Mechanistic studies of the 5-iodouracil chromophore relevant to its use in nucleoprotein photo-cross-linking

Article abstract of DOI:10.1021/ja9607852

The photoreactivity of the 5-iodouracil chromophore was investigated toward understanding photo-cross-linking of nucleic acids bearing the chromophore to functionality in associated proteins. Irradiation of 5-iodouridine (IU) in the presence of a 10-fold excess of N-acetyltyrosine N-ethylamide (1) at 308 nm with a XeCl excimer laser or at 325 nm with a HeCd laser yields uridine (U) and N-acetyl-m-(5-uridinyl)tyrosine N-ethylamide (2) in a 1:2 mole ratio. In the presence of N-acetylphenylalanine N-ethylamide, uridine and analogous ortho, meta, and para regioisomeric adducts (3o, 3m, and 3p) were formed in a similar U to adduct mole ratio. The primary photochemical process leading to products was established as carbon-iodine bond homolysis in the first excited singlet state from a deuterium labeling experiment, photoacoustic calorimetry, and quantum yield measurements. Photoreduction of IU in 2-propanol-d solvent gave U with no deuterium incorporation. Photoacoustic calorimetric measurements established that triplet benzophenone transferred energy to IU with a rate constant of 2 x 10⁹ M^-1 s^-1. Further, the reaction of IU with 1 to form 2 was sensitized by benzophenone; however, comparison of quantum yields upon direct and sensitized excitation indicated that, at most, only a small portion of the reactions occurred via the triplet state. With direct excitation of IU, quantum yields as a function of the concentration of 1 showed that U and adduct 2 resulted from a common intermediate proposed to be the 5-uridinyl radical. Uridine formation was enhanced by the presence of hydrogen atom donors at the expense of formation of 2. Quantum yields were independent of excitation wavelength in the region 310-330 nm but not the reaction medium. The quantum yield of uridine formation but not adduct formation was approximately an order of magnitude higher in 90% acetonitrile - 10% water than in pH 7 water. The results are discussed in terms of high-yield cross-linking of nucleic acids bearing the 5-iodouracil chromophore to associated proteins in light of cocrystal X-ray structural data.

Full text of DOI:10.1021/ja9607852

5796
J. Am. Chem. Soc. 1996, 118, 5796-5803
Mechanistic Studies of the 5-Iodouracil Chromophore Relevant
to Its Use in Nucleoprotein Photo-Cross-Linking
Christopher L. Norris, Poncho L. Meisenheimer, and Tad H. Koch*
Contribution from the Department of Chemistry and Biochemistry, UniVersity of Colorado,
Boulder, Colorado 80309-0215
ReceiVed March 11, 1996X
Abstract: The photoreactivity of the 5-iodouracil chromophore was investigated toward understanding photo-cross-
linking of nucleic acids bearing the chromophore to functionality in associated proteins. Irradiation of 5-iodouridine
(IU) in the presence of a 10-fold excess of N-acetyltyrosine N-ethylamide (1) at 308 nm with a XeCl excimer laser
or at 325 nm with a HeCd laser yields uridine (U) and N-acetyl-m-(5-uridinyl)tyrosine N-ethylamide (2) in a 1:2
mole ratio. In the presence of N-acetylphenylalanine N-ethylamide, uridine and analogous ortho, meta, and para
regioisomeric adducts (3o, 3m, and 3p) were formed in a similar U to adduct mole ratio. The primary photochemical
process leading to products was established as carbon-iodine bond homolysis in the first excited singlet state from
a deuterium labeling experiment, photoacoustic calorimetry, and quantum yield measurements. Photoreduction of
IU in 2-propanol-d solvent gave U with no deuterium incorporation. Photoacoustic calorimetric measurements
established that triplet benzophenone transferred energy to IU with a rate constant of 2 × 109 M-1 s-1. Further, the
reaction of IU with 1 to form 2 was sensitized by benzophenone; however, comparison of quantum yields upon
direct and sensitized excitation indicated that, at most, only a small portion of the reactions occurred via the triplet
state. With direct excitation of IU, quantum yields as a function of the concentration of 1 showed that U and adduct
2 resulted from a common intermediate proposed to be the 5-uridinyl radical. Uridine formation was enhanced by
the presence of hydrogen atom donors at the expense of formation of 2. Quantum yields were independent of
excitation wavelength in the region 310-330 nm but not the reaction medium. The quantum yield of uridine formation
but not adduct formation was approximately an order of magnitude higher in 90% acetonitrile-10% water than in
pH 7 water. The results are discussed in terms of high-yield cross-linking of nucleic acids bearing the 5-iodouracil
chromophore to associated proteins in light of cocrystal X-ray structural data.
Introduction
purpose of achieving specific photo-cross-linking to associated
protein and that it can be superior to its predecessor, 5-bro-
mouracil, in this application. In RNA, 5-iodouracil replaces
uracil and in DNA, thymine, and consequently, the substitution
produces minimal spatial perturbation of the nucleoprotein
complex of interest, especially with DNA. The three nucle-
oprotein complexes which have been characterized using the
technique are a variant of a small RNA hairpin loop within the
genome of the bacteriophage R17 bound to the R17 coat
protein,2 the single-stranded DNA telomeric sequence of the
ciliated protozoan, Oxytricha noVa, bound to its telomere binding
protein,3 and a variant of a stem loop of the U1 snRNA bound
to the N-terminal RNA binding domain of human U1A snRNP
protein.4 Sequencing has established cross-linking to specific
tyrosine,2,3,5 histidine,2,3 and phenylalanine4 residues. In all three
systems the cross-linking yield with 5-iodouracil substitution
was 3-5 times higher than with corresponding 5-bromouracil
substitution.
Photo-cross-linking is an established technique for identifying
regions of nucleoprotein complexes which are in proximity.1-5
It can also have potential for locating an unknown protein for
which binding to a known nucleic acid sequence is proposed
to have biological significance. The information content of the
photo-cross-linking experiment, however, is dependent upon the
specificity of the photoreaction, the level of perturbation of the
complex caused by introduction of molecular changes needed
to achieve photo-cross-linking, and the potential for identifica-
tion of the cross-link position. A major problem associated with
utilizing the technique has been the cross-linking yield. When
the yield is only a few percent, the specificity is in question.
Did the cross-linking result from a nonspecific encounter of
the partners of the complex? Photodamage to other sites within
the nucleoprotein complex can also be competitive and affect
subsequent photo-cross-linking and/or sequencing.
Three studies now indicate that 5-iodouracil is an excellent
chromophore for introduction into both RNA and DNA for the
A new application of nucleoprotein photo-cross-linking,
which depends in a very significant way on the cross-linking
yield, is photoSELEX. PhotoSELEX is a methodology for
identification of a nucleic acid, bearing one or more photore-
active nucleotides, from a large combinatorial library which will
bind with high affinity to a target protein and photo-cross-link
to the target protein in high yield. The methodology is based
upon the SELEX protocol, selective evolution of ligands by
exponential enrichment.6 A successful photoselection was
recently demonstrated with the iodouracil chromophore and
HIV-1 Rev as the target protein.7
X Abstract published in AdVance ACS Abstracts, June 1, 1996.
(1) Budowsky, E. I.; Abdurashidova, G. G. Prog. Nucl. Res. 1989, 37,
1. Abdurashidova, G. G.; Tsvetkova, E. A.; Budowsky, E. I. Nucl. Acids
Res. 1991, 19, 1909. Allen, T. D.; Wick, K. L.; Matthews, K. S. J. Biol.
Chem. 1991, 266, 6113. Wower, J.; Hixson, S. S.; Zimmermann, R. A.
Biochemistry 1988, 27, 8114. Lee, D. K.; Evans, R. K.; Blanco, J.;
Gottesfeld, J.; Johnson, J. D. J. Biol. Chem. 1991, 266, 16478. Sontheimer,
E. J. Mol. Biol. Rep. 1994, 20, 35.
(2) Willis, M. C.; Hicke, B. J.; Uhlenbeck, O. C.; Cech, T. R.; Koch, T.
H. Science 1993, 262, 1255.
(3) Hicke, B. J.; Willis, M. C.; Koch, T. H.; Cech, T. R. Biochemistry
1994, 33, 3364.
(4) Stump, W. T.; Hall, K. B. RNA 1995, 1, 55.
(5) Willis, M. C.; LeCuyer, K. A.; Meisenheimer, K. M.; Uhlenbeck,
O. C.; Koch, T. H. Nucleic Acids Res. 1994, 22, 4947.
(6) Turek, C.; Gold, L. Science 1990, 249, 505.
S0002-7863(96)00785-8 CCC: $12.00 © 1996 American Chemical Society

Mechanistic Studies of the 5-Iodouracil Chromophore
J. Am. Chem. Soc., Vol. 118, No. 24, 1996 5797
Earlier mechanistic studies of the 5-bromouracil chromophore
revealed two primary photochemical processes with potential
for photo-cross-linking, carbon-bromine bond homolysis and
photoelectron transfer.8,9 Further, partitioning between the two
processes was excitation wavelength dependent.10 Signatures
for the two processes were respective deuterium incorporation
in the product uracil upon irradiation in variously-deuterium-
labeled 2-propanol. With 2-propanol-d solvent, 5-deuteriouracil
formation was the signature for electron transfer, and uracil
formation for bond homolysis; with 2-deuterio-2-propanol
solvent the signatures were reversed. Irradiation of 5-bromou-
racil at 254 nm in 2-propanol-d solvent gave approximately two-
thirds uracil and one-third 5-deuteriouracil; irradiation at 308
nm with a XeCl excimer laser gave predominantly 5-deuterio-
uracil. Further, deuterium incorporation with 2-propanol-d
solvent was quenched by cis-piperylene. These and other
observations were interpreted in terms of bond homolysis in
the higher energy π,π* singlet state, little or no bond homolysis
in the lower energy n,π* singlet state, and intersystem crossing
from both singlet states to the triplet state which reacts by
electron transfer.
Figure 1. Comparison of UV absorption by 5-bromouridine (‚‚‚),
5-iodouridine (s), N-acetylphenylalanine N-ethylamide (-‚‚‚-), and
N-acetyltyrosine N-ethylamide (- - -) in 20 mM, pH 7 phosphate buffer.
primary photochemical process with excitation at the long-
wavelength tail of the absorption band was also photoelectron
transfer, possibly with the 5-iodouracil chromophore in the
triplet state. However, a number of studies with 5-iodouracil,14-16
5-iodouridine,17 and even oligonucleotides18 and nucleic ac-
ids19,20 bearing 5-iodouracil have concluded that the primary
photochemical process of this chromophore is carbon-iodine
bond homolysis. Indeed, carbon-iodine bond homolysis is a
general photoreaction of organic iodides,21,22 including vinyl
iodides.23 Because of the emerging importance of the 5-iodou-
racil chromophore in the characterization of nucleoprotein
complexes and photoSELEX and a perceived inconsistency of
the accepted carbon-iodine bond homolysis mechanism with
high-yield photo-cross-linking, we reinvestigated the photore-
activity, focusing on the primary photochemical process or
processes leading to photo-cross-linking. We now describe the
results of time-resolved photoacoustic calorimetric measure-
ments, photoreduction in 2-propanol-d solvent, and quantum
yield measurements of photoreaction in the presence of a
tyrosine derivative.
We perceived photoelectron transfer to be the more desirable
mechanism for achieving high-specificity photo-cross-linking
of nucleoprotein complexes because reactivity should depend
upon the proximity of the halouracil to electron rich functionality
in the protein. Bond homolysis should not depend upon the
environment and has potential for promiscuity. Fortuitously,
the photoelectron transfer mechanism can be selected with
excitation at longer wavelengths. Model studies established
cross-linking of bromouracil to derivatives of tyrosine, histidine,
tryptophan,11 and cystine12 upon 308 nm excitation. Further,
improved yields of nucleoprotein cross-linking with the 5-bro-
mouracil chromophore have been achieved with 308 nm
excitation.13
Results and Discussion
A recent mechanistic study of the photoreaction between
5-bromouridine and N-acetyltyrosine N-ethylamide to produce
an adduct as well as uridine points to a further variation on
photoelectron transfer initiation of cross-linking.14 Quantum
yield measurements together with careful UV absorption
measurements suggested that the majority of product formation
resulted from excited tyrosine donating an electron to bromo-
uridine rather than excited bromouridine abstracting an electron
from the ground state tyrosine derivative. Presumably, this
mechanism extends to the tryptophan chromophore but not to
the histidine chromophore which is transparent except at
wavelengths below 250 nm.
Absorption and Emission. The UV absorption by IU is
compared with absorption by 5-bromouridine (BrU) and deriva-
tives of the amino acids tyrosine and phenylalanine in Figure 1
in pH 7 buffered water. Of particular importance are absorptions
in the region 300-330 nm. 5-Iodouridine absorbs more
intensely in this region than BrU or any of the wild-type
nucleosides or amino acid residues with a molar extinction
coefficient of 165 L mol-1 cm-1 at 325 nm. This is particularly
significant because monochromatic 325 nm light is available
at reasonable intensity and cost from a helium-cadmium
(15) Rupp, W. D.; Prusoff, W. H. Biochem. Biophys. Res. Commun. 1965,
18, 158.
(16) Gilbert, E.; Schulte-Frohlinde, D. Z. Naturforsch., Teil B, 1970, 25,
492. Gilbert, E.; Wagner, G.; Schulte-Frohlinde, D. Z. Naturforsch., Teil
B, 1971, 26, 209. Gilbert, E.; Wagner, G.; Schulte-Frohlinde, D. Z.
Naturforsch., Teil B, 1972, 27, 501.
(17) Go¨rner, H. J. Photochem. Photobiol., A 1993, 72, 197.
(18) Sugiyama, H.; Tsutsumi, Y.; Fujimoto, K.; Saito, I. J. Am. Chem.
Soc. 1993, 115, 4443.
(19) Rahn, R. O.; Sellin, H. G. Photochem. Photobiol. 1983, 37, 661.
(20) Wood, T. G.; Marongiu, M. E.; Prusoff, W. H. Biochem. Pharmacol.
1991, 41, 439.
The high nucleoprotein photo-cross-linking yields achieved
with 5-iodouracil substituted nucleic acids suggested that the
(7) Jensen, K. B.; Atkinson, B. L.; Willis, M. C., Koch, T. H.; Gold, L.
Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 12220.
(8) Ito, S.; Saito, I.; Matsuura, T. J. Am. Chem. Soc. 1980, 102, 7535.
(9) Swanson, B. J.; Kutzer, J. C.; Koch, T. H. J. Am. Chem. Soc. 1981,
103, 1274.
(10) Dietz, T. M.; Trebra, R. J. von; Swanson, B. J.; Koch, T. H. J. Am.
Chem. Soc. 1987, 109, 1793.
(11) Dietz, T. M.; Koch, T. H. Photochem. Photobiol. 1987, 46, 971.
(12) Dietz, T. M.; Koch, T. H. Photochem. Photobiol. 1989, 49, 121.
(13) Gott, J. M.; M. C, W.; Koch, T. H.; Uhlenbeck, O. C. Biochemistry
1991, 30, 6290.
(14) Norris, C.; Meisenheimer, K. M.; Koch, T. H. Photochem. Photobiol.
1996, submitted.
(21) Kropp, P. J.; Worsham, P. R.; Davidson, R. I.; Jones, T. H. J. Am.
Chem. Soc. 1982, 104, 3972.
(22) Slocum, G. H.; Kaufmann, K.; Schuster, G. B. J. Am. Chem. Soc.
1981, 103, 4625.
(23) Kropp, P. J.; NcNeely, S. A.; Davis, R. D. J. Am. Chem. Soc. 1983,
105, 6907.

5798 J. Am. Chem. Soc., Vol. 118, No. 24, 1996
Norris et al.
(HeCd) laser. 5-Iodouridine in pH 7 buffered water showed
little or no fluorescence. A measurement of the fluorescence
quantum yield placed an upper limit on the quantum yield at 1
× 10-3, suggesting a short singlet lifetime.
the acoustic waves to match more closely that of the calibrant;
the calibrant deposited all of its photon energy on a time scale
faster than the apparatus could respond to it. Rate constant fits
to the photoacoustic waves gave data for a Stern-Volmer plot
for iodouracil quenching of benzophenone. The plot was linear
with a slope, (1.88 ( 0.03) × 109 M-1 s-1, equal to the rate
constant for energy transfer. The photoacoustic experiment also
provided a measure of the benzophenone triplet energy, 68.4
( 0.6; agreement with the literature value of 69 kcal/mol
provided an internal check of the experiment. The rate constant
for energy transfer to IU is about an order of magnitude below
the rate constant for diffusion in acetonitrile, consistent with a
triplet state energy close to that of benzophenone. This estimate
is in agreement with one made some years ago from phospho-
rescence excitation spectra in the solid state.24 As expected the
triplet energy is below that of 5-bromouracil which has been
estimated at about 72 kcal/mol.17,24 Further, the photoacoustic
measurements indicate that no enthalpic shortfall or excess is
observed in the total amount of heat released. Thus, the excited
triplet state of 5-iodouracil undergoes no photochemistry aside
from direct relaxation to ground state iodouracil. An alternative,
albeit unlikely, possibility (vide infra) is that triplet IU undergoes
photochemistry to thermoneutral products on a time scale faster
than the minimum time resolution of the photoacoustic tech-
nique, which in this case is 200 ns.
Quantum Yield Measurements. Direct irradiation of 1 mM
5-iodouridine with 8 mM N-acetyltyrosine N-ethylamide in
buffered pH 7 aqueous solution at 325 nm with a HeCd laser
gave uridine (U) and N-acetyl-m-(5-uridinyl)tyrosine N-ethyl-
amide (2) with quantum yields of destruction and formation as
shown in Table 2 (entry 1). The quantum yield of destruction
and the sum of the quantum yields of formation were in the
range of 1.5%; the low quantum yields are consistent with the
lack of an observable enthalpic shortfall in the photoacoustic
experiment in this medium. Direct irradiation of 5-iodouridine
alone gave rise to a similar, if not slightly greater, iodouridine
destruction quantum yield (Table 3, entry 1). Most but not all
of this destruction was accounted for by uridine formation, with
many other products present in the HPLC chromatograms. The
effect of the concentration of the tyrosine derivative 1 on the
partitioning between uridine formation and adduct 2 formation
is shown in Figure 2. At concentrations of 1 above 10 mM,
the product ratio (1:2) does not change and quantum yields
become constant. Hence, at higher concentrations of 1, reduc-
tion and adduct formation are coupled and result from reaction
of a common intermediate, such as the uridinyl radical, with 1.
Further, at concentrations of the reactants employed, the
common intermediate must not be able to re-form significant
amounts of starting material.
Photoreactions. Irradiation of IU at 308 nm with a xenon
chloride (XeCl) excimer laser or at 325 nm with a HeCd laser
in the presence of an excess of N-acetyltyrosine N-ethylamide
(1) in pH 7 aqueous medium yielded primarily uridine and
N-acetyl-m-(5-uridinyl)tyrosine N-ethylamide (2) in a 1:2 mole
ratio. The reaction was carried only to 50% destruction of IU
because 2 was somewhat photolabile with irradiation in the
region 300-330 nm. Adduct 2 was isolated by preparative
reversed phase HPLC and characterized from spectroscopic data
as reported in the Experimental Section and Table 1. The
1
assignment of the H NMR signals was made from splitting
patterns and homonuclear COSY spectra. NOE difference
spectroscopy established the regioconnectivity of the cross-link
between the 5-position of iodouridine and the meta position of
the tyrosine analog. Adduct 2 was identical to the adduct from
irradiation of a pH 7 solution of 5-bromouridine and 1.14
Similar irradiation of IU in the presence of an excess of
N-acetylphenylalanine N-ethylamide at 308 nm gave uridine
(30%) and three regioisomeric adducts, N-acetyl-o-(5-uridinyl)-
phenylalanine N-ethylamide (3o), N-acetyl-m-(5-uridinyl)phen-
ylalanine N-ethylamide (3m), and N-acetyl-p-(5-uridinyl)phen-
ylalanine N-ethylamide (3p) in the ratio 1:2:1 (65% total yield
by HPLC). The para isomer was separated from the ortho and
meta isomers by preparative reversed phase HPLC, and all three
1
isomers were characterized from H NMR and mass spectral
data reported in Table 1 and the Experimental Section.
Irradiation at 308 nm of IU in 2-propanol-d solvent to 50%
destruction gave uridine as the sole product. Isolation of the
uridine and characterization by 1H NMR spectroscopy showed
no deuterium incorporation at the 5-position. In contrast, an
analogous experiment with 5-bromouracil gave uracil with
approximately 80% deuterium incorporation at the 5-position.9
This result is consistent with carbon-iodine bond homolysis
in contrast with electron transfer as the primary photochemical
process.
Photoacoustic Calorimetry. Irradiation of 5-iodouridine in
a 90:10 acetonitrile-water mixture (v/v) gave rise to an enthalpy
release shortfall versus the calibrant of 9 ( 3% in argon-
saturated solution with excitation at 310 nm. Thus, a small but
significant fraction of the originally-absorbed photon energy was
retained in the form of endothermic products on a time scale
longer than the maximum kinetic lifetime detectable by the
apparatus (about 5 µs). No evidence of kinetic events within
the 200 ns to 5 µs lifetime resolution window was found. In
buffered aqueous solution, however, no statistically significant
enthalpic shortfall was detected, assuming that all of the
photoacoustic signal that arises is truly enthalpic in origin.
Irradiation in the presence of the tyrosine derivative did nothing
to change this observation. The effect of the medium on
photoreactivity was also apparent from quantum yield measure-
ments (vide infra).
Irradiation of IU and 1 in a 90:10 (v/v) acetonitrile-water
mixture gave rise to a similar quantum yield of adduct formation
but an unexpectedly high quantum yield of reduction to U as
reported in Table 3 (entry 6). The uridine to adduct 2 ratio
was almost 10:1, and the IU destruction quantum yield was
about a factor of 10 greater than that in aqueous solution. In
this experiment the analysis for U was complicated by the
presence of acetonitrile in the HPLC injection solvent which
caused significant broadening of the uridine peak. Identification
of the broad peak as U was established by comparison with the
peak from injection of a sample of U in 90:10 acetonitrile-
water. The assumption that the integral of this broad peak
between 0.5 and 2 min was a measure of uridine formation was
supported by a resulting destruction quantum yield approxi-
mately equal to the product formation quantum yield. The
Photoacoustic calorimetry was also used to study the triplet
state of 5-iodouridine produced by benzophenone sensitization
in 90:10 acetonitrile-water. Higher IU concentrations resulted
in more prompt heat deposition, which shifted the phases of
(24) Rothman, W.; Kearns, D. R. Photochem. Photobiol. 1967, 6, 775.

Mechanistic Studies of the 5-Iodouracil Chromophore
J. Am. Chem. Soc., Vol. 118, No. 24, 1996 5799

5800 J. Am. Chem. Soc., Vol. 118, No. 24, 1996
Norris et al.
Table 2. Effect of Triplet Quenchers and Radical Scavengers on the Quantum Yields of Destruction of 5-Iodouridine (IU) and Formation of
Uridine (U) and Tyrosine Adduct 2 with Solutions Containing 1 mM IU and 8 mM N-Acetyltyrosine N-Ethylamide and Excitation at 325 nm
Except As Noted
entry
solvent
quencher or scavenger
Φ (-IU)
Φ (U)
Φ (2)
1
2
3
4
5
6
7
8
H2O, pH 7a
none
0.015 ( 0.003 0.0042 ( 0.0007 0.0081 ( 0.0013
0.030 ( 0.005 0.0076 ( 0.0012 0.0078 ( 0.0012
H2O, pH 7a
H2O, pH 7a
H2O, pH 7a
H2O, pH 7a
2-propanol, 10 mM
2-propanol, 100 mM
acetonitrile, 100 mM
acetonitrile, 1 M
0.018 ( 0.004 0.015 ( 0.003
0.0024 ( 0.0004
0.015 ( 0.004 0.0046 ( 0.0011 0.0073 ( 0.0012
0.011 ( 0.004 0.0080 ( 0.0012 0.0062 ( 0.0010
90% MeCN-10% waterb acetonitrile
0.13 ( 0.03
0.12 ( 0.03
0.011 ( 0.002
H2O, pH 7a
H2O, pH 7a
potassium sorbate, 10 mM
0.020 ( 0.003 0.0022 ( 0.0003 0.0029 ( 0.0004
potassium sorbate, 10 mM, + 2-propanol, 10 mM 0.027 ( 0.004 0.0031 ( 0.0005 0.0026 ( 0.0004
a 0.05 M phosphate buffer. b Excitation wavelength 330 nm.
Table 3. Effect of Triplet Quenchers and Radical Scavengers on
the Quantum Yields of Destruction of 5-Iodouridine (IU) and
Formation of Uridine (U) with Irradiation at 325 nma
Table 4. Effect of Excitation Wavelength on the Quantum Yields
of Destruction of 5-Iodouridine (IU) and Formation of Uridine (U)
and Tyrosine Adduct 2 in 0.05 M pH 7 Phosphate Buffer
Containing 8 mM N-Acetyltyrosine N-Ethylamide
entry
quencher or scavenger
none
2-propanol, 10 mM
Φ (-IU)
Φ (U)
wavelength [IU]
1
2
3
4
0.017 ( 0.004 0.010 ( 0.002
0.027 ( 0.006 0.014 ( 0.002
(nm)
(mM)
Φ (-IU)
Φ (U)
Φ (2)
310
320
330
0.046 0.009 ( 0.003 0.008 ( 0.004 0.011 ( 0.002
0.20 0.014 ( 0.004 0.003 ( 0.003 0.009 ( 0.003
potassium sorbate, 10 mM 0.022 ( 0.006 0.0012 ( 0.0002
potassium sorbate, 10 mM, 0.023 ( 0.005 0.0025 ( 0.0004
+ 2-propanol, 10 mM
2.1
0.019 ( 0.008 0.006 ( 0.002 0.009 ( 0.002
a Solutions contained 1 mM IU in 0.05 M pH 7 phosphate buffer.
quenching constant of 2.1 × 109 M-1 s-1 25
.
Assuming that
this value applies under our conditions, two-thirds of the
benzophenone triplets are actually quenched by the tyrosine
analog and the rest by 5-iodouridine in a solution 15 mM in IU
and 30 mM in 1. If all of the reactivity occurred via the
5-iodouridine triplet state, then the cross-linking quantum yield
would go up to approximately 0.01. The quantum yield of
iodouridine triplet-induced cross-linking from direct irradiation
is smaller than this value by a factor equal to the iodouridine
intersystem crossing quantum yield, which has never been
measured at room temperature; it has been predicted to be low
on the basis of a low phosphorescence quantum yield in a glass
at 77 K.26 The quantum yield for adduct formation in 90%
acetonitrile-10% water solution upon direct excitation is 0.011
(Table 2, entry 6). If we assume that the intersystem crossing
yield lies somewhere between 0.1 and 0.01, then between about
9% and 0.9% of all cross-linked molecules formed by direct
irradiation originate from the triplet state of iodouridine; thus,
the triplet state reactivity of iodouridine at most comprises only
a small contribution to the formation of a uridine-tyrosine
adduct.
Figure 2. Observed quantum yields of formation of uridine (•) and
the photoadduct 2 (o) as a function of increasing N-acetyltyrosine
N-ethylamide (1) concentration. Solutions of 5-iodouridine (1 mM) and
N-acetyltyrosine N-ethylamide in phosphate buffer (20 mM, pH 7) were
irradiated at 325 nm.
increased quantum yield of U formation in this medium is also
consistent with the 9% enthalpic release shortfall observed by
photoacoustic calorimetry.
The 5-bromouracil chromophore shows excitation wavelength
dependent photoreactivity in 2-propanol solvent with predomi-
nance of C-Br bond homolysis at shorter wavelengths and
predominance of intersystem crossing followed by electron
transfer reactivity at longer wavelengths.9,10 Further, reaction
of BrU with 1 in an aqueous medium showed an apparent
wavelength dependence when reactivity was assumed to result
from excited BrU. This apparent wavelength dependence,
however, appears to result from a predominance of product,
resulting from excited 1.14 To test for a possible wavelength
dependence for IU photoreactivity, which might serve as a
signature for triplet state reactivity and/or reactivity of excited
1, a tunable Nd:YAG-pumped frequency-doubled dye laser was
used to irradiate solutions of 5-iodouridine and the tyrosine
derivative along iodouridine’s long-wavelength absorption tail
at 310, 320, and 330 nm. The resulting quantum yields from
these experiments are reported in Table 4. No wavelength
dependence on the cross-linking quantum yield was observed
within the experimental error.
The role of the triplet state was explored further using three
diene quenchers: cis-piperylene, 2,4-hexadien-1-ol, and potas-
sium sorbate. cis-Piperylene was used as an effective quencher
of electron-transfer-mitigated photoreduction of 5-bromouracil
by 2-propanol in an earlier study.7,8 Insignificant quenching
of adduct formation was observed; however, interpretation of
the experiment was complicated by the reactivity of the
quenchers as π-acceptors and hydrogen atom donors toward
radicals (vide infra).
Triplet reactivity of 5-iodouridine was also explored using
benzophenone sensitization with excitation at 355 nm with a
frequency-tripled Nd:YAG laser. As mentioned above (see
Photoacoustic Calorimetry in this section), 5-iodouridine was
shown to efficiently quench excited triplet benzophenone with
a second-order quenching constant of 1.9 × 109 M-1 s-1 in
90% acetonitrile-10% water. The sensitized reaction of IU
with 1 in the same medium gave adduct 2, and the quantum
yield was 3.1 × 10-3, assuming that complete energy transfer
took place between benzophenone and iodouridine. However,
tyrosine also is known to quench benzophenone excited triplet
states in 1:4 (v/v) acetonitrile-water solution with a reported
(25) Battacharyya, S. N.; Das, P. K. J. Chem. Soc., Faraday Trans. 2
1984, 80, 1107.
(26) Go¨rner, H. J. Photochem. Photobiol., B 1990, 5, 359.

Mechanistic Studies of the 5-Iodouracil Chromophore
J. Am. Chem. Soc., Vol. 118, No. 24, 1996 5801
Scheme 1
Table 5. Relative Second-Order Rate Constants for Quenching of
To confirm the importance of C-I bond homolysis on
initiating cross-linking between iodouridine and the tyrosine
derivative, a variety of hydrogen donors were tested as radical
scavengers. These included 2-propanol and acetonitrile, as well
as potassium sorbate and 2,4-hexadien-1-ol, which were orig-
inally intended for use as triplet quenchers. These compete with
the tyrosine derivative and IU as sources of hydrogen atoms.
Resulting quantum yields for all quenching experiments are
shown in Table 2. In general, reasonably high concentrations
of these radical scavengers were capable of significantly
quenching the formation of the uridine-tyrosine adduct, sug-
gesting that the predominance of cross-linking arises from initial
C-I bond homolysis. An increase in the quantum yield of
uridine formation paralleled the decrease in the quantum yield
of adduct formation. Reaction via C-I bond homolysis is also
consistent with quantum yield studies on 5-iodouridine in the
absence of the tyrosine derivative, where approximately the
same, or slightly greater, destruction quantum yield was
observed as with the tyrosine present (Table 3).
The observations of insignificant quenching of uridine-
tyrosine adduct 2 formation in the presence of diene triplet
quencher, insignificant sensitization of cross-link formation
using benzophenone, insignificant deuterium incorporation upon
iodouridine photoreduction in 2-propanol-d, and the lack of a
wavelength dependence on cross-linking efficiency led us to
the conclusion that the 5-iodouridine triplet state/electron transfer
mechanism makes up a minimal contribution to the photochem-
istry of iodouridine. Easily, less than 10% of adduct formation
with N-acetyltyrosine N-ethylamide arises from this mechanism.
The most logical explanation for cross-linking with 5-iodo-
uridine is via absorption followed by irreversible C-I bond
homolysis, giving rise to a uridin-5-yl radical which undergoes
radical substitution with the tyrosine derivative 1 via addition
at the position ortho to the phenolic hydroxyl substituent as
shown in Scheme 1. Uridine results from competitive hydrogen
atom abstraction from 1. This is borne out by the results of
irradiations carried out in the presence of various radical
quenchers. Of these quenchers, acetonitrile is the most unam-
biguous, as it is a pure hydrogen atom donor with no expected
influences on triplet state or electron transfer chemistry. It is,
however, only a weak hydrogen atom donor, requiring concen-
trations of 1 M in order to change the uridine:adduct 2 product
ratio from 0.5:1 to 1.3:1. Experiments at 100 mM acetonitrile
and 90% acetonitrile-10% water (v/v) give rise to results
consistent with the quenching observed at 1 M acetonitrile (see
Table 2).
Uridin-5-yl Radical (U•)
reaction
krelative
U• + tyrosine derivative 1 f adduct 2
U• + tyrosine derivative 1 f U
U• + 2-propanol f U
1.0 (defined)
0.53
0.42
0.006
0.25
2.3
U• + acetonitrile f U
U• + sorbate f U
U• + sorbate f U-sorbate adduct?
The radical quencher 2-propanol at 10 mM concentration was
found to double the quantum yield of U formation without
affecting the quantum yield of adduct formation (Table 2, cf.
entries 1 and 2). This contrasts somewhat with the result at
100 mM 2-propanol, where the quantum yield of U formation
was 4 times higher but adduct formation was 3 times smaller
(Table 2, cf. entries 1 and 3). The results of a study of
photoreduction of IU in the presence of 2-propanol by Go¨rner
may provide an explanation.17 He showed that when 2-propanol
was present at even modest concentrations, photoreduction of
IU occurred in part via a radical chain mechanism. The chain-
propagating step was an electron transfer from 2-propanol-2-yl
radical (the hydrogen-abstracted product of 2-propanol) to a
ground state 5-iodouridine, giving rise to a 5-iodouridine radical
anion. The radical anion then ejected iodide to generate more
uridin-5-yl radical. Operation of the chain mechanism increased
the quantum yield for the production of uridin-5-yl radical. In
the experiment described here, the increased quantum yield of
production of uridin-5-yl radical in 10 mM 2-propanol appears
to cancel the decrease in the expected quantum yield of adduct
formation due to radical quenching, leaving no apparent
influence on the quantum yield under these conditions. 2-Pro-
panol has been proposed to undergo electron transfer with the
triplet state of 5-bromouracil,9,10 making it potentially a more
ambiguous radical quencher than acetonitrile. However, as
stated above, iodouridine shows no significant triplet state or
electron transfer photochemistry.
The effect of radical quenchers on the quantum yields in an
aqueous medium, shown in Tables 2 and 3, is also completely
consistent with products arising from C-I bond homolysis. A
set of relative second-order rate constants for all radical
quenching processes resulting from fitting the quantum yield
data to the bond homolysis mechanism are given in Table 5.
From quantum yields of U and adduct 2 formation, the quantum
yield of iodouridine homolysis appears to range from 1.2% in
the presence of tyrosine derivative 1 to up to 1.7% upon addition
of 100 mM 2-propanol, with the increase due to the phenomenon

5802 J. Am. Chem. Soc., Vol. 118, No. 24, 1996
Norris et al.
explained above. With sorbate present, additional products,
assumed to be sorbate adducts, replace U and adduct 2 without
a significant change in the quantum yield of destruction.
homolysis depending upon the specific nature of the π-complex.
Although the model study with IU and 1 could not address
reactivity in a π-complex, it did establish the mode and level
of reactivity of the iodouracil chromophore in a non-π-stacking
environment.
Concluding Remarks. Electronically excited 5-iodouridine
reacts in an aqueous medium with N-acetyltyrosine N-ethyl-
amide (1) to yield uridine and adduct 2 in a 1:2 mole ratio and
with N-acetylphenylalanine to yield uridine and three regio-
isomeric adducts, 3o, 3m, and 3p, also in a 1:2 mole ratio.
Product studies, photoacoustic calorimetric measurements,
deuterium labeling experiments, quantum yield measurements,
and radical quenching studies together indicate that photore-
duction and adduct formation are initiated by irreversible
carbon-iodine bond homolysis in the singlet state followed by
hydrogen atom abstraction from 1 and radical addition to 1,
respectively.
A predominance of acetonitrile in the medium appears to have
an additional effect on uridin-5-yl radical production, with a
quantum yield of 1.4% at 1 M acetonitrile, and continuing
beyond 10% at 90% acetonitrile-10% water (v/v) (Table 2,
entry 6). The results of a recent study of diarylmethyl chloride
photochemistry by Peters and co-workers suggests a rationale
for the inefficiency of C-I bond homolysis in an aqueous
medium versus acetonitrile medium.27 They observed decay
of an initially formed diarylmethyl-chlorine geminate radical
pair to the ground state surface by electron transfer in competi-
tion with radical cage escape. Partitioning between re-formation
of starting material and a contact ion pair then occurred.
Possibly, an aqueous medium facilitates electron transfer decay
within the uridinyl-iodine geminate radical pair to the ground
state surface in competition with radical cage escape. On the
ground state surface, re-formation of starting material must be
favored over formation of a contact ion pair since products from
an ionic intermediate are not apparent.
Relevance to Nucleoprotein Photo-Cross-Linking. The
model cross-linking reaction, which requires diffusion, appears
different from the macromolecular cross-linking reaction. The
results with the model study suggest that about one-third of all
iodouridines should undergo photoreduction, while two-thirds
will undergo cross-linking. In some macromolecular systems,
cross-linking yields of a 5-iodouracil base to a tyrosine residue
can exceed 90%. Why does such a discrepancy exist between
the model experiments and macromolecular experiments? Ori-
entational limitations in some nucleoprotein complexes may
highly favor cross-linking over reduction. However, a more
elaborate explanation seems necessary to account for the
following observations. First, the irradiation time needed to
achieve high-yield nucleoprotein cross-linking varies substan-
tially as a function of the nucleoprotein complex, and we assume
that irradiation time bears some resemblance to quantum yield.
Second, a photoSELEX experiment starting with a combinatorial
library of 1014 sequences bearing multiple IUs converged to a
few sequences which bound with high affinity and photo-cross-
linked in good yield to HIV-1 Rev protein.7 Such convergence
requires high-yield cross-linking with the iodouracil chro-
mophore in a favorable photo-cross-linking environment and
photostability with the iodouracil chromophore in an unfavorable
photo-cross-linking environment. Possibly, a particular orienta-
tion of the cross-linking groups promotes an additional mech-
anism. In the two examples of nucleoprotein photo-cross-
linking using the iodouracil chromophore in which X-ray
cocrystal data are available, the iodouracil of the nucleic acid
and the aromatic ring of the amino acid residue of the protein
are located in a π-stacking arrangement.28,29 Excitation of the
iodouracil chromophore with an electron donor amino acid
residue nearby or possibly direct excitation of an iodouracil-
amino acid π-complex within a nucleoprotein complex may
result in electron transfer to form a radical ion pair. High-yield
cross-linking could then result from reaction of the radical ion
pair via bond formation followed by loss of HI or ejection of
iodide and subsequent bond formation via radical combination.
Such a mechanism could have a quantum yield more than 10
times higher than the 1.5% quantum yield for simple C-I bond
Experimental Section
General Remarks. 5-Iodouracil and 5-iodouridine (Sigma), as well
as ferrocene, 2-propanol-d, potassium chromate, and potassium phos-
phate buffer (Aldrich), were used as received without further purifica-
tion. N-Acetyltyrosine N-ethylamide was synthesized from N-acetyl-
tyrosine as previously described9 and recrystallized in ethyl acetate
before use. Methanol and acetonitrile (HPLC grade, Burdick and
Jackson) and water (Millipore, 18 MΩ cm) were used as received. 1H
NMR data were collected with a Bruker AM-400 spectrometer operating
at 400 MHz and UV-vis spectra with a Hewlett-Packard 8452A
spectrometer. HPLC analyses were performed with a Hewlett-Packard
1090 chromatograph equipped with a UV-vis diode array detector and
a Hewlett-Packard 5 µm, C-18 reversed phase, microbore column (2.1
mm × 10 cm). Detection was typically carried out at 280 nm with a
60 nm bandwidth. Mixtures of methanol and either water or 0.001 M
pH 6.8, phosphate buffer were used as eluents.
Emission Spectroscopy. Fluorescence quantum yields were mea-
sured with an apparatus consisting of a 1 kW xenon arc lamp, a SPEX
1320 0.5 m double excitation monochromator with 1.5 mm slits, a SPEX
1702 0.75 m emission monochromator with 1.5 mm slits, a Centronic
Series 4283 Model S25 photomultiplier tube housed in a Bailey
Instruments CHA-1 air-cooled housing, and SPEX digital single photon
counting electronics interfaced to an IBM-compatible computer.
Emitted light was typically collected at a 14° angle relative to the
incident light. 9,10-Diphenylanthracene (Aldrich, Gold Label) dissolved
in n-pentane (Aldrich) was used as the fluorescence standard.
Photoacoustic Calorimetry. The apparatus for photoacoustic
calorimetry has been previously described.30 Briefly, a cuvette equipped
with a stainless-steel-encased PZT (lead zirconate-lead titanate)
acoustic sensor (0.5 MHz resonance frequency) clamped to its side in
acoustic contact was irradiated at right angles to the sensor with a
nanosecond laser. The laser sources used was a PRA LN1000/102
nitrogen-pumped dye laser (Exciton BPBD dye, emission maximum
at 365 nm, 1.5 Hz repetition rate, 0.5 ns pulse width), a Lambda-Physik
EMG-101 XeCl excimer laser (308 nm, 2-10 Hz repetition rate, 10
ns pulse width), or a Spectra Physics DCR/PDL frequency-doubled
Nd:YAG-pumped dye laser (Exciton DCM dye, tunable output from
310 to 330 nm, 10 Hz, 4 ns pulse width). A Gould 4072 digital storage
oscilloscope was used to record the acoustic waveforms at 10 ns per
point, which were stored on an IBM PC. Laser Precision Rj-7000
pyrolytic energy probes were used to monitor both the laser energy
and the transmittance of the solutions to the excitation beam. Typically,
100 laser shots were averaged in cases where the solutions were not
stirred, while 20 shots were averaged in cases where a microflea stir
bar was used to briefly stir the solution in between individual laser
shots. A negligible difference was found in the results with and without
stirring.
(27) Lipson, M.; Deniz, A. A.; Peters, K. S. J. Phys. Chem. 1996, 100,
3580.
(28) Valegard, K., Murray, J. B., Stockley, P. G., Stonehouse, N. J.;
Liljas, L. Nature 1994, 371, 623.
The data analysis for photoacoustic calorimetry has been described
previously.31 An iterative nonlinear least squares procedure was used
(29) Oubridge, C.; Ito, N.; Evans, P. R.; Teo, C. H.; Nagai, K. Nature
1994, 372, 432.
(30) Peters, K. S.; Snyder, G. J. Science 1988, 241, 1053.
(31) Norris, C. L.; Peters, K. S. Biophys. J. 1993, 65, 1660.

Mechanistic Studies of the 5-Iodouracil Chromophore
J. Am. Chem. Soc., Vol. 118, No. 24, 1996 5803
to determine the best fit parameters for amplitude factors and rate
constants (if applicable). Ferrocene was used as the calibration
compound in organic solvents, while potassium chromate was used in
aqueous solution.
YAG-pumped frequency-doubled dye laser described above (see
Experimental: Photoacoustic Calorimetry in this section) was used to
obtain light from 310 to 330 nm. To obtain laser light for the
benzophenone sensitization quantum yield measurements at 355 nm,
the Spectra-Physics DCR Nd:YAG laser, used to pump the PDL dye
laser, was used alone, and the output was frequency-tripled.
Photoreduction of 5-Iodouridine in 2-Propanol-d. 5-Iodouridine
(13.5 mg, 0.036 mmol) was dissolved in 2 mL of D2O to exchange
acidic protons for deuterons. The D2O was removed in vacuo, and the
solid contents were dissolved in 5 mL of 2-propanol-d. A 1.5 mL
aliquot was transferred to a quartz cuvette and irradiated with a Lambda
Physik EMG-101 XeCl excimer laser (308 nm, 10 Hz, 40 mJ/pulse)
for 35 min. Approximately 50% of the 5-iodouridine was reduced to
uridine as indicated by analytical reversed phase HPLC. The solvent
was removed in vacuo, and the resulting crude yellow solid was
analyzed by 1H NMR spectroscopy in D2O. No detectable deuterium
incorporation was apparent at the 5-position of the uridine.
Photoreaction of 5-Iodouridine with N-Acetyltyrosine N-Ethyl-
amide. 5-Iodouridine (111 mg, 0.3 mmol) and N-acetyltyrosine N-ethyl
amide (325 mg, 1.3 mmol) were added to a 100 mL volumetric flask
and diluted to the mark with ultra pure water. The homogeneous
solution was transferred to a 100 mL quartz photocell bearing a
magnetic stirbar. The solution was degassed for 15 min with argon
and irradiated with stirring and continuous argon flow at 10 °C for 7
h with 308 nm monochromatic light (XeCl, 90 mJ/pulse, 10 Hz). The
yield of N-acetyl-m-(5-uridinyl)tyrosine N-ethylamide (2) as determined
by analytical HPLC was 30% (60% after correction for recovered
starting material). Adduct 3 was isolated by preparative reversed phase
HPLC on a Rainin 100 Å spherical pore C18 column, 10 mm × 25
cm, eluting with a methanol-water gradient (100% H2O to 100%
MeOH in 30 min at 4.3 mL/min). Solvent was removed in vacuo to
Actinometry was carried out using either a Scientech 362 power
meter or two Laser Precision Rj-7000 pyrolytic energy probes arranged
in a transmission/reflection orientation relative to a beam splitter. With
the energy probe orientation, absorbances to the laser light could be
monitored during the experiment. Screw top-sealed Spectrocell SUV
cuvettes were used and were degassed in most cases by bubbling
prepurified argon for at least 5 min through a 1 mL solution of starting
materials. Because the absorbances during most irradiations tended
to increase as the starting material was destroyed due to product
absorption, most irradiations were carried out to <15% destruction of
the limiting reagent, which was usually the iodouridine. The iodo-
uridine absorbance versus time plot assumed a linear decay of
iodouridine in order to subtract out the absorption caused by the
photoproducts. The power absorbed by the iodouridine was then
calculated for each absorbance-monitoring time point, and this power
was then trapezoid-rule-integrated to determine the total energy
absorbed by the iodouridine. For experiments with the Nd:YAG-
pumped dye laser, which suffered from severe power drift and a need
for constant retuning, a computer program written in C was employed
to make the actinometry calculation. Due to the variability in the
calibration of the power or energy meters as well as the number of
assumptions made in subtracting out non-starting-material absorption,
the typical actinometry error was estimated conservatively at 15%.
Quantification of reactant depletion and product buildup was followed
by HPLC at 280 nm.
Quantum Yields of Formation of Uridine and Adduct 2 as a
Function of N-Acetyltyrosine N-Ethylamide Concentration. 5-Io-
douridine (18.9 mg, 0.050 mmol) was diluted to 25 mL in phosphate
buffer (20 mM, pH 7) to give a 2 mM stock 2× solution. N-
Acetyltyrosine N-ethylamide (25 mg, 0.10 mmol) was diluted to 2.0
mL in phosphate buffer to give a 50 mM stock solution. The tyrosine
stock solution was serially diluted to afford 50, 36, 20, 10, 6, 2, 1, 0.5,
and 0 mM tyrosine 2× solutions. Individual reaction samples were
prepared by mixing 0.5 mL of iodouridine 2× stock solution with 0.5
mL of the appropriate tyrosine 2× stock solution. A 750 uL aliquot
of each sample was irradiated in a quartz cuvette for 4 min with a
HeCd laser (325 nm, 0.0395 W). Light absorbance (A ≈ 0.016) was
monitored by UV spectroscopy. Irradiated samples were analyzed by
reversed phase analytical HPLC.
give a white solid: mp 175-178 °C; UV (H2O) λmax 270 nm, ꢀ280
)
6970, ꢀ308 ) 2480, ꢀ325 ) 752 L mol-1 cm-1; mass spectrum (FAB-)
m/z 491, (FAB+) m/z 493 (calculated M + 1, 493), exact mass 493.1935
(calculated M + 1, 493.1934); 1H NMR, Table 1; 1H NOE difference
spectrum, irradiation at the resonance frequency for the proton at C6
gave a 2% enhancement of the resonance signal for the proton at o′
and 0% enhancement of the signal for the protons at the â-position
(see the structure associated with Table 1 for the numbering scheme).
Photoreaction of 5-Iodouridine with N-Acetylphenylalanine N-
Ethylamide. 5-Iodouridine (110 mg, 0.3 mmol) and N-acetylphenyl-
alanine N-ethyl amide (304 mg, 1.3 mmol) were added to a 100 mL
volumetric flask and diluted to the mark with ultrapure water. The
homogeneous solution was transferred to a 100 mL quartz photocell
bearing a magnetic stirbar. The solution was degassed for 15 min with
argon and irradiated with stirring and continuous argon flow at 4 °C
for 4 h with 308 nm monochromatic light (XeCl, 90 mJ/pulse, 10 Hz).
HPLC analysis showed less than 5% IU, 30% U, and the balance as a
mixture of the ortho, meta, and para N-acetyl(5-uridinyl)phenylalanine
N-ethylamides, 3o, 3m, and 3p, in the ratio 1:2:1. The adducts were
isolated in a combined yield of 15% by preparative reversed phase
HPLC as described above for the isolation of 2. The ortho and meta
adducts coeluted as a 1:2 molar mixture and were analyzed without
further separation. Solvent was removed in vacuo to give a white
solid: mass spectrum for all three adducts (FAB-) m/z 475, (FAB+)
Acknowledgment. We thank the Council for Tobacco
Research, the National Science Foundation, the RNA Center at
the University of Colorado, and Nexstar Pharmaceuticals, Inc.,
for financial assistance. T.H.K. also thanks the University of
Colorado Council on Research and Creative Work for a Faculty
Fellowship. We thank Mary Kasparian for assistance with some
initial photoacoustic calorimetric experiments, Steve Leone for
the use of a dye laser, Kevin Peters for the use of a Nd:YAG
laser and photoacoustic calorimeter, Josef Michl for the use of
fluorescence equipment, and Marc Schottelius for help with the
fluorescence quantum yield measurements.
1
m/z 477 (calculated for M + 1, 477); H NMR, Table 1.
Quantum Yield Measurements. Laser light was utilized for all
quantum yield measurements. For light at 308 nm, either the Lambda
Physik excimer laser described above or an MPB PSX-100 XeCl
excimer laser (10-100 Hz repetition rate, 2.5 ns/pulse) was used. Light
at 325 nm was obtained from an Omnichrome Series 74 continuous-
wave HeCd laser. For wavelength dependence studies, the same Nd:
JA9607852