PROBING PROTEIN STRUCTURE AND INTERACTIONS USING FUNCTIONALIZED NAPHTHALIMIDES L. Kelly, B. Abraham, M. Mullan Department of Chemistry and Biochemistry, University of Maryland, Baltimore County

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PROBING PROTEIN STRUCTURE AND INTERACTIONS USING
FUNCTIONALIZED NAPHTHALIMIDESL. Kelly, B.
Abraham, M. Mullan Department of Chemistry and
Biochemistry, University of Maryland, Baltimore
County
iNTRODUCTION. Biomolecular interactions,
particularly those involving nucleic acids and
proteins with each other or with small molecules,
are ubiquitous and critical to maintaining
structure and function. Work in our laboratory
has focused on using water soluble
1,8-naphthalimides and 1,4,5,8-naphthalene
diimides as protein structural probes. The
compounds initiate a number of photochemical
reactions. It is of interest to develop small
molecules that will cleave, crosslink, or
photoaffinity label proteins and DNA. In this
study, the fundamental reactions with amino acids
and proteins are studied to understand the
mechanisms of protein modification.
Laser Flash Photolysis Studies. Transient
spectroscopy was used to identify the specific
amino acids that are oxidized by the
naphthalimide excited states. The transient
spectrum shown in Figure 2 illustrates that the
oxidation of both tyrosine and tryptophan is
mediated by the 1,8-naphthalimide triplet excited
states (eq 1). When the experiment is conducted
in an air-saturated aqueous solution, the spectra
of the amino acid radicals are cleanly observed.
The rate constants for triplet-mediated electron
transfer were measured using individual amino
acids. Tyrosine and tryptophan were found to be
the only amino acids that could be oxidized by
the triplet state of compound (II). The native
proteins, bovine serum albumin (BSA) and lysozyme
also quenched the triplet state. Radicals were
observed as lysozyme quenching products. The
rate constants for the reaction shown in eq 1
were determined from the bimolecular quenching
plots shown in Figure 3. The rate constants and
radical (cage escape) yields are summarized in
Table 1 (ref. 2).
Amino Acid kq (x10-9 M-1s-1) Radical Yield
Tryptophan 2.60 0.16 0.52 0.04
Tyrosine 0.89 0.03 0.33 0.05
BSA 0.42 0.02 Not observed
Lysozyme 0.76 0.01 0.22 0.01
(a)
(c)
15 ms
EXPERIMENTAL RESULTS. Naphthalene imides and
diimides are synthesized from commercially
available aromatic anhydrides and primary amines.
To date, the anionic, cationic, and polyamine
derivatives shown in Figure 1 have been
synthesized.
0.5 ms
(b)
(d)
FIGURE 3. Bimolecular quenching plots for
reaction of the triplet excited states of
compound (II) with tryptophan (red) and tyrosine
(blue). Inset Triplet-state decay with added
(0 50 mM) tryptophan.
FIGURE 1. Synthetic Structural Probes
15 ms
TABLE 1. Summary of bimolecular rate constants
for and radical yields from the reaction of 3II
with amino acids and proteins.
FIGURE 2. Transient absorption spectrum of (II)
(10 mM) measured in an air-saturated aqueous
solution of (a) 2.25 mM tyrosine and (b)
tryptophan. The spectrum observed at long times
(15 ms) shows similar spectral features as the
known spectrum of the tyrosyl and tryptophan
radicals ((c) and (d), respectively (From ref.
1)).
Photochemistry of Naphthalene Diimides. The
redox properties of the naphthalene imides and
diimides are known. 1,4,5,8-naphthalene diimides
((III) and (IV)) have one-electron reduction
potentials that are ca. 0.5 V more positive than
their 1,8-naphthalimide counterparts (I and II).
Thus, it was of interest to compare their
photoreactivity with amino acids and proteins.
The fluorescence emission spectra of compounds
III and IV were compared. These are shown in
Figure 4. As seen from the figure, the relative
intensity of compound IV (and other carboxylated
naphthalene diimides) is significantly smaller
than that of compound III. The quantum yields
are indicated on the figure.
Photoaffinity Labels. Benzophenone-ligand (drug)
conjugates have been used to probe protein-ligand
interaction sites. Production of the n-p
excited state, followed by hydrogen atom
abstraction and radical recombination, gives rise
to a covalent adduct between the protein and
benzophenone. This is shown in Figure 7. Protein
degradation and fragment analysis is then used to
identify the ligand-protein interaction site.
Unfortunately, benzophenone has a very small
molar extinction coefficient above 300 nm and
requires a high concentration to produce a
reasonable absorption cross-section.
FIGURE 7. Benzophenone-initiated protein
photoaffinity labeling.
(IV) (f 0.017)
(III) (f 0.0009)
Preliminary evidence (Figure 5) suggests that
carboxyalkyl-functionalized 1,4,5,8-naphthalene
diimide derivatives may be well-suited for
photoaffinity labeling reagents. These compounds
have molar extinction coefficients in excess of
20,000 M-1cm-1. The proposed mechanism for
protein photoaffinity labeling is given in Scheme
III.
FIGURE 5. HPLC chromatograph showing separation
of (III) from the protein BSA. Upon irradiation
(l gt 320 nm 0 20 min as indicated) of (III) in
the presence of BSA, the peak at ca. 15 minutes
shows the UV absorption spectrum of (III).
Irradiation of (III), followed by mixing with a
solution of BSA, did not indicate attachment of
the naphthalene diimide to the protein.
FIGURE 4. Fluorescence emission spectra of
aqueous buffered solutions of compounds III and
IV. The solutions were optically matched at the
excitation wavelength of 382 nm. The absolute
fluorescence quantum yields are given.
SCHEME III.
Prospects as Protein Structural Probes.
Compounds (I) (IV) have shown diverse
photochemical reactivity with proteins. As shown
in Figures 2 and 3, the naphthalene imide (II)
predominantly reacts with proteins and amino
acids via one-electron amino acid oxidation in
the absence of oxygen. Gel electrophoresis
confirms that oxidative cross-linking is the
major protein modification product (Figure 6).
The reaction is believed to occur following
oxidation of either tyrosine or tryptophan,
deprotonation to product the neutral radical, and
radical-radical recombination.
The data shown in Figure 4, along with other
experiments (ref. 3) indicate that the singlet
state of compound III is rapidly quenched via
intramolecular electron transfer (Scheme I). By
analogy to published work on phthalimides, we
propose that, following intramolecular electron
transfer, homolytic bond cleavage produces CO2
and the carbon-centered radical. Preliminary
evidence (Figure 5) suggests that this
carbon-centered radical may covalently attach to
a protein.
CONCLUSION. We have shown that naphthalene
imides and diimides can be light-activated to
initiate a variety of protein modification event.
Photoinduced cleavage, cross-linking, and
affinity-labeling are useful tools to probe the
structure and dynamics of proteins. The
development of new photoaffinity labeling agents
with large extinction coefficients offers the
possibility of using these reagents at mM
concentrations.

• ACKNOWLEDGMENTS AND REFERENCES. This work was
  supported by NSF Grant CHE-9984874
• Wagenknecht, H. A. Stempt, E. D.A. Barton, J.
  K. Biochemistry 2000, 39, 5483-5491.
• Abraham, B. Kelly, L. A. J. Phys. Chem. B 2003,
  107, 12534 12541.
• Abraham, B. McMasters, S. Mullan, M. A.
  Kelly, L. A. J. Am. Chem. Soc. 2004, 126,
  4293-4300.

FIGURE 6. Separation of protein photoproducts
using gel electrophoresis (20 SDS/PAGE,
post-stained with coomassie blue). Solutions
contain 80 mM of lysozyme and 50 mM of (II). In
all cases, lanes 1 and 2 contain only lysozyme
(no (II)) samples run in lanes 1 and 3 have been
kept in the dark. Lane 2 has been exposed to
light for the entire duration of photolysis.
Lanes 4, 5, 6, 7 represent lysozyme samples that
have been irradiated for 0.5, 1, 2, and 3 hours
respectively, under nitrogen-saturated
conditions.