96 (t, J=8.0 Hz, 9H), 0.98 (t,
J=7.9 Hz, 9H), 1.18 (s, 3H), 1.85–1.98 (m, 3H), 2.10–2.18 (m, 1H), 2.62
(d, J=14.1 Hz, 1H), 2.68 (d, J=14.1 Hz, 1H), 2.91 (d, J=13.0 Hz, 1H),
3.11 (d, J=13.0 Hz, 1H), 3.60 (dd, J=5.2, 10.4 Hz, 1H), 3.68 (dd, J=3.3,
10.7 Hz, 1H), 3.74 (dd, J=5.0, 10.7 Hz, 1H), 3.77–3.83 (m, 2H), 3.86–
3.92 (m, 1H), 4.29–4.33 (m, 1H), 4.39–4.44 (m, 1H), 4.55 (d, J=12.4 Hz,
1H), 4.61 (d, J=12.4 Hz, 1H), 4.66 (d, J=12.1 Hz, 1H), 4.69 (d, J=
12.1 Hz, 1H), 5.32 (s, 2H), 5.47 (s, 2H), 6.30 (t, J=6.6 Hz, 1H), 6.33 (t,
J=7.1 Hz, 1H), 7.20–7.27 (m, 6H), 7.27–7.32 (m, 2H), 7.32–7.37 (m,
2H), 7.49 ppm (s, 1H); 13C NMR (CDCl3): d = À5.3, À5.2, À4.8, À4.6,
4.4, 4.8, 6.8, 6.9, 18.0, 18.5, 22.1, 25.8, 26.1, 32.4, 36.9, 40.2, 43.0, 44.2, 63.0,
63.1, 70.9, 71.9, 72.0, 72.2, 72.3, 84.4, 85.4, 86.4, 87.6, 108.6, 127.5, 127.6,
127.7, 128.3, 128.4, 138.1, 138.5, 139.3, 150.8, 152.4, 163.6, 173.7 ppm;
ESI-MS (positive ion): m/z: calcd for C60H101N4O12Si4+: 1181.7 [M+H]+,
found 1181.7; 5S-5: 1H NMR (CDCl3): d = 0.09 (s, 12H), 0.58–0.66 (m,
12H), 0.90 (s, 18H), 0.97 (t, J=7.9 Hz, 18H), 1.11 (s, 3H), 1.88–2.00 (m,
3H), 2.28 (dd, J=4.7, 12.7 Hz, 1H), 2.43 (d, J=14.2 Hz, 1H), 2.78 (d, J=
13.0 Hz, 1H), 2.79 (d, J=14.2 Hz, 1H), 3.18 (d, J=13.0 Hz, 1H), 3.59
(dd, J=4.9, 10.4 Hz, 1H), 3.65–3.80 (m, 3H), 3.81–3.87 (m, 1H), 3.90–
3.96 (m, 1H), 4.30–4.35 (m, 1H), 4.35–4.39 (m, 1H), 4.61 (s, 2H), 4.65 (s,
2H), 5.25 (d, J=9.9 Hz, 1H), 5.37 (d, J=9.9 Hz, 1H), 5.44 (s, 2H), 6.23
(t, J=6.7 Hz, 1H), 6.37 (t, J=7.0 Hz, 1H), 7.20–7.33 (m, 8H), 7.33–7.38
(m, 2H), 7.41 ppm (s, 1H); 13C NMR (CDCl3): d = À5.4, À5.2, À4.7,
À4.6, 4.4, 4.8, 6.8, 6.9, 18.0, 18.4, 20.8, 25.8, 26.0, 32.5, 37.2, 40.8, 42.3,
44.7, 63.4, 63.5, 70.7, 71.0, 71.9, 72.2, 72.8, 72.9, 85.0, 86.3, 86.8, 88.1,
108.6, 127.4, 127.5, 127.6, 127.7, 128.3, 128.4, 138.0, 138.1, 138.9, 150.7,
152.5, 163.4, 173.5 ppm; ESI-MS (positive ion): m/z: calcd for
C60H101N4O12Si4+: 1181.7 [M+H]+, found 1181.7.
1
oil (990 mg, 75%). H NMR (CDCl3): d = 0.09 (s, 3H), 0.10 (s, 3H), 0.11
(s, 3H), 0.12 (s, 3H), 0.90 (s, 9H), 0.92 (s, 9H), 1.06 (s, 3H), 1.08 (s, 9H),
1.90–1.97 (m, 1H), 2.00–2.06 (m, 2H), 2.16 (ddd, J=3.2, 6.1, 13.4 Hz,
1H), 2.44 (d, J=3.9 Hz, 1H), 2.53 (d, J=14.2 Hz, 1H), 2.60 (d, J=
14.2 Hz, 1H), 2.92 (d, J=13.1 Hz, 1H), 3.04 (d, J=13.1 Hz, 1H), 3.72
(dd, J=5.4, 11.1 Hz, 1H), 3.75–3.85 (m, 4H), 3.88–3.92 (m, 1H), 4.36–
4.44 (m, 2H), 4.54 (d, J=12.4 Hz, 1H), 4.60 (d, J=12.4 Hz, 1H), 4.64 (d,
J=12.2 Hz, 1H), 4.67 (d, J=12.2 Hz, 1H), 5.29 (d, J=10.0 Hz, 1H), 5.33
(d, J=10.0 Hz, 1H), 5.43 (s, 2H), 6.29 (dd, J1 =J2 =7.2 Hz, 1H), 6.34 (dd,
J1 =J2 =7.0 Hz, 1H), 7.17–7.25 (m, 6H), 7.25–7.31 (m, 2H), 7.32–7.35 (m,
2H), 7.36–7.43 (m, 6H), 7.44 (s, 1H), 7.66–7.71 ppm (m, 4H); 13C NMR
(CDCl3): d = À5.3 (2C), À4.8, À4.7, 18.0, 18.4, 19.3, 21.9, 25.8, 26.0, 27.0,
32.4, 36.5, 40.2, 42.7, 44.7, 62.9, 64.3, 70.8, 70.9, 71.9, 72.1, 72.2, 83.9, 84.6,
85.4, 87.5, 108.4, 127.4, 127.6, 127.7, 127.9 (2C), 128.3 (2C), 129.9, 130.0,
132.9, 133.2, 135.5, 135.6, 137.9, 138.4, 139.1, 150.7, 152.4, 163.5,
173.4 ppm; ESI-MS (positive ion): m/z: calcd for C64H91N4O12Si3+: 1191.6
[M+H]+, found 1191.7.
Synthesis of 8: A solution of 7 (910 mg, 0.76 mmol), Ac2O (3.60 mL) and
AcOH (1.15 mL) in DMSO (5 mL) was stirred at room temperature for
50 h. The reaction was quenched by addition of an aq. saturated
NaHCO3 solution (20 mL) at 08C and stirred for another 1 h. The aque-
ous phase was extracted with Et2O (4ꢃ30 mL). The combined organic
layers were washed with water (5ꢃ50 mL) and dried with Na2SO4. After
filtration and concentration under vacuum, the residue was purified by
flash chromatography (hexane/EtOAc 4:1) to afford 8 as a white solid
1
(830 mg, 87%). H NMR (CDCl3): d = 0.08 (s, 3H), 0.09 (s, 3H), 0.10 (s,
3H), 0.11 (s, 3H), 0.90 (s, 9H), 0.92 (s, 9H), 1.09 (s, 3H), 1.10 (s, 9H),
1.90–1.98 (m, 2H), 2.04 (ddd, J=2.3, 6.0, 13.8 Hz, 1H), 2.10 (s, 3H), 2.16
(ddd, J=3.1, 6.0, 13.4 Hz, 1H), 2.55 (d, J=14.2 Hz, 1H), 2.61 (d, J=14.2,
Hz, 1H), 2.93 (d, J=13.0 Hz, 1H), 3.07 (d, J=13.0 Hz, 1H), 3.71–3.81
(m, 4H), 3.88–3.91 (m, 1H), 3.92–3.95 (m, 1H), 4.40–4.43 (m, 1H), 4.47–
4.51 (m, 1H), 4.56 (d, J=12.4 Hz, 1H), 4.58 (d, J=11.6 Hz, 1H), 4.61 (d,
J=12.4 Hz, 1H), 4.62 (d, J=11.6 Hz, 1H), 4.64 (d, J=12.5 Hz, 1H), 4.67
(d, J=12.5 Hz, 1H), 5.30 (d, J=10.0 Hz, 1H), 5.34 (d, J=10.0 Hz, 1H),
5.44 (s, 2H), 6.28 (dd, J=4.0, 5.5 Hz, 1H), 6.30 (t, J=5.7 Hz, 1H), 7.18–
7.27 (m, 6H), 7.27–7.32 (m, 2H), 7.32–7.36 (m, 2H), 7.37–7.43 (m, 6H),
7.44 (s, 1H), 7.67–7.72 ppm (m, 4H); 13C NMR (CDCl3): d À5.3, À5.2,
À4.8, À4.7, 13.8, 18.0, 18.5, 19.3, 21.9, 25.8, 26.0, 27.0, 32.4, 33.5, 40.2,
42.8, 44.5, 62.9, 63.8, 70.7, 70.9, 71.9 (2C), 72.2, 73.5, 76.0, 83.1, 84.3, 85.4,
87.5, 108.5, 127.4, 127.6 (2C), 127.7, 127.9, 128.3 (2C), 129.9 (2C), 133.0,
Selective deprotection of 5R-5 to yield 6: Compound 5R-5 (2.00 g,
1.69 mmol) was dissolved in CH3CN (30 mL) and cooled to À208C. A
HF·pyridine solution (1.70m in acetonitrile, 20.3 mmol, 11.9 mL) was
added and the reaction mixture was stirred at À208C for 4 h. The reac-
tion was quenched by saturated aqueous NaHCO3 (40 mL). The mixture
was extracted with EtOAc (3ꢃ150 mL). The collected extracts were
Chem. Eur. J. 2011, 17, 9658 – 9668
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9665

L. Li et al.
133.2, 135.6 (2C), 138.0, 138.4, 139.1, 150.7, 152.4, 163.4, 173.5 ppm; ESI-
MS (positive ion): m/z: calcd for C66H95N4O12SSi3+: 1251.6 [M+H]+,
found 1251.6.
3H), 2.92 (d, J=13.2 Hz, 1H), 3.11 (d, J=14.4 Hz, 1H), 3.30 (d, J=
13.2 Hz, 1H), 3.50 (d, J=10.9 Hz, 1H), 3.66–3.72 (m, 1H), 3.77 (dd, J=
3.2, 11.7 Hz, 1H), 3.82–3.86 (m, 1H), 3.96 (dd, J=2.3, 11.7 Hz, 1H),
4.08–4.15 (m, 1H), 4.14 (d, J=10.9 Hz, 1H), 4.55–4.61 (m, 1H), 4.73 (d,
J=7.3 Hz, 1H), 4.77 (d, J=7.3 Hz, 1H), 6.28 (t, J=7.2 Hz, 1H), 6.32
(dd, J=4.3, 8.0 Hz, 1H), 7.19 (s, 1H), 7.34–7.39 (m, 4H), 7.39–7.43 (m,
2H), 7.63–7.68 (m, 4H), 9.11 (s, 1H), 10.50 ppm (s, 1H); 13C NMR
(CDCl3): d = À4.8, À4.6, 18.0, 19.4, 24.1, 25.7, 27.0, 36.4, 40.0, 40.5, 46.5,
62.0, 64.7, 69.8, 77.6, 81.6, 82.1, 82.7, 84.3, 96.3, 110.9, 127.7, 127.8, 130.0
(2C), 133.1, 133.2, 135.5, 135.6, 137.5, 150.2, 154.0, 164.2, 173.0 ppm; ESI-
MS (positive ion): m/z: calcd for C43H61N4O10Si2+: 849.4 [M+H]+, found
849.4.
Mono-deprotection of 8 to yield 9: A solution containing 8 (1.10 g,
0.88 mmol) and pyridinium p-toluenesulfonate (0.90 g, 3.58 mmol) in
MeOH (30 mL) was stirred at 08C for 1 h. The reaction mixture was al-
lowed to warm to room temperature and stirred for overnight. After
quenched by ice-cooled saturated aqueous sodium bicarbonate (20 mL),
the reaction mixture was diluted with CH2Cl2 (200 mL), washed with sa-
turated aqueous sodium bicarbonate (3ꢃ10 mL) and dried over anhy-
drous sodium sulfate. After filtration and concentration under vacuum,
the residue was purified by flash chromatography (hexane/EtOAc 2:1) to
afford 9 as a colorless oil (0.61 g, 62%). 1H NMR (CDCl3): d = 0.09 (s,
6H), 0.91 (s, 9H), 1.12 (s, 12H), 1.95 (ddd, J=2.5, 7.0, 13.6 Hz, 1H), 2.05
(ddd, J=1.9, 5.7, 13.6 Hz, 1H), 2.09–2.16 (m, 1H), 2.13 (s, 3H), 2.33
(ddd, J=2.8, 6.2, 13.6 Hz, 1H), 2.51 (d, J=14.2 Hz, 1H), 2.70 (d, J=
14.2 Hz, 1H), 2.87 (d, J=13.1 Hz, 1H), 3.11 (d, J=13.1 Hz, 1H), 3.15 (t,
J=4.9 Hz, 1H), 3.70 (ddd, J=3.1, 4.9, 12.0 Hz, 1H), 3.76 (dd, J=5.6,
11.0 Hz, 1H), 3.81 (dd, J=4.3, 11.0 Hz, 1H), 3.94 (ddd, J=2.4, 4.8,
12.0 Hz, 1H), 3.94–3.99 (m, 1H), 4.00–4.03 (m, 1H), 4.41–4.45 (m, 1H),
4.50–4.55 (m, 1H), 4.59 (d, J=12.5 Hz, 1H), 4.61 (d, J=11.6 Hz, 1H),
4.65 (d, J=11.6 Hz, 1H), 4.68 (d, J=12.5 Hz, 1H), 4.71 (s, 2H), 5.32 (s,
2H), 5.49 (d, J=10.2 Hz, 1H), 5.51 (d, J=10.2 Hz, 1H), 6.16 (dd, J1 =
J2 =6.7 Hz, 1H), 6.30 (dd, J=5.7, 9.0 Hz, 1H), 7.21–7.35 (m, 8H), 7.35–
7.39 (m, 2H), 7.39–7.47 (m, 6H), 7.71–7.75 (m, 4H), 7.80 ppm (s, 1H);
13C NMR (CDCl3): d = À4.8, À4.7, 13.9, 18.0, 19.3, 21.1, 21.7, 25.8, 27.0,
32.1, 33.5, 41.8, 42.8, 44.6, 62.1, 63.9, 70.7, 71.0, 72.2, 72.3, 72.5, 73.5, 76.1,
83.2, 84.3, 87.7, 88.6, 107.6, 127.6 (2C), 127.7 (2C), 127.9, 128.2, 128.3,
129.9 (2C), 133.0, 133.1, 135.6 (2C), 138.0, 138.2, 139.7, 150.6, 152.3,
De-protection of 11 to yield 5R-CH2SP: To a solution of 11 (85 mg,
0.10 mmol) in THF (10 mL) was added TBAF (1.0m in THF, 0.30 mL)
and stirred for overnight. After the evaporation of the solvent under
vacuum, the residue was then purified by reverse phase HPLC in the gra-
dient mode using water/CH3CN as solvent. The compound 5R-CH2SP
was isolated as colorless micro-crystals (45 mg, 90%). 1H NMR
([D6]DMSO): d = 1.09 (s, 3H), 2.09–2.22 (m, 3H), 2.30 (ddd, J=3.4, 7.8,
14.2 Hz, 1H), 2.55 (d, J=14.9 Hz, 1H), 2.59 (d, J=14.9 Hz, 1H), 3.13 (d,
J=12.9 Hz, 1H), 3.18 (d, J=12.9 Hz, 1H), 3.40 (dd, J=4.1, 12.1 Hz, 1H),
3.49 (ddd, J=2.9, 4.1, 7.3 Hz, 1H), 3.54 (dd, J=3.7, 10.9 Hz, 1H), 3.58
(dd, J=2.9, 12.1 Hz, 1H), 3.81–3.87 (m, 2H), 3.99–4.06 (m, 1H), 4.32–
4.39 (m, 1H), 4.72 (d, J=7.0 Hz, 1H), 4.74 (brs, 1H), 4.75 (d, J=7.0 Hz,
1H), 5.31 (brs, 1H), 5.95 (dd, J=5.9, 8.6 Hz, 1H), 5.99 (dd, J=3.3,
8.6 Hz, 1H), 7.59 (s, 1H), 10.03 (s, 1H), 11.28 ppm (brs, 1H); 13C NMR
([D6]DMSO): d = 23.2, 34.8, 35.2, 38.7, 40.0, 45.1, 60.3, 66.3, 69.4, 76.6,
82.0, 82.1, 83.6, 84.6, 95.4, 109.7, 138.2, 150.6, 153.3, 163.4, 174.3 ppm;
ESI-MS (positive ion): m/z: calcd for C21H29N4O10+: 497.2 [M+H]+,
found 497.2.
+
163.6, 174.2 ppm; ESI-MS (positive ion): m/z: calcd for C60H81N4O12SSi2
: 1137.5 [M+H]+, found 1137.5.
Synthesis of TCH T: As shown in Scheme 3, the dinucleotide TCH T was
2
2
synthesized via a modified procedure used in the synthesis of UCH U orig-
Preparation of 10: To a cooled (08C) solution of 9 (420 mg, 0.37 mmol)
and molecular sieve in anhydrous 1,2-dichloroethane (8 mL) was added a
freshly mixed solution of N-iodosuccinimide (NIS) (90 mg, 0.40 mmol)
and TfOH (5.00 mL, 55.5 mmol) in 1,2-dichloroethane/diethyl ether 1:1
(5 mL). After 30 min, the reaction mixture was filtered, diluted with
CH2Cl2 (50 mL), washed with aq. saturated Na2S2O3/NaHCO3 solution
(5 mL). The organic layer was dried over anhydrous sodium sulfate.
After filtration and concentration under vacuum, the residue was purified
by flash chromatography (hexane/EtOAc=2/1) to afford 10 as a white
solid (170 mg, 42%). 1H NMR (CDCl3): d = 0.10 (s, 3H), 0.11 (s, 3H),
0.91 (s, 9H), 1.05 (s, 9H), 1.09 (s, 3H), 1.98 (ddd, J1 =J2 =7.4 Hz, J3 =
13.0 Hz, 1H), 2.23 (ddd, J=5.2, 7.5, 13.0 Hz, 1H), 2.32 (d, J=14.3 Hz,
1H), 2.37–2.46 (m, 2H), 2.80 (d, J=14.3 Hz, 1H), 2.90 (d, J=13.0 Hz,
1H), 3.07 (d, J=13.0 Hz, 1H), 3.48 (dd, J=2.0, 11.0 Hz, 1H), 3.74–3.82
(m, 2H), 3.91–3.98 (m, 2H), 4.13–4.20 (m, 2H), 4.52–4.56 (m, 1H), 4.60–
4.67 (m, 4H), 4.72 (d, J=7.2 Hz, 1H), 4.77 (d, J=7.2 Hz, 1H), 5.28 (d,
J=9.8 Hz, 1H), 5.32 (d, J=9.8 Hz, 1H), 5.39 (s, 2H), 6.07 (dd, J=5.7,
8.5 Hz, 1H), 6.33 (dd, J=4.9, 7.9 Hz, 1H), 7.20–7.25 (m, 3H), 7.25–7.30
(m 5H), 7.32–7.38 (m, 9H), 7.62–7.67 ppm (m, 4H); 13C NMR (CDCl3):
d = À4.8, À4.6, 18.0, 19.4, 22.7, 25.8, 27.1, 36.3, 41.0, 41.1, 45.3, 62.6,
65.8, 70.7, 71.0, 71.1, 71.9, 72.3, 77.8, 82.1, 83.2, 84.9, 85.3, 96.4, 109.3,
127.5, 127.6, 127.7, 127.9 (2C), 128.3 (2C), 130.0 (2C), 132.9, 133.2, 135.5,
135.6, 136.5, 138.1, 138.5, 150.6, 153.8, 162.8, 173.2 ppm; ESI-MS (positive
ion): m/z: calcd for C59H77N4O12Si2+: 1089.5 [M+H]+, found 1089.5.
2
inally adopted by Carell and co-workers.[9a]
5’-O-Pivaloylthymidine (13): To
a solution of thymidine (12, 5.00 g,
20.7 mmol) in pyridine (70 mL) was added trimethylacetyl chloride
(PivCl) (2.26 mL, 24.8 mmol) at 08C. The reaction was allowed to warm
to room temperature and stirred overnight. After quenching with MeOH
(5 mL), the solvent was removed under vacuum. The residue was purified
by flash chromatography (hexane/EtOAc/MeOH 1:1:0.2) to afford com-
1
pound 13 as a colorless oil (5.30 g, 78%). H NMR (CDCl3): d = 1.23 (s,
9H), 1.91 (s, 3H), 2.08 (ddd, J1 =J2 =6.6 Hz, J3 =13.8 Hz, 1H), 2.52 (ddd,
J=2.6, 5.7, 13.8 Hz, 1H), 4.22–4.29 (m, 2H), 4.33 (brs, 1H), 4.37–4.44
(m, 2H), 6.33 (dd, J=6.0, 7.7 Hz, 1H), 7.33 (s, 1H), 10.32 ppm (s, 1H);
13C NMR (CDCl3): d = 12.5, 27.2, 38.9, 40.5, 64.1, 71.5, 84.7, 85.1, 111.3,
135.2, 150.8, 164.4, 178.5 ppm; ESI-MS (positive ion): m/z: calcd for
C15H23N2O6+: 327.2 [M+H]+, found 327.2.
5’-O-Pivaloyl-3’-O-methylthiomethylthymidine (14): Compound 13
(6.50 g, 19.9 mmol) was dissolved in DMSO (40 mL), Ac2O (27 mL) and
AcOH (8.50 mL) and stirred for 3 d. After the completion of the reac-
tion, ice cold saturated sodium bicarbonate solution (100 mL) was added
and the mixture was stirred for 1 h. The aqueous solution was extracted
with Et2O (3ꢃ80 mL). The combined organic phases were washed with
water (4ꢃ100 mL) and dried with Na2SO4. After concentration under
vacuum, the residue was purified by flash chromatography (hexane/
EtOAc 1:1) to afford 14 as
a
white solid (6.15 g, 80%). 1H NMR
(CDCl3): d = 1.25 (s, 9H), 1.93 (s, 3H), 2.05 (ddd, J=6.6, 6.7, 14.0 Hz,
1H), 2.15 (s, 3H), 2.50 (ddd, J=2.6, 5.9, 14.0 Hz, 1H), 4.22–4.29 (m, 2H),
4.34–4.40 (m, 1H), 4.42–4.47 (m, 1H), 4.62 (d, J=11.8 Hz, 1H), 4.70 (d,
J=11.8 Hz, 1H), 6.26 (dd, J=5.9, 7.9 Hz, 1H), 7.28 (s, 1H), 10.09 ppm (s,
1H); 13C NMR (CDCl3): d = 12.5, 13.7, 27.2, 37.4, 38.8, 63.6, 73.7, 75.5,
82.0, 85.1, 111.2, 134.7, 150.5, 164.1, 178.0 ppm; ESI-MS (positive ion):
m/z: calcd for C17H27N2O6S+: 387.2 [M+H]+, found 387.2.
De-benzyloxylation of 10 to yield 11:
A mixture of 10 (140 mg,
0.13 mmol) and DDQ (325 mg, 1.43 mmol) in wet 1,2-dichloroethane
(40 mL) was stirred at 558C for 1 day. The reaction solution was diluted
with CH2Cl2 (150 mL), and washed with aq. saturated NaHCO3 solution
(3ꢃ10 mL). The organic layer was dried over anhydrous sodium sulfate.
After filtration and concentration under vacuum, the residue was purified
by flash chromatography (hexane/EtOAc/MeOH 1:1:0.1). The collected
crude product was dissolved in a CH2Cl2/iPr2NEt solution (20:1, 10 mL)
and refluxed overnight. After concentration under vacuum, the residue
was purified by flash chromatography (hexane/EtOAc/MeOH 1:1:0.1) to
afford 11 as a white solid (92 mg, 84%). 1H NMR (CDCl3): d = 0.10 (s,
3H), 0.11 (s, 3H), 0.90 (s, 9H), 1.05 (s, 9H), 1.18 (s, 3H), 2.13 (ddd, J1 =
J2 =7.9 Hz, J3 =13.5 Hz, 1H), 2.19 (d, J=14.4 Hz, 1H), 2.26–2.38 (m,
Preparation of 16: To a mixture of 14 (1.50 g, 3.88 mmol), 15 (1.10 g,
3.88 mmol) and molecular sieve in dichloromethane (80 mL) was added
TfOH (51 mL, 0.58 mmol) and NIS (921 mg, 4.11 mmol) at room temper-
ature.[32] After 5 min, the reaction mixture was filter, diluted with di-
chloromethane (100 mL), washed with saturated aq. Na2S2O3 (10 mL)
and saturated aq. NaHCO3 (10 mL). The organic phase was dried with
9666
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ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2011, 17, 9658 – 9668

A Dinucleotide Spore Photoproduct Analogue
FULL PAPER
Na2SO4. After concentration under vacuum, the residue was purified by
flash chromatography (hexane/EtOAc/MeOH 1:1:0.2) to afford com-
pound 16 as a white solid (1.82 g, 77%). 1H NMR (CDCl3): d = 1.23 (s,
9H), 1.92 (s, 6H), 2.01–2.09 (m, 1H), 2.12 (s, 3H), 2.22–2.30 (m, 1H),
2.43 (ddd, J=1.9, 6.0, 13.4 Hz, 1H), 2.52–2.59 (m, 1H), 3.82 (dd, J=2.7,
10.9 Hz, 1H), 3.92 (dd, J=3.4, 10.9 Hz, 1H), 4.16–4.20 (m, 1H), 4.25–
4.34 (m, 3H), 4.37–4.43 (m, 1H), 4.82 (d, J=7.1 Hz, 1H), 4.86 (d, J=
7.1 Hz, 1H), 5.24–5.31 (m, 1H), 6.24 (dd, J=6.0, 7.9 Hz, 1H), 6.36 (dd,
J=6.1, 8.1 Hz, 1H), 7.28 (s, 1H), 7.41 (s, 1H), 10.20 (s, 1H), 10.23 ppm
(s, 1H); 13C NMR (CDCl3): d = 12.4, 12.7, 20.9, 27.1, 37.3, 38.0, 38.7,
63.9, 68.2, 74.2, 82.6, 83.2, 84.6, 85.2, 94.8, 111.2, 111.3, 134.8, 135.1, 150.5,
150.7, 164.0, 164.1, 170.5, 177.9 ppm; ESI-MS (positive ion): m/z: calcd
for C28H39N4O12+: 623.3 [M+H]+, found 623.2.
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Deprotection of 16 to yield TCH T: Compound 16 (830 mg, 1.37 mmol)
2
was stirred with aqueous NH3 solution (25%, 20 mL) at 808C for over-
night. The solvent was removed in vacuum. The residue was then purified
by reverse phase HPLC in the gradient mode using water/CH3CN as sol-
vent. The compound TCH T was isolated as a white solid (440 mg, 65%).
2
1H NMR ([D4]MeOH): 1.86 (s, 3H), 1.87 (s, 3H), 2.20–2.33 (m, 3H), 2.43
(ddd, J=2.4, 5.6, 13.5 Hz, 1H), 3.76 (dd, J=3.3, 11.9 Hz, 1H), 3.78–3.87
(m, 3H), 4.01–4.06 (m, 1H), 4.06–4.09 (m, 1H), 4.40–4.47 (m, 2H), 4.84
(d, J=7.1 Hz, 1H), 4.88 (d, J=7.1 Hz, 1H), 6.23 (t, J=6.9 Hz, 1H), 6.38
(t, J=6.6 Hz, 1H), 7.65 (s, 1H), 7.77 ppm (s, 1H); 13C NMR
([D4]MeOH): d = 12.5, 12.7, 38.8, 40.8, 62.7, 68.9, 72.2, 78.6, 86.2, 86.3,
86.7, 86.8, 95.9, 111.5, 111.6, 137.8, 138.0, 152.2 (2C), 166.2 ppm (2C);
ESI-MS (positive ion): m/z: calcd for C21H29N4O10+: 497.2 [M+H]+,
found 497.2.
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Acknowledgements
The authors thank the National Institute of Environmental Health Scien-
ces (NIH; R00ES017177, L.L.), IUPUI startup fund (L.L. and J.P.),
IUPUI RSFG fund (L.L.) as well as IUCRG grant (L.L.) for financial
support. The NMR and MS facilities are supported by the National Sci-
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Published online: July 20, 2011
9668
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Chem. Eur. J. 2011, 17, 9658 – 9668