Photoinduced electron transfer between non-native donor-acceptor moieties incorporated in synthetic polypeptide aggregates.

Article abstract of DOI:10.1021/ol026656+

A series of analogous photoactive polypeptides that form helical homo and hetero aggregates in aqueous media were prepared. A pyrenyl ketone (the principal chromophore and an electron acceptor) was attached to the N-termini. An electron donor, amidoethylcarbazole, was introduced as a side chain of a non-native amino acid, NCb, 14 residues away. Photoinduced electron-transfer rate constants of the order of 10(8) s(-1) between remote pyrene and carbazole were measured. [structure: see text]

Full text of DOI:10.1021/ol026656+

ORGANIC
LETTERS
2002
Vol. 4, No. 23
4001-4004
Photoinduced Electron Transfer between
Non-Native Donor−Acceptor Moieties
Incorporated in Synthetic Polypeptide
Aggregates
,†
Guilford Jones, II* and Valentine I. Vullev*
Department of Chemistry and Photonics Center, Boston UniVersity,
Boston, Massachusetts 02215
jones@chem.bu.edu
Received August 1, 2002
ABSTRACT
A series of analogous photoactive polypeptides that form helical homo and hetero aggregates in aqueous media were prepared. A pyrenyl
ketone (the principal chromophore and an electron acceptor) was attached to the N-termini. An electron donor, amidoethylcarbazole, was
introduced as a side chain of a non-native amino acid, NCb, 14 residues away. Photoinduced electron-transfer rate constants of the order of
108 s-1 between remote pyrene and carbazole were measured.
Investigation of synthetic photoactive polypeptides is im-
portant to the understanding of the mechanism of initial
charge separation in photosynthesis and of other redox
processes in natural systems.1 Here, we describe the design
and photophysical properties of a 32-residue amphipathic
polypeptide, TT2o (Scheme 1).2 The principal chromophore,
a pyrenyl ketone (OPy), was placed at the N-terminus. An
electron donor, 3-amido-9-ethylcarbazole, was introduced as
a side chain of a non-native asparagine derivative, N4-(9-
ethyl-9H-carbazol-3-yl)-L-asparagine (NCb), 14 residues (49
σ-bonds) away from the pyrenyl ketone (see Supporting
Information for synthetic details). For comparative electron-
transfer studies, polypeptide analogues lacking an electron
donor (containing asparagine (Asn), instead of NCb) were
prepared (e.g., TT2oX15N).
At physiological pH, the 32-residue TT2o contains nine
positive and nine negative charges. Three of the positively
charged residues (two Orn and a Lys) are situated at the
N-terminus, while the rest of the charged amino acids, are
distributed throughout the polypeptide sequence (Scheme 1).
Although such a surface charge distribution creates a field
co-directional with the helix dipole and destabilizes the
expected secondary structure, concomitantly, it will reinforce
the role of the helix dipole moment in propelling photo-
induced electron transfer toward the N-terminus.3
* Email for V.I.V.: vullev@fas.harvard.edu.
† Present address: Department of Chemistry and Chemical Biology,
Harvard University, Cambridge, MA 02138.
(1) For reviews see: Vullev, V. I.; Jones, G, II. Res. Chem. Interm. In
press. Ogawa, M. Y. Mol. Supramol. Photochem. 1999, 4, 113. Mutz, M.
W.; Wishart, J. F.; McLendon, G. L. AdV. Chem. Ser. 1998, 254, 145-
159. Rabanal, F.; Gibney, B. R.; DeGrado, W. F.; Moser, C. C.; Dutton, P.
L. Inorg. Chim. Acta 1996, 243, 213.
(2) The name TT (i.e., Two Towers) is assigned to series of polypeptides
designed to form co-directional helix dimers, e.g., TT1, TT2, etc. The lower-
case letter corresponds to the moiety that caps the N-terminus, e.g., “p” for
pyrene, “o” for oxopyrene, “b” for butyrate, etc.
The leucine zipper (the hydrophobic residues, located at
positions a/a′ and d/d′ in Scheme 1) contains isoleucines and
(3) Fox, M. A.; Galoppini, E. J. Am. Chem. Soc. 1997, 119, 5277.
Galoppini, E.; Fox, M. A. J. Am. Chem. Soc. 1996, 118, 2299.
10.1021/ol026656+ CCC: $22.00 © 2002 American Chemical Society
Published on Web 10/23/2002

gregation of the principal chromophore occurs, characterized
by band broadening, a 5-10 nm red shift, and a decrease in
molar extinction coefficient (Figure 1). Concurrently, upon
aggregation of pyrenyl ketone groups, the monomer fluo-
rescence band (∼450 nm) is quenched and a broader,
significantly weaker band appears at ∼545 nm (Figure 1).7
Larger extinction coefficient values obtained for TT2o in
comparison to those of TT2oX15N at wavelengths shorter
than ∼370 nm are due to absorption of the carbazole moiety.
The aggregation patterns of the principal chromophores of
the polypeptides suggest that their N-termini are in proximity
to each other.8-10
Scheme 1. Wheel Diagram,4-6 Amino Acid Sequences and
Formal Charges for TT2o and Analogous Polypeptides
The two minima (∼207 and 220 nm) observed in the
circular dichroism (CD) spectra of TT2o and its analogues
are characteristic of the high helical content of the polypep-
tides (Figure 2a). The higher molar ellipticity obtained for
TT2o, in comparison with that of TT2oX15N, is ascribed to
an induced CD signal originating from the carbazole moiety,
suggesting that the electron donor is uniquely oriented in
proximity to the polypeptide helices as opposed to a more
loose arrangement of the normally flexible NCb side chain.
CD spectroscopy was employed for examination of the
formation of hetero-aggregates from oppositely charged
polypeptide analogues (Figure 2b). Despite the low helical
content of TT2oX15N, the mixture of the two polypeptides
exhibits a strong CD signal at ∼222 nm. Similar evidence
for aggregation between oppositely charged TT2o analogues
was gathered from fluorescence data (see Supporting Infor-
mation for details).
leucines arranged to favor the formation of a parallel helix
dimer.4 Indeed, size-exclusion chromatographic analysis
showed the presence of only dimers, at micromolar polypep-
tide concentrations (see Supporting Information).
Furthermore, the charges of the residues located next to
the helical hydrophobic interface, i.e., positions e/e′ and g/g′
on the wheel diagram (Scheme 1), were used for electrostatic
reinforcement of helix dimer formation.5 In addition to the
homo-dimers of TT2o and TT2oX15N (formed when e/e′
and g/g′ are positively and negatively charged, respectively)
hetero-dimers, TT2o( and TT2o(X15N, were expected to
be favored structures for equimolar mixtures of oppositely
charged polypeptide analogues (Scheme 1).6
Alkylpyrene derivatives have a high aggregation propen-
sity in aqueous media10,11 and have been favorite units for
photo labeling.3,9,12 The selected pyrenyl ketone, the principal
chromophore, shows a red-shifted absorption relative to the
parent pyrene and serves as a better electron acceptor
(E0OPy/OPy-• ) -1.65 V vs SCE) (see Supporting Informa-
tion for electrochemical data).13,14 The driving force for the
photoinduced electron transfer involving the electron donor,
Comparison between the absorption spectra of γ-oxo-1-
pyrenebutanoic acid (OPBA) and TT2oX15N indicated that
for the polypeptides dissolved in water, ground-state ag-
Figure 1. Absorption and emission properties of TT2o, TT2oX15N, and OPBA in water and DMF. The aqueous samples contained 100
mM phosphate buffer, pH 7, and the DMF samples contained 1 M guanidinium chloride. Inset: fluorescence spectra of TT2o and TT2oX15N,
λex ) 390 nm. OPBA ) γ-oxo-1-pyrene butanoic acid.
4002
Org. Lett., Vol. 4, No. 23, 2002

Table 1. Fluorescence Properties of OPBA and TT2o
Analogues in Trifluoroethanol (TFE) and Aqueous Media
a
d
e
Φfl
τc
(ns)
knr × 108 ket × 108
(æa)b
(s-1
)
(s-1
)
OPBA
TT2o
TFE
0.32
0.20
0.61
1.93h
0.956h
3.30h
2.54h
6.90i
3.46h
2.43h
3.5
8.3
1.2
1.4
waterf
TFE
waterf 0.017
0.70
2.1
(0.59)
0.57
TT2oX15N
TFE
waterf 0.048
1.2
0.70
(0.62) 13.8i
TT2o(
waterg 0.058
2.68h
2.81i
2.66h
6.89i
3.5
(0.50)
0.11
TT2o(X15N waterg
1.4
(0.56)
a Total fluorescence quantum yield, λex ) 390 nm. b The portion of the
total emission assigned to the aggregate fluorescence, æa ) (Saggregate
total)(Atotal/Aaggregate), obtained from deconvolution of the corresponding
/
S
emission spectra, where A is the absorption at λex and S is the integrated
c
d
fluorescence intensity (see Supporting Information). λex ) 337 nm. knr
e
Figure 2. CD spectra of TT2o and its analogues in aqueous
media: (a) TT2o and TT2oX15N, 8 µM, in the presence of 100
mM phosphate buffer, pH 7; (b) TT2o-X15N (10 µM),
TT2o+X15N (10 µM), and TT2(X15N (i.e., 5 µM TT2o-X15N
and 5 µM TT2o+X15N), in the presence of 1 mM phosphate buffer,
pH 6.
) (1 - Φfl)/τ, or for aggregate, knr ) (1 - æaΦfl)/τ. ket ) knr(TT2o) -
knr(TT2oX15N) f 100 mM phosphate buffer, pH 7. g 1 mM phosphate
h
i
buffer, pH 6. λem ) 450 nm. λem ) 580 nm.
pyrenyl ketone moiety behaves identically in TT2o and
TT2oX15N, regardless of the presence of the carbazole.21
For polypeptides aggregates, however, (i.e., in aqueous
media) a more than 2-fold increase in the value of the
nonradiative rate constants was observed when the electron
donor is present.22 We ascribe this alteration of knr to long-
range electron entrainment from NCb to OPy (Scheme 1);
the corresponding charge-transfer rate constants, ket, were
calculated from the difference between knr obtained for
polypeptides with and without an electron donor (Table 1).
In polypeptide media (i.e., â ≈ 1.4 Å-1)23 the observed
electron-transfer rates (i.e., ket ∼108 s-1) can be achieved
when the donor-acceptor distance is about 8-10 Å.
However, if through-space electronic coupling is excluded
as a possibility, the charge-transfer pathways should involve
at least four hydrogen bonds,24 suggesting a highly organized
polypeptide structure in the region of the redox moieties.
(see Supporting Information for details).25
amidoethylcarbazole, and the principal chromophore is ∆Get
) - 0.54 eV.13-15
Significant quenching of the red-shifted fluorescence
(ascribed to the pyrenyl ketone aggregate) was observed for
TT2o analogues in water, when the carbazole moiety is
present (Figure 1, inset). For organic solvents, where the
polypeptides exist only as monomers, this quenching phe-
nomenon was not detected. The fluorescence quantum
yields16-18 and lifetimes19,20 for the various polypeptide
analogues and OPBA in trifluoroethanol (TFE) and buffered
water are summarized in Table 1. The calculated values for
nonradiative decay rate constants, knr, indicate that the
(4) Harbury, P. B.; Zhang, T.; Kim, P. S.; Alber, T. Science 1993, 262,
1401.
(5) McLachlan, A. D.; Stewart, M. J. Mol. Biol. 1975, 98, 293. Lumb,
K. J.; Kim, P. S. Science 1995, 268, 436.
(6) O’Shea, E. K.; Lumb, K. J.; Kim, P. S. Curr. Biol. 1993, 3, 658.
(7) Armbruster, C.; Knapp, M.; Rechthaler, K.; Schamschule, R.; Parusel,
A. B. J.; Ko¨hler, G.; Wehrmann, W. J. Photochem. Photobiol., A 1999,
125, 29.
(8) Daugherty, D. L.; Gellman, S. H. J. Am. Chem. Soc. 1999, 121, 4325.
(9) Jones, G., II.; Vullev, V. I.; Braswell, E.; Zhu, D. J. Am. Chem. Soc.
2000, 122, 388.
(10) Jones, G., II.; Vullev, V. I. Org. Lett. 2001, 3, 2457.
(11) Jones, G., II.; Vullev, V. I. J. Phys. Chem. A 2001, 105, 6402.
(12) Garc´ıa-Echeverr´ıa, C. J. Am. Chem. Soc. 1994, 116, 6031.
(13) The Rehm Weller equation14 was used to estimate ∆Get: E00(OPy)
≈ 3.1 eV.; the oxidation potential of the donor, E0(Cb+•/Cb) ) 0.91 V vs
SCE.15
(14) Rehm, D.; Weller, A. Israel J. Chem. 1970, 8, 259.
(15) Ambrose, J. F.; Carpenter, L. L.; Nelson, R. F. J. Electrochem.
Soc. 1975, 122, 876.
To test if electron transfer, yielding radical-ion intermedi-
ates, is indeed responsible for the increase in nonradiative
(19) Although for TT2o and TT2o( at 337 and 355 nm up to ∼20% of
the excitation energy is absorbed by the electron donor, i.e., the carbazole
(Cb), we have a reason to believe that the energy (or an electron) is
transferred from Cb to the pyrenyl ketone in less than a nanosecond since
no carbazole emission or triplet formation has been detected.
(20) James, D. R.; Siemiarczuk, A. ReV. Sci. Instrum. 1992, 63, 1710.
(21) The emission of OPBA appears to be quenched, probably because
of the carboxylate that is 3 σ-bonds away from the chromophore.
(22) From emission data it was deduced that in aqueous medium more
than ∼85% of the principal chromophore of the polypeptides is in aggregated
form and, hence, almost all of the excitation energy is absorbed by
aggregated pyrenyl ketone. Therefore, the analysis of the fluorescence
quenching is concentrated solely on the red-shifted band of the emission
spectra (see Supporting Information for details).
(16) The fluorescence quantum yields were calculated from emission
data of samples with known optical density at the excitation wavelength,
λex ) 390 nm.17 Coumarin 102 in ethanol, Φfl ) 0.95,18 was applied as a
standard.
(23) Moser, C. C.; Dutton, P. L. Biochim. Biophys. Acta 1992, 1101,
171.
(17) Demas, J. N.; Crosby, D. A. J. Phys. Chem. 1971, 75, 991.
(18) Jones, G., II.; Rahman, M. A. J. Phys. Chem. 1994, 98, 13028.
(24) Beratan, D. N.; Skourtis, S. S. Curr. Opin. Chem. Biol. 1998, 2,
235.
Org. Lett., Vol. 4, No. 23, 2002
4003

decay rates, transient absorption studies were conducted using
nanosecond laser flash photolysis.26 When TT2o(X15N was
flash-photolyzed, only the OPy triplet (at ∼440 nm) was
observed (Figure 3a and c). However, identical photoexci-
Information for assignments of the transient spectra.) The
radical transients decayed in ∼20 µs, with a simultaneous
rise in the OPy triplet absorption at 440 nm (Figure 3d).
Then both signals peaking at 440 and 500 nm decayed with
rates comparable to that of the loss of the OPy triplet
signature (Figure 3c and d).27 These observations suggest
that the back electron transfer in TT2o systems leads to
pyrenyl ketone triplet formation, a finding that is energeti-
cally feasible since the OPy triplet state lies about 2.2 eV7
above the ground state and 0.36 eV below the charge-
separated state.
In summary, we have demonstrated that the medium
provided by a polypeptide helical aggregate can mediate an
efficient electron transfer between redox moieties that are
well separated along a 32-residue polypeptide chain. Charge
recombination that leads to a triplet-excited state is observed.
Included also is the first report of a convenient pyrenyl ketone
macromolecular photoprobe, as well as a straightforward
procedure for preparation and introduction of a chromphoric
amino acid, NCb, in a polypeptide via solid-phase synthesis
(see Supporting Information).
Acknowledgment. This research has been supported by
the Department of Energy, Division of Basic Energy Sci-
ences. We also extend our gratitude to Ms. Yiping Li for
her involvement with the gel filtration analysis.28
Supporting Information Available: Experimental sec-
tion and additional photochemical and electrochemical data
on various pyrene and carbazole derivatives. This material
is available free of charge via the Internet at http://pubs.acs.org.
Figure 3. Transient absorption data for TT2o( and TT2o(X15N
in argon-purged aqueous medium. Transient spectra of (a)
TT2o(X15N, 12 µM; and (b) TT2o(, 8 µM. The corresponding
decays were fit to biexponential functions: (c) for TT2o(X15N,
τ1 ) 150 µs, τ2 ) 830 µs; (d) for TT2o(, at 440 nm, τrise ) 19 µs
and τfall ) 820 µs, and at 500 nm τ1 ) 23 µs and τ2 ) 780 µs. (1
mM phosphate buffer, pH 6; λex ) 355 nm, 8 ns pulse, 10 mJ/
pulse).
OL026656+
(25) In such systems, the electronic coupling and the nature of the
chromophore aggregates (e.g., geometry and hydrogen bonding) can be
strongly influenced by even slight conformational changes explaining 2-fold
difference in emission lifetimes and the 3-fold difference in ket observed
for analogous homo and hetero polypeptide aggregates (Table 1).
(26) Jones, G., II.; Vullev, V. I. Photochem. Photobiol. Sci. In press.
Vullev, V. I. PhD Dissertation, Boston University, 2001, pp 205-209 and
571-582.
(27) Note that the absorbances of OPy triplet and radical-anion overlap
(Figure 3). Hence, the fast component at 500 nm is ascribed to the radical
decay, and the slow component to the triplet (Figure 3b and d).
(28) This is article No. 13 in the series Photoactive Polypeptides from
Boston University.
tation of TT2o( yielded, at early times, a strong transient
(λmax ∼500 nm) and a broad band at ∼650 nm, which are
ascribed to the pyrenyl ketone radical-anion and carbazole
radical-cation, respectively (Figure 3b).19 (See Supporting
4004
Org. Lett., Vol. 4, No. 23, 2002