Long-lived charge-separated species observed on flash photolysis of peptide conjugates. Interplay of local and radical ion pair triplet states

Article abstract of DOI:10.1021/jo981241g

Peptides composed of alanine (Ala) and tryptophan (Trp), modified with the (nitro)pyrenesulfonyl chromophore (Pyr and NPyr) at the N-terminus have been examined by nanosecond laser flash photolysis. A common phototransient for Pyr-AlaOEt and Pyr-Ala-TrpOEt was observed that exhibited broad absorption at 410-550 nm and decay time constants in the range, τ(1/2) = 20- 40 μs. This species was assigned to the triplet excited state that is local to the pyrene chromophore (³Pyr). For the conjugates having a stronger electron acceptor group at the N-terminus, NPyrAla-TrpOEt and NPyr-Ala-Ala- TrpOEt, the local triplet was replaced with a phototransient whose principal feature is a sharp band at 440 nm (assigned to the NPyr^-. radical anion). The radical ion transients for the NPyr peptide derivatives were assigned to intermediates that result from the intramolecular electron transfer quenching of NP excited species by pendant groups (i.e., the indole ring of tryptophan). The lifetimes observed for the radical ion transients associated with the NPyr series were relatively long (extending to ca. 400 ns) and depended in an interesting way on the structure of the peptide linkage. A mechanism of electron transfer in the singlet manifold and recombination yielding a local Pyr triplet state is important for the Pyr series.

Full text of DOI:10.1021/jo981241g

8938
J . Org. Chem. 1998, 63, 8938-8945
Lon g-Lived Ch a r ge-Sep a r a ted Sp ecies Obser ved on F la sh
P h otolysis of P ep tid e Con ju ga tes. In ter p la y of Loca l a n d Ra d ica l
Ion P a ir Tr ip let Sta tes1
Guilford J ones II* and Lily N. Lu
Department of Chemistry, and the Center for Photonics, Boston University,
Boston Massachusetts 02215
Received J une 25, 1998
Peptides composed of alanine (Ala) and tryptophan (Trp), modified with the (nitro)pyrenesulfonyl
chromophore (Pyr and NPyr) at the N-terminus have been examined by nanosecond laser flash
photolysis. A common phototransient for Pyr-AlaOEt and Pyr-Ala-TrpOEt was observed that
exhibited broad absorption at 410-550 nm and decay time constants in the range, τ1/2 ) 20-40 µs.
This species was assigned to the triplet excited state that is local to the pyrene chromophore
(3Pyr). For the conjugates having a stronger electron acceptor group at the N-terminus, NPyr-
Ala-TrpOEt and NPyr-Ala-Ala-TrpOEt, the local triplet was replaced with a phototransient whose
principal feature is a sharp band at 440 nm (assigned to the NPyr-• radical anion). The radical
ion transients for the NPyr peptide derivatives were assigned to intermediates that result from
the intramolecular electron transfer quenching of NP excited species by pendant groups (i.e., the
indole ring of tryptophan). The lifetimes observed for the radical ion transients associated with
the NPyr series were relatively long (extending to ca. 400 ns) and depended in an interesting way
on the structure of the peptide linkage. A mechanism of electron transfer in the singlet manifold
and recombination yielding a local Pyr triplet state is important for the Pyr series.
In tr od u ction
In recent communications, we have reported on the
synthesis and photochemical properties of a series of
modified peptides composed of alanine (Ala) and tryp-
tophan (Trp) amino acid residues.6-8 This series was
constructed for the purpose of investigating photoinduced
intramolecular electron transfer that occurs between an
electron acceptor group (and light-absorbing unit) that
is attached to the N-terminus of selected peptides having
1-3 amino acid links and pendant electron donors. In
several of these examples, the tryptophan residue played
a direct role as electron donor due to the electroactivity
of its side-chain indole ring [E° (Trp+• f Trp) ) 1.0 V vs
saturated calomel electrode (SCE)]. Particular attention
was paid to the rates of back electron transfer for the
series:
A variety of systems that invoke the peptide link for
connecting electron donors and acceptors have been
reported in recent years.2-7 A principal finding in much
of this work is that a low-lying excited state or redox
intermediate associated with a metal center or an organic
chromophore is involved in a forward electron transfer
step. For the organic chromophores, singlet quenching
(and a presumed formation of singlet radical ion pairs)
has been the most common mechanism. In several
peptide systems, particularly those involving transition
metal centers, the lifetimes of electron-transfer interme-
diates have been investigated, and dependences on
donor-acceptor distance, thermodynamic driving force,
temperature, and other experimental variables have been
obtained.4 The issue of spin multiplicity for either
precursor or radical(ion) pair states, however, is not
readily assessed for the donor-acceptor systems having
heavy metal atoms, either because radical pair states are
not involved or because of the complication that results
from strong mixing of spin states for such systems.
Pyr-• - peptide - donor+•
f
Pyr - peptide - donor (1)
Reported separately7,8 are results having to do with
intramolecular electron transfer that occurs between
pyrenesulfonyl (electron acceptor) chromophores (e.g.,
Pyr) and donor groups for a family of peptides, including
Trp residues, investigated using flash photolysis methods
and conducted on the picosecond time scale. The general
trends are consistent with a moderate falloff in intra-
molecular electron-transfer rate with (through-bond)
distance along the peptide backbone, a dependence on
driving force, and a modest dependence on the presence
of Ala vs Trp in the peptide sequence.7,8 Decay times for
(1) Paper no. 8 in the series, Photoactive Peptides.
(2) Isied, S. S.; Ogawa, M. Y.; Wishart, J . F. Chem. Rev. 1992, 92,
381; Kozlov, G. V.; Ogawa, M. Y. J . Am. Chem. Soc. 1997, 119, 8377;
Ogawa, M.. Y.; Wishart, J . F.; Young, Z.; Miller, J . R.: Isied, S. S. J .
Phys. Chem. 1993, 25, 569.
(3) Fox, M. A.; Galoppini, E. J . Am. Chem. Soc. 1997, 119, 5277;
Inai, Y.; Sisido, M.; Imanishi, Y.; J . Phys. Chem. 1991, 95, 3847; Sisido,
M.; Inai, Y.; Imanishi, Y. Macromolecules 1990, 23, 1665; Schanze, K.
S.; Cabana, L. A. J . Phys. Chem. 1990, 94, 2740.
(4) See, for example, Slate, C. A.; Striplin, D. R.; Moss, J . A.; Chen,
P.; Erickson, B. W.; Meyer, T. J . J . Am. Chem. Soc. 1998, 120, 4885;
Ogawa, M. Y.; Moreira, I.; Wishart, J . F.; Isied, S. S. Chem. Phys. 1993,
176, 589.
(5) Werner, U.; Wiessner, A.; Kuhnle, W.; Staerk, H. J . Photochem.
Photobiol., A 1995, 85, 77; Anglos, D.; Bindra, V.; Kuki, A. J . Chem.
Soc., Chem. Commun. 1994, 213; Basu, G.; Anglos, D.; Kuki, A.
Biochemistry 1993, 32, 3067.
(6) J ones, G., II.; Farahat, C. W.; Oh, C. J . Phys. Chem. 1994, 98,
6906; J ones, G. II; Farahat, C. Res. Chem. Intermed. 1994, 20, 855
(7) J ones, G., II.; Lu, L. N.; Vullev, V.; Gosztola, D. J .; Greenfield,
S. R.; Wasielewski, M. R. Bioorg. Med Chem. Lett. 1995, 5, 2385.
(8) J ones, G., II.; Lu, L. N.; Oh, C.; Mari, F.; Gosztola, D. J .;
Greenfield, S. R.; Wasielewski, M. R, in preparation.
10.1021/jo981241g CCC: $15.00 © 1998 American Chemical Society
Published on Web 10/29/1998

Species Observed on Flash Photolysis
J . Org. Chem., Vol. 63, No. 24, 1998 8939
radical ion pair states varied for this series of measure-
ments in the range of 10 ps to 1 ns. Data of this type
provide reference points regarding the formation and
decay of radicals derived from tryptophan, intermediates
that been identified in studies of native proteins and
other model systems containing Trp residues.9
We report here phototransient data for a peptide series
that is engaged in long-range electron transfer and for
which the effects of spin for the precursor and radical
ion pair states can be readily distinguished. Laser flash
photolysis on the nanosecond time scale has confirmed
that electron transfer takes place for a series of simple
di- and tripeptides containing the Trp residue and
bearing the pyrenesulfonyl chromophore. The appear-
ance of long-lived intermediates depends critically on the
relative energies of the local triplets [for substituted
pyrenes, ET ) 46-48 kcal/mol (ca. 2.1 eV)] and the
radical ion pair species (e.g., Pyr-• - link- Trp+•). For
the derivatives bearing the 8-nitro-1-pyrenesulfonamide
(NP) moiety, we find that a larger driving force for
electron transfer results in a positioning of the radical
pair state below the local triplet. For peptides in which
donor and acceptor groups are separated nominally by
1.0-1.3 nm (average edge-to-edge distances associated
with randomly distributed conformations7,8), the decay
times for radical pair intermediates are relatively long,
reaching the microsecond domain. The present series has
special value, because the singlet and triplet routes for
electron transfer and the consequent influence of spin
multiplicity on the lifetime of radical-pair intermediates
are delineated.
F igu r e 1. Absorption spectra for 10 µM Pyr-AlaOEt and 20
µM NPyr-AlaOEt in acetonitrile.
Ta ble 1. F lu or escen ce a n d P h ototr a n sien t Da ta for
(Nitr o)P yr en esu lfon a m id e Con ju ga tes
transient
(nm)b
k-et
compound
Pyr-AlaOEt
Pyr-Ala-TrpOEt
NPyr-AlaOEt
Φfa
τ (τ1/2), µs (106 s-1
)
0.31
430 (3Pyr)
(38)
(20)
0.014 430 (3Pyr)
0.0040 450 (3NPyr) 12.2 (6.5)
NPyr-Ala-TrpOEt
0.0006 440 (NPyr-•
)
)
0.40
0.16
0.20
2.5
6.2
5.0
NPyr-Ala-Ala-TrpOEt 0.0009 440 (NPyr-•
515 (Trp+•
)
a
Resu lts a n d Discu ssion
Aerated samples (10-20 µM in CH3CN, 20 °C); λexc ) 330-
b
350 nm. Nd/YAG laser (355 nm) irradiation of 10-20 µM
conjugates in Ar-purged CH3CN.
P yr en esu lfon a m id e Con ju ga tes: Absor p tion a n d
F lu or escen ce P r op er ties. The selected peptides, as
C-terminal ethyl esters, were prepared from commer-
cially available N- and C-terminal protected L-amino
acids, using standard peptide coupling methodology and
1-pyrenesulfonyl chloride as the agent for conjugation of
the pyrene chromophore at N-termini.10 The nitro-
pyrenesulfonyl series was prepared starting with mono-
nitration of Pyr-AlaOEt which introduced (preferentially)
displayed UV spectra that showed a small perturbation
arising from introduction of the Trp moiety (ca. 280 nm)
and prominent bands associated with Pyr moieties
(Figure 1, for Pyr-AlaOEt, ꢀ350 ) 16,000 M-1 cm-1
,
acetonitrile solvent). The fluorescence observed for the
Pyr conjugates (λmax ) 382 and 400 nm) revealed an
emission quantum efficiency for acetonitrile solutions
that depended on the tethering of Trp residues (Table
1). The emission data are consistent with the quenching
of the singlet state for those peptides containing an
electroactive substituent (the indole ring of tryptophan,
Trp).6-8 The introduction of a nitro group in the 8-posi-
tion of the 1-pyrenesulfonamide chromophore led to
substantial alteration in photophysical properties, in-
cluding red-shifts in the principal absorption maxima
(Figure 1). Fluorescence appears with λmax ) 430 nm for
the NPyr series, with quantum efficiencies that are
diminished (Table), consistent with the established
effects of a nitro substituent on the parent pyrene
chromophore and related hydrocarbons (more efficient
intersystem crossing, vide infra).11,12
(9) Burdi, D.; Aveline, B. M.; Wood, P. D.; Stubbe, J .; Redmond, R.
W. J . Am. Chem. Soc. 1997, 119, 6457; Kim, S. T.; Sancar, A.;
Essenmacher, C.; Babcock, G. T. Proc. Natl. Acad. Sci. U.S.A. 1993,
90, 8023; Bobrowski, J .; Holcman, J .; Poznanski, J .; Ciurak, M.;
Wierzchowski, K. L. J . Phys. Chem. 1992, 96, 10036; Lee, H.; Faraggi,
M. H. Biochim. Biophys. Acta 1992, 1159, 286; Mishra, A. K.;
Chandrasekar, R.; Faraggi, M.; Klapper, M. H. J . Am. Chem. Soc. 1994,
116, 1414; Faraggi, M.; DeFellipis, M. R.; Klapper, M. H. J . Am. Chem.
Soc. 1990, 112, 5640; J ones, G., II.; Vullev, V.; Lu, N. L., in preparation.
(10) Hirashima, Y. et al. Biochim Biophys. Acta 1990, 1047, 35.
a nitro group at the 8-position of the pyrene chromophore
(see Experimental Section for details). The conjugates
of 1-pyrenesulfonic acid, Pyr-AlaOEt and Pyr-Ala-Trp,

8940 J . Org. Chem., Vol. 63, No. 24, 1998
J ones and Lu
F igu r e 2. Transient absorption spectra for (a) 10 µM Pyr-AlaOEt and (b) 20 µM NPyr-AlaOEt in Ar-purged acetonitrile (λex
355 nm).
)
F la sh P h otolysis of P yr en esu lfon a m id e Con ju -
ga tes. Pulsed laser excitation of Pyr-AlaOEt at 355 nm
(Nd:YAG laser, 80 mJ /7-ns pulses) resulted in the spectra
shown in Figure 2a. The decay half-life for this transient
observed at 430 nm for Ar-purged solutions was about
40 µs; aeration of the acetonitrile samples resulted in a
diminution of the decay time to the submicrosecond
regime (a first-order fit of the decay data for aerated
solutions yielded τ ) 0.23 µs). The data for Pyr-AlaOEt
are consistent with the assignment of the phototransient
observed in the 100 ns-100 µs regime to the local triplet
excited state associated with the pyrene chromophore
(λmax ) 420 nm11-13). Notably absent in the transient
spectra for Pyr-AlaOEt is an absorption that has been
identified with the radical anion species, Pyr-•; this
transient, a narrow band centered at 490 nm,7,8 in turn
is similar to the radical anion of the parent hydrocarbon,
pyrene (λmax ) 490; ꢀ ) 49,000 M-1 cm-1) and several
simple pyrene derivatives.13
Spectra obtained on flash photolysis of Pyr-Ala-TrpOEt
contrasted with those for Pyr-AlaOEt; an inspection of
the earliest possible time domain that could be inter-
rogated with nanosecond resolution was informative
(Figure 3). The transient behavior observed in the first
200-ns time interval after the laser pulse showed (1) a
bleach of the absorption due to the ground state of Pyr-
Ala-TrpOEt (at 350 nm); (2) sharp bands associated with
residual (local pyrene) fluorescence at 380 nm that
dissipate completely within 50 ns; (3) a small feature at
490 nm in the early time frame that is likely to be due
to absorption by Pyr-•; and (4) growth of a broader
transient absorption (λmax ) 425 nm) and other features
that appear similar to those observed for the longer time
domains for Pyr-AlaOEt (those assigned to the local
triplet, Figure 2a).
F igu r e 3. Transient absorption spectra for 10 µM Pyr-Ala-
TrpOEt in air-saturated acetonitrile. Negative ∆OD corres-
ponds to fluorescence decay at 380 nm for shorter time domain;
the longer time domain featuring triplet absorption at 430 nm
appears in the inset (λex ) 355 nm).
positioning of electronic state energies, as shown in
Scheme 1. Unlike the simply substituted model com-
pound, Pyr-AlaOEt, which gives rise to triplets via direct
intersystem crossing, Pyr-Ala-TrpOEt participates in
electron transfer in the singlet manifold leading to a
decrease in fluorescence yield and lifetime [for Pyr-
AlaOEt and Pyr-Ala-TrpOEt (acetonitrile), lifetimes of
11.5 and 0.88 ns have been obtained8,14]. The radical ion
pairs thus formed (accounting for >90% of nonradiative
singlet decay) lie in energy above the locally excited
The transient behavior for the Pyr series can be
understood in terms of a mechanism of electron transfer
and charge recombination suggested by the relative
(14) The fluorescence lifetime observed for Pyr-Ala-TrpOEt only
partially reflects the degree to which singlet quenching by the remote
Trp leads to the radical ion pair state. According to pulse-probe
measurements with subpicosecond resolution,7,8 the rise times for the
radical pair state via the singlet manifold can be shorter than 10 ps.
The variations are due either to the distribution of conformations for
the peptides, some of which undergo more rapid electron transfer,5 or
to direct excitation of a weak CT absorption observed at longer
wavelengths around 400 nm corresponding to direct population of the
radical ion pair state.8
(11) Scheere, R.; Henglein, A. Ber. Bunseng.-Ges. Phys. Chem. 1977,
12, 1234.
(12) Borg, R. A. J . J . Phys. Chem. 1994, 98, 11439.
(13) Gratzel, M.; Kalyanasundaram, K.; Thomas, J . K. J . Am. Chem.
Soc. 1974, 96, 7869.

Species Observed on Flash Photolysis
J . Org. Chem., Vol. 63, No. 24, 1998 8941
Sch em e 1
3
triplet species, Pyr*. Intersystem crossing for singlet
radical ion pairs is possible either via direct conversion
to the local triplet or via (an equilibrium with) the triplet
radical ion pair state. For the latter path, decay to the
local triplet occurs by way of a spin-allowed charge
recombination.
This type of electron-transfer-induced intersystem
crossing has been reported previously for linked electron
donor-acceptor systems.15 The relative energies re-
quired for the mechanism shown in Scheme 1 are derived
from the following parameters: (1) the singlet energy
associated with the pyrenesulfonamide chromophore
(1Pyr*) (3.37 eV) obtained from the frequency at the
intersection of absorption and emission spectra; (2) the
reduction potential for Pyr-AlaOEt, obtained from cyclic
voltammetry of the alanine conjugate (E1/2 ) -1.69 V vs
SCE16); and (3) the tryptophan reduction potential (for
Trp+• f Trp, E° ) 1.0 V vs SCE) derived from data for
radical(ion) equilibria measured by pulse radiolysis.17
Flash P h otolysis of Nitr opyr en esu lfon am ide Con -
ju ga tes. On flash excitation NPyr-AlaOEt provided a
broadly absorbing transient (Figure 2b), the yield of
which was higher than that observed for Pyr-AlaOEt, in
accord with a higher yield of intersystem crossing for the
nitro compound.11 The half-life for this transient was 6.5
µs for Ar-purged acetonitrile solutions; oxygen again was
effective in quenching this species (τ ) 0.27 µs for air-
saturated acetonitrile solutions). The broadly absorbing
transient observed on flash photolysis of NPyr-AlaOEt
was compared with that reported for irradiation of
1-nitropyrene (a discernible peak at 450 nm and a broad
absorption feature at 520-620 nm with ꢀ530 ) 10,000 M-1
cm-1)11; the strong similarities confirmed the assignment
F igu r e 4. Transient absorption spectra for (a) 20 µM NPyr-
Ala-TrpOEt and (b) 20 µM NPyr-AlaOEt with 10 mM Cbz-
TrpOEt (Ar-purged acetonitrile; λex ) 355 nm).
nm (Figure 4a)] and (first-order) decay times in the
submicrosecond regime (τ ) 0.40 and 0.16 µs, respec-
tively; vide infra). These bands appeared to be virtually
identical with that observed for the radical anion of
nitropyrene (ꢀ440 ) 24,000 M-1 cm-1).11 Less well resolved
were weaker bands that appeared at 510-580 nm for
NPyr-Ala-TrpOEt and for NPyr-Ala-Ala-TrpOEt.
A
summary regarding the identification of transients and
decay data for the various peptide derivatives is pre-
sented in the Table.
Several control experiments further verified that the
transients that appeared in the microsecond time domain
were correctly ascribed to electron transfer intermediates.
Flash photolysis of NPyr-AlaOEt in the presence of a
tryptophan derivative, benzyloxycarbonyltryptophan
ethyl ester (Cbz-TrpOEt), gave rise to the spectra shown
in Figure 4b. The small feature that is discernible at
515 nm is most readily associated with the radical species
that results from one-electron oxidation of tryptophan
(Trp).17,18 The data are consistent with the formation of
nitropyrenesulfonamide triplet for NPA in relatively high
yield (a quantum yield of intersystem crossing, Φisc ) 0.6,
has been reported for 1-nitropyrene11) and effective
bimolecular electron-transfer quenching by Cbz-TrpOEt.
For the tryptophan moiety, the absorption properties of
3
of the triplet NPyr* intermediate.
Spectral data for the NPyr conjugates with the elec-
troactive amino acid substituent (Trp) led again to
significantly different results. Laser irradiation of
acetonitrile solutions at 355 nm of NPyr-Ala-TrpOEt and
NPyr-Ala-Ala-TrpOEt resulted in strongly absorbing
phototransients having well-defined maxima [λmax ) 440
the radical-cation (λmax ) 560 nm; ꢀ ) 3,000 M-1 cm-1
)
have been distinguished from the deprotonated radical
species (λmax ) 510 nm; ꢀ ) 2,000 M-1 cm-1) in pulse
radiolysis and other transient studies of aqueous solu-
tions, for which a pKa of the Trp+• species in water (4.3)
has been determined.17,18 The present data regarding the
weaker absorbing transients for NPyr peptide derivatives
in the 500-600 nm range are less compelling for precise
(15) See Roest, M. R.; Oliver, A. M.; Paddon-Row, m. N.; Verhoeven,
J . W. J . Phys. Chem., A 1997, 101, 4867; van Willigen, H.; J ones, G.
II; Farahat, M. S. J . Phys. Chem., A 1996, 100, 3312; Liddell, P. A.;
Kuciauskas, D.; Sumida, J . P.; Nash, B.; Nguyen, D.; Moore, A. L.;
Moore T. A.; Gust, D. J . Am. Chem. Soc. 1997, 119, 1400; Hasharoni,
K.; Levanon, H.; Greenfield; Gosztola, D. J .; Svec, W. A.; Wasielewski,
M. R. J . Am. Chem. Soc. 1996, 118, 10228, and refs cited therein.
(16) J ones, G. II; Vullev, V. unpublished results.
(17) De Felippis, M. R.; Murthy, C. P.; Broitman, F.; Weinraub, D.;
Faraggi, M.; Klapper, M. H. J . Phys. Chem. 1991, 95, 3416. Harriman,
A. J . Phys. Chem. 1987, 91, 6102.
(18) Merenyi, G.; Lind, J .; Shen, X. J . Phys. Chem. 1988, 92, 134.
Bryant, F. D.; Santus, R.; Grossweiner, L. I. J . Phys. Chem. 1975, 79,
2711

8942 J . Org. Chem., Vol. 63, No. 24, 1998
J ones and Lu
assignment with regard to state of protonation. Contri-
butions from both radical and radical cation forms in the
time domain near 1 µs are possible; we are unaware of
an independent assignment for the Trp radical(ion)
species for dry acetonitrile solvent.20
The feasibility of the electron-transfer step involving
NPyr excited states and Trp can be assessed from
parameters that include the triplet state energy for the
nitropyrenesulfonyl chromophore [after the value for
nitropyrene, ET ) 46 kcal/mol (2.0 eV)],16 and the
reduction potential recorded for NPyr-AlaOEt (E1/2
)
-0.73 V vs SCE)16 and for Trp (vide supra). The
resulting energetics data (Scheme 1) show that the
driving force for electron transfer is clearly more favor-
able for the combination, NPyr-AlaOEt and Trp vs
Pyr-AlaOEt and Trp. The effectiveness of excited singlet
state quenching of NPyr-AlaOEt by Cbz-TrpOEt (in a
bimolecular process) was assessed from fluorescence
quenching data. The quenching of the emission of NPyr-
AlaOEt at 430 nm on addition of Cbz-TrpOEt (1.8-20
mM, acetonitrile solvent) yielded as Stern-Volmer plot
with slope ) 0.062 M-1. Using a lifetime for the singlet
of NPyr-AlaOEt, τ ) 8.0 ps,8 a bimolecular quenching
constant was determined, k ) 7.7 × 109 M-1 s-1, a value
near that expected for a diffusion-limited rate.
The fate of radical(ion) species observed on nanosecond
flash photolysis of NPyr-Ala-TrpOEt and NPyr-Ala-Ala-
TrpOEt can be understood most readily in terms of return
to ground state species via back electron transfer. The
peptide conjugates were relatively resilient to photo-
bleaching on repetitious pulsed photolysis. Within a
3-fold concentration range, the decay times were not
dependent on peptide concentration. The depletion of the
440-nm transient for the peptides was examined in detail
using trial fits of the decay data to first and second order
and multicomponent decay parameters. The most sat-
isfactory fits, those associated with the simple first-order
rate law, yielded lifetimes and rate constants (in the
range of 106 s-1) as shown in the Table. The decay of
transients at 440 and 515 nm appeared to be simulta-
neous, again consistent with a mechanism of depletion
for the transients involving charge recombination.
Notably, the decay of the radical ion pair intermediate
was somewhat faster for the longer peptide. Also, for
solutions of NPyr-Ala-TrpOEt, the 440-nm transient
decayed more readily when aerated (τ ) 0.12 µs); for
undegassed samples the ratio of the absorbances at 440/
(510-560) decreased also, consistent with the intercep-
tion of the radical ion pair state by molecular oxygen (and
electron transfer from NPyr-• to O2).21
F igu r e 5. Histogram displaying distributions of relative
energies (top) and center-to-center distances (bottom) of Pyr-
Ala-TrpOEt conformations from computer simulations. (a)
Aerated samples (10-20 µM in CH3CN, 20 °C); λexc ) 330-
350 nm. (b) Nd:YAG laser (355 nm) irradiation of 10-20 µM
conjugates in Ar-purged CH3CN.
arrays of energies for individual conformations, or group-
ings in histogram form according to energy and critical
distances. The important distances between putative
electron donor and acceptor groups, defined in terms of
spans through space between points “center-to-center”
(c-c) for aromatic rings and distances through bonds
between these same groups, “edge-to-edge” (e-e) (C1 of
pyrene and C3 of the indole ring) were computed as an
average to be 0.88 and 1.2 nm, respectively. A similar
set of population/energy/distance data was obtained for
the homologous peptide, Pyr-Ala-Ala-TrpOEt, which also
served as a model for the nitro derivative; the simulations
provided for the homologous peptide c-c and e-e dis-
tances of 1.1 and 1.5 nm, respectively.
The modeling results tended to confirm that long-range
ordering of the chains (e.g., as with R-helices) or favor
for particular conformations (i.e., folded vs extended) is
not important for the short peptide chains under inves-
tigation, in accord with 2D-NMR (nuclear Overhauser
effect) findings that are reported separately.8 This
determination is important because folded forms would
introduce a mechanism of electron transfer that could be
dominated by through-space interaction if distances
between electroactive pendants for highly favored con-
formations were as close as 5 Å.22 Mechanisms that use
through-bond interaction have been shown to be quite
effective in providing pathways for electron transfer via
P ep tid e Con for m a tion a l An a lysis. The peptide,
Pyr-Ala-TrpOEt, was selected for molecular modeling,
particularly with regard to the discovery of any confor-
mational preferences shown by the peptide series.
Geometries and distance relationships with respect to the
Pyr chromophore and the potential electron donor group,
the indole ring of tryptophan residues, were established.
The distribution of conformations for Pyr-Ala-TrpOEt
obtained from the simulations shown in Figure 5 includes
(19) Creed, D. Photochem. Photobiol. 1984, 39, 537.
(20) A detailed examination of spectra at higher resolution for the
model system, NPyr-AlaOEt and Cbz-TrpOEt in dry acetonitrile,
showed only the single distinct (although weak) feature at 515 nm at
20 µs and longer times after a 355-nm laser pulse; Lu, N., Dissertation,
Boston University, 1995, chap 4.
(21) See, for example, J ones, II, G.; Malba, V.; Bergmark, W. R. J .
Am. Chem. Soc. 1986, 108, 4214.

Species Observed on Flash Photolysis
J . Org. Chem., Vol. 63, No. 24, 1998 8943
long-range orbital overlaps or tunneling paths.22,23 In
terms of previous studies on peptides, rate data for
electron transfer between terminal groups held via
oligopeptide spacers (e.g., prolines) have been consistent
with a mechanism of through-bond interaction via pep-
tide backbone linkages extending over distances of 1.0-
4.0 nm.2,4 The most important finding for the present
peptide models is that a very large number of favorable
conformations lie in energy within a range of 2-3 kcal/
mol and that the terminal and side-chain groups (as well
as chain segments) are oriented in largely random
fashion (with a probable time constant of a few nanosec-
ond for conformational averaging24).
controlling this ISC step. There have been several
reports of molecules in which donor and acceptor subunits
are held together by a tether of significant length
(commonly 5-15 σ bond separations), for which radical
ion pair states that remain spin-correlated evolve on
photoexcitation.27 A distinction between singlet and
triplet paths for charge separation across peptide bonds
could be made for only a few cases in which amino acid
derivatives provide short links between donor and
acceptor groups (e.g., glycine spacers or a diketopipera-
zine bridge,5 or a thiourea or thiohydantoin-linked N-
terminal group6), coupled with the observation of radical
pair intermediates that live in the microsecond time
domain.6
Su m m a r y. The more important general features of
the electron-transfer mechanisms for the pyrenesulfon-
amide derivatives are illustrated with the aid of Scheme
1. For the linkage of tryptophan (Trp) and pyrene-
sulfonamide (Pyr), electron transfer is a dominant path
for the singlet manifold; significant fluorescence quench-
ing is observed. Charge recombination, either with or
after radical pair intersystem crossing leads to the local
pyrene triplet (3Pyr*) and ultimate nonradiative decay.
The alternative involves the nitropyrene chromophore
that enters the triplet manifold of states, either by an
accelerated rate of intersystem crossing that is inherent
to the nitroaromatic stage or via fast singlet electron
transfer and intersystem crossing at the radical pair
stage.25 In either event the lower lying species is the
triplet radical ion pair for the NPyr series. The lifetimes
for these species (abbreviated NP-.A(A)W+., Scheme 1)
are impressively long for charge-separated intermediates
having relatively short links and through-bond distances
of separation of 12-15 Å. A direct comparison can be
made with picosecond flash photolysis data for the Pyr-
Ala (Ala)TrpOEt series for which return electron transfer
from radical ion pair states in the singlet manifold has
been associated with shorter decay times, 0.76 and 3.1
ns for the peptides with one and two alanine spacers,
respectively.7,25 Thus, the slow rate of charge recombina-
tion for the NPyr series may be controlled by the rate of
intersystem crossing (ISC) for the linked radical ion pair
The interesting finding that the longer peptide link,
NPyr-Ala-Ala-TrpOEt, is as effective (or slightly more
effective) in producing a shorter lived radical ion pair may
be the result of a requirement that through-space inter-
action between remote groups contribute at least mod-
erately to the ISC mechanism. Models show that the
longer peptide, Pyr-Ala-Ala-TrpOEt (and presumeably
the nitro derivative), provides a similarly large number
of low-energy conformations that have a moderate dis-
tance of separation for through-space interaction (<0.8
nm, analogous to data shown in Figure 5), even though
the average center-to-center distance between electron-
transfer sites is somewhat larger than that for Pyr-
AlaTrpOEt.
Recent refinements regarding the mechanism of long-
range electronic coupling that leads to ISC for radical
pairs focus on the role of random motion of spacer and
substituent groups which modulates the through-space
exchange interaction that is assumed to have an expo-
nential falloff with distance.28 Another recent view29 of
long-distance exchange electronic coupling envisions a
“through excluded volume” mechanism to which there
can be through-bond, through-vacuum, and through-
solvent contributions, with evidence of some favor for the
through-bond component.
Exp er im en ta l Section
3[NPyr-• - Link - Trp+•] f
1[NPyr-• - Link - Trp+•] (2)
Ma ter ia ls a n d Gen er a l P r oced u r es. The benzyloxycar-
bonyl-protected amino acids were obtained from Bachem
Bioscience Inc. The amino acid ethyl esters were obtained
from Sigma. The deuterated NMR solvents were purchased
from Cambridge Isotope Laboratories. The high-performance
liquid chromatography (HPLC) grade acetonitrile was obtained
from Fisher Scientific. Palladium on activated carbon (pal-
ladium content, 10%), along with other reagents and solvents,
was obtained from Aldrich. The Silica Gel 60 (230-400 mesh,
E. Merck Inc.) was used as the stationary phase for flash
column chromatography (typically, a 2.5 cm ID × 32 cm
column for a 0.5-1.0-g sample of crude product);30 solvent
composed of 5-50% ethyl acetate in petroleum ether, which
gave an Rf for product of 0.2-0.3 on thin-layer chromatography
a rate that will be influenced by several factors that
include spin-orbit and hyperfine interaction.26 The
spacer length (number of intervening residues) and the
distribution of conformations for the peptide backbone
and side chains are no doubt important factors in
(22) Beratan, D. N.; Onuchic, J . N.; Betts, J . N.; Bowler, B. J .; Gray,
H. B. J . Am. Chem. Soc., 1990, 112, 7915.
(23) For a review, see J ordan, K. D.; Paddon-Row, M. N. Chem. Rev.
1992, 92, 395.
(24) Fasman, G. D. Prediction of Protein Structure and the Principles
of Protein Conformation; Plenum Press: New York, 1989.
(25) The values for radical pair decay time for the Pyr series
correspond to faster components from transient decay data from double
exponential fits on fs laser flash photolysis - acetonitrile solvent).7,8
An investigation of the fast processes that involve electron transfer
quenching of the local singlet state of NPyr chromophores is presently
underway (note fluorescence quantum yield trends in the table). The
early data from flash experiments show formation and at least partial
decay of radical ion pairs in the 1-50-ps time domain; J ones, G. II;
Lu, L. N.; Gosztola, D. J .; Greenfield, S. R.; Wasielewski, M. R.,
unpublished results.
(27) See, for example, Wasielewski, M. R. Chem. Rev. 1992, 92, 435;
van Dijk, S. I.; Groen, C. P.; Hartl, F.; Brouwer, A. M.; Verhoeven, J .
W. J . Am. Chem. Soc. 1996, 118, 8425; Hakamura, H.; Usui, S.;
Matsuda, Y.; Matsuo, T.; Maeda, K.; Azumi, T. J . Phys. Chem. 1993,
97, 534; Staerk, H.; Kuhnle, W.; Treichel, R.; Weller, A. Chem. Phys.
Lett. 1985, 118, 19.
(28) Staerk, H.; Busmann, H. G.; Kuhnle, W.; Treichel, R. J . Phys.
Chem. 1991, 95, 1906; Shafirovich, V. Y.; Batova, E. E.; Levin, P. P. J .
Phys. Chem. 1993, 97, 4877; Schulten, K.; Bittl, R. J . Chem. Phys. 1986,
85, 228.
(26) Steiner, U. E.; Wolff, H. J . In Photochemistry and Photophysics;
Rabek, J . F., Ed.; CRC Press: Boca Raton, 1991; Vol. 14; J enks, W.
S.; Turro, N. J . Res. Chem. Intermed. 1990, 13, 237.
(29) Forbes, M. D. E.; Ball, J . D.; Avdievich, N. I. J . Am. Chem. Soc.,
1996, 118, 4707.
(30) Still, W. C.; Kahn, M.; Mitra, A. J . Org. Chem. 1978, 43, 2923.

8944 J . Org. Chem., Vol. 63, No. 24, 1998
J ones and Lu
(TLC) (Silica Gel 60 F254), was used for elution of amino acid
or peptide derivatives.
methanol removed by rotary evaporation to yield 0.64 g of a
light yellow solid (93%). The product, L-alanyl-L-tryptophan
ethyl ester (0.64 g, 2.1 mmol), was used directly without
further purification, starting with dissolution in 8 mL THF
with 2 mL of triethylamine. 1-Pyrenesulfonylchloride (0.6 g,
2.1 mmol) in 12 mL THF was added dropwise, and the reaction
was stirred at room temperature for 18 h. The precipitate was
filtered and the solvent removed by rotary evaporation to yield
a yellow solid. The product was purified by flash column
chromatography and recrystallized from ethyl acetate/petro-
leum ether to yield 0.66 g (57%) of white crystals (mp 204-
205 °C): 1H NMR (400 MHz, CDCl3, with one drop of DMSO-
d6) δ 8.93 (br, W-H-3), 8.85 (d, J ) 9.4 Hz, H-10), 8.57 (d, J
) 8.2 Hz, H-2), 8.21 (d, J ) 7.6 Hz, H-8), 8.20 (d, J ) 7.6 Hz,
H-6), 8.16 (d, J ) 9.4 Hz, H-9), 8.12 (d, J ) 8.9 Hz, H-5), 8.06
(d, J ) 8.2 Hz, H-3), 8.01 (t, J ) 7.6 Hz, H-7), 7.99 (d, J ) 8.9
Hz, H-4), 7.23 (d, J ) 7.7 Hz, W-H-7), 7.23 (d, J ) 8.1 Hz,
W-H-7), 7.06 (td, J ) 8.1, 7.0, 1.1 Hz, W-H-5), 7.00 (d, J )
7.4 Hz, A-NH), 6.95 (td, J ) 7.6, 7.0, 1.0 Hz, W-H-6), 6.88
(d, J ) 8.1 Hz, W-NH), 6.85 (d, J ) 2.3 Hz, W-H-2), 4.51 (m,
W-CH), 3.84 (m, OCH2CH3), 3.75 (m, A-CH), 3.07 (dd, J )
14.0, 5.5 Hz, W-CH2-Ha), 3.02 (dd, J ) 14.0, 6,3 Hz, W-CH2-
Hb), 1.00 (t, J ) 7.1 Hz, OCH2CH3), 0.94 (d, J ) 7.1 Hz,
A-CH3); 13C NMR (100 MHz, CDCl3) δ 171.3 (W-CdO), 171.2
(A-CdO), 136.1 (W-C8), 133.9 (C1), 132.8 (C2a), 130.5 (C5a),
129.8 (C9), 129.7 (C8a), 129.2 (C5), 127.3 (C10c), 127.0 (C6,
C7, C8), 126.9 (C4), 126.8 (W-C9, C2), 124.2 (C10a), 123.9
(C3), 123.8 (C2), 123.7 (C10), 123.2 (C10b), 121.0 (W-C7),
118.4 (W-C5), 117.9 (W-C6), 111.4 (W-C4), 109.0 (W-C1),
60.2 (OCH2CH3), 53.0 (W-CH), 51.4 (A-CH), 26.8 (W-CH2), 19.0
(A-CH3), 13.7 (OCH2CH3); LRMS (EI, 70 eV) m/z (relative
intensity) 567 (M+, 19), 352 (17), 217 (23), 216 (11), 215 (75),
207 (14), 202 (39), 201 (69), 200 (38). 143 (11), 131 (13), 130
(100); HRMS (EI, 70 eV) m/z 567.1826 (M+, calcd for
N-(1-P yr en esu lfon yl)-L-a la n in e Eth yl Ester (P yr -Ala O-
Et). 1-Pyrenesulfonyl chloride10 (0.80 g, 2.8 mmol) in 15 mL
tetrahydrofuran (THF) was added dropwise to a mixture of
the hydrochloride salt of L-alanine ethyl ester (0.43 g, 2.8
mmol) in 10 mL of THF and 2 mL of triethylamine. The
solution was stirred at room temperature for 5 h. The
precipitate that formed was filtered and the solvent removed
by rotary evaporation to obtain a yellow solid. The latter
product was purified by flash column chromatography on silica
gel and recrystallized from ethyl acetate/petroleum ether to
yield 0.64 g (62%) of yellow cubic crystals (mp 104-105 °C):
1H NMR (400 MHz, CDCl3) δ 8.95 (d, J ) 9.5 Hz, H-10), 8.66
(d, J ) 8.2 Hz, H-2), 8.30 (d, J ) 7.8 Hz, H-8), 8.28 (d, J ) 9.5
Hz, H-9), 8.26 (d, J ) 7.8 Hz, H-6), 8.17 (d, J ) 8.8 Hz, H-5),
8.16 (d, J ) 8.2 Hz, H-3), 8.08 (t, J ) 7.8 Hz, H-7), 8.04 (d, J
) 8.8 Hz, H-4), 5.63 (d, J ) 8.3 Hz, A-NH), 3.98 (qd, J ) 7.1,
8.3 Hz, A-CH), 3.6 (m, OCH2CH3), 1.27 (d, J ) 7.1 Hz,
A-CH3), 0.78 (t, J ) 7.2 Hz, OCH2CH3); 13C NMR (100 MHz,
CDCl3) δ 171.5 (CdO), 134.0 (C1), 132.6 (C3a), 130.5 (C5a),
130.0 (C9), 129.7 (C8a), 129.3 (C5), 127.3 (C10c), 127.1 (C6,
C8), 127.0 (C7), 126.8 (C4), 126.7 (C3), 124.2 (C10a), 124.0 (C2),
123.7 (C10), 123.1 (C10b), 60.3 (-OCH2CH3), 51.2 (A-CH), 18.3
(A-CH3), 13.2 (-OCH2CH3). LRMS (EI, 70 eV) m/z (relative
intensity) 381 (M+, 33), 308 (11), 256 (38), 217 (30), 202 (38),
201 (100), 200 (34); HRMS (EI, 70 eV) m/z 381.1032 (M+, calcd
for C21H19NO4S 381.1035).
N-Ben zyloxyca r bon yl-L-a la n in e-p-n itr op h en yl Ester .
A solution of N-benzyloxycarbonyl-L-alanine (5.0 g, 0.022 mol)
and p-nitrophenol (3.3 g, 0.024 mol) in 200 mL ethyl acetate
was cooled to 0 °C, and dicyclohexylcarbodiimide (DCC) (4.6
g, 0.022 mol) in 20 mL of ethyl acetate was added dropwise.
The solution was stirred at 0 °C for 2 h and left in the
refrigerator overnight. The precipitate that formed was
filtered, and the filtrate was washed successively with solu-
tions of 5% Na2CO3, 5% HCl, and 5% NaCl and dried over
anhydrous sodium sulfate. The solution was concentrated by
rotary evaporation until 10 mL remained. The concentrated
solution was added dropwise to 200 mL of petroleum ether to
C
32H29N3O5S 567.1813).
N-[1-(8-Nit r op yr en esu lfon yl)]-L-a la n in e E t h yl E st er
(NP yr -Ala OEt). The conjugate, Pyr-AlaOEt (50 mg, 0.13
mmol) in 5 mL acetic anhydride was cooled to 0 °C, and nitric
acid (8.4 µL, 69% in water) in 1 mL of acetic anhydride was
added dropwise. The reaction was stirred at 0 °C for 2 h and
at room temperature for 10 min. When the reaction mixture
was added slowly to ice water, an oily yellow solid separated.
The solid was washed with water. The product was purified
by flash chromatography and recrystallized from ethyl acetate/
petroleum ether to yield 20 mg (35%) of yellow crystals (mp
186-187 °C), Rf ) 0.22 (ethyl acetate/petroleum ether, 1:3);
1H NMR (400 MHz, CDCl3) δ 9.24 (d, J ) 10.0 Hz, H-9), 9.08
(d, J ) 10.0 Hz, H-10), 8.78 (d, J ) 8.0 Hz, H-7), 8.74 (d, J )
8.4 Hz, H-2), 8.35 (d, J ) 8.0 Hz, H-6), 8.33 (d, J ) 8.4 Hz,
H-3),8.28 (s, H-4, H-5), 5.56 (d, J ) 8.3 Hz, A-NH), 4.05 (m,
A-CH), 3.68 (m, OCH2CH3), 1.30 (d, J ) 7.2 Hz, A-CH3), 0.88
(t, J ) 7.1 Hz, OCH2CH3); 13C NMR (100 MHz, CDCl3) δ 171.7
(CdO), 144.1 (C8), 134.7 (C1), 134.6 (C5a), 133.6 (C3a), 130.3
(C4), 129.6 (C5), 128.3 (C6), 127.2 (C8a), 127.0 (C3), 126.4 (C9),
126.2(C10), 124.4 (C10a), 124.3 (C10c), 124.2 (C2), 123.5
(C10b), 123.4 (C7), 61.7 (OCH2CH3), 51.8 (A-CH), 19.8 (A-CH3),
13.6 (OCH2CH3); LRMS (EI, 70 eV) m/z (relative intensity) 426
(M+, 71), 368 (93), 310 (92), 262 (46), 246 (100), 237 (83), 220
(38), 200 (50), 97 (29), 81 (31), 69 (58), 57 (40), 55 (38), 43 (36);
HRMS (EI, 70 eV) m/z 426.0864 (M+, calcd for C21H18N2O6S
426.0885).
form
a white precipitate. The product was collected by
filtration and dried under vacuum to yield 6.9 g (89%) of a
white powder (mp 78-79 °C). 1H NMR (400 MHz, CDCl3) δ
8.26 (d, J ) 8.5 Hz, ΦNO2-2H), 7.35 (s, Φ-5H), 7.27 (d, J ) 8.5
Hz, ΦNO2-2H), 5.25 (d, J ) 5.6 Hz, A-CH), 5.13 (s, Φ-CH2),
4.61 (m, A-CH), 1.58 (d, J ) 7.3 Hz, A-CH3).
N-Ben zyloxyca r bon yl-L-a la n yl-L-tr yp top h a n eth yl es-
ter (Cbz-Ala -Tr p OEt). A solution of N-benzyloxycarbonyl-
L-alanine, p-nitrophenyl ester (0.90 g, 2.6 mmol) in 25 mL
pyridine (freshly distilled) was cooled to 0 °C, and 0.8 mL of
triethylamine was added. The hydrochloride salt of L-tryp-
tophan ethyl ester (0.70 g, 2.6 mmol) in 5 mL pyridine was
added dropwise. The mixture was stirred at 0 °C for an hour
and at room temperature overnight. After evaporation of
pyridine under a vacuum the resulting oil was dissolved in 80
mL of ethyl acetate. The ethyl acetate solution was washed
successively with solutions of 5% Na2CO3, 5% HCl, and 5%
NaCl, and dried over anhydrous sodium sulfate. After evapo-
ration of most of the ethyl acetate under vacuum, the resulting
liquid was added to 80 mL of petroleum ether which led to
the formation of a white precipitate. The product was collected
by filtration and dried under vacuum to yield 0.97 g (85%) of
a white powder (mp 123-124 °C): 1H NMR (400 MHz, CDCl3)
δ 8.05 (s, W-H-3), 7.49 (d, J ) 7.8 Hz, W-H-7), 7.30 (s, Φ-5H),
7.30 (d, W-H-4, overlap with Φ-5H), 7.14 (t, J ) 8.2, 7.1 Hz,
W-H-5), 7.07 (t, J ) 7.8, 7.1 Hz, W-H-6), 6.93 (s, W-H-2),
6.49 (d, J ) 6.8 Hz, A-NH), 5.25 (d, J ) 6.8 Hz, W-NH), 5.03
(m, Φ-CH2), 4.85 (m, W-CH), 4.18 (m, A-CH), 4.09 (m, OCH2-
CH3), 3.28 (d, J ) 5.4 Hz, W-CH2), 1.29 (d, J ) 6.8 Hz,
A-CH3), 1.19 (t, J ) 7.1 Hz, OCH2CH3).
The major product (above) (Rf ) 0.22) was accompanied by
a significant minor product, Rf ) 0.24 [TLC, ethyl acetate and
petroleum ether (1:3) as eluant]. Formation of these mono-
substituted products is consistent with nitration at positions
1
6 and 8.31 A comparison of the H spectra of the Pyr-AlaOEt
with NPyr-AlaOEt showed that the major product is indicative
of nitration at position 8 (see Supporting Information). Nuclear
Overhauser effect observed between protons assigned to posi-
tions 9 and 10 further supported the identification of the
8-nitro isomer.
N-(1-P yr en esu lfon yl)-L-a la n yl-L-tr yp top h a n Eth yl Es-
ter (P yr -Ala -Tr p OEt). A mixture of N-benzyloxycarbonyl-
L-alanyl-L-tryptophan ethyl ester (1.0 g, 2.3 mmol) in 12 mL
of methanol and 0.1 g of 10% Pd/C was stirred under hydrogen
provided by a balloon for 4 h. The Pd/C was filtered and the
(31) Furniss, B. S.; Hannaford, A. J .; Smith, P. W. G.; Tatchell, A.
R. In Vogel’s Textbook of Practical Organic Chemistry, 5th ed.; J ohn
Wiley & Sons: New York, 1989, chp 6.

Species Observed on Flash Photolysis
J . Org. Chem., Vol. 63, No. 24, 1998 8945
N-[1-(8-Nit r op yr en esu lfon yl)]-L-a la n yl-L-t r yp t op h a n
Eth yl Ester (NP yr -Ala -Tr p OEt). A solution of N-[1-(8-
nitropyrenesulfonyl)]-L-alanine ethyl ester (200 mg, 0.47 mmol)
in 10 mL ethanol water (1:1) with 1 mL 2 N NaOH was stirred
at 60-80 °C for 3 h. The solution was neutralized with
concentrated HCl to pH 3 and extracted with ethyl acetate (3
× 10 mL). The ethyl acetate layer was dried over anhydrous
sodium sulfate followed by rotary evaporation to yield a yellow
solid. The dried solid was used directly in the following step.
N-[1-(8-Nitropyrenesulfonyl)]-L-alanine (187 mg, 0.47 mmol)
was dissolved in 20 mL THF and cooled to 0 °C. 1-Hydroxy-
benzotriazole (HOBt, 127 mg, 0.94 mmol), N-methylmorpho-
line (0.1 mL), and the hydrochloride salt of L-tryptophan ethyl
ester (189 mg, 0.71 mmol) were added successively. DCC (97
mg, 0.47 mmol) in 3 mL of THF was added dropwise. The
reaction was stirred at 0 °C for 2 h and then at room
temperature overnight. The precipitate was filtered and the
solvent removed by rotary evaporation to yield a yellow solid.
The product was purified by flash column chromatography and
recrystallized from ethyl acetate/petroleum ether to yield 104
mg (36%) of yellow crystals (mp 193-194 °C): 1H NMR (400
MHz, CD3COCD3) δ 10.05 (s, W-H-3), 9.37 (d, J ) 9.9 Hz,
H-9), 8.92 (d, J ) 9.9 Hz, H-10), 8.76 (d, J ) 8.6 Hz, H-7),
8.69 (d, J ) 8.2 Hz, H-2), 8.51 (d, J ) 8.6 Hz, H-6), 8.38 (d, J
) 8.8 Hz, H-5), 8.30 (d, J ) 8.8 Hz, H-4), 8.38 (d, J ) 8.2 Hz,
H-3), 7.38 (d, J ) 8.0 Hz, A-NH, W-NH), 7.35 (d, J ) 8.1
Hz, W-H-7), 7.23 (d, J ) 7.9 Hz, W-H-4), 7.08 (t, J ) 7.9, 7.0
Hz, W-H-5), 7.01 (d, J ) 2.4 Hz, W-H-2), 6.93 (t, J ) 8.1, 7.0
Hz, W-H-6), 4.14 (m, A-CH, W-CH), 3.83 (m, OCH2CH3), 2.82
(W-CH2-Ha overlap with solvent), 2.68 (dd, J ) 14.5, 6.7 Hz,
W-CH2-Hb), 1.15 (d, J ) 7.1 Hz, A-CH3), 0.96 (t, J ) 7.1
Hz, OCH2CH3); LRMS (EI, 70 eV) m/z (relative intensity) 612
(M+, 18), 582 (6), 310 (92), 269 (6), 216 (10), 215 (89), 131 (7),
130 (100); HRMS (EI, 70 eV) m/z 612.1677 (M+, calcd for
tions, Inc., including the conformational search subroutine that
interfaces with the CHARMm molecular mechanics program.
Additionally, a semiempirical quantum mechanical program,
MOPAC (version 6.0; QCPE 455), was used in determining
charge distributions. To arrive at an array of low-energy (most
probable) conformations for a particular peptide, the sequence
of steps began with a random selection of all the flexible
dihedral angles. The resulting high-energy conformation was
thoroughly energy-minimized by the adopted-basis Newton-
Raphson algorithm. The energy-minimized conformation was
used to initiate the next sequence in a cumulative manner.
For the computations certain assumptions or constraints were
made: (1) the solvent dielectric constant was assumed to have
a value of 37 and a radial distance dependence; (2) the amide
carbonyl and amide proton positions were assumed to be
antiparallel and coplanar. Typical runs provided simulations
of 500 low-energy conformations whose distribution, in terms
of population and energy, was determined. The “average”
parameters, such as average energy and average distance,
were obtained by arithmetic averaging.
La ser F la sh P h otolysis. Nanosecond phototransient ex-
periments were carried out using a Nd:YAG laser system and
detection methods described previously,33 with principal com-
ponents consisting of a Quantel YG-581-10 Nd:YAG laser, a
LeCroy 6880A 1.35 gigasamples/second waveform digitizer, a
LeCroy 6010 MAGIC controller, a pulsed xenon monitoring
lamp, a H-20 monochromator (version V grating) from Instru-
ments SA, and a Hamamatsu R928 PMT. The samples for
flash photolysis experiments were 1.0 × 10-5 M in concentra-
tion for the pyrene series (Pyr) and 2.0 × 10-5 M for the
nitropyrene derivatives (NPyr) and prepared by injecting 5-10
µL of a 1.0 × 10-2 M stock solution of peptide in DMF into 5
mL of acetonitrile. All experiments were performed at room
temperature with Ar-purged or air-saturated solutions as
indicated. Excitation was carried out at 355 nm with 7-ns
pulses using 2.2 cm × 1.0 cm rectangular Pyrex cells. Each
measurement of decay time was obtained as an average of
transient decay data for at least five pulse sequences and two
independent samples. Decay times were shown not to be
dependent on pulse energy (40-80 mJ /pulse) or peptide
concentration (10-30 µM).
C
32H28N4O7S 612.1679).
N-[1-(8-Nitr op yr en esu lfon yl)]-L-a la n yl-L-a la n yl-L-tr yp -
top h a n Eth yl Ester (NP yr -Ala -Ala -Tr p OEt). In a similar
fashion, N-[1-(8-nitropyrenesulfonyl)]-L-alanine (187 mg, 0.47
mmol) was coupled to L-alanyl-L-tryptophan ethyl ester (213
mg, 0.71 mmol). The product was purified by flash chroma-
tography and recrystallized from ethyl acetate/petroleum ether
to yield 103 mg (32%) of yellow crystals (mp 273-274 °C): 1H
NMR (400 MHz, DMSO) δ 10.79 (s, W-H-3), 9.29 (d, J ) 9.9
Hz, H-9), 8.84 (d, J ) 9.9 Hz, H-10), 8.84 (d, J ) 8.7 Hz, H-7),
8.70 (d, J ) 8.4 Hz, H-2), 8.6 (m, overlap of H-6, H-3, A1-
NH), 8.5 (s, overlap of H-4, H-5), 8.01 (d, J ) 7.2 Hz, W-NH),
7.86 (d, J ) 7.5 Hz, A2-NH), 7.38 (d, J ) 8.0 Hz, W-H-7),
7.27 (d, J ) 8.3 Hz, W-H-4), 7.03 (d, J ) 2.3 Hz, W-H-2),
7.00 (t, J ) 8.3, 7.4 Hz, W-H-5), 6.91 (t, J ) 8.0, 7.4 Hz, W-H-
6), 4.29 (m, W-CH), 3.99 (m, A1-CH), 3.90 (m, OCH2CH3),
3.73 (m, A2-CH), 2.98 (dd, J ) 14.0, 5.7 Hz, W-CH2-Ha),
2.94 (dd, J ) 14.0, 8.3 Hz, W-CH2-Hb), 0.97 (m, A1-CH3
overlap with OCH2CH3), 0.63 (d, J ) 6.9 Hz, A2-CH3); LRMS
(EI, 70 eV) m/z (relative intensity) 683 (M+, 7), 217 (15), 215
(55), 130 (100), 111 (16), 97 (20), 85 (15), 84 (56), 83 (15), 70
(18), 44 (24), 43 (23); HRMS (EI, 70 eV) m/z 683.2073 (M+,
calcd for C32H28N4O7S 683.2050).
Ack n ow led gm en t. Support of this research by the
Department of Energy, Office of Basic Energy Sciences,
Division of Chemical Sciences, is gratefully acknowl-
edged. We also thank Valentine Vullev and Dr. Churl
Oh for valued technical assistance.
Su p p or tin g In for m a tion Ava ila ble: Copies of 1H and 13C
NMR spectra (5 pages). This material is contained in micro-
fiche, immediately follows this article in the microfilm version
of the journal, and can be ordered from the ACS; see any
current masthead page for ordering information.
J O981241G
(32) Clark, T. In Handbook of Computational Chemistry; J ohn
Wiley: New York, 1985.
(33) Malba, V.; J ones, G. II; Poliakoff, E. Photochem. Photobiol. 1985,
42, 451; J ones, G., II.; Oh, C. J . Phys. Chem. 1994, 98, 2367.
Molecu la r Mod elin g.32 The software used was based
primarily on the QUANTA package from Molecular Simula-