The photochemistry of Uracil: A reinvestigation

Article abstract of DOI:10.1111/j.1751-1097.2010.00826.x

Results from a re-examination of the photochemical reactions undergone by uracil (Ura) are presented. Irradiation of Ura in frozen aqueous solution at -78.5°C produces two diastereomeric (6-4) products, namely the cis and trans isomers of 5-hydroxy-6-4'-(pyrimidin-2'-one)-5,6-dihydrouracil. Upon heating in 0.1 m HCl, each of these compounds decomposes to form 6-4'-(pyrimidin-2'-one) uracil. In addition, evidence for production of a hydrate of a trimer of Ura is presented, Irradiation of this compound at 254 nm forms Ura and a (6-4) adduct as products. The compounds 5-4'-(pyrimidin-2'-one)uracil and 6-hydroxy-5,6-dihydrouracil were also present after Ura was irradiated in frozen aqueous solution. Cyclobutane dimers (CBDs) are formed when Ura is irradiated in the frozen aqueous state, in fluid aqueous and acetonitrile solution and in the presence of photosensitizers (e.g. acetone). Published information, concerning the identity and relative quantitative importance of the four CBD isomers (cis-syn, cis-anti, trans-syn and trans-anti) formed in these photochemical systems, is incomplete and often in substantial disagreement. Using chemical methods in conjunction with HPLC, the identity and relative amounts of the four dimers have been determined in each of these systems. Consequently, a number of inconsistencies found in the literature concerning dimer product identity and quantitative distribution have been resolved.

Full text of DOI:10.1111/j.1751-1097.2010.00826.x

Photochemistry and Photobiology, 2011, 87: 82–102
The Photochemistry of Uracil: A Reinvestigation
Martin D. Shetlar* and Vladimir J. Basus§
Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, CA
Received 29 June 2010, accepted 18 September 2010, DOI: 10.1111/j.1751-1097.2010.00826.x
say in the context of ribosomes (see, e.g. Zhirnov and
ABSTRACT
Wollenzien (6) and references therein). The presence of Ura
in the genomes of RNA viruses implies that photoreactions
of Ura must be considered in studies of UV-induced genetic
damage, such as inactivation of infectivity (see, e.g. Smirnov
et al. [7]).
Results from a re-examination of the photochemical reactions
undergone by uracil (Ura) are presented. Irradiation of Ura in
frozen aqueous solution at )78.5ꢀC produces two diastereomeric
(6-4) products, namely the cis and trans isomers of 5-hydroxy-6-
4¢-(pyrimidin-2¢-one)-5,6-dihydrouracil. Upon heating in 0.1 M
HCl, each of these compounds decomposes to form 6-4¢-
(pyrimidin-2¢-one)uracil. In addition, evidence for production of
a hydrate of a trimer of Ura is presented, Irradiation of this
compound at 254 nm forms Ura and a (6-4) adduct as products.
The compounds 5-4¢-(pyrimidin-2¢-one)uracil and 6-hydroxy-5,6-
dihydrouracil were also present after Ura was irradiated in
frozen aqueous solution. Cyclobutane dimers (CBDs) are formed
when Ura is irradiated in the frozen aqueous state, in fluid
aqueous and acetonitrile solution and in the presence of
photosensitizers (e.g. acetone). Published information, concern-
ing the identity and relative quantitative importance of the four
CBD isomers (cis-syn, cis-anti, trans-syn and trans-anti) formed
in these photochemical systems, is incomplete and often in
substantial disagreement. Using chemical methods in conjunction
with HPLC, the identity and relative amounts of the four dimers
have been determined in each of these systems. Consequently, a
number of inconsistencies found in the literature concerning
dimer product identity and quantitative distribution have been
resolved.
The main photoreactions that Ura undergoes in the
absence of external additives lead to production of CBDs
and a photohydrate. Four CBDs are formed, namely the cis-
syn (c,s), cis-anti (c,a), trans-syn (t,s) and trans-anti (t,a)
stereoisomers (8) (displayed as Ia, Ib, Ic and Id in Scheme 1)
while photohydration (9) yields 6-hydroxy-5,6-dihydrouracil
(II in Scheme 1). Similar types of photoproducts are formed
by the two other major nucleobases, cytosine and thymine
(Thy) (for reviews, see [1,2,8–10]), while the c,s, c,a and t,s
CBDs of 5-methylcytosine, a minor base occurring in mam-
malian DNA, have also been isolated and characterized (11).
Analogous photohydration and photodimerization reactions
have been observed when Ura is incorporated into nucleo-
sides, nucleotides, oligonucleotides, polynucleotides and nu-
cleic acids (for reviews, see [1,2,8–10]). A number of other
studies have looked at the photoinduced reactions of Ura with
species added to its aqueous environment. Among the
reactions for which products have been isolated and charac-
terized are those resulting from photo-induced reaction of this
compound with hydrogen cyanide in aqueous solution (12),
alcohols (see [13,14] for reviews), amines (15) and the amino
acid cysteine (16). Survey studies have suggested that Ura,
either as a nucleobase (17) or when incorporated into
polyuridylic acid (18), is reactive with a number of amino
acids other than cysteine.
Another type of reaction that appears to be characteristic of
the pyrimidine bases, either when irradiated in the frozen state
as the nucleobase or nucleoside or when incorporated into
dinucleotides, oligonucleotides and nucleic acids and irradi-
ated in solution, is formation of so-called (6-4) adducts (for
reviews, see [1,2,19]). The photoreaction of Ura (or related
compounds) to form a corresponding (6-4) adduct, namely 5-
hydroxy-6-(4¢-pyrimidin-2¢-one)-5,6-dihydrouracil (see, e.g.
structure IIIa) has not been directly demonstrated. However,
the occurrence of this reaction has been inferred through the
identification of a putative dehydration product, 6-4¢-(pyrim-
idin-2¢one)uracil (IV) (20). It has been generally assumed that
the parent (6-4) adduct is very labile, undergoing fast spon-
taneous dehydration to form IV ([19], p 333). As acid
hydrolysis of one of the chromatographic fractions obtained
in the study of the photoreaction of uridine in frozen aqueous
solution produced material with the properties of IV, Varghese
INTRODUCTION
The photochemistry of uracil (Ura), one of the nucleobases
found in RNA and occasionally in DNA, has received
considerable attention over the past 50 years. (For compre-
hensive reviews of nucleic acid photochemistry, as well as
that of nucleobases, nucleosides, nucleotides, dinucleotides
and oligonucleotides, see [1,2]; for reviews of more recent
work, see [3,4]). One reason for this interest in Ura
photochemistry is that it is one of the four main nucleobases
found in cellular RNA. Photodamage to such RNA (e.g.
messenger RNA) can have consequences for cellular function,
particularly with respect to functions involving protein
synthesis (5). However, photoreactions in RNA can also be
used to gain information about structural aspects of RNA,
§Current address: Vladimir J. Basus, Caixa Postal 007, Extrema, Minas Gerais
37640-006, Brazil.
*Corresponding author e-mail: shetlar@cgl.ucsf.edu (Martin D. Shetlar)
ꢁ 2010 The Authors
Photochemistry and Photobiology ꢁ 2010 The American Society of Photobiology 0031-8655/11
82

Photochemistry and Photobiology, 2011, 87 83
O
H
N
O
O
O
O
O
H
N
H
H
H H
H H
H
H
H
H H
H H
O
H
H
O
H
H
H
N
N
H
N
N
N
N
N
N
H
O
O
O
H
O
O
O
N
H
N
H
N
H
N
H
H N
H H
N
H
O
H
O
(t,s)
Ic
(t,a)
Ia (c,s)
Ib (c,a)
Id
O
O
O
O
OH
H
H
N
H
H
4
H
N
H
N
N
3
5
6
H
H
OH
H
2
1
O
N
OH
O
O
N
H
O
N
H
H
H
H
H
H
N
H
4'
N
N
H
H
5'
6'
3'
2'
N
II
IIIb
IIIa
IV
1'
N
H
O
O
N
O
H
N
H
O
H
H
N
OH
CH3
H
H
N
H
H
H
H
N
N
O
O
O
O
N
O
H
N
H
N
N
CH3
H
H
H
H
OH
N
VI
O
H
V
VII
O
N
N
H
O
N
H
H
Scheme 1.
(21) concluded that a precursor (6-4) adduct is formed in this
system as well.
MATERIALS AND METHODS
General Aspects. Uracil and Thy were obtained from Sigma (St. Louis,
Mo). HPLC solvents were from Fisher (Fair Lawn, NJ), while NMR
solvents were provided by Aldrich (Milwaukee, WI). Chemicals used
in various methylation procedures were from Aldrich. Preparative
In spite of the extensive previous work done on the
photochemistry of Ura itself, there are a number of outstand-
ing questions. One of these questions concerns whether a Ura
(6-4) adduct would be sufficiently stable to allow its isolation
and characterization. A second question deals with unresolved
issues in the literature concerning the identity and quantitative
distribution of the CBDs of Ura formed in various photo-
chemical reaction systems. As pointed out in 1976 by Fisher
and Johns (8) (pp. 248–252), there are significant disagree-
ments in the literature concerning these matters, both in
directly induced reactions (i.e. in ice and in fluid solution) and
in reactions photosensitized by acetone; since that time, there
have been no published reports that resolve these contradic-
tions.
In this paper, we show that two isomeric (6-4) adducts of
Ura can be isolated and characterized after this nucleobase is
irradiated in the frozen aqueous state. In addition, we have
looked in detail at the products of the Ura photodimerization
reactions occurring in four systems, namely in fluid aqueous
and acetonitrile solution, in frozen aqueous solution and when
the photoreactions of Ura are sensitized by acetone in aqueous
solution. Using chemical methods, we have definitively estab-
lished the identities of the dimers formed and, using HPLC, we
have studied the quantitative distribution of the four CBDs
(c,s, c,a, t,s and t,a) formed in each of these four systems.
These results provide a considerably more complete descrip-
tion of the photochemical reactions occurring in these systems
than has been previously available. They also resolve a number
of contradictions found in the literature.
separations were accomplished on
a Shiseido Capcell UG120
10 · 250 mm column (5 mm particle size, Yokohama, Japan) (Column
A), while analytical HPLC separations were carried out using a Capcell
UG120 4.6 · 150 mm column (5 mm particle size) (Column B), a
Microsorb Phenyl column (4.6 · 250 mm, 5 mm particle size; Varian,
Walnut Creek, CA) (Column C) and a Microsorb Amino column
(4.6 · 250 mm, 5 lm particle size) (Column D). In some cases, final
purification was done on Column C. Column E, used in some
experiments, was a Capcell UG120 4.6 · 250 mm column (5 mm
particle size). The HPLC system employed was a Rainin binary
gradient pumping system (Emeryville, CA) coupled to a Hewlett-
Packard 1040A diode array detector (Palo Alto, CA). Prior to injection,
all HPLC samples were subjected to spin filtration on Costar Spin-X
microcentrifuge tubes containing a 0.2 lm nylon filter (Corning
Incorporated, Corning, NY). Rotatory evaporations were done on a
Buchi R-200 Rotovapor (New Castle, DE) with vacuum provided by a
¨
mechanical pump; two dry ice traps were placed between sample and
pump.
NMR spectra were run at 600 MHz on a Varian INOVA NMR
spectrometer (Palo Alto, CA). MALDI mass spectra were obtained on
an Applied Biosystems 4700 Mass Spectrometer (Foster City, CA),
running in the reflector mode, while ESI mass spectra were run on
either a Waters Micromass ZQ4000 instrument (Beverly, MA) or
a
Brucker Fourier Transform 9.4T spectrometer (Rheinstetten,
Germany).
Unfiltered Spectronics BLE IT155 lamps, with output mainly at
254 nm and housed in Spectroline XX-15A lamp housings (Westbury,
NY), were used for irradiations of Ura solution in the ice phase or to
induce reaction in fluid aqueous solution; Spectronics BLE IT158
lamps, with output centered at 312 nm and contained in the same
housing, were used to photosensitize reactions in solutions containing
acetone.

84 Martin D. Shetlar and Vladimir J. Basus
Irradiations in the frozen state were done on 250 mL batches of
aqueous Ura (2 mM). Each batch was placed in a 13 · 9 inch non-stick
baking pan (Bradshaw International, Rancho Cucamonga, CA) and
frozen on two 25 · 25 cm dry ice slabs ()78.5ꢀC) placed side by side.
During freezing the pan was covered with a 15 · 10 inch Pyrex baking
dish, so as to minimize frosting of the surface of the frozen solution.
Four lamps, contained in two Spectroline X-Series lamp housings,
were placed on top of the pan containing the solutions; irradiation was
done with the pan resting on the dry ice slabs. The exposure at the
surface of the frozen aqueous layer in the pan averaged around
25 J m)2 s)1, as measured using a Spectronics DM-254N UV Meter
(Westbury, NY).
Irradiations of Ura contained in acetonitrile solution were carried
out on a small scale. A 3 mL portion of parent solution was placed in a
12 mm diameter flat-bottomed cylindrical quartz tube (75 mm in
length) with a 14 ⁄ 35 quartz taper seal outer joint fused to its end. The
tube was than fitted with a T-shaped adaptor fitted with a 14 ⁄ 35 taper
seal inner joint and two screw joints compatible with Mininert valves
suitable for Reactivials (Pierce, Rockford, IL). The taper seal and one
of the screw joints were attached to the ends of the top bar of the tee.
After fitting the screw joints with Mininert valves, an 8 inch long
needle (Wilmad, Buena, NJ) was inserted through the septum of
the Mininert valve attached to the top bar of the tee and pushed to the
bottom of the tube; a short bleed needle pierced the septum of the
second valve. The sample was deoxygenated with 99.997% pure N2.
After needle removal and valve closure, the UV spectrum of the sample
was measured using acetonitrile in the same tube as a blank. During
irradiation, the sample was placed next to an unfiltered BLE IT155
lamps.
Preparation and fractionation of the noncyclobutane dimer
photoproducts produced by irradiation of Ura in aqueous frozen
solution. In this section, we describe the steps used in workup of
aqueous solutions of Ura irradiated on dry ice, with a primary focus
on putative (6-4) adducts(s) and trimer hydrate.
Preparation, characterization and workup of solutions of Ura
irradiated at dry ice temperature. In a typical experiment, 1 L of
solution (2 mM) was irradiated in 250 mL batches for 60 min. After
thawing, 150 lL of the resulting yellow-green solution was diluted to
3 mL, its UV spectrum was measured and the absorbance at 258 nm
was determined; a similar measurement were made with the parent
solution and it was estimated that about 62% of the parent Ura had
reacted. The 20-fold diluted irradiated sample displayed absorbance to
the red of the parent Ura peak with a maximum at 304 nm and an
absorbance of 0.014. Using an approximate value for e304 of 4900 as
the molar absorption coefficient of uracil (6-4) adducts (based on an
analogous value of e316 for the corresponding thymine-thymine adduct
([19], p. 341), it was calculated that a total of about 57 lM of (6-4)
product was present in the original irradiated 1 L of solution and that
about 9% of the Ura consumed had been converted to such
compounds. This corresponds to about 12.8 mg of total (6-4) product
of MW 224 being produced by irradiation of 1 L of solution. Using
relative areas of HPLC peaks corresponding to Ura, the percent
conversion was calculated to be about 59%. The calculated conversion
varies by a couple of percentage points from run to run, as might be
expected for situations where the reflective properties of the surface of
the frozen solution being irradiated may not be identical for each run.
The HPLC properties of freshly irradiated solution were examined
using Column A. A representative analytical chromatogram from a
500 lL injection is shown in Fig. 1; this chromatogram was obtained
using the following water ⁄ methanol gradient at a flow rate of
5 mL min)1: 0 min, 0% MeOH; 4 min, 0%; 5 min, 15%; 8 min,
15%; 8.5 min, 0%; 14 min, 0%. The peaks labeled 2 and 3 elute with
retention times corresponding to the putative (6-4) adducts of interest;
Irradiations involving direct absorption of light by Ura in aqueous
fluid solution, or photosensitized reactions of Ura in aqueous solution
containing acetone, were carried out as described in the on-line
Supporting Information section, which contains a discussion of the
preparation of the CBDs of 1,3-dimethyluracil.
Abs.
Abs.
Time
Figure 1. The HPLC chromatogram resulting from injection of 500 lL of a freshly prepared 2 mM solution of uracil that was frozen on dry ice and
irradiated for 60 min as described in Materials and Methods. Panel A displays the chromatogram seen with detection at 225 nm, while panel B
corresponds to that observed with detection at 304 nm. The chromatogram was obtained on a Shiseido Capcell UG120 10 · 250 mm column using
the following water ⁄ methanol gradient at a flow rate of 5 mL min)1: 0 min, 0% MeOH; 4 min, 0%; 5 min, 15%; 8 min, 15%; 8.5 min, 0%;
14 min, 0%. Detailed identification of the peaks is discussed in Materials and Methods; however, the peaks labeled 2 and 3 in Panel B correspond
to (6-4) adducts.

Photochemistry and Photobiology, 2011, 87 85
however, the main type of product eluting in peak 2 is cyclobutane
dimer in nature. Peak 4 corresponds to unreacted Ura, although CBD-
type product coelutes with the parent compound. The materials
and 3.4 min, contained material with little UV absorbance above
250 nm, while F2 (3.4–3.9 min) was a compound with a slight peak of
UV absorbance with a maximum at 304 nm, similar to that expected
for (6-4) type products; this fraction also contained a large amount of
other material, found to be cyclobutane dimer. Fraction F3 (3.9–
4.5 min) contained a second compound with (6-4) adduct-like absor-
bance, along with another compound without UV absorbance above
250 nm and F4 (4.5–5.6 min) contained several products present only
in small amounts. The fraction F5 (5.6–6.6 min) contained Ura and F6
(6.6–16 min) contained a variety of compounds present in small
amounts, including the known (6-4) dehydration product IV. We gave
our initial attention to characterization of the compounds with (6-4)-
like absorbance contained in F2 and F3 and the material contained in
F1. The final purification and characterization of these compounds is
described in Results and Discussion.
corresponding to peaks 1, 2,
3 and 4 were the focus of the
investigations described below. The material eluting in Peak 5 shows
a UV spectrum with a broad absorption band centered at 313 nm; the
spectrum and elution time are different than those of the previously
characterized 6-4¢-(pyrimidin-2¢one)uracil (IV) (20). The compound
responsible for this peak is lost during workup and was not
characterized. The products responsible for the peaks denoted by x
and y were likewise not identified in our studies of this reaction system.
The complex of peaks denoted by g is artifactual and characteristic of
the eluting gradient, being present even in the absence of injected
sample.
The liter of irradiated solution was reduced to 20 mL by rotatory
evaporation at 40ꢀC, placed in the cold (8ꢀC) overnight, and then
filtered through a Whatman HAWP filter to remove the precipitated
material (mostly Ura CBDs). This precipitate had a yellow-tan cast.
The precipitate was washed with 5 mL of distilled water; the wash
solution was added to the parent solution and, after appropriate
dilution, this solution was assayed for (6-4) adduct by UV spectros-
copy. About 85% of the material absorbing at 304 nm was recovered
in this solution. A UV absorbing tail extending out to 375 nm was
present in the original photolyzed solution, but not in the diluted assay
sample; this suggests that one or more additional compounds
contribute to the 304 nm absorbance in the original irradiated solution
(see below for discussion of the nature of such compounds). Indeed, an
HPLC assay indicated that about 97% of total putative (6-4) adduct
remained in the concentrated solution. The 25 mL of concentrate was
further concentrated to 3 mL by rotatory evaporation and filtered; the
precipitated material was washed with five 1 mL portions of distilled
water. The wash solution was added to the filtrate, along with an
additional 2 mL of distilled water, to give a total of 10 mL of
concentrated solution. An HPLC assay of this concentrate indicated
that about 94% of the original putative (6-4) adducts had been
recovered.
An alternate method for preparation of concentrates of Ura photo-
products. A modified approach was explored as an alternative for
preparation of concentrates of putative (6-4) adducts from Ura
solutions irradiated in ice. In a typical run using this procedure,
990 mL of solution, irradiated as described above, was concentrated to
about 46 mL. The resulting concentrate was cooled on ice and filtered.
The filtrate was then taken to dryness using rotatory evaporation and
10 mL of methanol was added. The precipitate was scraped from the
sides of the evaporation flask and the resulting slurry was placed in a
15 mL Falcon plastic centrifuge tube. After centrifugation in a
swinging bucket rotor, the supernatant was removed by decanting to
give extract E1. This same procedure was repeated two times to give E2
and E3. Assay of the UV absorbance of each of these extracts at
304 nm, after 10-fold dilution with distilled water, indicated that the
absorbances of the three extracts had a ratio of 81.5:14.5:4. HPLC
analysis of the extracts using Column C (see above), after removal of
methanol by rotatory evaporation, dissolution of the residue in water
and filtration, indicated that much of the cyclobutane dimeric product
had been removed and that the extracts contained the putative (6-4)
products of interest; however, Ura was the predominant component of
each extract. (The analytical gradient used was as follows: 0 min, 0%
MeOH; 3.75 min, 0%; 6 min, 20%; 7.5 min, 20%; 7.8 min, 0%;
12 min, 0%, flow rate: 1.6 mL min)1). This gradient has the desirable
attribute that it is able to resolve the peak containing putative (6-4)
adduct from the main cyclobutane dimer peak; however, it is unable to
resolve the two putative (6-4) adducts. Comparative analysis of the
solution prepared by the approach described in the previous section
and the solution prepared by extraction indicated that the former
contained about 2.5-fold more CBD, while the amounts of (6-4)
adduct were almost identical. The material corresponding to Peak 1 in
Fig. 1 is present in samples prepared by either approach.
Preparations and procedures relevant to identification and
quantitation of the cyclobutane dimers of Ura. A second focus in our
study of the photochemistry of Ura was identification and quantitation
of the CBDs formed when the parent compound was irradiated under
several conditions. Our approach to establishing the identities of the
various CBDs of Ura involved conversion of purified dimers into
methylated forms that had previously been well characterized via work
published in the literature. This approach requires that we have
authentic samples of the various CBDs of 1,3-dimethyluracil (DMU)
and 1,3-dimethylthymine (DMT). The methods used for preparing
these dimers are appropriately modified and modernized versions of
those already published in the literature; the details of these procedures
are described in Supporting Information. We also required samples of
the various mixed dimers of DMU and DMT and knowledge of the
chromatographic and chemical behaviors of these various dimers. By
extending the methodology originally developed by Taguchi and Wang
(22,23), as described below, we were able to satisfy this need, as well as
verify the identity of various DMU and DMT dimers. We then
describe how this methodology was used to assign a stereochemical
identity to each Ura CBD. Finally, we describe chromatographic
conditions that permit separation and quantitation of the each of the
Ura CBDs when produced under various conditions of interest. (It
should be noted that, while the c,s and t,a isomers of the Ura CBDs
exist as single meso forms, the corresponding c,a and t,s forms of the
Ura CBDs are, in fact, racemic mixtures of two optical isomers.
Consequently, the results presented below, say for the t,s CBD, in
actuality pertain to the corresponding racemic mixture of optical
isomers.)
Preparation of mixed DMU-DMT cyclobutane dimers and
verification of the stereochemistry of the DMT cyclobutane dimers. To
determine (or confirm) the stereochemistry of each of the DMT CBDs,
as well as prepare
a reaction mixture containing each of the
corresponding mixed DMU-DMT dimers, we utilized a modified
version of the C-methylation methodology described by Taguchi and
Wang (22,23). (The preparation of the various DMU and DMT CBDs
used in the following protocols is described in Supporting Informa-
tion.)
A slurry containing 320 mg of Ag2O per mL of N,N-dimethylfor-
mamide (DMF) was prepared and stored in a glass stoppered flask. To
two samples of each of the four DMU dimers of known stereochem-
istry, each sample containing about 1 mg of purified dimer and
enclosed in a 1.5 mL screw top plastic microcentrifuge tube (Sarstedt,
Newton, NC), was added 260 lL of slurry and 140 lL of methyl
iodide. After mixing on a vortex mixer (Fisher, Tustin, CA), the
resultant slurry was placed on the top of a Labline Orbital Shaker
(Melrose Park, IL) and agitated at ambient temperature; one sample
was removed after 3.5 h and the second after 16 h. Immediately after
removal from the shaker, 270 lL of the slurry was removed and
diluted with 230 lL of DMF. The resultant slurry was filtered using a
Costar nylon spin tube and the filtrate was placed in a 15 mL plastic
centrifuge tube. To this tube was added 2.5 mL of CHCl3; this resulted
in immediate formation of a white precipitate. After shaking, the tube
was centrifuged and the supernatant was decanted from the compacted
precipitate at the bottom of the tube. The CHCl3 layer was shaken
with 1 mL of water; after separation of the layers, 0.5 mL of the
CHCl3 layer was removed and placed in the bottom of a 1.5 mL
Sarstedt screw top microcentrifuge tube. This sample was taken to
dryness via rotatory evaporation in two stages. The chloroform was
removed on a water aspirator driven apparatus at room temperature
First pass separation of components of the photoproducts produced
when Ura is irradiated in frozen solution at dry ice temperature. The
concentrate obtained using the first approach described above was
chromatographed in 1 mL batches on Column A using the following
water ⁄ methanol gradient with a flow rate of 5 mL min)1: 0 min, 0%
MeOH; 4 min, 0%; 5 min, 15%; 8 min, 15%; 8.5 min, 0%; 16 min,
0%. Six fractions were collected. Fraction 1 (F1) eluting between 2.3

86 Martin D. Shetlar and Vladimir J. Basus
and then drying was continued at 60ꢀC on the Buchi Rotovapor; this
the above conditions was DMT; c,a DMT CBD was the dominant
minor product. Small amounts of additional products, most likely
incompletely methylated dimers (26), were formed in each of the
methylation reactions. We also found, as observed previously (26), that
the t,a cyclobutane dimer of Thy does not methylate to form the
corresponding tetramethlylated dimer.
¨
latter step removes most of the DMF remaining after the water
extraction of the CHCl3. (The procedure used here omits a step
included in the previous protocols [22–24], namely treatment with
aqueous sodium cyanide; we found the end results were the same
without it.) During the rotatory evaporation, the Sarstedt tube was
fitted to the taper seal 24 ⁄ 40 joint of an antiflash rotatory evaporator
trap using a Fisherbrand Sav-It 16 mm tube closure pierced by several
holes. (The top of this type of closure fits snugly inside the taper joint,
while the bottom of the closure fits tightly around the screw portion of
the microcentrifuge tube.) The dried material was dissolved in 0.5 mL
of water and filtered through a spin tube. The resulting solutions were
chromatographed on Column B using the following water ⁄ MeOH
gradient (Gradient D) run at 2 mL min)1: 0 min, 7% MeOH; 4 min,
7%; 6.5 min, 40%; 8 min, 40%; 8.3 min, 7%; 13 min, 7%. In line with
a previous finding by Taguchi and Wang, we observed that the DMU
c,s dimer is resistant to methylation to form a corresponding c,s DMT
homodimer, even after 16 h of incubation. However, Taguchi and
Wang also indicated that a mixed dimer was formed in the methylation
reaction. In agreement with their observations, we found a peak,
corresponding to the mixed c,s DMU-DMT dimer, that eluted at
8.2 min in the reaction mixture that had been run for 16 h. Each of the
other DMU CBDs reacts to form both a DMT dimer and a mixed
dimer. The retention times of the four DMU dimers, and of the various
dimers formed in the methylation reactions of each of the DMU
CBDs, are shown in Table 1; the HPLC conditions corresponded to
those of Gradient D on Column B, as described above. These data
confirm that the order of elution of the DMT dimers using Gradient D
as being c,s; c,a; t,s; t,a; the same order of elution also holds for the
DMU dimers and the mixed DMU-DMT dimers.
Methylation of the CBDs of Ura and identification of individual dimer
stereoisomers. The CBDs of Ura have very low solubilities and are
difficult to separate by HPLC (particularly the c,s and c,a isomers [Ia
and Ib]). In addition, the c,a and t,a isomers (Ib and Id) are somewhat
labile when heated in aqueous solution or treated with acid or base.
These properties present challenges to the investigator desiring pure
samples of these materials for identification by use of structural
techniques (e.g. by IR or NMR spectroscopy). Although IR spectros-
copy has been used to identify the various Ura dimers, based on
comparison of the resultant spectra with those of a synthesized sample
of each isomer, application of this technique has also led to
contradictory identifications. It was for this reason that we developed
an independent means of assigning the stereochemical nature of each
dimer, namely identification of the products produced by methylating
the various dimers using protocols based on those used by Taguchi and
Wang (22,23) for methylation of the DMU dimers. Information
resulting from the use of such methylation reactions provides three
pieces of evidence pointing to the correct assignment of the structure of
a particular dimer (two for the c,s isomer). For example, methylation
of the Ura t,s dimer should yield a reaction mixture that, upon
examination by HPLC, would contain compounds that eluted with
retention times and UV spectral characteristics corresponding to those
of the t,s DMU CBD, the t,s DMU-DMT dimer and the t,s DMT-
DMT CBDs, as tabulated in Table 1.
As a further confirmation that the c,s, c,a and t,s DMT dimers,
isolated as described in Supporting Information, were assigned
correctly, we methylated authentic samples of each of the correspond-
ing CBDs of Thy. For this purpose, we used a modified version of the
procedure described by Blackburn and Davies (25). We dried samples
of each isomer, suspended the residue in 1 mL of 1 M NaOH (0.4 M
NaOH in the case of the c,a dimer) and added 100 lL of dimethyl
sulfate. The resulting heterogeneous mixture was heated in boiling
water for about a minute, after which the solution was homogeneous.
Then 0.5 mL of CHCl3 was added and the solution was shaken, so as
to extract the methylated dimer into the chloroform phase. About
0.4 mL of the organic phase was removed and taken to dryness by
rotatory evaporation at 40ꢀC. The residue was taken up in 500 lL of
water and 50 lL was chromatographed on column B, using Gradient
D as described previously. In each case, the methylated dimer eluted at
the same time as the corresponding authentic DMT dimer, while the
absorption spectrum of each dimer could be superimposed on that of
its corresponding DMT dimer. These results provide additional
evidence that the identities of the c,s, c,a and t,s dimers of DMT are
correctly assigned. It should be noted that the predominant product
resulting from action of dimethyl sulfate on the c,a Thy dimer under
Elad and coworkers (24) found that methylation of the c,s CBD of
Ura to form the corresponding DMU dimer went smoothly when it
was treated with silver oxide and methyl iodide in DMF. In our work
we used the modified procedure, outlined above for C-methylation of
DMU CBDs, to carry out a similar reaction on fractions containing
isolated Ura dimers, or dimer mixtures, to form the corresponding
DMU, DMU-DMT and DMT dimers. Once the identities of the
individual dimers were established, we could quantitatively evaluate
the distribution of dimers formed under various conditions (see Results
and Discussion below).
We used the acetone-photosensitized reaction of Ura to provide the
products required for identification of the various Ura CBDs; evidence
has been previously presented indicating that all four dimers are
formed in this system (27). A volume of 500 mL of Ura (2 mM) was
photolyzed in 167 mL batches in 80 ⁄ 20 water ⁄ acetone under flowing
nitrogen for 45 min, as described in Supporting Information for the
analogous reaction of DMU. The reaction mixture was rotatory
evaporated to dryness and the resulting precipitate was resuspended in
5 mL of water. After sonication, the solution was filtered and the
precipitate was set aside. The supernatant was chromatographed in
1 mL portions on Column A using the following water ⁄ methanol
gradient running at 5 mL min)1: 0 min, 0%; 3 min, 0%; 4 min, 12%,
5.5 min, 12%; 9.5 min, 20%; 12.5 min, 20%, 12.8 min, 0%; 17 min,
0%. Six fractions were collected, corresponding to the major peaks in
the chromatogram; these are denoted as A1–A6. Fraction A1 eluted
between 3.2 and 3.8 min, A2 between 4.0 and 5.0 min, A3 between 5.7
and 6.2 min, A4 between 8.7 and 9.3 min, A5 between 12.5 and
13.1 min and A6 between 13.6 and 14.1 min. Fractions A1, A2 and A4
had UV absorption spectra characteristic of those expected for CBDs,
while the spectrum of A3 suggested that it was a mixture of products
that contained Ura as one of the components. When the absorption
spectra of appropriately diluted samples of A1, A2 and A4, each
contained in a 3 mL quartz cuvette, were followed as a function of
irradiation time at 254 nm, the resultant spectra in each case showed
gradual conversion to the spectrum of Ura. A similar study of A3
showed the amount of absorption due to Ura increased significantly
with irradiation time. HPLC of the reaction mixtures, produced by
irradiation of A1 and A4, indicated that the parent peak in each case
was diminished in area, while the corresponding peak for Ura was
greatly increased in size; no other products were noted. However, a
similar study on A2 indicated that it contained at least one major
component that was resistant to photoreaction. (Column E was used
for this set of studies, in conjunction with 93 ⁄ 7 water ⁄ MeOH flowing
at a rate of 2 mL min)1.) The last two fractions (A5 and A6) may be
Table 1. HPLC retention times for the cyclobutane dimers of 1,3-
dimethyluracil (DMU-DMU), 1,3-dimethylthymine (DMT-DMT) and
the mixed dimers of DMU and DMT (DMU-DMT).
Dimer
DMU-DMU
DMU-DMT
DMT-DMT
c,s
c,a
t,s
5.5 min
7.5 min
8.7 min
9.1 min
8.2 min
8.9 min
9.4 min
10.0 min
8.7 min*
9.4 min
9.9 min
t,a
10.5 min
Retention times were measured from chromatograms obtained from
runs on a Shiseido Capcell UG120 column (4.6 · 150 mm) using the
following water ⁄ MeOH gradient flowing at 2 mL min)1: 0 min, 7%
MeOH; 4 min, 7%; 6.5 min, 40%; 8 min, 40%; 8.3 min, 7%; 13 min,
7%.
*The retention time of the c,s DMT dimer was determined using an
authentic sample prepared as described in the Supporting Information
accompanying the paper. This dimer is not formed when the c,s DMU
dimer is methylated.

Photochemistry and Photobiology, 2011, 87 87
acetone adducts of Ura, one of which has been previously reported
(28). Upon irradiation at 254 nm, the product in each of these
fractions reverts to Ura and at least one other component (as revealed
by UV spectroscopy).
HPLC protocols that would be effective in providing this information
for underivatized reaction mixtures. (It should be noted that the
methylation protocols described above were not designed for quan-
titative application.) While we were unable to find a single HPLC
column that permitted separation of all four isomers in one run, we
did develop methods that allowed us to separate the peaks corre-
sponding to Ura and the (c,s + c,a), t,a and t,s CBDs in one run and
the peaks associated with Ura and the (t,a + t,s), c,a and c,s dimers in
a second run. One set of conditions utilized Column C, a Microsorb
Phenyl column, with water flowing at 1.6 mL min)1 being the eluent.
The second separation protocol used Column D, a Microsorb Amino
column using acetonitrile–water mixtures as eluents. While 95% ⁄ 5%
acetonitrile ⁄ water flowing at a rate of 3 mL min)1 was most often
used for elution, the eluent composition was varied between
90% ⁄ 10% and 96% ⁄ 4% CH3CN ⁄ H2O, as required in individual
circumstances. As an example of the types of separations achieved, the
chromatograms in Fig. 2 were obtained when the reaction mixture
from the acetone-photosensitized reaction of Ura (2 mM, 20% acetone
in aqueous solution, 45 min photolysis under N2, 99% of Ura reacted)
was injected on these two columns; on column D, the eluent was 95 ⁄ 5
CH3CN ⁄ H2O at a flow rate of 3 mL min)1. (In general, before
injection, a few mL of the solution was rotatory evaporated to dryness
and, immediately after the last visible moisture had disappeared,
redissolved in the same volume of water as that of the original sample.
For acetone sensitization experiments, where redissolution of insolu-
ble dimers is a problem, the protocol was slightly different; see Results
and Discussion.) After spin filtration, the redissolved sample was
injected directly on Column C; prior to injection on Column D, a
portion of the redissolved sample was diluted 10-fold with acetonitrile.
As can be seen in Panel B, the Microsorb Amino column possesses the
capability of separating the t,a and t,s dimers in this particular system,
as well as the c,a and c,s isomers. However, when the concentration of
Ura in the injected photoreaction mixture is high, the quality of the
separation on this column is significantly diminished; reliable quan-
titative measurements of peak areas for the t,a and t,s peaks are then
difficult to obtain.
Heating of samples containing the various dimers in boiling
water, followed by HPLC analysis of the resulting solutions using
the same conditions, led to disappearance of those peaks in Fig. 2
corresponding to the c,a and t,a CBDs and increases in the area
corresponding to Ura; corresponding decreases in the areas of peaks
assigned solely to c,s and t,s dimers were not observed. This thermal
behavior correlates with similar behavior observed by Jennings et al.
(27) after they subjected the c,a and t,a CBDs of Ura to heating at
100ꢀC. These observations support the peak assignments made in
Fig. 2.
The results of the experimental work described in the sections
immediately above provide the tools needed to identify the CBDs
formed and to quantitatively analyze the amounts of each CBD
present in photoreaction mixtures formed by irradiation of Ura under
a variety of conditions. The results obtained from such qualitative and
quantitative analysis of CBD content for a number of different systems
will be described in Results and Discussion. The results from these
analyses are useful in resolving a number of disagreements concerning
product identification and quantitation that are present in the
literature on Ura photochemistry.
To assess the stability of the products in the various fractions to
heating, a portion of each fraction was placed in a screw-top tube and
heated at 100ꢀC for 15 min (or longer). In the case of A1, the sample
was heated for 15, 30, 45, 60 and 75 min; after each 15 min period of
heating the solution was chilled on ice and a sample was withdrawn.
HPLC of each sample was then run on Column A, using 93 ⁄ 7
water ⁄ methanol flowing at
a . Under these
rate of 5 mL min)1
conditions, the parent dimer(s) eluted after 2.9 min and Ura at
4.1 min. The area corresponding to the dimer decreased with heating,
while the amount of Ura in the sample correspondingly increased.
Between 60 and 75 min, however, relatively little change in the peak
areas for Ura and dimer occurred; around 50% of the original peak
area corresponding to dimer was lost after 75 min of heating. These
results suggest that A1 contains two dimers, one of which is stable to
heating and the second of which is unstable to reversion to Ura.
Analogous heating of A4 (elution time: 7.1 min) did not lead to
noticeable decomposition of the component dimer, while boiling of A2
(elution time: 3.5 min) for 15 min led to production of a small amount
of Ura and sharpening of the parent peak, as compared with the
unheated sample; these results for A2, along with those from study of
the effect of irradiation with 254 nm light (see above) suggest that A2
contains at least two components.
To determine which of the above fractions contained the various
CBDs of interest, we methylated samples of A1, A2, A3 and A4,
using a procedure similar to that described above for the DMU
dimers. In each case, the residue from a 1 mL portion of the
concentrated fraction of interest was contained in a Sarstedt 1.5 mL
screw-top microcentrifuge tube. To each residue was added the
methylation reagents as described above for methylation of the DMU
dimers; the resultant slurries were agitated for 16 h at room
temperature. After work up, the resultant aqueous solutions of
methylated reaction products were analyzed as described for the
analogous reaction mixtures obtained from methylation of the DMU
CBDs. Analysis of the reaction mixture obtained from methylation of
A1 using Gradient D (see above) indicated that the predominant
peaks had elution times of about 5.8 and 7.5 min. In separate runs,
coinjection of authentic c,s and c,a DMU CBDs with the methylated
sample resulted in increased areas for these two peaks and confirmed
that these two peaks corresponded to the c,s and c,a DMU dimers.
Additional peaks eluting at 8.2, 8.9 and 9.3 min, corresponding to the
elution times for the c,s DMU-DMT CBD, the c,a DMU-DMT CBD
and the c,a DMT dimer (see Table 1), provided confirmatory
evidence that A1 was a mixture of the c,s and c,a CBDs of Ura
(Ia and Ib).
Similar analysis of methylated A3 produced peaks at 5.1 and
9.1 min. Coinjection of DMU lead to enhancement of the 5.1 min
peak, while coinjection of the t,a DMU cyclobutane dimer led to
increased peak area for the 9.1 min peak. In addition, a peak eluting at
9.9 min can be assigned as the t,a DMU-DMT dimer, which supports
the conclusion that A3 is a mixture of Ura and the t,a cyclobutane
dimer (Id) of Ura. HPLC analysis of methylated A4 indicated that the
predominant product eluted at 8.8 min. Coinjection with authentic, t,s
DMU CBD led to a single peak eluting at 8.7 min with enhanced area,
while coinjection with authentic t,a DMU dimer produced a chro-
matogram with two peaks, one eluting at 8.8 min and the other at
9.1 min. Thus, it can be concluded that A4 contains the t,s CBD of
Ura (Ic). It is interesting to note that the elution order of the t,s and t,a
Ura CBDs is reversed from that observed for the corresponding DMU
dimers.
Preparation of the (6-4) adduct of thymine. For several experiments
in which the behavior of the thymine (6-4) adduct (5-hydroxy-6-4¢-(5¢-
methylpyrimidin-2¢-one)-5,6-dihydrothymine (V) [29]), was compared
with that of the uracil (6-4) adducts, we required an authentic sample
of this compound. This compound was prepared as described in
Supporting Information.
When the reaction mixture from methylation of A2 was analyzed by
HPLC, nine peaks (three major and six minor) appeared in the
chromatogram. It appears that this fraction is a mixture of several
products, none of which is a Ura cyclobutane dimer. We made no
attempt to further characterize the components of A2.
HPLC conditions for separation of the Ura c,s and c,a CBDs from
one another and for separating the t,a cyclobutane dimer from Ura. In
order to efficiently identify each of the Ura cyclobutane dimer
components in various reaction mixtures and to be able to obtain a
quantitative measure of the amount of each CBD produced under
various photochemical reaction conditions, it is desirable to have
RESULTS AND DISCUSSION
Here we will deal with two main topics. The first is establish-
ment of the structures of those water-soluble photoproducts
formed when Ura is irradiated in the frozen state at dry ice
temperature. The second is identification of the Ura CBDs and
provision of relative quantitative measures of the amounts of
these CBDs formed when Ura is irradiated under a variety of
conditions.

88 Martin D. Shetlar and Vladimir J. Basus
Abs.
Abs.
Time
Figure 2. The HPLC chromatograms resulting from injection of the mixture resulting from acetone-photosensitized reaction of uracil (2 mM) on a
Microsorb Phenyl column (panel A) and a Microsorb Amino column (panel B). Preliminary sample preparation and elution conditions are
described in Materials and Methods. The traces at three different wavelengths are overlapped in each chromatogram.
distilled water (1.6 mL min)1) as eluent. P2 eluted after
Photochemistry in the aqueous Ura system in the frozen state:
2.4 min, while residual dimer came off after 2.7 min. The
Purification of (6-4) adducts from F2 and F3 and a hydrated
resulting P2 was repurified using the same conditions.
form of a uracil trimer from F1
The minor putative (6-4) adduct, P3, contained in F3, was
rechromatographed on a Column D using 85 ⁄ 15 acetoni-
In the following paragraphs, we describe the isolation and
characterization of the two (6-4) adducts and a trimer hydrate
trile ⁄ water flowing at 4 mL min)1 as eluent. Before chroma-
of uracil. The work described below uses as a starting point the
fractions F1, F2 and F3 that were isolated during the first-pass
fractionation of the photoreaction mixture resulting from
irradiation of aqueous Ura in the frozen state, as described in
Materials and Methods.
tography, F3 was taken to dryness by rotatory evaporation
and redissolved in 1 mL of 80 ⁄ 20 acetonitrile ⁄ water. Then
100 lL injections of F3 were made on Column D; the desired
compound eluted at 2.5 min, while various impurities eluted
after the main peak. The predominant impurity, eluting at
3.0 min, was the uracil photohydrate II (9), identified by
comparison of its properties with those of an authentic sample.
To accomplish the separation of the components of F1,
100 lL portions of concentrated F1 were diluted with 200 lL
of acetonitrile and the resulting 300 lL samples were chroma-
tographed on Column D using 70 ⁄ 30 acetonitrile ⁄ water
flowing at 3 mL min)1. Two main peaks, eluting at 1.2 min
(F1-1) and 1.8 min (F1-2), were collected. Irradiation of F1-1
at 254 nm yielded only Ura as a product, indicating that F1
was contaminated by cyclobutane dimer. Upon analytical
Typical protocols for purification of the products contained in
F1, F2 and F3. Fraction F2 contained the major putative (6-4)
adduct, termed P2, along with significant amounts of cyclob-
utane dimer impurity. While difficult to remove using a
Capcell UG120 column, an excellent separation of P2 from
dimer was achieved using a phenyl column (Column C). After
rotatory evaporation of F2 to dryness and redissolution in
2 mL of doubly distilled water, the resultant solution was
rechromatographed, using 200 lL injections with doubly

Photochemistry and Photobiology, 2011, 87 89
HPLC of the reaction mixture after irradiation of F1-2 at
254 nm, using Column D, both Ura and a peak with the same
elution time as P2 (see above) came off the column; some IV
was present as well. Only very small amounts of F1-2 could be
isolated.
The purified products P2 and P3 were each subjected to
study by mass spectrometry, NMR spectroscopy and UV
spectroscopy. The high resolution ESI mass spectrum of P2
was obtained on a Brucker Fourier Transform 9.4 T instru-
ment in the positive ion mode. This spectrum yielded a
molecular mass of 225.06451, which corresponds to a proton-
ated species containing two uracils with a molecular formula of
C8H9N4O4. Interestingly, we were unsuccessful in obtaining a
molecular mass of P2 using MALDI mass spectrometry,
possibly due to facile decomposition of the adduct in the acidic
matrix. However, MALDI-MS of P3, using a-cyano-4-
hydroxycinnamic acid as a matrix, yielded a parent ion peak
with a mass of 225.060 based on internal calibration, again
corresponding to the molecular mass expected for a proton-
ated dimeric form of uracil.
Proton NMR data are consistent with both P2 and P3 being
(6-4) adducts, with one having the structure shown as IIIa and
the other with the structure indicated by IIIb. The relevant
chemical shift and splitting constants are given in Table 2. All
of the expected protons are present. The resonances for the C5
and C6 protons of P3 are very close in chemical shift; the
resulting two proton multiplet spectrum is quite complex,
complicated by coupling of C6H with NH1. Upon addition of
a small amount of D2O, this multiplet was transformed into a
quartet, which could be analyzed as an AB spectrum, as
described by Becker (31), p. 136. The chemical shifts of the C5
and C6 protons for both P2 and P3 are consistent with those
expected for protons located in a saturated uracil ring, while
those of the C5¢ and C6¢ are similarly consistent with
expectation for protons located at in an unsaturated uracil
ring.
Determination of the nature of the photoproducts P1-2, P2 and
P3. We used UV spectroscopy, mass spectrometry and chem-
ical studies to establish the nature of P1-2 and, in addition,
used NMR spectroscopy to study P2 and P3.
The available evidence suggests that P1-2 is an adduct of a
trimeric form of Ura with water. Because of the very small
amounts of this product that were isolated, we were unable to
establish the detailed structure of this compound; however,
two lines of evidence support the description of this compound
as being trimeric in nature. As mentioned above, photolysis of
P1-2 at 254 nm yields two main products; these were identified
by HPLC as being Ura and a compound having the properties
of P2 (which is shown to be a (6-4) adduct in the next
paragraph). This is similar to the behavior observed when the
water adduct of the thymine trimer is similarly irradiated (30).
The ESI mass spectrum of P1-2, run in the positive ion mode,
shows peaks at 377.26 and 393.34 corresponding to the sodium
and potassium adducts of a compound with the molecular
formula C12H8N6O7, the chemical formula of a trimer of uracil
to which has been added a molecule of water. A weak peak
corresponding to the protonated form of the parent trimer
hydrate is also present, as is a peak corresponding to the
protonated form of the trimer itself. Early mass spectral
evidence, indicating a polymeric compound with a molecular
mass of 370 is formed when Ura is irradiated in the frozen state
at dry ice temperature, was obtained by Khattak and Wang
(20); they suggested that this compound was a trimeric species.
As the observed molecular mass of this compound differs
significantly from the value obtained here and, as it is indicated
that the putative trimer reverts to uracil upon irradiation (20)
and is extremely labile (see Wang [19], p. 334), the relationship
of the putative trimer of Khattak and Wang to the one
described here is unclear. Indeed, Jennings et al. (27) sug-
gested, based on IR studies, that the substance identified as
a trimer by Khattak and Wang contained a mixture of the
c,s and c,a dimers of Ura (see below for further discussion).
The presence of three exchangeable protons in the spectra
obtained for P2 and for P3, as shown by the disappearance of
the resonances for these protons after addition of small
amounts of D2O to the NMR samples contained in d6-DMSO,
is also in agreement with predictions from the proposed
structures IIIa and IIIb. In the case of P2, we were able to
measure the coupling constant for the interaction of C6H with
N1H as being 3 Hz. We also obtained a 13C NMR spectrum
for P2. The chemical shift data are shown in Table S1, along
with the corresponding 13C data that we obtained for V.
(While this latter (6-4) adduct has been extensively studied {see
Table 2. Proton NMR data for the (6-4) adducts of uracil.
(6-4) adduct
C5¢H
C6¢H
7.87
(broadened)
8.07 (d, 6.2)
7.99 (d, 6.0)
C5H
C6H
N1H
N3H
N1¢H
IIIa (d6-DMSO)
6.27 (d, 6.6)
4.45 (d, 6.8)
4.51 (dd, 6.8,3)
7.72
(broadened)
11.9
(v. broad)
10.1
(broadened)
IIIa (D2O)
IIIb (d6-DMSO)
6.71 (d, 6.2)
6.41 (d, 6.0)
4.85 (d, 6.9)
4.23 (complex
multiplet)
4.95 (d, 6.9)
4.23 (complex
multiplet)
7.88
11.9
(broadened)
10.2
(broadened)
IIIb (d6-DMSO
+ D2O)
6.42 (d, 6.0)
7.99 (d, 6.0)
4.230 (d, 6.6) or
4.253 (d, 6.6)*
4.230 (d, 6.6) or
4.253 (d, 6.6)*
Proton NMR spectra were run at 600 MHz on a Varian INOVA NMR spectrometer. TMS was used as an internal standard in DMSO, while TSP
was used for this purpose in D2O. Each of the peaks gave an appropriate integration area for the number of protons expected. Coupling constants
in Hz are given in parentheses after the chemical shifts of the corresponding CH proton in ppm. In general, the resonances observed for protons
attached to nitrogen in both IIIa and IIIb in DMSO-d6 either disappeared or became greatly diminished in peak area after addition of small
amounts of D2O. The connectivity of C5¢H and C6¢H in IIIb was verified via a COSY experiment.
*Upon addition of D2O, to the d6-DMSO solution of IIIb, the broadened quartet-like multiplet centered at d = 4.23 simplified to a distinct quartet
centered near d = 4.24, reflecting loss of coupling of C6H to the exchangeable N1H proton. The d values given for C5 and C6 were calculated using
standard methods for analysis of AB spectra; it is not possible to assign either of these two resonances to a specific proton.

90 Martin D. Shetlar and Vladimir J. Basus
[19] for leading references}, the 13C NMR spectral data for V
has not been previously published.) As can be seen, the
chemical shifts for the corresponding carbons in the two
structures are reasonably close to one another; this provides
additional support for the assignment of P2 as a (6-4) adduct.
All of the above spectroscopic data are consistent with the
assignment of the structures of P2 and P3 as (6-4) adducts, as
described by structures IIIa and IIIb. However, chemical
studies also provide strong supporting evidence for these
structural assignments. Boiling an aqueous 0.1 M HCl solu-
tion of each of these compounds for 5 min results in its
conversion to compound IV; this product differs from IIIa and
IIIb in that it has lost the elements of a water molecule from
positions C5 and C6. (Dehydration of P2 and P3 is much
slower in boiling water in the absence of acid.) The structure of
IV has been previously established by Khattak and Wang (20),
based on UV, NMR and mass spectral evidence. This result
rules out an alternative possibility, namely that either P2 or,
alternatively, P3 is a (5-4) adduct, similar to that shown in VI.
Dehydration of VI would lead to a compound with the
structure shown by VII. However, the UV spectrum of VII is
available in the literature (32); comparison of the UV spectrum
of VII (Fig. 5 in Bergstrom and Leonard [32]) with that of IV,
shown in Fig. 2 in Khattak and Wang (20), shows that the two
spectra are very different. This disproves the hypothesis that
either P2 or P3 is a (5-4) adduct.
Compound VII is present in small amounts in solutions
arising from irradiation of 2 mM Ura on dry ice as described
above. When such a solution is chromatographed using 92 ⁄ 8
(10 mm sodium phosphate, pH 7.6) ⁄ MeOH at 2 mL min)1 on
Column B, a compound elutes after 3 min with an absorption
maximum at 353 nm. Using a procedure similar to that of the
previous workers (32), we prepared authentic VII by irradiat-
ing 10 mL of a deoxygenated solution containing 2 mM uracil
and 1 mM 4-thiouracil in a 20 mL Pyrex tube with black ray
lamps (Spectronics BLE-1800B) for 1 h. In agreement with
expectation from the results of the previous work, HPLC of
the resulting solution on Column B, using 92 ⁄ 8 (10 mm
sodium phosphate, pH 7.6) ⁄ MeOH at 2 mL min)1, showed
that two main products were present, namely compounds with
absorption spectra similar to those provided by Bergstrom and
Leonard (32) for VII and 5-4¢-(pyrimidin-2¢-one)-4-thiouracil.
The first of the compounds eluted at 2.9 min and had an
absorption spectrum identical to that of the corresponding
peak arising from HPLC of the irradiated uracil solution. The
second eluted at 5.0 min and had an absorption spectrum
similar to that given previously for 5-4¢-(pyrimidin-2¢-one)-
4-thiouracil (32). As expected, the compounds assigned as VII
coeluted when the irradiated Ura and 4-thiouracil-Ura solu-
tions were coinjected. As observed by Bergstrom and Leonard
(32), VII has a low solubility in aqueous solution and
precipitates when irradiated solutions are concentrated.
However, when the pH was fixed by use of 10 mM phosphate
buffer (pH 7.6) as the aqueous component of the eluent, then
the absorption spectral profiles became identical.
Solutions of Ura, freshly irradiated on dry ice and then
thawed, are always slightly yellow-green in color. However,
none of the compounds discussed above have absorption tails
extending into the visible region. We therefore carefully
examined the absorption spectrum of freshly irradiated 2 mM
uracil solutions in the visible region and found evidence for a
weak absorption band with a maximum around 427 nm. Upon
allowing unconcentrated solutions to stand in the cold for
several weeks, some cyclobutane dimer precipitates; however,
this dimer precipitate has a yellowish cast, suggesting that this
compound (hereafter termed Z) has a limited solubility.
Concentration of freshly irradiated solution by rotatory
evaporation leads to precipitation of uracil CBD on the sides
of the flask holding the solution. While there is an increase in
absorbance of the supernatant at k = 427 nm as concentra-
tion proceeds, a substantial portion of Z appears to be lost by
coprecipitiation with uracil cyclobutane dimer. We were
unable to isolate enough of Z for characterization. However,
we were able to characterize its HPLC retention properties and
obtain its absorption spectrum using the spectral capture
capability of the diode array detector. Using 92 ⁄ 8 (10 mm
sodium phosphate, pH 7.6) ⁄ MeOH at 2 mL min)1 on Column
B, Z elutes at 1.4 min; using these same elution conditions,
uracil elutes at 0.95 min, VII elutes at 3.0 min and a composite
peak corresponding to cyclobutane dimer and (6-4) adducts
elutes at 0.85 min. In addition to a broad maximum in the
spectrum of Z at 427 nm, there are two shallow minima at
287 and 345 nm between which lies a second maximum at
320 nm. Approximate ratios of the absorbances, obtained
using the diode array detector, have the following values:
A427 ⁄ A320 = 4.9; A427 ⁄ A287 = 5.9; A427 ⁄ A345 = 5.8.
An effort was made to obtain a rough estimate of the
relative amounts of combined (6-4) adduct (IIIa + IIIb) and
VII. To do this, we measured the absorbances of a freshly
irradiated Ura solution (2 mM) at 304, 357 and 427 nm. After
subtraction of spectral background at the wavelengths of
interest, the residual absorbance of a freshly irradiated
solution of 2 mM Ura at 304 nm is 0.207, the absorbance at
353 nm is 0.037 and the absorbance at 427 nm is 0.017. From
the measured spectrum of Z, the ratio of the absorbance at 353
to that at 427 nm is A353 ⁄ A427 = 0.54, while the correspond-
ing value of A304 ⁄ A427 is about 0.20. Using these ratios, we can
calculate the contribution of the absorbance of Z to the
measured absorbance values at 304 and 357 nm and correct
these two values to those that would be measured at these
wavelengths in the absence of Z. The corrected values are 0.028
at 354 nm and 0.204 at 304 nm. As the (6-4) adducts have
negligible absorbance at 353 nm, we can use an estimated
value of 22 900 (based on an average of the values for emax
given by Bergstrom and Leonard [32] of 23 500 at pH = 2 and
22 300 at pH 10) to estimate a value of 1.2 lM for the
concentration of VII. From the published absorption spectrum
(Fig. 5 in Bergstrom and Leonard [32]), it can be estimated
that the e value of VII at 304 nm is about 7900. Using the
calculated concentration of VII, the contribution of VII to the
absorbance of the solution at 304 nm can be evaluated as
0.0096. Thus, the value of the combined absorbance at 304 nm
due to IIIa and IIIb can be evaluated as being about 0.190.
The exact profile of the spectrum of VII with respect to
wavelength is sensitive to the pH of the eluting solvent. In our
initial runs, using doubly distilled water as the aqueous
component of the eluting solvent, we found that there was
some variability from run to run of the diode array spectra
corresponding to putative VII; consequently, it was difficult to
establish with certainty that the spectrum of the putative VII
formed in the uracil solution irradiated on dry ice was identical
to that produced via irradiation of 4-thiouracil with Ura.

Photochemistry and Photobiology, 2011, 87 91
Using an estimated value of emax = 4900 at 304 nm (that of
the thymine 6-4 adduct) and assuming as an approximation
that IIIa and IIIb have the same e value, we calculate the
concentration ([IIIa] + [IIIb] to be 38.9 lM. Thus, it can be
estimated that the concentration of 6-4 adduct is about 32-fold
greater than that of VII. Assuming that all VI produced (VI
being the putative precursor of VII) indeed decomposes to VII,
then this calculation suggests that production of (6-4) adducts
is a considerably more facile reaction that that leading to (5-4)
adduct when frozen aqueous solutions of uracil are irradiated
on dry ice.
selectivity. Explanation of the selective elimination of water
from P2, as compared with P3, by invoking an E2 mechanism
requires that P2 be able to adopt a conformation that is not
accessible to the P3 molecule (see below). Viewing the amino
column-induced reaction in this context, an unprotonated
alkyl amine, bound to the silica column packing, would act as
a catalytic attacking base, removing hydrogen from C6H, and
the hydroxyl group at C5 would depart simultaneously with
the hydrogen at C6. This is shown schematically in Fig. 3, in
which departure of hydroxyl is assisted by the availability of
protonated amine. For this mechanism to be feasible, a
fraction of amino groups must exist in the unprotonated state
within the Microsorb Amino column. While the pKa of
propylamine is 10.57 ([35], p. 163), Varian (the column
manufacturer) indicates that the pKa of the NH2 packing is
9.8 (Varian Catalog of Chromatography and Spectroscopy
Products and Accessories, 2003–2004, p. 9). The actual
percentage of amino groups present as free amine within the
column packing is difficult to assess, as it probably depends on
the history of the column. Our column has been used mainly
with eluents that were mixtures of acetonitrile and double
distilled water (generally degassed under vacuum, filtered and
stored under air in sealed Pyrex bottles until used), although it
has also been used with 25 mM ammonium formate as the
aqueous component of the eluent. In view of the unknowns
concerning the state of amino groups within the column
packing, it is probably not fruitful to draw conclusions about
the mechanism of dehydration of P2, based solely on the
observation that this reaction occurs during passage through
the Mircrosorb Amino column. Instead, we carried out
experiments with another aliphatic amine in solution, namely
methylamine (MeA), to determine if P2 was again selectively
dehydrated, as compared with P3. The theoretical basis for
believing such selective dehydration might occur for P2,
assuming it is indeed IIIa, is given immediately below. This
development is followed by a discussion of our experimental
results.
Suggestive evidence that P2 and P3 have the structures IIIa and
IIIb, respectively. The spectroscopic and chemical evidence
presented above strongly supports the hypothesis that both P2
and P3 are (6-4) adducts of uracil. However, this evidence does
not provide a definitive indication as to which product has the
structure given by IIIa (in which the hydroxyl group and the
2¢-pyrimidone ring are cis with respect to one another in their
attachment to the dihydropyrimidine ring) and which corre-
sponds to IIIb (in which the OH group and the 2¢-pyrimidone
ring are trans with respect to each other). Indeed, definitive
assignment of the structures must await X-ray diffraction
studies, such as was done in the assignment of the conforma-
tion of the corresponding known thymine (6-4) adduct (V) to a
structural type analogous to IIIa (33). One could reason from
analogy that P2 corresponds to IIIa, based on the fact that it is
the predominant form produced in the photoreaction and that
this is the only form that, thus far, has been reported to form
when thymine is irradiated under similar conditions at dry ice
temperature. However, there are experimental observations
that, combined with quantum mechanical calculations,
strongly support the hypothesis that P2 has the structure IIIa
and, therefore, that P3 can be assigned the structure IIIb. Our
initial observation was that when purified P2 is chromato-
graphed on Column D (a Microsorb Amino column packed
with silica particles to which aminopropyl groups are bonded)
using 85% acetonitrile ⁄ 15% water as eluent, a significant
fraction of the injected parent material is lost. In its place, a
significant sized broad peak elutes with UV spectral properties
identical to those of IV. A plausible reason for this instability
of P2 is that interaction of this molecule with free amine
groups, associated with the column packing, in some manner
induces loss of water in the C5-C6 region of the dihydropyr-
imidine portion of the molecule. An analogous reaction is not
observed when P3 is chromatographed. Indeed the injected P3
can be quantitatively recovered. Thus, the amino column
appears to induce selective elimination of water from P2, as
compared with P3. (It was for this reason that a Microsorb
Phenyl column was used to purify P2, but a Microsorb Amino
column could be used for final purification of P3.)
As indicated above, if an E2 mechanism is invoked to
explain the selective dehydration of P2 (with accompanying
identification of this compound as IIIa), as compared with P3
(which then would be identified as IIIb), an important
stereochemical question must be answered; this question deals
with the range of conformations available to IIIa, as compared
with IIIb. Facile E2 mechanisms require that the hydrogen
removed by base be able to take an anti-periplanar orientation
+
O
O
H NH2R
OH
H
H
H
H
N
N
+
H2O
:NH2R
H
H
A generalized description of the above reaction is elimina-
tion of a molecule HX from a parent molecule to form a
double bond. While a number of types of mechanism have
been proposed to explain such elimination reactions, the so-
called E2 mechanism is usually invoked to explain conforma-
tion-selective behavior of the type described above. (For a
detailed discussion of elimination reactions, see Chapter 17 in
Smith and March [34]; the E2 mechanism is discussed on pp.
1478–1486.) Other types of elimination mechanism are not
generally regarded as displaying such a high degree of
O
O
N
H
N
H
H
N
N
IV
IIIa
H
H
O
O
N
N
H
H
Figure 3. Depiction of the E2 mechanism as a pathway for the
decomposition of the product P2 (putatively IIIa) to IV, as induced by
aminopropyl groups associated with the packing of Microsorb Amino
columns and by aqueous 500 mM methylamine at pH 10.1. Discussion
is given in the text.

92 Martin D. Shetlar and Vladimir J. Basus
with respect to the leaving group (the hydroxyl group). Thus, if
P2 is to be identified as IIIa, then it must be shown that the
species IIIa can take up an anti-periplanar conformation and
that IIIb does not have access to this same conformation.
Considering torsional strain induced by the groups attached to
C5 and C6 bonds in the dihydropyrimidine moiety of IIIa,
could such an anti-periplanar conformation indeed be acces-
sible to IIIa and not available in IIIb? To answer to this
question, we used Spartan ‘08 for the Macintosh to calculate
the torsional energy profile of IIIa as a function of the dihedral
angle between the C5O and C6H bond of the dihydropyrim-
idine ring in IIIa. These calculations were done using the
Hartree-Fock model with a 3-21G basis set and the protocol
detailed in the Tutorial and Users Guide for Spartan ‘08 ([36],
pp. 98–101). (The dihedral angle in this situation is defined as
the angle between two planes, both passing through the length
of the bond between C5 and C6, with one plane passing
through the C5O and the other through the C6H bond. An
angle of )180ꢀ [or 180ꢀ] corresponds to the conformation
where the C5O bond and the C6H bond are anti-periplanar,
while a value of 0ꢀ [or )360ꢀ] describes the case where these
two groups are periplanar.) The energy profile generated by
the set of calculations is displayed in Fig. 4. These results for
IIIa indicate there are two energy minima, one near )180ꢀ (the
anti-periplanar conformation) and a significantly deeper one at
about )65ꢀ. Thus, the calculations indicate that the anti-
periplanar conformation of IIIa is not only accessible, but is
located at a torsional energy minimum. Similar calculations
performed on IIIb indicate that the deepest minimum in the
torsional energy is at about )55ꢀ, that there is a shallow
minimum at 55ꢀ and that the torsional energy is very high in
the anti-periplanar conformation at 180ꢀ (see Fig. 4). The
periplanar conformation at 0ꢀ is available for IIIb, although at
an energy that is elevated above the corresponding energy
curve minimum. While syn-elimination can occur from peri-
planar conformations via the E2 mechanism, it is usually
considerably less facile then anti-elimination and occurs only
when the anti-periplanar conformation cannot be achieved
(Smith and March [34], p. 1482). As it is unlikely that IIIb can
take up an anti-periplanar conformation under any conditions,
it would be expected to be considerably less labile than IIIa
towards elimination of water via an E2 mechanism in the
presence of bases, such as aliphatic amines. Assuming an E2
mechanism is indeed operative, observation of facile elimina-
tion for P2 and absence of elimination (or considerably slower
elimination) would strongly suggest that P2, the major (6-4)
adduct, is IIIa and P3 is IIIb.
200
100
0
U(6-4)U (cis)
T(6-4)T (cis)
U(6-4)U (trans)
E(rel) (kJ)
-100
-200
-300
-300
-200
-100
0
100
200
Dihedral Angle (degrees)
Figure 4. The potential (torsional) energies of IIIa, IIIb and V,
displayed as a function of the dihedral (torsional) angle about the
bond between C5 and C6 in the dihydropyrimidine partner in these
adducts. The dihedral angle in this situation is defined as the angle
between two planes, both passing through the length of the bond
between C5 and C6, with one plane passing through the C5O and the
other through the C6H bond. An angle of )180ꢀ (or 180ꢀ) corresponds
to the conformation where the C5O bond and the C6H bond are anti-
periplanar, while a value of 0ꢀ (or )360ꢀ) describes the case where these
two groups are periplanar. These three potential energy curves was
generated using Spartan ‘08 for the Macintosh to calculate the
torsional energy profile of the three molecules as a function of the
dihedral angle between the C5O and C6H bond of the dihydropyr-
imidine ring portions of IIIa, IIIb and V. These calculations were done
using the Hartree-Fock model with the 3-21G basis set and the
protocol detailed in the Tutorial and Users Guide for Spartan ‘08 ([36],
pp. 98–101). In general, these calculations are initiated by constraining
the relevant dihedral angle to a particular initial value and then seeking
the equilibrium geometry of the molecule under this constraint; the
relevant output for the purpose of the graph is the total energy of the
molecule when in this equilibrium geometry. The constraint is then
changed by a user selected angle increment and a new equilibrium
geometry and its corresponding energy are evaluated. This is repeated
until a user selected final value of the dihedral angle is reached. The
relative energies appearing on the graph are those calculated by
subtracting the calculated total energy of the initial conformation from
the total energies of various constrained conformations for which
calculations were done. Care was taken to select initial and final
dihedral angles, such that Spartan was able to carry out all calculations
without providing an ‘‘error’’ message at any stage of the sequence of
calculations; such signals usually signify the presence of unacceptable
steric overlaps. The body of the paper contains detailed discussion of
the implications of the information contained in this figure.
It should be noted that the quantum chemical results
calculated via Spartan, including the minima in the potential
energy curve displayed in Fig. 4, correspond to the situation
predicted in vacuum at 0 K; the relative energies of the various
conformations of IIIa in solution (e.g. in the aqueous MeA
reaction system) or when interacting with the aminopropyl
column packing could have significantly different values than
those calculated. However, because steric factors forbid a
molecule with structure IIIb from taking up an anti-periplanar
structure, conclusions about the unlikelihood of a species with
this structure undergoing reaction via an E2 mechanism should
be unaltered in these situations.
(MeA), (pKa = 10.62 [35]), can induce selective dehydration.
An initial study was made, in which P2 and P3 were adjusted
to the same absorbance (0.92) before making a 1 ⁄ 1 dilution
with 1 M MeA (pH 10.1). Injections of 20 lL samples, taken
after varying times of incubation at 19ꢀC, were made on
Column B using 90%(10 mM sodium phosphate buffer, pH
7.6) ⁄ 10% MeOH flowing at 2 mL min)1 as eluent; the
temperature was controlled during the reaction by placing
the aqueous incubation medium in a Dewar flask. Attention
was focused on material eluting at about 1.6 min, correspond-
ing to IV. The striking end result of this experiment was that
over a 48 min time interval in which P2 was incubated, the
measured peak area for IV (at 304 nm) increased from an
As indicated above, we carried out exploratory experiments,
in which we examined the question of whether methylamine

Photochemistry and Photobiology, 2011, 87 93
undetectable level to 90 area units; the corresponding mea-
sured area of IV in the incubation of P3 increased from 2 to 5
area units. A control experiment was carried out using the
same conditions, in which P2 was incubated in 0.1 mM NaOH;
the pH of the 0.2 mM parent solution was adjusted, so after
two-fold dilution its pH was 10.1. After 36 min incubation, the
amount of IV detected at 304 nm increased from 0 to 8 area
units. This result indicates that MeA is responsible for most of
the dehydration occurring at pH 10.1, rather than hydroxide
anion coming from reaction of MeA with water.
were measured, using detection at 304 nm. In our analysis, we
used the fact that, in 1st order kinetics, quantities proportional
to concentration can be used in lieu of concentrations
themselves to determine rate parameters. We found that the
rate of disappearance of peak area corresponding to the parent
compound obeyed 1st order kinetics; the value of the rate
constant for disappearance of P2, calculated from the slope of
a linear plot (q = 0.992) of the natural log of the area
corresponding to [P2] vs time, was 0.014 min)1. When the
peak areas corresponding to IV were plotted as a function of
time, the resultant graph had the shape expected for a
It should be noted that IV elutes as a pair of broad
overlapping peaks, the major peak eluting at 1.51 min and the
minor peak at 1.71 min, both peaks having identical absorp-
tion spectra; the ratio of peak areas, measured at 304 nm, was
about 8 ⁄ 1. No other peaks were doubled. The same behavior
was noted in experiments made with Column C, described
below; however, with the phenyl column, the corresponding
ratio of peak areas was about 3 ⁄ 2. The reason for this
chromatographic behavior of IV appears to be associated with
incomplete neutralization of MeA in the injected sample by the
10 mM phosphate buffer used as eluent (see below). When IV,
prepared by heating of P2 with 0.1 M HCl for 10 min, followed
by rotatory evaporation to dryness and dissolution in distilled
water, was injected on the phenyl column using 95%(10 mM
sodium phosphate buffer, pH 7.6) ⁄ 5% MeOH flowing at
1.6 mL min)1 as eluent, a single sharp peak eluted. When a
sample of this material was diluted two-fold with 1 M MeA,
pH 10.1, and injected, two overlapping broader peaks resulted;
the material in these two peaks had identical absorption
spectra. In this paper, the sums of the two peak areas
corresponding to IV were used for analyses requiring such
data.
The results described above, taken in aggregate, suggest that
selective dehydration of P2, as compared with P3, can indeed
be induced by alkyl amines. However, Column B was unable
to optimally separate certain MeA associated impurity peaks,
eluting near the retention times of P2 and P3, from these two
compounds; thus, we elected to use another column to do
studies on the kinetics of disappearance of these two com-
pounds. For this purpose, we used Column C (a Microsorb
Phenyl column), which led to excellent resolution of P2 and P3
from impurities and reaction products. In experiments with P2,
we incubated 140 lL samples of this compound (contained in
1.5 mL plastic snap-top tubes) in 500 mM MeA (pH = 10.1)
for varying lengths of time at 19ꢀC. In a representative
experiment, the sample for incubation was prepared by
diluting 70 lL of aqueous parent adduct solution
(OD304 = 3.36) with 70 lL of 1 M MeA (pH 10.1), followed
by vortexing and then starting the timer. The concentration of
amine can be regarded as constant during the course of the
reaction and, therefore, the second order kinetics predicted by
the E2 mechanism can be simplified to pseudo first order
kinetics. After withdrawal and injection of an initial sample of
10 lL after 0.5 min of incubation, succeeding samples of
10 lL were taken after appropriate intervals (spaced in the
neighborhood of 12 min) over a 64 min time period and
subjected to analytical HPLC on Column C; the eluent was
95%(10 mM sodium phosphate buffer, pH 7.6) ⁄ 5% MeOH
flowing at 2 mL min)1. Using these elution conditions, P2
eluted at about 1.8 min and the dehydration product IV eluted
at 3.6 min. The areas of the peaks corresponding to P2 and IV
product produced by a 1st order reaction (AreaIV = [AreaIV 0
]
{1)exp[)kt]}). These experiments were conducted two more
times, using a different elution condition (97%[10 mM sodium
phosphate buffer, pH 7.6] ⁄ 3% MeOH flowing at
1.6 mL min)1). In both runs, the first order plots were linear
(q > 0.99) and the values of the two calculated rate constants
for disappearance of P2 were both calculated to be
0.014 min)1
.
Analogous studies were done with P3; in these experiments,
it was found that the parent compound was relatively stable. In
a typical experiment, we diluted 200 lL of P3 (OD304 = 0.30)
with 200 lL of MeA (1 M, pH 10.1) and incubated as above for
a total of 58 min. The reaction mixture was sampled five times
over the 58 min time interval and the withdrawn samples
(50 lL) were analyzed using HPLC on Column C (95%[10 mM
sodium phosphate buffer, pH 7.6] ⁄ 5% MeOH flowing at
2 mL min)1). Plotting the natural log of the peak area,
measured at 304 nm, as a function of time yielded a straight
line (q = 0.999); the rate constant for disappearance of P3 was
calculated from the slope to be 0.003 min)1. However, only
barely detectable amounts of IV were formed during this time
interval. The production of traces of IV could be interpreted as
being due to P3 undergoing a slow syn-elimination process;
however, we cannot rule out the possibility that this IV is
formed from residual P2 impurity remaining in P3, even after
careful purification. Repetition of the same experiment with P3
several times, as outlined above, led to similar results each
time.
We did one further set of experiments to determine if the
rate of reaction does indeed depend on amine concentration.
When incubation of P2 was done in 100 and 250 mM MeA,
both at pH 10.0 and 19.0ꢀC, and the resultant solutions were
analyzed as above on Column C after appropriate time
intervals, the pseudo first order rate constants for disappear-
ance of P2 were evaluated to be 0.006 and 0.010 min)1
,
respectively. These results do indicate a dependence of the rate
of reaction on amine concentration dependence; however,
rather than the expected ratio of 2.5 for the rate constant in
250 mM solution (in a pure E2 mechanism), compared with
that in 100 mM solution, the observed ratio is 1.67. The
corresponding ratio for the rate constants obtained for 500 mM
(pH 10.1) and 250 mM are 1.50, instead of 2.
The results of the experiments on P2 and P3 described in the
above paragraphs are remarkable, in that they give a clear-cut
indication that dehydration to form IV in 500 mM MeA
solution at pH 10.1 is highly selective for P2, as compared with
P3. The results of the quantum chemical calculations,
described above, also indicate IIIa should be much more
susceptible to facile dehydration by an E2 mechanism than
IIIb. Thus, assuming an E2 elimination mechanism (or E2-like

94 Martin D. Shetlar and Vladimir J. Basus
mechanism; see below) is operative, the dehydration behavior
of P2, compared with P3, is consistent with the identification
of P2 as IIIa and P3 as IIIb. We have assumed this is the
correct assignment in labeling the NMR data given in Table 2
and Table S1 and in the discussion that follows below. These
results also lend credence to the idea that the observed selective
dehydration of P2 (IIIa) on the amino column is due to
interactions with the free amino groups contained within the
column packing.
Our results provide a starting point for further detailed
studies of the mechanism of dehydration of P2. It must be
considered that the mechanism may, in fact, not be pure E2,
but instead may lie close to E2 in the ‘‘E1-E2-E1cB spectrum’’,
as discussed by Smith and March (34) (p. 1494); this is
important due to the fact that OH is a poor leaving group and
in view of our preliminary evidence suggesting that the
dependence of the rate of the dehydration reaction is nonlinear
in unprotonated amine concentration. Results from further
experiments (e.g. studies of the dependence of the kinetics on
amine concentration and on pH; studies with other amines
having different pKa values; studies of kinetic isotope effects)
will be required to draw more definitive conclusions about the
mechanistic pathway for dehydration of IIIa induced by
amines.
Upon first consideration, it might be thought that the
thymine (6-4) adduct (V), being analogous to IIIa, should be
similarly unstable towards decomposition upon the Microsorb
Amino column. Calculations for V using the same protocol,
employing the Hartree-Fock model and 3-21G basis set used in
the calculations for IIIa, indicate that there are minima in the
torsional energy profile at about the same values of the
dihedral angle as for IIIa (see Fig. 4). Thus, the results of these
calculations suggest that V can take indeed up the anti-
periplanar conformation that is desirable for facile elimination
via an E2 mechanism. Experiment, however, shows that V is
stable to HPLC on this column. In addition, we found V to be
stable when incubated in MeA solution (500 mM, pH 10.1,
19ꢀC) under the same conditions where IIIa underwent facile
elimination of water. If the E2 mechanism is to be given weight
as a possible mechanism for amine-induced decomposition of
IIIa, then there must be a reason for stability of V under the
same conditions. A possible explanation can be found in the
greater accessibility of the C6 hydrogen to free amine groups
on the column (or in MeA solutions) in the case of IIIa, as
compared with the accessibility of the corresponding hydrogen
in V. Examination of the structures of IIIa and V, both in the
anti-periplanar conformation, indicates that the two methyl
groups present in V, but not in IIIa, offer a degree of
protection of the C6 hydrogen from interaction with free
amine groups in the column packing or in solution. Similar
protection of C6H is not evident in the corresponding
structure for IIIa. (These two structures, shown side-by-side
in Figure S1 in color, were generated as side products of the
torsional energy calculations described above.)
exclusion of nucleobase from water during the freezing
process. (For a review of the physical processes that occur
in the freezing of aqueous solutions, including formation of
solute aggregates, and the chemistry and photochemistry that
can occur in the aggregates contained in such frozen solutions,
see Montenay-Garestier et al. [37].) It appears that thermal
reactions of primary oxetane photoproducts can result in their
conversion to secondary products upon warming of frozen
solutions containing photolyzed aggregates. For example,
Rahn and Hosszu (38) have studied the conversion of putative
oxetane precursor, produced by irradiation of Thy in ice at
)196ꢀC, to Thy(6-4)Thy as a function of temperature and
concluded that, upon annealing the frozen irradiated solution
for 10–20 min at various temperatures, this conversion pro-
cess could occur at temperatures as low as )80ꢀC. The rate of
conversion increased with increasing temperature between )80
and 0ꢀC. As irradiations on dry ice occur at about )78.5ꢀC, it
is likely that conversion of oxetane precursor to Thy(6-4)Thy
adduct occurs continuously throughout the period that frozen
solutions containing Thy are irradiated at this temperature.
One possible interpretation of these results is that as the
temperature of annealing is increased, shells containing
increasing amounts of mobile interfacial water are formed
around the Thy microcrystallites containing the oxetanes;
such mobile water would then be available to catalyze the
conversion of precursor oxetane to Thy(6-4)Thy. Another
possibility is that water trapped within microcrystallites might
become increasingly available for reaction as temperature
increases. While we have not conducted similar studies of
temperature dependence of conversion of putative oxetane
precursor to Ura(6-4)Ura in irradiated frozen solutions of
Ura, it seems reasonable that the same type of behavior could
be expected in this system as well. It is interesting to note that
Ura photohydrate II (6-hydroxy-5,6-dihydrouracil [9]) was
found as a minor product in the photoreaction of Ura in dry
ice (see the discussion concerning the purification of P3
above). This suggests that mobile water may be available to
photoexcited uracils within microcrystallite lattices at this
temperature.
Figure 5 displays a plausible reaction scheme via which
compounds with structures IIIa and IIIb could be formed from
a common oxetane intermediate. In this figure, two Ura
molecules react photochemically to form an intermediate
oxetane (Int-1). (A more realistic visualization of the structural
framework of Int-1, albeit as the optical isomer, is shown in
Fig. 6; for sake of clarity, all hydrogens have been removed
except those at C5 and C6 on the dihydropyrimidine ring.)
This intermediate can react with the oxygen of an incoming
nucleophile (e.g. water) in two ways. A nucleophilic backside
attack (relative to the oxetane oxygen [labeled Ox] at the
carbon labeled 4¢ in Fig. 6) would lead to breaking of the
oxetane ring bond to the 2-pyrimidone ring; loss of a proton
from the added water and protonation of the oxygen formerly
associated with the oxetane would lead to Int 2a (Fig. 5), in
which the cis stereochemical configuration of this oxygen,
relative to the 2-pyrimidone ring, is retained. Loss of
water from the 2-pyrimidone ring would then lead to forma-
tion of IIIa. Alternatively, nucleophilic backside attack (again
relative to the oxetane oxygen) by a water molecule on the
carbon labeled 5 in the dihydropyrimidine ring of Int-1 (see
Figs. 5 and 6) would eventually lead to IIIb. The resultant
Plausible mechanistic pathways describing formation of the (6-4)
isomers IIIa and IIIb. It is thought that CBDs and the (6-4)
adducts (or the putative oxetane [or azetidine] precursors [19])
of the pyrimidine nucleobases and related compounds in the
frozen state are produced within microcrystallites of these
compounds; these microcrystallites are formed as a result of

Photochemistry and Photobiology, 2011, 87 95
O
O
OH
OH
H
H
N
N
H
H
H
H
_
H2O
OH
O
O
N
H
N
H
H
H
H
H
H
N
N
H2O
O
O
H
O
O
H
H
N
H
N
H
H
H
N
N
O
h
B
A
2
Int-2a
IIIa
O
H
O
H
N
H
N
H
N
O
O
H
O
N
H
H
OH
H
H
H2O
H
H
N
N
OH
H
_
H2O
O
H
Int-1
O
O
N
H
N
H
H
H
H
H
H
N
N
O
O
N
N
H
H
Int-2b
Figure 5. Reaction pathways leading to formation of IIIa and IIIb from the common oxetane intermediate Int-1, as discussed in the text. A more
IIIb
realistic rendering of the structure of Int-1 in its alternative enantiomeric form is given in Fig. 6.
displacement of the oxygen of the oxetane ring would lead it to
becoming associated with the pyrimidone ring. The incoming
oxygen from the water, after deprotonation to form a hydroxyl
group, would have a stereochemical configuration in which it is
trans to the 2-pyrimidone ring. Loss of water from the
pyrimidone ring then leads to IIIb.
to definitive elucidation of the processes occurring in the
conversion of oxetane to two forms of (6-4) adduct.
There are no reports in the literature indicating that the
trans configuration of the Thy(6-4)Thy adduct has been
isolated and characterized. It is therefore interesting to ask
the question ‘‘Why does not thymine photoreact to form a
similar trans form of the Thy(6-4)Thy adduct?’’ This question
can be addressed by comparing the relative accessibility of C5
in Int-1, the oxetane precursor to Ura(6-4)Ura, to that of C5 in
the thymine analog of Int-1 (see Fig. 5). Quantum mechanical
calculations of the equilibrium conformations of these two
compounds yielded the structures shown in color in Figures S2
and S3, these figures show both ball and tube and space filling
models of these two compounds. (Details of the calculations,
carried out using Spartan 08, are given in the caption of
Fig. 6.) Examination of Figure S2 indicates that access to the
C-5 carbon in the Ura version of Int-1 is very open, while
corresponding access to C-5 in the thymine oxetane interme-
diate is highly restricted, due to blockage by the 5-methyl
group on the dihydropyrimidine ring. However, when the 4¢-
carbon is examined, access to water is equivalent in the two
structures (see Figure S3). This suggests that the enhanced
reactivity of Ura towards formation of the trans form of
Ura(6-4)Ura, as compared with reactivity to produce the
corresponding Thy product, is due to steric blockage of access
of water to C5 in the latter case.
Another possibility to consider is that species, other than
water, capable of inducing oxetanes to undergoing ring-
opening reactions could be concentrated in a mobile water
shell surrounding microcrystallites containing oxetanes, or,
alternatively, contained as contaminants within the microcrys-
tallites. (In this regard, it is interesting to note that irradiation
of frozen solutions containing a 2 ⁄ 1 mixture of Ura ⁄ Thy at
dry ice temperature resulted in the formation of the Thy(6-
4)Ura adduct [39], indicating that microcrystallites formed
upon freezing do not necessarily contain a single species.) Such
contaminant species might be hydrogen or hydroxide ions and
their counterions. For example, hydrogen ion could protonate
O4¢ of the oxetane; this could be followed by N3¢-lone pair
assisted conversion of this protonated species to IIIa. Alter-
natively, OH) could act as a nucleophile in a manner similar to
the mechanism illustrated above with water. Concentration of
such species, along with their counterions, would occur
because the freezing process in water excludes solutes (such
as Thy or Ura) and impurities. Solute and impurity molecules
would be concentrated into smaller and smaller volumes as
freezing of the parent solution proceeded to completion;
presumably volumes of water containing solutes and impuri-
ties would be the last to freeze (40). Kinetic and other studies
of frozen aqueous nucleobase systems, to which deliberate
additions of varying amounts of acidic or basic species (e.g.
HCl or ammonia) have been made, might yield insights leading
It is interesting to note that the C5 position in the putative
oxetane or azetidine precursors to Cyt(6-4)Thy and Cyt(6-
4)Cyt adducts would be predicted to be more accessible to
nucleophilic attack than C5 in the Thy(6-4)Thy adduct. It is
thus possible that both forms of these two adducts would be
formed under suitable UV-irradiation conditions, perhaps

96 Martin D. Shetlar and Vladimir J. Basus
and the relative yields (based on peak area) in which they are
formed for a number of photoreaction systems.
Identification and determination of the relative yields of the
dimers formed when uracil is irradiated in frozen solution. In the
early 1960s, a number of workers noted that irradiation of
Ura in the frozen state at dry ice temperature yields product(s)
that were shown to have the character of CBDs (40–43).
However, there was not uniform agreement among these
researchers concerning the number and properties of the
dimers formed in this photochemical reaction. For example,
Wacker and coworkers (42) indicated that one isomer was
formed, while Smith (43) found that two products appeared
after paper chromatography, one with an Rf of 0.12 and the
other with an Rf of 0.01. Smith noted that the faster moving
material converted into the slower moving material upon
standing, while the slower moving compound could be
reconverted to the faster moving dimer by treatment with
acid, alkali or heat. (Demonstration that two crystalline forms
of the c,s Ura dimer exist was accomplished by Varghese [21]
and independently by Jennings et al. [27]; one form is much
less soluble than the other. Insoluble dimer spotted at the
origin probably accounts for the Rf = 0.01 spot.) The findings
of Smith and of Wacker et al. diverged somewhat in another
way, as the latter workers found that the dimer they observed
was unstable to boiling in water for 15 min and to treatment
with acid or base. Smith, in agreement with results from
Smietanowska and Shugar (41), found that the dimeric
material he studied was stable under similar conditions. Later
work also contained suggestive evidence that more than one
dimer was formed. For example, Blackburn and Davies (44)
found that initially isolated dimer was partially degraded by
treatment with formic acid, leaving a residual amount of
undecomposed material identical to the starting material.
After methylation of the ‘‘ice dimer’’ of Ura, Elad et al. (24)
noted that the end product was contaminated with the c,a
dimer of DMU. Varghese (21) indicated, in his study of the
photoreaction of Ura in the frozen state, that about 10% of
the dimer formed was the c,a isomer, the remainder being the
c,s form. Structural identification of the c,s dimer has been
carried out using chemical studies in conjunction with NMR
spectrometry (44), by X-ray diffraction study (45) and by
chemical degradation studies (46). The structure of the c,a
isomer has been derived from a X-ray diffraction study. This
crystalline dimer, obtained after irradiation of Ura in the
frozen state, was obtained by crystallization of the c,a
cyclobutane dimer from the product mixture resulting from
boiling a sample containing a putative uracil trimer (47); this
c,a form of the dimer was thought to be a trimer decompo-
sition product. (See, however, Jennings et al. [27], p. 489,
where an alternative interpretation of the crystallization of the
c,a cyclobutane dimer from solutions containing the putative
trimer is given.)
Figure 6. Two views of a ball-and-stick model of the 4¢R,5S,6S isomer
of the putative oxetane precursor of the (6-4) adduct of uracil. This is
the enantiomer of Int-1 displayed in Fig. 5. All hydrogens, except
those located at C5 and C6, are hidden. Oxygen atoms are denoted by
black balls, nitrogens by light gray balls and carbon atoms are coded
by medium gray. The oxygen of primary interest is labeled Ox. As
described in the text, backside attack of water at C4¢ in line with the
C4¢-Ox bond would lead to IIIa and analogous attack at C5 would
produce IIIb. This model was constructed using Spartan ‘08 for the
Macintosh to calculate the equilibrium ground state conformation.
The sequence of calculations used was as follows. A set of conformers
was sought via molecular mechanics calculations using the MMFF
force field. Only one low energy conformer was found. The equilibrium
geometry of this conformer was determined using semiempirical
calculations utilizing the PM3 model. The equilibrium conformation
determined in this round of calculation was then reoptimized using the
Hartree-Fock model with the 3-21G basis set. The equilibrium
conformation was then given a final optimization using the Hartree-
Fock model and the 6-31G* basis set. Informative alternative views of
this compound are displayed in color in Figures S2 and S3.
even in a biological context. Similarly, one might expect two
forms of the Ura(6-4)Thy and Ura(6-4)Cyt adduct could be
prepared under appropriate conditions.
Identification and measurement of the relative yields of
the cyclobutane dimers of Ura formed under various
irradiation conditions
In Materials and Methods, we discussed the results of studies
of the HPLC behavior of the Ura CBDs. We indicated that
through use of two columns, namely a Microsorb Phenyl
column (Column C) and a Microsorb Amino column (Column
D), separations can be obtained such that the peak corre-
sponding to each dimer is reasonably well resolved from the
peaks corresponding to the other components of a reaction
mixture containing all of the dimers and Ura. In this section,
we apply this methodology to identifying the dimers produced
When we analyzed the photoreaction solution, resulting
from irradiation of aqueous Ura (2 mM) in the frozen state for
1 h, using the two-column approach described towards the end
of Materials and Methods, it was found that, in fact, all four
CBDs are formed. Peak area measurements for the HPLC
chromatogram, obtained using the Microsorb Phenyl column,
showed that 90% of the total integrated area at 225 nm for
CBD peaks was found under the (c,s + c,a) peak, while 6.5%

Photochemistry and Photobiology, 2011, 87 97
was under the t,a peak and 3.5% was under the t,s peak. When
the same reaction mixture was run on the Microsorb Amino
column, the c,s peak and c,a peaks contained 63% and 37%,
respectively, of the total area under these two peaks. There-
fore, it can be calculated that the distribution of areas for the
four peaks corresponding to cyclobutane dimer is 57%
c,s ⁄ 33% c,a ⁄ 3.5% t,s ⁄ 6.5% t,a. (In general, the percentages
listed here, as well as in the following paragraphs, are based on
average peak areas calculated from integrated peak area values
for several runs.) These results indicate that the fraction of c,a
dimer formed is somewhat higher than previously estimated by
Varghese (21), who used ion exchange chromatography to
separate the two isomers. The formation of the two trans
isomers when Ura is irradiated in the frozen state has not been
previously reported. (It should be emphasized that the relative
yields we present for the various CBDs may differ from those
that would be obtained via methods that directly yield
measures of molar concentrations of these products. To
convert our results to this type of measure, we would need
to know e values for the various CBDs at 225 nm; such
information is not presently available for these compounds.)
These workers also found that a precipitate formed during
irradiation for each acetone-sensitized reaction they studied.
Their analysis of each individual precipitate implied that it
consisted of predominately t,s dimer with some t,a CBD
contamination; only the t,s form remained after the precipitate
was treated with boiling water. They also found that at least
one component of the precipitate was unstable to treatment
with ammonia; about 13% of the weight of the precipitate was
converted to Ura, the product that they determined was
formed by treatment of the t,a dimer with ammonium
hydroxide. Thus, there is significant conflict between the
results of Varghese and those of Jennings et al. concerning the
nature of the dimers making up precipitates resulting from the
photoreaction of Ura using acetone-sensitization.
New experimental results suggest that the Ura t,s CBD is
produced in greater amounts than the t,a CBD in acetone
sensitized reactions. To help resolve the disagreement between
the results of Varghese and those of Jennings et al., we
conducted two studies; one looked at the composition of the
freshly prepared reaction mixture resulting from acetone-
sensitized reaction of Ura and then examined the composition
of the same mixture after a precipitate had been allowed to
form. The second study determined the composition of the
precipitate formed when the acetone-sensitized reaction was
run as described by Varghese in his study. Specifically, our first
study was designed to determine the ratios of yields of the
various dimers formed when the photoreaction of Ura (2 mM)
was sensitized by acetone (20% V ⁄ V). About 175 mL of
solution was irradiated for 45 min under flowing 99.997%
nitrogen that bubbled through the solution. (Prior to flowing
into the reaction vessel, the nitrogen was bubbled through a
solution that was of the same composition as the reaction
medium.) We then analyzed the dimer content of this solution
immediately after irradiation and after standing overnight at
room temperature. (After standing, a significant amount of
precipitate could be observed in the flask containing the
irradiated solution.) Samples (0.5 mL) of the freshly reacted
solution were placed in Sarstedt screw-top microcentrifuge
tubes and most of the acetone was removed by rotatory
evaporation on a water aspirator, as described in Materials
and Methods. The remaining solution was rediluted to 0.5 mL,
injected successively on the Microsorb Phenyl and Microsorb
Amino columns and analysis of the resulting chromatograms
was carried out as described above. The following area ratios
were obtained for the various dimers: 19% c,s ⁄ 18% c,a ⁄ 39%
t,s ⁄ 24% t,a. The corresponding ratios for the solution that had
stood overnight were: 29% c,s ⁄ 27% c,a ⁄ 20% t,s ⁄ 24% t,a. It
can be seen that a considerable loss of the t,s CBD from the
parent solution occurred on standing, along with a smaller loss
of the t,a dimer. (If loss of t,a CBD had not occurred from
solution upon standing, the percentage of this dimer in
solution would have been higher than 24%, rather than
remaining constant at this value.) These results imply that the
precipitate formed upon standing contains both the t,s and the
t,a Ura CBDs. (In experiments with freshly irradiated solu-
tions, care must be taken that precipitation does not start
before or during sample workup. Otherwise, there can be
significant variation from the results described above. This
variability is likely due to partial precipitation of the relatively
insoluble trans dimers prior to injection; this particularly true
Identification and distribution of the yields of dimers formed
when photoreaction of Ura is sensitized by acetone in fluid
aqueous solution. Krauch et al. (48) and Greenstock and
Johns (49) published early indications that all four CBDs of
Ura are formed when acetone is used to photosensitize the
reaction of the parent compound in aqueous solution. Based
on experiments in deoxygenated solution (unspecified concen-
tration of Ura) using radiotracer techniques, Greenstock and
Johns found that the various isomers were produced in
comparable amounts and provided quantitative measurements
of the amount of each dimer formed, while Krauch et al.
identified one of the dimers as the c,s form. The chemical
identities of the dimers corresponding to each measured yield
were not established by Greenstock and Johns; however, data
from experiments done by the same workers with Ura in
frozen solution permits identification and quantitation of the
relative amounts of the c,s and c,a isomers formed. From their
data, it can be calculated that the ratio of yields of c,s CBD to
c,a CBD is about 60 ⁄ 40. Later work by Varghese (21),
employing 50% acetone and Ura at a concentration of 2 mM,
measured the amounts of dimer found in each of two phases.
After 125-fold concentration of the original photolyzed
solution, the precipitate formed was filtered off and the yields
of the various CBDs in the supernatant were found to be 68%
c,s ⁄ 29% c,a ⁄ 0% t,s ⁄ 3% t,a. Infrared spectroscopic examina-
tion of the precipitate, after washing with methanol to remove
Ura, indicated to Varghese that it contained the t,a dimer as
the predominant product. Based on calculations that took into
account the weight of the precipitate, he concluded that the t,a
dimer was the major form of CBD produced in the acetone-
sensitized photoreaction of Ura. In work published at about
the same time, Jennings and coworkers (27) presented their
findings on the acetone-sensitized reactions of Ura. While they
did not evaluate quantitative yields of the various dimers, they
did identify those dimers that dominated after irradiation of
10 mM Ura in water ⁄ acetone mixtures of compositions ranging
from 5% to 83% V ⁄ V acetone ⁄ water. In agreement with
Varghese, the supernatant was found to contain predominately
the c,s and c,a forms of the dimer, as well as some t,a CBD.

98 Martin D. Shetlar and Vladimir J. Basus
of the t,s CBD. Such precipitation causes the calculated
amounts of the trans dimers to be lowered, as compared with
the cis dimers.)
reactions contain a mixture of both trans dimers as indicated
by Jennings and co-workers.
Based on the above discussion, along with careful consid-
eration of the protocols and results of the two previous
workers, some retrospective suggestions can be made about
reasons why Varghese and Jennings et al. differed in some of
their conclusions. Both Varghese (21) and Jennings et al. (27)
used IR spectroscopy as their prime tool for identification of
the dimer(s) present in the precipitates resulting from their
photoreactions. Jennings and co-workers published IR spectra
for each of the dimers they obtained. They indicated that the
identity of the isomer corresponding to each spectrum was
verified by comparisons with IR spectra provided to them by
Professor E. Fahr, in whose laboratory authentic samples of
each of the four dimers had been prepared via nonphoto-
chemical synthetic methods (50,51). Varghese similarly pub-
lished four IR spectra that he designated as being for the a
and b forms of the c,s dimer, the c,a dimer and the t,a
cyclobutane dimer of Ura and indicated that the latter dimer
had been synthesized using the procedure of Richter and Fahr
(50); the IR spectrum of the t,s CBD was not included in the
paper. However, comparison of the IR spectra given in the
two papers indicates that the spectrum that Jennings et al.
(27) identify as being that of the t,s isomer is labeled as being
the t,a isomer by Varghese (21). Varghese indicates that his
conclusion that the precipitate was composed mainly of t,a
cyclobutane dimer was based on comparison of the IR
spectrum of the precipitate he obtained, after irradiation of
2 mM Ura in the presence of 50% acetone, with the IR of the
compound that he identified as the t,a isomer. Thus, in
contrast to the identification made by Jennings and cowork-
ers, Varghese concluded that the precipitate was made up
mainly of t,a CBD. This juncture of disagreement led to other
disagreements in interpretation. Varghese indicated that
column chromatography of the substances contained in the
precipitate, using 0.1 N ammonia as eluent, resulted in
destruction of about 90% of the chromatographed material
and suggested that the product produced was a cyclobutane
dicarboxylic acid. In contrast, Jennings and co-workers, after
studying the stability of the compound they identified as the
t,a CBD to treatment with concentrated ammonia, concluded
that the reaction product was uracil. They found that similar
treatment of the compound they identified as the t,s dimer
produced an unidentified compound with an IR band at
1400 cm)1. (It can be noted that the carboxylate ion func-
tional group has a symmetric stretching mode vibration at
1400 cm)1 (see, e.g. Pasto and Johnson [52], p. 127)). In
another experiment, Varghese found that heating his putative
t,a CBD at 100ꢀC for 15 min had no effect (see Table 1 in
Varghese [21]). In contrast, Jennings and co-workers found
that refluxing of the dimer they identified as t,a in boiling
water for 24 h resulted in conversion of this compound to
uracil, while the t,s isomer were stable to this treatment. The
agreement of this behavior with the previously reported
analogous thermal properties for these two dimers (50) was
used by Jennings et al. to confirm the identifications of the t,a
and t,s isomers based on IR spectra.
In the second study, we irradiated 2 mM Ura in 50%
acetone under nitrogen and worked up the resulting photore-
action mixture as described by Varghese (21). The resulting
precipitate, after washing with methanol, was dried in air. We
then methylated a weighed amount of this precipitate with
CH3I in a slurry containing Ag2O in DMF as described in
Materials and Methods. After workup as described previously,
the HPLC of the methylated sample was run using the same
column and gradient as previously described. While quantita-
tive assessments cannot be made, the resulting chromatogram
showed strong peaks corresponding to both the t,s and t,a
DMU-DMU dimers. Also present was a peak eluting at the
retention time of the t,s DMU-DMT dimer and a peak
corresponding to the overlapping retention times of the t,s
DMT dimer and t,a DMU-DMT CBD (see Table 1). There
was not any evidence for the presence of methylated c,s or c,a
CBDs of Ura in the sample. In a second experiment, the
precipitate was heated for 1 h in boiling water, the resulting
mixture was taken to dryness and the resultant solid material
was methylated as above and subjected to HPLC. Analysis of
the chromatogram produced indicated that the peaks corre-
sponding to the methylated dimers of the t,a isomer were
greatly diminished in area, as compared to the corresponding
peaks assigned as belonging solely to methylation products of
the t,s dimer. This is agreement with expectation, as Richter
and Fahr (50) remarked on the greater thermal lability of the
t,a cyclobutane dimer of Ura, as compared to the t,s form.
This is also in agreement with the thermal behavior of the t,a
(and c,a) CBDs formed in photoreactions of thymine; the half-
life of the t,a CBD of thymine at 100ꢀC in neutral solution,
with respect to reversion to thymine, is only a few minutes (see,
e.g. Fig. 11 in Fisher and Johns [8]). Summarizing, our results
imply that both t,s and t,a CBDs are significant components of
the precipitate that was isolated using the workup methods
employed by Varghese to study mixtures of Ura CBDs
produced via sensitization with 50% acetone; our results
confirm that the c,s and c,a CBDs remain in solution during
precipitate formation.
The conflicting conclusions of Varghese (21) and of
Jennings et al. (27), regarding the photochemistry occurring
in acetone-sensitized solutions of Ura, have been resolved by
the information obtained in our work. Our results indicate
that the precipitate studied by Varghese, instead of consisting
of mainly one dimeric form (the t,a CBD) as he proposed, was
a mixture containing significant amounts of both the t,s and
t,a isomers. While done in more concentrated solutions of Ura
(10 mM vs 2 mM, as used in the experiment of Varghese and in
our replication of his experiment), Jennings et al. also
observed that the precipitates formed during irradiation
contained both of the trans dimers. Furthermore, our deter-
mination of the composition of freshly photosensitized reac-
tion mixtures show that the t,s dimer is the product present in
largest amount; in the supernatant of solutions that had stood
overnight, the t,s dimer was present in the smallest amount,
while some t,a dimer had also been lost. This lost material
must have ended up in the precipitates that were formed upon
standing. All of these observations strongly support the
conclusion that precipitates formed in acetone-sensitized
Identification and distribution of the yields of dimers formed
when uracil is irradiated in fluid aqueous solution. The photo-
reactions of Ura in fluid aqueous solution have received a

Photochemistry and Photobiology, 2011, 87 99
significant amount of study, although much of this attention
has been focused on the reaction to form the hydrate of Ura
(see the discussion in Fisher and Johns [9]). However, there is
also published information concerning the formation of CBDs.
Greenstock et al. (53) indicated that irradiation in fluid
aqueous solution produced several dimers, while Brown and
Johns (54) found that all four dimers were produced. Using
radiotracer techniques, Greenstock and Johns (49) measured
the relative yields of the four dimers when Ura was directly
irradiated in deaerated aqueous solution at an unspecified
concentration. As the yields of the c,s and c,a dimers can be
determined (based on identification of these two compounds
from a study of reactions of Ura in the frozen state reported in
the same paper), the ratio of the amounts of these two isomers
can be evaluated as 54 ⁄ 46. Relevant data from other sources is
also available. Unpublished work by Varghese (cited in Fisher
and Johns [8], p. 244) indicated that the ratio of dimer product
yields was 35% c,s ⁄ 15% c,a ⁄ 0% t,s ⁄ 50% t,a (irradiation done
at an unspecified Ura concentration) while Fendler and Bogan
(55), using radiolabeled Ura and paper chromatography to
two sets of experiments. Consideration of the arguments given
in the discussion of the experiments of Varghese (21) on the
acetone-sensitized photoreaction of Ura (see above) suggests
that the value of 50% t,a CBD measured in his unpublished
work may, in fact, reflect contributions from both the t,s and
t,a forms of the cyclobutane dimer.
There is considerable discrepancy between the results of
Fendler and Bogan and the results published here for the
situation when a 10)4 M solution of Ura is irradiated in fluid
solution. Fisher and Johns (8) (p. 249) suggested that the
disagreement in observed product ratios, between the work of
Varghese (done at unspecified concentration) and that of
Fendler and Bogan, could be due to three possible reasons:
mistaken identification of the dimers, to the effects of
concentration on the product ratios or to changes in dimer
ratios with increasing time of irradiation. Comparison of
results from the present work (15% c,s ⁄ 24% c,a ⁄ 26% t,s ⁄ 35%
t,a) in which about 79% of the DMU in an actinometric
solution had been converted to product, to those published by
Fendler and Bogan (15% c,s ⁄ 78% c,a ⁄ 3.5% t,s ⁄ 3.5% t,a) in
which 77% of the DMU in a similar solution was converted to
product suggests that the latter two reasons can be ruled out as
causes for major changes in product ratios and that misiden-
tification of photoproducts is a more plausible explanation. It
is worth noting that failure to resolve the c,a, t,s and t,a
dimers, with accompanying identification of the composite
paper chromatography spot as the c,a CBD, would lead to an
artificially high apparent c,s ⁄ c,a ratio of 15 ⁄ 85. Similar
addition of the area contributions of the c,a, t,s and t,a dimers
observed in our work, to obtain the area of a composite spot,
would give an analogous c,s ⁄ c,a ratio of 15 ⁄ 85. Under these
circumstances, the products identified by Fendler and Bogan
as t,s and t,a dimers would be minor noncyclobutane dimer
photoproducts. In support of the idea that other unidentified
products are produced when 10)4 M Ura is irradiated in
aqueous solution, our solutions contained an unidentified
product that, upon HPLC using Column C with distilled water
as eluent, eluted between the peak for Ura and the peak
corresponding to t,a dimer; the absorption spectrum corre-
sponding to this peak was similar to those observed for the c,s,
c,a and t,a CBDs of Ura. (The uracil hydrate elutes before Ura
under these elution conditions.) The percentage contribution
of the area for this peak (monitored at 225 nm) to the total
area observed for all product peaks (excluding the hydrate of
Ura) was about 6%; this can be compared with the value of
7% observed for the sum of the contributions of the putative
t,a and t,s dimers to the total dimer yield, as evaluated by
Fendler and Bogan. If the scenario described here should be
valid, it would help account for the discrepancy between the
results of the work of Fendler and Bogan and the other results
discussed above.
study the photochemistry of Ura at a concentration of 10)4
M
in degassed aqueous solution, found this ratio to be 15%
c,s ⁄ 78% c,a ⁄ 3.5% t,s ⁄ 3.5% t,a after DMU, in an actinometric
solution irradiated in parallel, had been converted to products
to an extent of 77.1%. Presence of oxygen in the solution of
Ura strongly suppressed formation of CBDs. (It should be
noted that product distribution in uracil systems directly
irradiated in solution with 254 nm light is potentially sensitive
to the percent conversion of parent to product as well as initial
concentration; as the percentage conversion of parent uracil to
product increases, dimeric products become increasingly able
to compete with the parent for light with consequent CBD
photoreversal. As a competing photoirreversible hydration
reaction occurs in the case of Ura, the amounts of the total and
individual cyclobutane dimer products present in solution may
increase and then decrease with increasing reaction time;
indeed, such behavior is observed in the data presented in
Table 1 of the paper by Fendler and Bogan.)
We examined the dimer distribution in two different
situations where Ura was irradiated at 254 nm in fluid solution
under flowing nitrogen. In the first, we irradiated 170 mL of
2 mM Ura for 3 h as described in Materials and Methods;
about 41% of parent Ura had reacted. Analysis of the results
from HPLC, as described above, indicated that in this system
the area ratios for the dimer distribution was 20% c,s ⁄ 26%
c,a ⁄ 22% t,s ⁄ 32% t,a. In the second experiment, we irradiated
170 mL of deoxygenated 10)4
M Ura concurrently with
170 mL of 10)4 DMU at 254 nm; the two solutions were
placed in equivalent positions in the lamp holder described in
Materials and Methods and irradiated for 12 min. After this
time, 78.6% of the DMU had disappeared, which is close to
that obtained in the experiment of Fendler and Bogan cited
above (77.1% DMU conversion). In this situation, the
corresponding area ratio was found to be 15% c,s ⁄ 24%
c,a ⁄ 26% t,s ⁄ 35% t,a.
Identification and distribution of the yields of the dimers formed
when uracil is irradiated in acetonitrile solution. There have
been two studies made of the photochemistry of Ura when
dissolved in acetonitrile. Both studies were designed to
examine questions relevant to understanding the excited
state(s) responsible for reaction and to gather kinetic data
describing the various excited state processes that Ura under-
goes when photolyzed in acetonitrile. The work of Lamola and
Mittal (56) in 0.7 mM solution indicted that two dimers were
The ratio of c,s ⁄ c,a of 44 ⁄ 56 calculated for the direct
reaction of 2 mM Ura is in reasonable agreement with the value
of 56 ⁄ 44 evaluated from the data of Greenstock and Johns (49)
(see above), particularly taking into account that different
methods of quantitation were employed and that considerably
different concentrations of Ura may have been used for the

100 Martin D. Shetlar and Vladimir J. Basus
Table 3. Distribution of the yields* of individual Ura cyclobutane dimers.
Uracil System
% c,s
% c,a
% t,s
% t,a
2 mM, frozen in dry ice†
57
19
29
20
15
18
14
33
18
27
26
24
18
14
3.5
39
20
22
26
32
52
6.5
24
24
32
35
32
20
2 mM, acetone photosensitized, freshly irradiated
2 mM, acetone photosensitized, stood overnight
2 mM, fluid aqueous solution
0.1 mM, fluid aqueous solution
0.17 mM in acetonitrile, 75% conversion to product
0.17 mM in acetonitrile, 17% conversion to product
*Yields were evaluated using HPLC peak areas as described in the text of the paper; these numerical values represent averages from several runs.
Peaks were detected at 225 nm.
†Solution was not deoxygenated. All other systems were flushed with 99.997% pure nitrogen prior to irradiation.
formed, while the work of Wagner and Bucheck (57) in
0.19 mM solution suggested that all four isomers might be
formed, based on analogy with results from the work of
Krauch et al. (48) on the photosensitized reaction of Ura in
aqueous solution.
Summarizing discussion. The results of our measurements of
the distribution of dimer yields, based on measurement of
chromatographic peak areas, are summarized in Table 3. As a
result of the studies on the CBDs produced when Ura is
irradiated under various conditions, the following general
conclusions can be drawn.
Using the protocols described above, we identified the
dimers formed and measured the peaks areas corresponding to
each dimer using a concentration of Ura approximating one of
those employed by Wagner and Bucheck. We prepared a stock
saturated solution of Ura in acetonitrile by stirring 56 mg of
Ura overnight in 500 mL of HPLC grade CH3CN. Making
the assumption that the emax for Ura in CH3CN is about the
same as that in water, we determined that the concentration of
the supernatant solution was 0.87 mM. We than diluted a
portion of the concentrated supernatant to a concentration of
0.17 mM and used this solution for our experiments. A 3 mL
portion of the 0.17 mM solution was placed a cylindrical
irradiation tube and prepared for irradiation as described in
Materials and Methods. The spectrum was measured against
the acetonitrile blank (see Materials and Methods) after being
irradiated for 0, 15, 30, 60, 90, 120 and 150 s. After 150 s, about
75% of the initial absorbance of Ura had disappeared. (It
should be noted that the orientation of the external surface of
the tube with respect to the entering light beam in the
spectrometer was the same for each spectral measurement
and in measurement of the blank; failure to do this resulted in
severe displacements of the baseline and consequent unusable
spectra.) When a similar run was done under air, the reaction
was much slower, with 16 min irradiation required to achieve
66% conversion of parent Ura to product. Analysis of
the product mixture resulting from irradiation under N2, using
the Microsorb Amino and Microsorb Phenyl columns as
described above, showed that all four CBDs are indeed formed
and that the distribution of areas for the product peaks after
150 s irradiation was 18% c,s ⁄ 18% c,a ⁄ 32% t,s ⁄ 32% t,a. The
reaction mixture obtained from 15 s irradiation (17% conver-
sion of Ura to products) was similarly studied; the correspon-
ding distribution was 14% c,s ⁄ 14% c,a ⁄ 52% t,s ⁄ 20% t,a. It is
interesting to note that the 15 s sample contains a relatively
high content of t,s dimer, as compared with the 150 s sample.
The probable reason for this is that the t,s dimer has a
somewhat higher molar absorbance at 254 nm than the other
three dimers (such is the case for the thymine CBDs; see
Table 13, p. 277, in Fisher and Johns [8]). As a result, the t,s
dimer is likely to be more susceptible to photoreversal at high
extents of conversion than the other three dimers.
1. Both the c,s and c,a CBDs are formed in major amounts
when Ura is irradiated in frozen aqueous solution with the
c,s dimer being the predominant dimer (57% yield) and the
c,a dimer being present to the extent of 33% of the total
dimer product formed (based on HPLC peak areas mea-
sured at 225 nm). The t,a and the t,s dimers were produced
in smaller yields, amounting in total to about 10% of the
total dimer product yield. These results resolve inconsis-
tencies in results reported in the literature, some suggesting
that only one dimer was formed and others that two were
produced when Ura is irradiated in ice. Our findings also
address the findings of some workers that heat or acid
instability was associated with the dimeric product(s)
formed; one of the major products produced, the c,s
isomer, is stable to heat and acid treatment, while the other
major product (the c,a CBD) is unstable to similar
treatment.
2. When Ura is irradiated in fluid solution (e.g. in water or
acetonitrile) all four CBD isomers are formed in significant
amounts under each of the conditions studied. This is also
true when the photoreaction of Ura is sensitized by acetone.
Our results, along with careful comparison of results and
analysis of experimental protocols used in previous work,
allowed us to make plausible suggestions about how several
inconsistencies in the literature can be resolved.
CONCLUDING REMARKS
It has been shown in this study that two diasteromeric (6-4)
adducts are formed when Ura is irradiated in frozen solution.
This is the first report indicating that a (6-4) product can be
formed, in irradiations of pyrimidine nucleobases and related
compounds, with the 5-hydroxy and 6-pyrimidone groups
trans with respect to one another A plausible mechanism is
proposed, showing how two diasteromeric forms can be
generated from the type of oxetane intermediate thought to
be a precursor of (6-4) adducts. It has also been shown that
photoreaction of aqueous Ura in the frozen state generates a
trimeric species as well as a (5-4) adduct (which was detected as
its dehydration product) and uracil hydrate. Also reported

Photochemistry and Photobiology, 2011, 87 101
4. Cadet, J., E. Sage and T. Douki (2005) Ultraviolet radiation-
mediated damage to cellular DNA. Mutat. Res. 571, 3–17.
5. Gordon, M. P., C.-W. Huang and J. Hurter (1976) Photochem-
istry and photobiology of ribonucleic acids, ribonucleoprotein,
and RNA viruses. In Photochemistry and Photobiology of Nucleic
Acids, Vol. 2 (Edited by S. Y. Wang), pp. 265–308. Academic
Press, New York.
6. Zhirnov, O. V. and P. Wollenzien (2003) Action spectra for UV-
light induced RNA-RNA crosslinking in 16S ribosomal RNA in
the ribosome. Photochem. Photobiol. Sci. 2, 688–693.
7. Smirnov, Y. A., S. P. Kapitulex and N. V. Kaverin (1992) Effects
of UV-irradiation upon Venezuelan equine encephalomyelitis
virus. Virus Res. 22, 151–158.
8. Fisher, G. J. and H. E. Johns (1976) Pyrimidine photodimers. In
Photochemistry and Photobiology of Nucleic Acids, Vol. 1 (Edited
by S. Y. Wang), pp. 225–294. Academic Press, New York.
9. Fisher, G. J. and H. E. Johns (1976) Pyrimidine photohydrates. In
Photochemistry and Photobiology of Nucleic Acids, Vol. 1 (Edited
by S. Y. Wang), pp. 169–224. Academic Press, New York.
10. Davies, R. J. H. (1995) Ultraviolet radiation damage in DNA.
Biochem. Soc. Trans. 23, 407–418.
11. Shetlar, M. D., V. J. Basus, A. M. Falick and A. Mujeeb (2004)
The cyclobutane dimers of 5-methylcytosine and their deamina-
tion products. Photochem. Photobiol. Sci. 10, 968–979.
12. Moore, A. M. (1959) Ultraviolet irradiation of pyrimidine deriv-
atives. III. Effect of HCN on the photolysis. Can. J. Chem. 37,
1281–1287.
here are definitive identifications of the CBDs formed in a
several types of photoreaction systems containing Ura (i.e. in
the frozen aqueous state, in fluid aqueous and acetonitrile
solution and in aqueous acetone-sensitized reaction systems).
Quantitative measures of the yields of the four dimers in each
of these systems are also given; these measures are based on
analysis of HPLC peak areas. Reports of the results of prior
work on the photochemistry of Ura are incomplete in their
description of the photochemistry occurring in these systems
and, in some cases, in disagreement. Based on the work
reported here, suggestions for resolving several contradictions
in the literature have been made and a fuller picture of the
photochemistry of Ura has emerged.
Acknowledgements—Research support from the National Science
Foundation (CHE-0131203) is gratefully acknowledged. We thank
Professor Arnold Falick and Dr. David King of the Mass Spectrom-
etry Laboratory, Howard Hughes Medical Institute, University of
California, Berkeley, CA and Michael Cohen of the Kip Guy
laboratory at the University of California, San Francisco, for running
mass spectra of the various adducts and Janet Chung of the Tom
James laboratory at the University of California, San Francisco for
running several proton NMR spectra. We also thank the two
anonymous reviewers for their careful critiques and useful suggestions
concerning various aspects of the original manuscript.
13. Shetlar, M. D. (1980) Cross-linking of proteins to nucleic acids by
ultraviolet light. Photochem. Photobiol. Rev. 5, 105–197.
14. Saito, I. and H. Sugiyama (1990) Photoreactions of nucleic acids
and their constituents with amino acids and related compounds. In
Bioorganic Photochemistry, Vol. 1 (Edited by H. Morrison), pp.
317–340. John Wiley and Sons, New York.
15. Hom, K., G. Strahan and M. D. Shetlar (2000) Ring opening
photoreactions of cytosine and uracil with ethylamine. Photochem.
Photobiol. 71, 243–253.
SUPPORTING INFORMATION
Additional Supporting Information may be found in the online
version of this article:
16. Varghese, A. J. (1974) Photoaddition products of uracil and cys-
teine. Biochim. Biophys. Acta 374, 109–114.
Appendix S1. Preparation of DMU and DMT cyclobutane
dimers and the (6-4) adduct of Thy
17. Smith, K. C. (1969) Photochemical addition of amino acids to 14C-
uracil. Biochem. Biophys. Res. Commun. 34, 354–357.
18. Shetlar, M. D., J. Carbone, E. Steady and K. Hom (1984) Pho-
tochemical addition of amino acids and peptides to polyuridylic
acid. Photochem. Photobiol. 39, 141–144.
Figure S1. Ball and stick (top) and space-filling (bottom)
depictions of IIIa and V in an anti-periplanar conformation.
Figure S2. Equivalent perspectives of the 4¢R,5S,6S enanti-
omers of the putative oxetane precursors of the (6-4) adduct of
uracil and the corresponding adduct of thymine, shown as
both ball and stick (top) and space-filling models (bottom).
Figure S3. Another pair of equivalent perspectives of the
4¢R,5S,6S enantiomers of the putative oxetane precursors of
the (6-4) adduct of uracil and the corresponding adduct of
thymine, shown as both ball and stick (top) and space-filling
models (bottom).
Table S1. 13C chemical shifts for the major (6-4) adduct of
uracil (IIIa) and the (6-4) adduct of thymine (V).
Please note: Wiley-Blackwell are not responsible for the
content or functionality of any supporting information sup-
plied by the authors. Any queries (other than missing material)
should be directed to the corresponding author for the article.
19. Wang, S. Y. (1976) Pyrimidine bimolecular photoproducts. In
Photochemistry and Photobiology of Nucleic Acids, Vol. 1 (Edited
by S. Y. Wang), pp. 295–356. Academic Press, New York.
20. Khattack, M. C. and S. Y. Wang (1969) Uracil photoproducts
from uracil irradiated in ice. Science 163, 1341–1342.
21. Varghese, A. J. (1971) Photochemistry of uracil and uridine.
Biochemistry 10, 4282–4290.
22. Taguchi, H. and S. Y. Wang (1976) Preparation and stereocon-
figurations of heterodimers of pyrimidines. Biochem. Biophys. Res.
Commun. 73, 356–363.
23. Taguchi, H. and S. Y. Wang (1977) C-Alkylation and deterium
exchange of cyclobutane-dipyrimidines. J. Org. Chem. 42, 3321–
3323.
24. Elad, D., I. Rosenthal and S. Sasson (1971) Solution photodimer-
ization of 1,3-dimethyluracil. J. Chem. Soc. (C) 2054–2057.
25. Blackburn, G. M. and R. J. H. Davies (1966) The photochemistry
of 1-methylthymine and 3-methylthymine. J. Chem. Soc. (C)
1342–1345.
26. Weinblum, D., F. P. Ottensmeyer and G. F. Wright (1968) The
structures of the isomeric thymine dimers as deduced from their
dipole moments. Biochim. Biophys. Acta 115, 24–31.
27. Jennings, B. H., S. Pastra-Landis and J. W. Lerman (1972)
Photosensitized dimerization of uracil. Photochem. Photobiol. 15,
479–491.
28. Varghese, A. J. (1975) Photocycloaddition of acetone to uracil and
cytosine. Photochem. Photobiol. 21, 147–151.
29. Varghese, A. J. and S. Y. Wang (1968) Thymine-thymine adduct
as a photoproduct of thymine. Science 160, 186–187.
30. Wang, S. Y. (1971) Thymine phototrimer. J. Amer. Chem. Soc. 93,
2768–2771.
REFERENCES
1. Cadet, J. and P. Vigny (1990) The photochemistry of nucleic acids.
In Bioorganic Photochemistry, Vol. 1 (Edited by H. Morrison), pp.
1–272. John Wiley and Sons, New York.
2. Ruzsicska, B. P. and D. G. E. Lemaire (1995) DNA photochem-
istry. In CRC Handbook of Organic Photochemistry and Photobi-
ology (Edited by W. M. Horspool and P.-S. Song), pp. 1289–1315.
CRC Press, Boca Raton, FL.
3. Cadet, J., S. Courdavault, J.-L. Ravanat and T. Douki (2005)
UVB and UVA radiation-mediated damage to isolated and cel-
lular DNA. Pure Appl. Chem. 77, 947–961.

102 Martin D. Shetlar and Vladimir J. Basus
31. Becker, E. D. (1980) High Resolution NMR: Theory and Chemical
Applications, 2nd edn. Academic Press, Orlando, FL.
45. Adman, E., M. P. Gordon and L. H. Jensen (1968) Structure of
the cis-syn-photodimer of uracil: A puckered cyclobutane. Chem.
Commun. 1019–1020.
32. Bergstrom, D. E. and N. J. Leonard (1972) Photoreaction of
4-thiouracil with cytosine. Relation to photoreactions in Escheri-
cia coli transfer ribonucleic acids. Biochemistry 11, 1–9.
33. Karle, I. L. (1969) Crystal structure of a thymine-thymine adduct
from irradiated thymine. Acta Crystallogr. B 25, 2119–2126.
34. Smith, M. B. and J. March (2007) March’s Advanced Organic
Chemistry: Reactions, Mechanisms and Structure, 6th edn. John
Wiley and Sons, Inc., New York.
46. Donges, K. H. and E. Fahr (1966) The action of irradiation
¨
on nucleic acid components. IV. Structure of dimeric uracil
formed by ultraviolet radiation of uracil. Z. Naturforsch. B 21,
87.
47. Konnert, J., J. W. Gibson, I. L. Karle, M. N. Khattack and S. Y.
Wang (1970) Crystal structure of dimers of uracil and 6-methyl-
uracil. Nature 227, 953–954.
35. Perrin, D. D. and B. Dempsy (1974) Buffers for pH and Metal Ion
Control. Chapman and Hall, London, UK.
36. Hehre, W. and S. Ohlinger (2008) Spartan ‘08 for Windows,
Macintosh and Unix. Wavefunction, Inc., Irvine, CA.
´
37. Montenay-Garestier, T., M. Charlier and C. Helene (1976)
Aggregate formation, excited-state interactions and photochemi-
cal reactions in frozen aqueous solutions of nucleic acid constit-
uents. In Photochemistry and Photobiology of Nucleic Acids, Vol. 1
(Edited by S. Y. Wang), pp. 381–417. Academic Press, New York.
38. Rahn, R. O. and L. Hosszu (1969) Photochemical studies of
thymine in ice. Photochem. Photobiol. 10, 131–137.
39. Rhoades, D. F. and S. Y. Wang (1970) Uracil-thymine adduct
from a mixture of uracil and thymine irradiated with ultraviolet
light. Biochemistry 9, 4416–4420.
40. Wang, S. Y. (1961) Photochemical reactions in frozen solution.
Nature 190, 690–694.
41. Smietanowska, A. and D. Shugar (1961) Photochemistry of model
oligo- and polynucleotides. V. Properties of photodimers of uracil
and thymine. Bull. Acad. Polon. Sci. Cl. II 9, 375–380.
42. Wacker, A., D. Weinblum, L. Trager and Z. H. Moustafa (1961)
¨
Photochemical reactions with uracil and uridine. J. Mol. Biol. 3,
790–793.
43. Smith, K. C. (1963) Photochemical reactions of thymine, uracil,
uridine, cytosine and bromouracil in frozen solution and in dried
films. Photochem. Photobiol. 2, 503–517.
48. Krauch, C. H., D. M. Kramer, P. Chandra, P. Mildner, H. Feller
and A. Wacker (1967) Photodimerization of uracil sensitized by
acetone. Angew. Chem. Int. Ed. Engl. 6, 956.
49. Greenstock, C. L. and H. E. Johns (1968) Photosensitized
dimerization of pyrimidines. Biochem. Biophys. Res. Commun. 30,
21–27.
50. Richter, P. and E. Fahr (1969) Synthesis of trans ⁄ syn- and
trans ⁄ anti-dimeric uracil. Angew. Chem. Internat. Edit. 8, 208–
209.
51. Richter, P. and E. Fahr (1970) Die synthese des cis ⁄ syn- und
cis ⁄ anti-dimeren uracils. Tetrahedron Lett. 11, 1921–1923.
52. Pasto, D. J. and C. R. Johnson (1969) Organic Structure Deter-
mination. Prentice-Hall, Englewood Cliffs, NJ.
53. Greenstock, C. L., I. H. Brown, J. W. Hunt and H. E. Johns
(1967) Photodimerization of pyrimidine nucleic acid derivatives in
aqueous solution and the effect of oxygen. Biochem. Biophys. Res.
Commun. 27, 431–436.
54. Brown, I. H. and H. E. Johns (1968) Photochemstry of uracil.
Intersystem crossing and dimerization in aqueous solution.
Photochem. Photobiol. 8, 273–286.
55. Fendler, J. H. and G. Bogan (1974) Micellar effects on uracil
photodimerization. Photochem. Photobiol. 20, 323–328.
56. Lamola, A. A. and J. P. Mittal (1966) Solution photochemistry of
thymine and uracil. Science 154, 1560–1561.
57. Wagner, P. J. and D. J. Bucheck (1970) Photodimerization of
thymine and uracil in acetonitrile. J. Amer. Chem. Soc. 92, 181–
185.
44. Blackburn, G. M. and R. J. H. Davies (1966) The structure of
uracil photo-dimer. Tetrahedron Lett. 7, 4471–4475.