Photohydrate-mediated reactions of uridine, 2′-deoxyuridine and 2′-deoxycytidine with amines at near neutral pH

Article abstract of DOI:10.1111/php.12069

Photohydrates are formed in high yield when uridine (Urd), 2′-deoxyuridine (dUrd), cytidine (Cyd) and 2′-deoxycytidine (dCyd) are irradiated with UVC in aqueous solution. The thermal reactions of the photohydrates of Urd with amines at pH values near pH 7.5 have been studied using UV spectroscopy, HPLC, mass spectrometry and, in some cases, NMR. It has been found that a number of amines (i.e. ethylenediamine, N,N′- dimethylethylenediamine, glycine, glycinamide, glycylglycine, glycylgylcylglycine, putrescine, spermidine and spermine) react thermally with such hydrates to form products with UV spectra characteristic of opened ring uridine-amine adducts. In general, these products display a strong absorption peak with λ_max in the range between 288 and 310 nm. Mass spectral studies of a number of the products indicate that they contain one molecule of parent nucleoside and one molecule of reactant amine. Upon standing in water these products revert to parent hydrate, while heating produces parent nucleoside. Less comprehensive studies indicate that photohydrates of dUrd and dCyd undergo analogous thermal reactions. Preliminary results suggest that UV-irradiated polyuridylic acid and polycytidylic acid undergo similar reactions. These results may have relevance for obtaining a complete understanding of the biological effects of producing Urd and dCyd photohydrates in a cellular environment. 2013 Wiley Periodicals, Inc. Photochemistry and Photobiology

Full text of DOI:10.1111/php.12069

Photochemistry and Photobiology, 2013, 89: 869–877
Photohydrate-Mediated Reactions of Uridine, 2′-Deoxyuridine and 2′-
Deoxycytidine with Amines at Near Neutral pH
Martin D. Shetlar*1, Kellie Hom1,2 and Vincent J. Venditto3
1Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, CA
2Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD
3Department of Bioengineering and Therapeutic Sciences, School of Pharmacy, University of California, San Francisco, CA
Received 24 October 2012, accepted 27 February 2013, DOI: 10.1111/php.12069
ABSTRACT
between uridine (Urd) and water is shown in Scheme 1, in which
Ia is Urd. Because of the asymmetric sugar attached at N1, the
photohydrate exists in the two epimeric forms shown as Ib and
Ib′. Presumably these two forms can interconvert via an aldehy-
dic isomer labeled Ib″ in Scheme 1 (5). While thymidine (Thd)
hydrates are formed with low quantum yields upon UV irradia-
tion of Thd solutions (Cadet and Wang, unpublished work cited
in [1], p. 46), these compounds have been synthesized by an
alternative method and their structures determined (7). The for-
mation of two diastereomeric cytidine (Cyd) photohydrates has
been demonstrated via NMR studies, although these compounds
have not been characterized individually (8). Knowledge of the
photohydrates of 2′-deoxycytidine (dCyd) is also not very
advanced; detailed structural studies of the two diastereomers
remain to be accomplished. These hydrates readily undergo
deamination processes to form dUrd hydrates (for a detailed dis-
cussion, see [9]). The photohydrates of the various pyrimidine
nucleobases, as well as those of the nucleosides and other related
compounds, are unstable to treatment with heat, acid and base,
usually reverting predominately to the parent compound (1,2).
In addition to water, other nucleophiles can photoreact with
pyrimidine nucleobases, nucleosides and related compounds. For
example the photochemistry of the pyrimidine nucleobases and
nucleosides in the presence of amines has received considerable
attention in the literature (10,11). It has been found that the
photoproducts formed in such reactions differ significantly in
structure from the photohydrates produced in reactions with
water. The photoreactions of thymine (Thy) and thymidine (Thd)
with a variety of amines (including lysine) were among the first
to be studied (12). The predominant reaction undergone by these
two compounds at basic pH is a ring-opening reaction in which
the reactant amine, say RNH2, becomes attached to C2 in the
parent pyrimidine ring while the N1 nitrogen, along with any
substituent (e.g. alkyl group or sugar), is found attached to the
C6 of the parent compound in the product. This reaction is
shown for Thd in Scheme 2 below.
Photohydrates are formed in high yield when uridine (Urd),
2′-deoxyuridine (dUrd), cytidine (Cyd) and 2′-deoxycytidine
(dCyd) are irradiated with UVC in aqueous solution. The
thermal reactions of the photohydrates of Urd with amines at
pH values near pH 7.5 have been studied using UV spectros-
copy, HPLC, mass spectrometry and, in some cases, NMR. It
has been found that a number of amines (i.e. ethylenedia-
mine, N,N′-dimethylethylenediamine, glycine, glycinamide,
glycylglycine, glycylgylcylglycine, putrescine, spermidine and
spermine) react thermally with such hydrates to form prod-
ucts with UV spectra characteristic of opened ring uridine-
amine adducts. In general, these products display a strong
absorption peak with kmax in the range between 288 and
310 nm. Mass spectral studies of a number of the products
indicate that they contain one molecule of parent nucleoside
and one molecule of reactant amine. Upon standing in water
these products revert to parent hydrate, while heating pro-
duces parent nucleoside. Less comprehensive studies indicate
that photohydrates of dUrd and dCyd undergo analogous
thermal reactions. Preliminary results suggest that UV-irradi-
ated polyuridylic acid and polycytidylic acid undergo similar
reactions. These results may have relevance for obtaining a
complete understanding of the biological effects of producing
Urd and dCyd photohydrates in a cellular environment.
INTRODUCTION
Additions of water to uracil (Ura) and related compounds were
among the earliest photoreactions of nucleic acid components to
be studied. These reactions involve addition of H2O across the
5,6-double bond of the parent compound. For example the prod-
uct formed by irradiation of uracil in water is 6-hydroxy-5,
6-dihydrouracil (1,2). Photohydration reactions of the pyrimidine
nucleobases cytosine (Cyt) and thymine (Thy) also received early
attention (1,2), while the hydrates of 5-methylcytosine (m5Cyt)
have been studied more recently (3). The pyrimidine nucleosides
of uracil and cytosine are particularly reactive towards photohy-
drate formation (for general reviews, see [1,2]).
Interestingly, upon treatment of the opened ring conjugate,
shown in the E configuration in Scheme 2, with base or heat
causes it to undergo ring closure to form a photoexchange prod-
uct (PEX) in which the reactant amine is incorporated into the
final product and the group originally present at N1 (e.g. N1dRib)
is expelled. The overall reaction can thus be viewed as a photo-
exchange reaction. The nature of these reactions was first eluci-
dated by Saito and co-workers; summaries of this early work are
available (10,11). Later work showed that Ura, Cyt, m5Cyt, and
Irradiation of uridine (Urd) (4,5) and 2′-deoxyuridine (6) in
aqueous solution induces rapid addition of water. The reaction
*Corresponding author email: shetlar@cgl.ucsf.edu (Martin D. Shetlar)
© 2013 Wiley Periodicals, Inc.
Photochemistry and Photobiology © 2013 The American Society of Photobiology 0031-8655/13
869

870 Martin D. Shetlar et al.
O
incubation of Urd hydrates with glycinamide. In less extensive
proton NMR studies, we have also characterized the ethylamine
and ethylenediamine adducts of dUrd and the ethylamine adduct
of dCyd. Finally, returning to the seminal observations of Fikus
and Shugar, we present evidence that, at least in part, the absor-
bance changes observed when uridine hydrates are incubated
with ammonia can be accounted for by production of an opened
ring Urd-ammonia adduct.
H
H
N
H
H
OH
O
N
hν
H2O O
O
H
N
Rib
H
4
Ib
N
H
N3
5
6
H
2
H
1
O
O
O
O
H
N
Rib
H
Ia
H2O
hν
Ib"
Rib
H
N
H
OH
H
O
MATERIALS AND METHODS
N
Rib
Ib'
General aspects. Urd, dUrd, Ura, 1-methyluracil (1-MeU) and dCyd were
purchased from Sigma (St. Louis, MO), as were ethylenediamine.2HCl, gly-
cine, glycinamide.HCl, glycylglycylglycine, putrescine.2HCl, cadaverine.2HCl,
spermidine.3HCl and spermine.4HCl. Methylamine, N,N′-dimethylethy-
lenediamine, 1,3-diaminopropane.2HCl and glycylglycine were obtained
from Aldrich (Milwaukee, WI). Ethylamine and ethylenediamine (EDA)
were from Fluka (Buchs, Switzerland). Polycytidylic acid (K+ salt) was
from P-L Biochemicals (Milwaukee, WI), while polyuridylic acid (K+ salt)
was obtained from Boehringer Mannheim. Ammonium hydroxide and
HPLC solvents were from Fisher (Fair Lawn, NJ); NMR solvents were
from Aldrich. Preparative separations were accomplished using a Shisei-
do Capcell UG120 10 9 250 mm reverse phase column (5 lm particle
size [Yokohama, Japan]); hereafter, this column is termed Column A.
Analytical HPLC was done on a Capcell UG120 4.6 9 150 mm reverse
phase column (5 lm particle size); in the following we term this Column
B. The HPLC system employed was a Rainin binary gradient pumping
system (Emeryville, CA) hooked up to a Hewlett-Packard 1040A diode
array HPLC detector (Palo Alto, CA). HPLC samples were filtered, using
Costar Spin-X micro-centrifuge filter tubes containing a 0.2 lm nylon fil-
ter (Corning Incorporated, Corning, NY). UV spectra were measured on
a Hewlett-Packard 8452A diode array spectrometer or by using the “on
the fly” spectral capture capability of the Hewlett-Packard diode array
HPLC detector. Rotatory evaporations were carried out using a B€uchi
R-200 Rotovapor (New Castle, DE) connected to dry ice traps and a
mechanical pump.
Electrospray ionization (ESI) mass spectra were run on either a
Waters Micromass ZQ4000 instrument (Beverly, MA) or a Sciex API300
triple quadrupole electrospray instrument (Toronto, Canada). Liquid sec-
ondary ionization mass spectra (LSIMS) were run on a Kratos MS-50
(Ramsey, NJ). NMR spectra were run at 600 MHz on a Varian INOVA
NMR spectrometer (Palo Alto, CA) or at 300 MHz on either a GE QE-
300 NMR spectrometer (Fremont, CA) or a Bruker Advance NMR spec-
trometer with Topspin software (Billerica, MA).
Irradiation methods. Production of the photohydrates of Urd, dUrd,
dCyd, Ura and 1-methyluracil (1MeU) were carried out in the cold with
254 nm radiation. This light was provided by unfiltered Spectronics
BLE-1T155 15 watt lamps (Spectronics, Westbury, NY) housed in
Spectroline XX-15A lamp holders. The parent solutions were placed in
187 mL cylindrical quartz vessels (47.6 cm length 9 2.6 cm OD) from
Southern New England Ultraviolet Company (Branford, CT). These ves-
sels fit snugly between the two lamps in a Spectroline XX-15A lamp
holder placed in a “lamps up” orientation and photoreactions were carried
out with 165 mL portions of solution with the irradiation vessels placed
in this position; one end of the lamp housing was elevated at about a 10°
angle to the horizontal to avoid leakage of the solution from the tops of
the stoppered vessels. During irradiation, the reaction vessels were stop-
pered with glass taper seal stoppers. Irradiations were done under air in a
cold room at about 5°C.
Scheme 1.
5-bromouracil (5BrU) (13–16) undergo similar photoinduced
ring-opening and thermal ring closure reactions, as do 5-bromo-
2′-deoxyuridine and 2′-deoxycytidine (dCyd) (15,17).
Early work by Fikus and Shugar (18) showed that solutions
of photohydrates of Urd made basic with NH3 (pH 11.8–12.0)
displayed transient appearance of a UV absorption spectrum with
kmax at 290 nm. This spectrum disappeared over a period of an
hour; during the course of the loss of absorbance at 290 nm, Urd
was regenerated in almost quantitative yield. It was suggested
that the reversion reaction of hydrate to uridine proceeds by two
pathways, one being a direct base catalyzed reaction and the sec-
ond a ring opening reaction, followed by ring closure and dehy-
dration. Similar results were obtained in solutions containing
added NaOH or KOH, except that reversion to Urd was signifi-
cantly less than quantitative, likely due to a third competing irre-
versible reaction in which ring opening at the 3,4 bond occurred.
It was proposed (18) that the structure of the product displaying
kmax = 290 nm was Ic (see Scheme 3), in which a hydroxyl
group is attached to C3. It can be noted that Ic is an isomeric
form of Ib″ in Scheme 1.
Can Urd (or dUrd) photohydrates be induced to undergo ring-
opening reactions with additives other than hydroxide or ammo-
nia? Do dCyd hydrates react in the same manner? We describe
here results of experiments with Urd (dUrd) hydrates and dCyd
hydrates that indicate ring-opening reactions do indeed occur
with a variety of amines (e.g. ethylamine, ethylenediamine, glyci-
namide, glycylglycine, spermine) and that a number of these
reactions occur readily at pH 7.5. The reaction products are
nucleoside-amine adducts; a number of these adducts are stable
enough to isolate and submit for mass spectrometric study. In
1
addition, utilizing a variety of H and 13C NMR techniques, we
have carried out an extensive study of adducts formed upon
O
O
Me
Me
H
H
N
N
RNH2
Irradiations in spectrophotometer cuvettes were done in self-masking
0.6 mL volume quartz cells (Precision Cells, Hicksville, NY); these were
Basic pH
hν
NHdRib
OR-E
NHR
O
O
(a)
N
dRib
Thd
Heat or Acid
(b)
O
O
1
H
Me
N
H
2
N
3
OH
O
O
NHRib
Ic
N
R
PEX
Scheme 2.
Scheme 3.

Photochemistry and Photobiology, 2013, 89 871
closed within Teflon stoppers and placed directly against one of the
lamps contained in the lamp holder described above. Thermal reactions
of the various photohydrates with amines were followed by UV spectros-
copy in the same cells.
Preparation of photohydrates. The protocols for photochemical syn-
thesis and isolation and purification of the various photohydrates are
described in Appendix A1 in the Supporting Materials. Using these pro-
cedures, we isolated pure samples of the photohydrates of Urd, dUrd,
dCyd, Ura and 1-MeU. In those experiments, described below, that used
purified nucleoside photohydrates as reactants, we usually used mixtures
of the two diastereomers. (Upon standing in the cold, gradual isomerization
of each purified diastereomer yields a mixture of two isomers; thus, after
purification of the individual diasteromers, we combined them to give the
solutions used for thermal incubations.)
mixtures of Urd hydrates (Ib and Ib′) (or dUrd hydrates) and
EDA. The pKa values for EDA are 6.85 and 9.93 (19). (Photohy-
drates were prepared as described in Materials and Methods and
in Appendix A1). Buffers containing a weak acid (e.g. a diproto-
nated amine) and its conjugate base (a monoprotonated amine)
display reasonably high buffer capacities over a pH range
between pKa Æ 1 ([19], Table 2.4). Thus, over the pH range
5.85 and 7.85, the equilibrium between diprotonated and mono-
protonated EDA provides good buffering action; as a result, the
pH in incubation mixtures containing Urd or dUrd hydrates, at
the concentrations used in this work (2 mM and smaller), and
EDA, present in significant excess at pH values within this
range, should be well protected against significant pH changes
during reaction. The progress of the ring-opening reaction of
mixtures of Ib and Ib′ in solutions containing EDA at pH 7.5
after short reaction times is shown in Fig. S2. As can be seen,
absorbance with a kmax at 294 steadily increases over the time
interval used. A pH profile for the rate of formation of putative
opened ring compounds was constructed by measuring the absor-
bance at 294 nm produced by incubation for set time intervals at
a number of pH values. This profile is shown in Fig. S3. As can
be seen, reaction can be detected at a pH value as low as 5.7; at
this pH, the percentage of EDA in the form of EDA.2HCl is
93%, while the percentage of total EDA in the form EDA.HCl is
7%. (The percentage of total EDA present as completely unprot-
onated amine is very small at pH 5.7). The profile displayed in
Fig. S3 confirms that the reaction involves a free amine moiety,
as the larger the concentration of unprotonated amine, the greater
the amount of hydrate converted to opened ring form in a given
reaction time.
The reaction of dUrd hydrate with EDA was also examined
to confirm that it behaves similarly to the Urd hydrate. Indeed,
spectrometric monitoring of the absorption of a mixture of epi-
meric dUrd hydrates and EDA (90 mM, pH 7.3) over a 16 min
period produced a set of curves very similar to that displayed in
Fig. S2. We carried out a considerably more extensive study of
purified reaction product in this system, using UV spectroscopy,
mass spectrometry and proton NMR as tools. The detailed results
of this study are described in Appendix A2a. In short, the prod-
uct isolated displayed spectroscopic properties consistent with it
being a mixture of the E and Z isomers of either IIc or IId (see
Scheme 4); The UV spectra of the two adducts, as obtained via
use of the spectral capture technique of the diode array detector,
are shown in Fig. S4. These two products, separable by HPLC,
were termed PEDA1 and PEDA2. (short for ProductEthyleneDi-
Amine with subscripts 1 and 2 denoting the two isomeric
forms).
RESULTS
Introduction: A puzzling observation
An impetus for the studies reported here was the observation of
baffling behavior when dUrd was irradiated at 254 nm in aqueous
solution containing EDA at near neutral pH. In addition to the
expected disappearance of the parent dUrd, due to formation of
photohydrates Ib and Ib′, there appeared an unanticipated red-
shifted band with a kmax at about 296 nm. Indeed, this absorption
band steadily increased in intensity, even after irradiation had
been terminated. (This behavior is displayed in Fig. S1 in the
Supporting Information). Further investigation showed that other
amines induced the same type of behavior. These observations,
coupled with those of Fikus and Shugar (18) on the appearance
of red-shifted absorption spectra when Urd hydrate is incubated
in basic ammoniacal solution (see above), suggested that thermal
reactions of photohydrates of Urd (and other pyrimidine nucleo-
side photohydrates) with amines at near neutral pH values merited
further investigation. In particular, these results suggested that
Urd and dUrd hydrates reacted thermally with appropriate amines
(e.g. EDA) to form either compounds with structures of the type
given by IIa for Urd adducts (or IIc for dUrd adducts) (see
Scheme 4) or, alternatively those of the type shown by IIb and
IId in Scheme 4. Below, we present evidence indicating that the
latter type of adduct, in which the reactive amine becomes
attached to C3, is the more likely product in the observed reac-
tions of hydrate with amine.
Thermal reaction of Urd and dUrd photohydrates with
ethylenediamine
Some of our most extensive studies of the thermal reactions of
pyrimidine nucleoside photohydrates with amines were done with
We were able to obtain a 1D proton NMR spectrum for this
mixture of adducts; however, because of the thermal instability
of these adducts, we were unable to carry out 13C and 2D NMR
studies to distinguish between structures of type IIc and IId. It
can be noted that chemical evidence, discussed in Appendix
A2a, favors assignment of the adducts as being E and Z isomers
of structure type IId. However, more definitive assignments
could be made if various 2D NMR techniques (e.g. heteronuclear
multiple bond correlation [HMBC] or correlation spectroscopy
[COSY]) were to be applied to adducts less labile than those iso-
lated after incubation of the dUrd hydrate-EDA system. Indeed,
we found that adducts arising from the incubation of Urd
hydrates with glycinamide (GlyAm) and the dUrd hydrates with
ethylamine satisfied this need for increased stability.
O
1
H
N
2
3
NHR'
NHR
O
IIa: R = NH(CH 2)2NH2, R' = Rib
IIb: R = Rib, R' = NH(CH 2)2NH2
IIc: R = NH(CH 2)2NH2, R' = dRib
IId: R = dRib, R' = NH(CH 2)2NH2
IIe: R = NHCH 2CONH2, R' = Rib
IIf: R = Rib, R' = NHCH 2CONH2
IIg: R = R' = Ethyl
IIh: R = dRib, R' = Ethyl
Scheme 4.

872 Martin D. Shetlar et al.
It should be noted that our work has not ruled out the possi-
bility that photo-induced reaction can also produce products with
structures similar to, say, IIb or IId when solutions of dUrd or
Urd are irradiated in the presence of EDA near neutral pH. Proof
that such adducts are formed only via thermal pathways will
require further research.
the configuration with J = 13 Hz can be assigned to be the E
configuration. The C2H proton resonances are present at
4.81 ppm (J = 13 Hz, 0.85 H, E configuration) and 4.63 ppm
(J = 8.1 Hz, 0.15 H, Z configuration). The observed integrated
areas for the protons corresponding to the E and Z isomers imply
that the ratio of E to Z in the NMR sample is 85/15. The glycina-
mide CH2 protons integrate to only 1.67 protons (84%) at
3.86 ppm with the remaining 16% being found under one of the
ribose protons peaks at 3.95 ppm. In the case of the dominant E
configuration, the width of the C2H and C3H peaks suggests that
interconvertible conformations of this isomer exist, each with
somewhat different environments for protons corresponding to
these peaks. The sharper corresponding signals for the Z isomer
suggest that only one major conformational form is accessible for
the C2H and C3H protons in this species. This explanation of the
broadness of the C3H and C2H NMR peaks for molecules in the
E configuration and the sharpness of the corresponding vinyl pro-
tons in the Z configuration draws support from the results of cal-
culations carried out with Spartan 10 for the Macintosh; Fig. S8
displays conformational snapshots of the three lowest energy con-
formations of the E configuration of the Urd-glycinamide adduct
and the single low energy conformation of the Z configuration, as
obtained via use of a Hartree-Fock 3-21G basis set for quantum
chemical calculations (for further details, see the caption for Fig.
S8). While these calculations of relatively low sophistication must
be regarded as giving only approximate results, they do indicate
that one of low energy conformations (Fig. S8-C), accounting for
an estimated 9% of the total population of E isomer, has a differ-
ent environment for C2H and C3H than that found in the other
two low energy conformations (Fig. S8-A and B) for the same
two protons; in particular the adducted glycinamide moiety dis-
plays a different conformation, relative to the vinyl group, than
the other two conformations. (The main difference in the other
two low energy conformations for the E isomer lies primarily in
the very different relationship of the ribose ring to the remainder
of the molecule.) The same set of calculations suggests that, due
to hydrogen bond stabilization of a six-membered ring containing
these moieties, the C3H and C2H in the Z isomer see essentially
one environment (see Fig. S8-D).
Thermal reactions of Urd hydrates with glycinamide
The reaction of Urd hydrates with glycinamide (GlyAm) pro-
duced adducts in good yields that are quite stable and that can
be readily purified via HPLC without using aqueous eluents con-
taining salt (thus eliminating potential product losses during a
desalting step). The protocol for preparation of sufficient
amounts of the desired GlyAm adducts for purposes of in-depth
NMR study is given in Appendix A2b, along with information
concerning their spectral properties. In short, incubation of Urd
hydrate with GlyAm produces both a major adduct (kmax
=
291 nm) and a minor adduct (kmax = 299 nm) that elute quite
close to one another; thus, semi-preparative HPLC on Column A
resulted in isolation of a mixture of two isomeric forms of the
Urd-GlyAm adduct (putatively E and Z in nature). ESI mass
spectrometry of the isolated mixture of adducts yielded a single
value of 319.1 for the molecular mass, consistent with a structure
containing one Urd and one GlyAm.
The characterization of the adduct mixture by NMR proved to
be quite informative. The results of these NMR studies show that
the adducts formed in the Urd hydrate-GlyAm system correspond
in structure to IIf in Scheme 4, rather than IIe. Furthermore, 2D
NMR studies show that both the E and Z isomers are present in
the NMR sample and suggest that the E isomer of the adduct
exists as interconvertible conformational forms. The various
NMR results, along with their implications, are discussed below;
the NMR spectra themselves are displayed in Figures S5–S7, S9
and S10, contained in the Supporting Information.
We first conducted NMR studies of the putative mixture of E
and Z adducts in DMSO-d6, so as to observe interactions
between the anomeric ribose C1′H (d ꢀ 5.32) and the NH pro-
ton generated by the ring opening reaction. Indeed, we observed
1
this interaction in the H NMR spectrum as a pair of doublets at
Additional information concerning the attachment site of the
ribose to the remainder of opened ring was obtained by study of
the 13C NMR and HMBC spectra in D2O. Weak carbon signals
are seen for the olefin and glycinamide moieties in the 13C spec-
trum of the Z isomer, while the corresponding signals are too
weak to be seen for the E isomer (Fig. S9-A). The DEPT 135
spectrum (Fig. S9-B) indicates that a weak signal at 50.5 ppm cor-
responds to the CH2 of the glycinamide moiety and that a strong
9.04 (Z configuration) and 9.25 ppm (E configuration)
(J = 9.4 Hz in both cases) integrating to 0.14 protons and 0.86
protons, respectively (Fig S5). The structure was further analyzed
by HMBC (see Fig. S6-A), so as to confirm that the NH proton
is adjacent to the ribose ring, as manifested by its interaction
with the C1′ and C2′ carbons (Fig S6-B). Little additional useful
structural information could be ascertained from the NMR spec-
tra in this solvent, due to spectral broadening and the inherent
complexity of the spectrum because of the presence of reso-
nances due to –OH and –NH sites.
To obtain more information, the adduct mixture was analyzed
in D2O, which not only decreased the number of observed pro-
tons, but also sharpened the signals for most of the other protons.
As expected, the resonances for the NHRib proton (see above)
are no longer present. The vinyl protons for the various adduct
forms could be readily observed. The C3H protons, next to the
attached glycinamide group (Scheme 4), could be observed as a
broad doublet at 7.64 ppm (J = 13 Hz, 0.85 H) and a sharp
doublet at 6.79 ppm (J = 8.1 Hz, 0.15 H) (see Fig. S7). As the
coupling constants for hydrogens that are trans to one another are
generally larger than those that are cis with respect to each other,
1
resonance at 61 ppm is that of C5′H2. (Summaries of the H and
13C NMR assignments for various resonances, seen in the spectra
discussed above, are given in Figs. S7 and S9 respectively).
Study of the same sample by HMBC (Fig. S10-A) confirms
correlation between the glycinamide carbon at 50.5 ppm and the
Z isomer C3 proton at 6.79 ppm (Fig. S10-B). In addition, the
resonance of the glycinamide methylene protons of the Z config-
uration is correlated with carbon resonances at 155 (C3) and
176 ppm (CONH2) (see Fig. S10-C). The glycinamide protons
in the E isomer (3.86 ppm) correlate with carbons that should
display resonances at 152 and 174 ppm; the absence of these
carbon peaks in the 13C spectrum suggests that their signals may
be broadened by the existence of readily interconvertible confor-
mations in the E isomer.

Photochemistry and Photobiology, 2013, 89 873
52.5 mM, k = 0.033 minÀ1 [P] = 95 mM, k = 0.039 minÀ1
Summing up, the data from detailed NMR study of the Urd-
GlyAm adducts in D2O and in DMSO-d6 shows that these com-
pounds exist as the general structural type shown as IIf, with both
the E isomer (major form) and Z configuration (minor form) of
this structure being present in the NMR sample; the alternative
general structure IIe can be ruled out. The NMR data is consistent
with the idea that interconvertible conformational states exist for
the E configuration of IIf in aqueous solution, but that a single
conformational state is dominant for the Z configuration.
;
.
When k is plotted versus [P], a slope of 0.117 L molÀ1 minÀ1 and
an intercept of 0.026 minÀ1 (r2 = 0.988) were evaluated. These
limited results suggest that phosphate species can participate in a
general acid or general base catalyzed reversal mechanism. These
results have similarities to those obtained for ring-closure of amine
adducts of Thy and Ura to form thymine and uracil hydrates (20).
When a similar incubation of the Urd-EDA adduct was car-
ried out in 0.1% TFA, reaction to form product(s) with an
absorption spectrum similar to that of Urd hydrates was complete
within 1 min.
Thermal reactions of Urd photohydrates with various amines
at near neutral pH
Scheme 5 displays a set of reactions that are consistent with
our results on the reactions of Urd photohydrates with amines at
near neutral pH to give Urd-amine adducts. This scheme assumes
that Ib”, the aldehydic form of photohydrate Ib, reacts reversibly
with base (in this case, RNH2) to form an opened ring compound
(Int). (As displayed in Scheme 1, Ib reversibly converts into its
epimer, namely Ib′ via Ib″; both forms would participate in the
reaction sequence displayed in Scheme 5, even though Ib′ is not
displayed.) Finally, Int undergoes reversible loss of water to
form the observed product (IIp). This scheme is consistent with
the observation that IIp reverts to photohydrates upon standing
in water in the absence of amine and is also consistent with the
data displayed in Fig. S3, in which the rate of reaction to form
IIp increases with pH (i.e. with the concentration of unprotonat-
ed amine groups present in solution).
Do other amines, besides EDA and GlyAm, also react with the
hydrates of Urd to yield adducts similar in nature to IIb and IIf?
To answer this question, we examined the reaction products
formed when a variety of amines were incubated at ambient tem-
perature with a mixture of the two diastereomeric uridine
hydrates at near neutral pH. In addition to EDA and GlyAm, we
examined the reactivity of glycine (Gly), glycylglycine (Gly)2,
glycylglycylglycine (Gly)3, N,N′-dimethylethylenediamine (DME-
DA), propane-1,3-diamine (PDA), putrescine (butane-1,4-dia-
mine) [Put], cadaverine (pentane-1,5-diamine) [Cad], spermidine
(N-(3-aminopropyl)butane-1,4-diamine) [Spd] and spermine (N,N
′-bis(3-aminopropyl)butane-1,4-diamine) [Sper]. (DMEDA can be
regarded as a model of the secondary amine moieties found in
Spd and Sper.) Most of these amines have a pKa (or set of pKa
values) that indicate a significant proportion of the total amine
concentration exists in a partially unprotonated state in aqueous
solution at pH 7.5 (see Table S1 in the Supporting Information).
Of the various compounds studied, only Cad failed to yield
appreciable amounts of product. The products from the various
reactions were isolated using HPLC and studied using UV spec-
trophotometry and ESI mass spectrometry. In all cases (except
for Gly), ESI mass spectrometry of the isolated adducts yielded
peaks with the predicted molecular mass values, while each of
the UV spectra closely resembled those observed for the Urd-
GlyAm and dUrd-EDA adducts. These results suggest that Urd
hydrates react with these other amines to form the same type of
adduct formed by GlyAm and EDA. The protocols used in the
preparation and isolation of these compounds, as well as details
concerning the UV spectral properties and molecular masses of
these compounds are given in Appendix A2c.
It can be noted that the results of preliminary studies,
described in Appendix A2c, suggest that both Ura photohydrate
and 1-MeU photohydrate react with EDA and GlyAm at near
neutral pH to form similar types of adducts (albeit in very small
amounts).
Exploratory studies of the thermal reactions of dCyd
photohydrates with various amines at near neutral pH
The observation of reactivity of Urd (and dUrd) photohydrates
with a variety of amines at near neutral pH suggested that dCyd
photohydrates might undergo similar reactions. Therefore we pre-
pared and isolated these hydrates (Appendix A1) and incubated
them with several of the same amines listed above (EDA, DME-
DA, GlyAm, (Gly)2, Put, Sper). Indeed, via monitoring of the
reaction by HPLC, we did observe that compounds with the
absorption spectra expected for dCyd adducts were formed in
these systems (see Fig. S11 for the spectra of two isomers of the
putative dCyd adduct with EDA). However, these products were
thermally unstable; the corresponding dUrd-amine adducts were
It is interesting to note that the purified adducts of the various
amines were quite stable in the aqueous frozen state (À20°C)
over a 3-month period. However, when incubated in distilled
water at room temperature, these compounds reverted to parent
hydrates of Urd or dUrd. To obtain some quantitative informa-
tion about the effect of phosphate buffer on the stability of ad-
ducts at physiological temperature and near physiological pH, we
studied the rate of loss of the Urd-EDA adduct at 37°C and
pH 7.6 at three different phosphate concentrations. For this
study, we used HPLC as a tool, examining the peak area corre-
sponding to the Urd-EDA adducts (measured at 291 nm) as a
function of incubation time in a temperature controlled water
bath. Column A was used for chromatography, using 92%
(100 mM NaCl)/8% MeOH flowing at 4 mL minÀ1 as eluent.
Under these conditions, both the E and Z forms of adduct eluted
at 3.8 min. The following pseudo-first order rate constants were
measured at various fixed values of [P], the phosphate concentra-
O
O
O
H
H
H
H
H
H
N
N
N
H
H
H
OH
H
H
NHR
RNH
+
2
HO
H
O
O
O
O
N
N
Rib
N
H
H
Rib
Rib
Ib"
Ib
+
H2O
Int
-H2O
O
H
H
N
H
N
H
NHR
O
Rib
IIp
tion in mmol LÀ1
:
[P] = 9.5 mM, k = 0.027 minÀ1
;
[P] =
Scheme 5.

874 Martin D. Shetlar et al.
among the compounds formed in their decomposition. In three of
the incubation mixtures, we found evidence for formation of trans-
amination products of the type produced when dCyd is incubated
with amines in the presence of bisulfite (21). Details concerning
these experiments and their results are given in Appendix A3.
ammoniacal solutions and likewise found that very similar UV
spectral changes occurred in both systems. Extending this work,
using HPLC analysis, we found that two products, both with UV
spectral characteristics expected of ring-opened adducts, were
formed in ammoniacal solutions of the hydrates of Urd and dUrd.
This work is described in Appendix A4. We did not, however,
answer the question as to whether these two compounds are
ammonia adducts, ring-opened forms of the hydrate or one of
each; In principle, questions concerning the nature of these two ad-
ducts can be answered via mass spectral and proton NMR studies
of pure samples of the two adducts; however, for practical reasons,
such studies could be quite difficult (see Appendix A4). We
instead focused on the question of whether ring-opened amine ad-
ducts are formed when photohydrates of dUrd or Urd are incu-
bated with a different amine at basic pH.
UV-C irradiated polyuridylic acid (poly rU) and polycytidylic
acid (poly rC) appear to form ring-opened adducts when
incubated at near neutral pH with DMEDA
It is known that the pyrimidine bases in both poly rU and poly
rC undergo photohydration when irradiated in aqueous solution
(22–24). We conducted a pair of exploratory studies to determine
if rU and rC hydrates, when incorporated into polynucleotides,
are also reactive towards adduct formation. The potassium salts
of poly rU (14 mg) and poly rC (12 mg) were dissolved with
stirring over a three day period in doubly distilled water to pro-
Thermal reaction of dUrd photohydrates with ethylamine at
basic pH
duce solutions at a polynucleotide concentration of 1 mg mLÀ1
;
UV spectroscopic measurements on diluted samples indicated
that the absorbances of the resulting solutions were 24.0 and
22.6 respectively. A solution made up of 0.5 mL of poly rU and
9.5 mL of distilled water was prepared in a stoppered quartz tube
and irradiated for 30 min as described in Material and Methods.
UV spectroscopy indicated that the absorbance at 260 nm had
gone from 1.30 to 0.08. Then 1.5 mL of this irradiated mixture
was placed in a 3 mL cuvette and 1.5 mL of 100 mM DMEDA
(pH = 7.5) was added. (DMEDA is one of the more reactive of
the amines utilized in the experiments described above.) Using
the H-P diode array spectrometer to monitor the progress of reac-
tion of poly rU at ambient temperature, we observed that the
absorbance at 301 nm increased from 0.045 to 0.23 over a
64 min period. After this solution had incubated for 8 days in a
refrigerator at 4°C, the absorbance at 301 nm had increased to
0.6. A similar study with poly rC indicated a lesser reactivity,
with the absorbance at 297 nm increasing from 0.068 to 0.09
over a 36′ incubation period at room temperature; after 8 days
incubation in the refrigerator, the absorbance at 297 nm had
increased to 0.144. These results suggest that incubation of both
irradiated poly rU and poly rC with DMEDA yields ring-opened
adducts. The reactivity of irradiated poly rC toward formation of
putative adducts is considerably smaller than that of irradiated
poly rU. This observation suggests, but does not prove, that uri-
dine hydrate-amine adducts, formed by incubation of RNA con-
taining photohydrates with appropriate amines, could be
quantitatively more important products than the corresponding
cytidine-amine adducts.
We next examined the HPLC of reaction mixtures in which dUrd
hydrate was incubated with ethylamine. A solution of dUrd
(167 mL, 1 mM) was irradiated in the cold at 254 nm for
60 min. UV spectroscopy of the irradiated solution that was
diluted 10-fold indicated that little dUrd remained. We then
added sufficient 1 M ethylamine to make the solution 100 mM in
this compound (pH = 10.7). We made 10-fold dilutions of
appropriate volumes of the samples taken after a number of incu-
bation times and measured the UV spectrum. The observed time
course of the overlaid spectral curves was very similar to the
time course displayed for the Urd hydrate-ammonia system (see
above). Portions of the samples were rotatory evaporated to dry-
ness, redissolved in distilled water and chromatographed on Col-
umn B using 80/20 H2O/MeOH flowing at 2 mL minÀ1 as
eluent. After 4 min incubation, two product peaks with UV spec-
tral properties expected of ring-opened adducts appeared with
elution times of 2.78 min (kmax = 297 nm) and 5.33 min
(kmax = 307 nm). The ratio of the peak areas, calculated using
values measured at kmax, was A2.78/A5.33 = 21. With continuing
time of incubation, the peak areas for the two putative adducts
decreased and the amount of dUrd in solution increased; this
behavior is similar to that seen in incubations of the photohy-
drates of Urd and dUrd with ammonia at basic pH.
An adduct formed after irradiation of dUrd in the presence
of ethylamine is identical to the thermal reaction product of
dUrd hydrate with ethylamine
In previously unpublished work, Hom (17) showed that after
dUrd (1 mM) was irradiated in 100 mM ethylamine solution at
pH 10.4, a mixture of ring-opened adducts were present as the
main products. The general structure of the photoadducts was
that displayed in Scheme 4 as IIh (the E-isomer of the adduct);
this structure was established by use of UV spectroscopy, mass
spectrometry and proton NMR spectroscopy (see details in
Appendix A5). We repeated this photochemical reaction, analyz-
ing the reaction mixture using the same HPLC conditions
described above; we observed two peaks corresponding to puta-
tive adducts. The retention times of the two adduct peaks seen
after incubation of dUrd hydrate with ethylamine and those of
the two adduct peaks produced when dUrd is irradiated in the
presence of ethylamine are almost identical. In addition, the UV
Thermal reaction of dUrd and Urd photohydrates with
ammonia at basic pH
As mentioned in the Introduction, Fikus and Shugar (18) found
that incubation of the Urd photohydrate in solution, adjusted to
pH = 12 with NaOH or KOH, induced formation of a ring-opened
product that subsequently reverted to Urd; a direct reaction of the
hydrate to form Urd and an irreversible reaction leading to another
product also occurred. They proposed that the reversible product
was Ic, an opened-ring form of the hydrate (Scheme 3). The same
reaction appeared to occur in 1 M ammoniacal solutions at
pH = 12. They also noted that the same type of reaction and
kinetic behavior occurred with dUrd hydrates. We repeated this
work for both Urd hydrates and dUrd hydrates in hydroxylic and

Photochemistry and Photobiology, 2013, 89 875
spectra of the material eluting in each of the corresponding
adduct peaks in the two experimental systems, were superimpos-
able, as shown by using the spectral capture feature of the diode
array detector. Thus, we can identify adducts formed by incuba-
tion of the dUrd hydrate as being of type IIh (in E and Z forms).
It appears likely that at least a portion (if not all) of the IIh
formed, upon irradiation of dUrd in the presence of ethylamine
at pH 10.4, is due to thermal reaction of the 2′-deoxyribose ana-
logs of Ib and Ib′ with ethylamine.
Analysis of the NMR spectrum of IIh in d6-DMSO (one-dimen-
sional and one-dimensional and correlated spectroscopy [COSY])
has been done and the results are presented in Appendix A5. It has
been found that multiple forms of IIh are present in the NMR
sample; indeed, the NMR data for the non-exchangeable protons
have similarities to the NMR data obtained for the EDA adduct
PEDA2 (see above and Appendix A2a).
mass spectrometric studies indicated that dCyd hydrates are, in
fact, formed upon irradiation of cellular DNA with UVC (9).
However, the yield was at least 100-fold less than the total
yield of dimeric type compounds (i.e. cyclobutane dimers and
(6-4) adducts). (It should be noted that the amounts of dCyd
hydrate formed were monitored by assessing the amounts of
dUrd hydrate present in digests of the irradiated DNA; thus, the
amount of dCyd hydrate that, instead, underwent reversion to
parent dCyd was not measured.) Gorelic (25) has shown that
Urd hydrates are formed when E. coli 30 S ribosomes are irra-
diated with UV light and that this reaction appears to be com-
petitive with UV light-induced RNA-protein cross-linking
reactions. Early studies, summarized in (26), suggest that Urd
photohydrates and, in some cases Cyd hydrates, are formed in
tRNA, mRNA and RNA viruses when irradiated with UVC
light. As one example (27), it was shown that irradiation of sin-
gle stranded genomic RNA, isolated from the R17 virus, with
monochromatic 280 nm light induced both Urd and Cyd
hydrate formation; it was determined that, under the irradiation
conditions used, the photoproducts consisted of about 57%
pyrimidine cyclobutane dimer, 38% Urd hydrate and 5% Cyd
hydrate. Based on the results referenced above, it is plausible
that UVC irradiation can produce photohydrates in single
stranded regions of irradiated cellular DNA, as well as in vari-
ous cellular RNA macromolecules and RNA macromolecular
complexes containing single stranded RNA, Such photohydrates
presumably would be available for reaction with polyamines to
form adducts. One important unanswered question for the pho-
tobiologist would be “Can treatment of cells with UVB light
induce such reactions (i.e. formation of Urd, rCyd and/or dCyd
hydrates, followed by reaction with polyamines)?”. Of relevance
to this question, dCyd displays a UV spectrum with significant
absorbance between 290 and 300 nm (see Fig. S12 in the Sup-
porting Information).
Budowsky and co-workers (28) have indicated that cytosine
hydrates, formed by UVC irradiation of the RNA bacteriophage
MS2, are responsible for the observed cross-linking of the coat
protein to the genomic RNA of this virus. These hydrates evi-
dently react thermally with lysines in the coat protein of this
phage, resulting in RNA-protein cross-linking. The proposed nat-
ure of this type of cross-linking is different than the general type
of cross-link that would be produced by the reactions described
in this paper. Acid digestion of the irradiated MS2 phage yields
a modified cytosine, namely e-N-(2-oxopyrimidyl-4)-lysine (29)
as one of the hydrolysis products. It was therefore concluded that
this conjugate (a cytosine transamination product) is responsible
for the observed UV-induced RNA-protein cross-linking in MS2
phage, as well as that observed when this same phage was trea-
ted with sodium bisulfite.
Thermal reaction of dCyd photohydrate with ethylamine at
basic pH leads to formation of an adduct analogous to IIh
After incubation of dCyd photohydrates with ethylamine at basic
pH (e.g. 10.5), compounds with HPLC characteristics and
absorption spectral profiles similar to those expected for opened
ring adducts appeared. As shown in Appendix A6, the predomi-
nant product formed in this incubation is (N-(N′-2′-deoxyribosy-
1′-yl)carbamoyl)-3-ethylaminoacrylamidine), which is analogous
in structure to IIh; this compound is also found as one of the
products when solutions containing dCyd and ethylamine are
irradiated at basic pH at 254 nm. More extensive information,
including UV spectra, the molecular mass and proton NMR
spectroscopic data, will be found in Appendix A6. The analo-
gous results for the corresponding dCyd-ethylamine adduct, found
after dCyd is irradiated in the presence of ethylamine at basic pH,
are given in Appendix A6. The adduct N-(N′-ethylcarbamoyl)-3-
(2′-deoxyribosy-1′-yl) aminoacrylamidine. which has a structure
analogous to IIc, is also produced in this irradiated system (17).
DISCUSSION
In the preceding Results section, we have described the produc-
tion of ring-opened amine adducts of Urd, dUrd and dCyd that
occurs when the photohydrates of these compounds are incu-
bated in the presence of the parent amine at near neutral pH. It
can be conjectured that similar reactions to form adducts occur
in UV-irradiated RNA or DNA in its cellular environment. In
such putative reactions, Spd, Sper and, possibly, Put would react
with RNA or DNA pyrimidine nucleosides that had been previ-
ously converted to hydrates by absorption of UV light. Proving
that such reactions actually occur at the cellular level could pres-
ent a worthy challenge for a resourceful photobiologist. One
obstacle to successful investigation could be the potential insta-
bility of adducts towards formation of parent hydrate (in the case
of Urd adducts) or deaminated adduct (in the case of dCyd or
Cyd adducts) after transferal from amine-containing medium (the
cellular milieu) to water, phosphate buffer or other media used
for in vitro investigations (see Scheme 5, Scheme A3-3 and the
accompanying discussions). If such reactions do occur in vivo,
delineation of the biological ramifications of such lesions could
provide interesting opportunities for investigation.
What kind of processes might be affected by formation of
adducts? Many in vitro studies have been made of the interactions
of polyamines with RNA of various types (e.g. ribosomal and
transfer RNAs) (30), as well as DNA (30,31). It is thought that
many of the same types of interactions occur in vivo as well and
are involved in the regulation of cellular processes, including, for
example chromatin remodeling and transcription (32). Formation
of dCyd adducts with Spd and Sper, followed by their conversion
to more stable dUrd adducts, could have effects on transactions
involving DNA, perhaps leading to undesirable effects on cellular
function. Similarly, formation of Urd-Spd (Cyd-Spd) or Urd-Sper
(Cyd-Sper) adducts in nuclear or cytoplasmic RNAs (e.g. transfer
Are photohydrates formed upon UV irradiation of cellular
structures containing DNA and RNA? In the case of DNA,

876 Martin D. Shetlar et al.
RNAs, ribosomal RNA, messenger RNA) could have adverse
consequence in processes involving these macromolecules. Pre-
sumably such biological effects would be mediated by alterations
in local structure and/or changes in accessibility of the modified
base toward proteins or other molecules involved in biochemical
processing activities. It should be reemphasized that a particular
adduct at a given site may have a transient existence. Adducts
appear to form in the presence of amine and revert to hydrate
when amine is removed (see above). This suggests that a dynamic
equilibrium exists between photohydrate and adduct (see
Scheme 5). Consider, for example a RNA population containing
several RNAs with hydrate lesions or a DNA containing several
hydrates. Unless equilibrium constant considerations strongly
favor adduct formation at the concentration of polyamine in the
RNA or DNA environment (i.e. nearly all hydrates are converted
to adducts), then an adduct decomposition-formation cycle would
be set up. As a result, it is possible that the existence of amine ad-
ducts might be transient at a particular site and that sites of adduct
formation could move around.
It is interesting to note that the level of two of the polyamines
studied here are present in the millimolar range in rat liver nuclei,
with measured values of 5.3 mmol kgÀ1 of tissue for Spd and
4.5 mmol kgÀ1 for Sper (33); the level for Put was determined to
be 58 lmol kgÀ1. (The concentration of Sper used in our incuba-
tions was about 22.5 mM, while that of Spd was 45 mM.) These
three polyamines are also present external to the nucleus, with
90% of total Spd and Sper being in extranuclear compartments
and 80% of total Put being there [33]). The effective concentra-
tions of the various polyamines in the neighborhood of DNA and
RNA molecules in the cell would be expected to be higher
because of attractive interactions between the negative charges on
the phosphate backbone of the nucleic acids and the multiple posi-
tive charges carried by polyamines at physiological pH.
Figure S1. The time course of the UV spectrum of a solution
(pH = 7.6) containing dUrd hydrate (0.2 mM) and ethylenediamine
(90 mM) upon irradiation at 254 nm and subsequent thermal reaction.
Figure S2. The initial part of the time course of the absorp-
tion spectrum of a mixture of Urd hydrates (0.8 mM) and EDA
(90 mM) at pH = 7.50.
Figure S3. A pH profile for the thermal reaction of a mixture
of diastereomeric photohydrates of uridine in solution with ethy-
lenediamine (EDA).
Figure S4. The UV spectra of the two forms of adduct pro-
duced upon incubation of a mixture of dUrd photohydrates with
ethylenediamine at pH 7.5.
1
Figure S5. The H NMR spectrum of Urd-GlyAm adducts in
DMSO-d6.
Figure S6. The HMBC spectra of the Urd-GlyAm adducts in
DMSO-d6.
1
Figure S7. The H NMR of the Urd-GlyAm adducts in D2O.
Figure S8. “Snapshots” of low energy conformations gener-
ated via quantum chemical calculations on the E and Z isomers
of the UrdGlyAm adduct.
Figure S9. The 13C NMR spectrum of the Urd-GlyAm ad-
ducts in D2O.
Figure S10. HMBC spectra of the Urd-GlyAm adducts in
D2O.
Figure S11. UV spectra of two adducts formed upon incuba-
tion of a mixture of dCyd photohydrates with ethylenediamine at
pH 7.5.
Figure S12. UV spectrum of a putative transamination pro-
duct produced via reaction of a mixture of dCyd hydrates with
glycinamide, along with the spectrum of dCyd itself.
Table S1. pKa values for the various protonated amines uti-
lized in reactions with the photohydrates of uridine, 2′-deoxyuri-
dine and 2′-deoxycytidine (at 25°C, unless otherwise specified).
Appendix S1. contains sections dealing with the (1) prepara-
tion of the photohydrates of Urd, dUrd and dCyd, Ura and
1-MeU, (2) preparation and characterization of reaction products
of dUrd and Urd hydrates with various amines, (3) results of
exploratory studies of reactions of dCyd photohydrates with var-
ious amines, (4) reaction products resulting from incubation of
the photohydrates of Urd and dUrd in ammonical solution and
(5) the results of spectroscopic studies of adducts produced via
incubation of dUrd hydrate and dCyd hydates with ethylamine.
Another possible reaction that could occur at physiological
pH is adduct formation via reaction of N-terminal amino groups
of proteins complexed with photohydrate-containing DNA or
RNA. The peptides (Gly)2 (pKa = 8.25) and (Gly)3 (pKa = 7.91)
can be viewed as models for N-terminal amino acid regions in
proteins. The observed reactivities of the amino groups of these
peptides with nucleoside photohydrates suggest that, under the
right circumstances, thermal adduct formation could occur in UV
irradiated nucleic acid-protein complexes. If so, this would lead
to formation of transient DNA-protein cross-links with unknown
biological consequences.
Acknowledgements—Research support from the National Science Foun-
dation (CHE-0131203) is gratefully acknowledged. We also gratefully
acknowledge the Bio-organic, Biomedical Mass Spectrometry Resource
(A. L. Burlingame, Director), supported by the NIH Division of Research
Resources Grant RR 01614; in particular, we thank David Maltby for
obtaining the required mass spectra in a timely manner. We thank Dr.
Janet Chung for running the proton NMR spectrum of the dUrd-EDA
adduct. The sections of the paper dealing with reactions of ethylamine are
based, in part, on a dissertation submitted by K. Hom in partial satisfaction
of the requirements for the Ph.D. in Pharmaceutical Chemistry at the
University of California, San Francisco. This latter work was supported by
a grant from the National Institutes of Health (Grant GM23526).
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