J. Phys. Chem. 1993,97,3956-3967
3956
ARTICLES Spin-Forbidden Excitation Transfer and Heavy-Atom Induced Intersystem Crossing in Linear and Cyclic Peptides Gautam Basu,+ Matthew Kubasik, Demetrios Anglos, and Atsuo Kuki' Cornell University, Department of Chemistry, Baker Laboratory, Ithaca, New York I4853 Received: October 30, 1992; In Final Form: January 20, 1993
Four dimeric peptides containing one fluorescent chromophore, j3-( 1'-naphthyl)-L-alanine or j3-( 1'-naphthyl)D-alanine, and one heavy atom perturber, p-bromo-L-phenylalanine, were synthesized as two pairs of diastereoisomers-one cyclic pair and one linear pair. The backbone atoms of the cyclic peptides form a stable six-membered diketopiperazine ring which provides a rigid molecular framework onto which the side chains are affixed cis or trans to each other. The fluorescence emission (steady state and time resolved) and the time-resolved triplet-triplet absorption of the naphthyl residue in these peptides were monitored, revealing remote electronic interactions between the naphthyl and the bromophenyl groups. All peptides exhibited fluorescence quenching. A nominally spin-forbidden singlet-triplet energy transfer and a heavy atom induced enhanced intersystem crossing were established as two simultaneously active mechanisms for the fluorescence quenching of the naphthyl residue. An understanding of the energy-transfer rate and the extraction of its electronic matrix element is straightforward. But the heavy atom enhanced intersystem crossing, which reflects the quantum mechanical participation of the bridging peptide groups, shows unexpected trends. Subtle factors arising from the detailed properties of the intervening peptide backbone evidently contribute to this remote heavy atom effect. Here we propose a perturbation analysis, including an integration over intermediary vibronic states, in which the remote heavy atom effect arises through two virtual energy transfer interactions. This provides a general model for radiationless relaxation processes driven by indirect electronic interactions. The electronic coupling responsible for the exchange-mediated remote heavy atom effect in the peptide series is examined. This electronic coupling is a measure of the very weak electronic delocalization common to all exchange-mediated interactions, including electron transfer.
Introduction The presence of a covalently linked heavy atom is known to induce a strong spin-orbit coupling in a molecule. Correspondingly the moleculartriplet state acquiresa partial singlet character, and as a consequence, the intersystem crossing rate is enhanced in the molecule. This is the internal heavy atom effect.' The heavy atom containing molecule may also perturb the nonradiative relaxation rate of the excited singlet state of a distinct chromophoric molecule that does not contain a heavy atom, known as the external heavy atom effect.2 In addition, if the lowest triplet state of the heavy atom containing perturber molecule lies below the lowest singlet of the distinct chromophore, intermolecular singlet triplet energy transfer can occur, an otherwise spin-forbidden proce~s.~ Both of these processes, the external heavy atom effect and the spin-forbidden excitation transfer, fall in the general category of remote electronic interactions between two distinct molecular partners. Such intermolecularinteractions can be studied as a bimolecular process or as a unimolecular process; in the latter case the perturber and the chromophore can be covalentlylinked in a nonconjugated fashion within a molecular framework of well defined geometry. Because the distance and the geometry between the chromophore and the perturber affect the extent of such interactions, the unimolecular studies yield maximum information about the nature and extent of the remote interaction in question. Furthermore, only unimolecular studies allow one to explore the extent to which two remote electronic
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'Current address: Department of Chemistry, Faculty of Science, Kyoto University, Kyoto 606, Japan.
0022-3654/93/2097-3956%04.00/0
subsystems within a single molecule are either distinct, that is, interacting in a way equivalent to the intermolecular external heavy atom effect at that distance, or brought into stronger electronic communication by the (nonconjugated) covalent coupling. With naphthalene as the chromophore and bromobenzene as the heavy atom perturber, bimolecular studies have shown4that both the external heavy atom effect and energy transfer are operative for this pair, with both processes active in reducing the fluorescencequantum yield of naphthalene. Unimolecular studies have also been performed596 between these two partners in an attempt to explore the remote heavy atom effect (RHAE) as a potentially useful tool for exploring remote electronicinteractions, including those that drive electron transfer, in biomolecules. Short octameric oligopeptides with rigid and well-defined backbone helical conformations7were used in these unimolecular studies for providing the molecular framework while naphthalene and bromobenzene (BrBz) were incorporated in the peptides as sidechains of guest amino acids. In this paper we report our investigation on the same chromophore and perturber pair in a larger set of peptides, which includes the helical peptides as well as dimers which achieve a well-defined backbone geometry through a different strategy. Peptides containing two amino acid residues can be cyclized to yield a stable six-membered diketopiperazine ring composed of the backbone atoms (see Figure 1). Furthermore, since the two amino acid residues can be cis or trans to each other, one can design two molecules covalently identical yet differing in the distanceand orientationbetween the two residues. We synthesized 0 1993 American Chemical Society
Excitation Transfer in Linear and Cyclic Peptides
The Journal of Physical Chemistry, Vol. 97, No. 16, 1993 3951
Br-LL-ldim Br-4,5-0ct
Br-D.L-ldim
Br-3,S-Oct
D,L-ldim
Figure 1. Structures of all peptides studied in the present work. (a) The dimeric peptides were prepared in diastereomeric sets D,L and L,L. The linear pair possesses conformational flexibility whereas the cyclic pair is conformationally constrained by the rigid diketopiperazine ring. Within the cyclic series, the two peptides Br-o,L-dim and Br-L,L-cdim differ from each other by the disposition of the Nap and the Bph group (trans or cis to each other). T h e octameric peptides are shown in their preferred helical backbone conformation (a-helical for Br-4,S-Oct and 310-helicalfor the Br-3,6-0ct and Br-3,5-0ct) as determined by NMR experiments. In Br-3,6-0ct the Nap and the Bph group face the same side of the 310-helixand can come within van der Waals distance of each other. In Br-4,S-Oct and Br-3,5-0ct, the two groups are constrained to be further away.
two such cyclic peptide dimers containing two guest @-aromatic amino acids, @-( 1’-naphthy1)alanine (Nap) and p-bromophenylalanine (Bph). One dimer contained D-Nap and L-Bph and thus is the trans diastereoisomer (Br-D,L-cdim) whereas the cis diastereoisomer contained L-Nap and L-Bph (Br-L,L-cdim).Two linear dimers were also synthesized that were diastereomeric, one containing D-Nap and L-Bph (Br-D,L-ldim) and the other containing L-Nap and L-Bph (Br-L,L-ldim). The linear peptides had substantial conformationalflexibilitywhile the corresponding cyclic dimers, due to the presence of the rigid diketopiperazine ring, were conformationally much more well defined; in Br-D,Lcdim the two aromatic partners sandwich the diketopiperazine ring, and in Br-L,L-cdim the two partners face each other in the same side of the ring.8 The four dimers hence comprise two sets of diastereoisomers: the floppy linear set and the rigid cyclo set. The covalent distance (through-bond) between the Nap and the Bph residue was constant throughout the set and only the noncovalent distance (through space) differed. Thecorresponding control peptide for optical studies was linear and contained D-Nap and L-phenylalanine (D,L-ldim).
dimers
sequence
o,L-ldim Br-o,L-ldim Br-L,L-ldim Br-o,L-cdim Br-L,L-cdim
t-Boc-(o-Nap)(L-Pk)-OMe t-Boc-(o-Nap) (L-Bph)-OMe t-Boc-( L-Nap)(L-Bph)-OMe c- [o-Nap,~-Bph] c-[~-Nap,~-Bph]
This set of peptides, combined with the helical series, provides seven interacting geometries in which to explore the remote heavy atom effect. The sequencesof the Aib-rich helical peptides from the previous work6 are also given below, and all the peptides are shown in Figure 1 where the dispositionsof the naphthyl and the bromophenyl groups are depicted. peptides
sequence
Br-3,6-0ct Br-3,S-Oct Br-4,S-Oct C-3,6-0ct
Ac-(Aib)(Aib)(Nap)(Aib)(Aib)(Bph)(Aib)(Aib)-NHMe Ac-(Aib)(Aib)(Nap)(Aib)(Bph)(Aib)(Aib)(Aib)-NHMe Ac-(Aib)(Aib)(Aib)(Nap)(Bph)(Aib)(Aib)(Aib)-NHMe Ac-(Aib)(Aib)(Nap)(Aib)(Aib)(Pk)(Aib)(Aib)-NHMe
Fluorescence quenching studies, both steady state and time resolved, were performed across this set to study the interaction of the bromophenyl group with the naphthalene chromophore. Time-resolved triplet-triplet (T-T) absorption experiments were
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The Journal of Physical Chemistry, Vol. 97, No. 16, 199'3
employed to reveal the mechanism of the fluorescence quenching by measuring the corresponding changes in naphthalene triplet yield (h). Both Nap triplet (TIN") producing fluorescence quenching due to RHAE and non-TINaPproducing fluorescence quenching due to the spin-forbidden excitation transfer to the bromophenyl T I state were observed. Results from the dimeric peptides showed overall a very similar trend to that of the octameric peptides.6 Experimental data for thecurrent set and the previously reported set of peptides are analyzed as a single set to obtain quantitative estimates of the electronic matrix elements for the RHAE. This requires an analysis of the relevant Franck-Condon weighted density of states. The native spin-orbit interaction from SI-TI in naphthalene and the internal heavy atom effect have been analyzed closely in single vibronic level emission spectral studies by Rice et al.,9 and a fair amount is known in detail about this parent chromophore. Yet the question posed here is not the spin-orbit coupling per se, but rather how the electrons within the naphthalenic component of the peptidesdetect the presence of the bromophenyl component. We may consider that the spin forbiddenness which greatly attenuates the intrachromophoric relaxation of SI -TI provides a keen sensitivity to very weak exchanges of spin angular momentum with other components in the naphthalene environment. Hence, the degree of electronic delocalization between the chromophore and the perturber controls the electronic matrix element in RHAE.596 Quantitative evaluation of the individual electronic matrix elements contributing to the overall RHAE is important since this subtle electronic delocalization which underliesthe exchange matrix element is fundamental to all remote exchange-mediated interactions,lOincluding triplet-triplet energy transfer and electron transfer.
Materials and Methods The Aib-rich octameric peptides were synthesized as described p r e v i ~ u s l y . Their ~ ~ ~ ~solution structures have been examined extensively by 1D and 2D IH NMR.' All of the peptide dimers were synthesized by solution-phasepeptidesynthesis techniques.' l a The amino acids Nap and Bph were obtained from Bachem Inc., and morpho-CDIllb was used as the peptide coupling reagent. The peptides were purified by flash chromatography along the synthetic route and were thoroughly characterized by IH-NMR spectroscopy and FAB mass spectroscopy. Prior to any optical studies the peptides were further purified by reversed-phase C I ~ HPLC. Detailed synthetic protocols for two of the peptides are given below, and others were synthesized in a similar fashion. t-Boc-D-Nap-L-Bph-OMe. A 150-mg portion of the free acid t-Boc-(D-Nap)-OH, 130 mg of the free amine H-(L-Bph)-OMe, 250 mg of morpho-CDI, and 75 mg of HOBT were stirred in 3 mL of CHzClz overnight. The precipitate which formed was removed by filtration, and the CH2ClZ layer was washed with 10% HC03-, 10% citric acid, and water. The contents of the organic layer were then subjected to flash chromatography (5% CH30H/CH2C12) yielding the desired peptide (R, = 0.48, 5% CH,OH/CH,Cl*; yield 190 mg; 70%). IH NMR (200 MHz, CDC13): 6 in ppm 8.2-7.3 (m, 7 H, Nap Ar), 7.15 (d, 2 H, Bph Ar), 6.4 (d, 2 H, Bph Ar), 6.05 (d, 1 H, Bph NH), 5.15 (d, 1 H , Nap NH), 4.75 (m, 1 H , BrPhe Ca-H), 4.5 (m, 1 H, Nap Ca-H), 3.6 (s, 3 H, CH3 ester), 3.5 (m, 2 H, NapCo-H),2.9(d/d, 1 H,Bph C@-H),2.45(m, 1 H,BphC@-H), 1.45 (s, 9 H, C H ~ B O C )FAB-MS: . 557.2, 555.2 (MH+, two bromine isotopes as required). Cyclo-(D-Nap-L-Bph). A 150-mg portion of the protected dimer was dissolved in 2 mL of 1:1 CH2C12/TFAand the solvent removed under vacuum. The residue was the dissolved in 5 mL of EtOAc and extracted twice with 10% HC03-. The organic layer was then dried, evaporated, and diluted with 50 mL of toluene and refluxed for 12 h. A precipitate formed which was collected, and this was confirmed to be the cyclic dimer (R, = 0.33, 5% CH30H/CH,C12; yield 84 mg; 75%).
A
.-0 Y)
3 a
0.6
-1s -d." 0.2 0.4
k
al
I
0.0
360 400 440 nanometers Figure 2. Excitation corrected steady-state fluorescence spectra for all the dimeric peptides, in acetonitrile. Relative to the control dimer (D,LIdim), all bromo dimers exhibited distinct fluorescence quenching. The D,L-dimers, both linear and cyclo, exhibited a greater quenching than the corresponding diastereomeric L,L-dimers. 320
1H-NMR (200 MHz, DMSO): 6 in ppm 8.2-7.2 (m, 9 H, Nap Ar, Nap NH, Bph NH), 7.35 (d, 2 H, Bph Ar), 7.05 (d, 2 H, Bph Ar), 3.75 (m, 1 H, Nap Ca-H), 3.5 (m, 1 H, Bph Ca-H), 3.4 (d, 1 H, Nap CB-H), 3.2 (d, 1 H, Nap C@-H),2.95 (d/d, 1 H, BrPheCB-H), 2.7 (d/d, 1 H, BrPhe Co-H). FAB-MS: 425.1, 423.1 (MH+). Solvents used for optical studies were of HPLC grade and were used as received. The concentrations of the peptides were measured by diluting a stock solution and monitoring the optical density of the naphthalene chromophore at 284 nm. The final concentrations used were 20 pM or less. UV-vis spectra were measured on a Varian DMS-300 spectrophotometer. Steadystate fluorescence spectra were measured on a SLM 8000 fluorimeter with 2-nm excitation slit, 8-nm emission slit, and sample excitation at the naphthyl absorption of 290 nm, which is of lower energy than the lowest bromophenyl singlet transition. All samples were freeze-pumpthawed at least four times before any yield or lifetime measurement. Uncorrected fluorescence spectra were used to measure the relative naphthalene fluorescence quantum yield of the peptides from the corresponding ratios of the emission maxima (340 nm) corrected for the ratio of (l-lO-Azw); all emission spectra were of identical shape and only differed in intensity. Naphthalene triplet yield measurements were performed by the nanosecond pump (290 nm) probe (420 nm) technique, and the details have been reported elsewhere.6 Fluorescence lifetimes were measured using the technique of time-correlated single-photon counting. Samples were excited at 292 nm by the frequency-doubled output (angle tuned BBO) of a cavity-dumped dye laser (Coherent 701-3CD, Rhodamine 590) synchronously pumped by the second harmonic of a CW mode-locked Nd3+:YAG laser (Coherent Antares 76-s). Naphthalene fluorescence photons were detected with a 12 pm Hamamatsu microchannelplate. Spectral resolution was obtained with a Spex 0.25-m monochromator with a 20-nm bandpass. The data were transferred toa Macintosh IIcxfor Marquardt nonlinear least-squares analysis.
Resuits Quenchingof Naphthyl Fluorescence. The parent chromophore, 1-methylnaphthalene (MNap) and all peptides containing the chromophoric amino acid Nap showed identical UV-vis spectra ruling out the formation of any ground-state complex between MNap and the BrBz group. The emission spectra were also identical in shape. All four dimers containing Bph showed fluorescence quenching activity in CH&N, as shown in Figure 2. Br-L,L-ldim was the least active (34% quenching) while BrD,L-cdim showed maximum fluorescencequenching (60% quenching). Results for all of the dimeric peptides are summarized in Table I along with the data reported earlier for the octameric
Excitation Transfer in Linear and Cyclic Peptides
The Journal of Physical Chemistry, Vol. 97, No. 16, 1993 3959
TABLE I: Fluorescence Yields, Triplet Yields, and the Relative Contributions from Triplet Yielding (At) and Nontriplet Yielding (k,) Quenching Rate Constants for the Isomeric Octamers and Diastereomeric Dimers ~~
peptides Br-4,5-0ct Br-3,5-0ct Br-3,6-0ct Br-D,L-ldim Br-D,L-cdim Br-L,L-ldim Br-L,L-cdim
~F/#F,?
bT/bT,?
0.86 f 0.02 0.89 f 0.02 0.32 f 0.01 0.54 f 0.01 0.40 f 0.02 0.76 f 0.01 0.60 f 0.02
0.91 f 0.03 1.01 f 0.03 0.85 f 0.02 0.98 f 0.03 0.73 f 0.02 0.94 f 0.01 0.98 f 0.01
analysisb TR TR
ss ss TR ss
TR
k m PS-'
kX,70%) was accompanied by a minor component. Interestingly, the two linear dimers exhibited clean single exponential decay despite the fact that they are by far theleast conformationally constrained (most floppy). It appears that rapid conformational interconversion is occurring in the linear dimers on this fluorescence time scale when the backbone is least constrained. The conformational states of the cyclo dimers depend upon substitution pattern. It has been established that in cyclic peptide dimers the presence of two bulky residues cis to each other (Br-L,L-cdim) forces the diketopiperazine ring to be planar in order to release steric strain.* A single backboneconformation is likely for the Br-L,L-cdim. On the other hand, in trans cyclodimers (Br-D,L-cdim) the diketopiperazine ring assumes the more favorable boat conformation-opening up the possibility of yet another conformational equilibrium, that between the two boat forms, each with a distinct decay rate. The rate of boat-boat interconversion is estimated to be slow if taken as similar to the known cyclohexane chair*hair interconversion rate of 1 X los s-1-12 The steady-state naphthyl fluorescence data, &/4~,, precisely matched the time resolved data, T F / T F , ,for each monoexponential peptide: Br-D,L-ldim, Br-L,L-ldim, Br-3,5-0ct, and Br-4,5-0ct. The steady-state data, 4~/&,, were then compared to the integrated time-resolved data as (E~c~TF,~)/TF,, for the nonmonoexponential peptides (ci and T F ,are ~ the fractional amplitudes and lifetimes of each component i ) : (0.37 vs 0.40) for Br-D,Ld i m , (0.58 vs 0.60) for Br-L,L-cdim, and (0.32 vs 0.32) for Br3,6-0ct. Hence, the yield measurements and the fits to the timeresolved data are in excellent accord. A more detailed analysis of the time-resolved fluorescence data in relation to peptide side-chain dynamics will be presented elsewhere. However, in summary, four peptides exhibited monoexponential decay while conformational heterogeneity was detectable for the trans cyclodimer Br-D,L-cdim, Br-L,L-dim, and Br-3,6-0ct, where the interacting partners were held close to each other by conformational constraint. The fluorescence data were analyzed to obtain the rate constants in two different ways depending on whether a peptide showed monoexponential decay or biexponential decay (see Table I). Naphthalene Triplet-Producingand Nontriplet-Producing Fluorescence Quenching. To further probe into the mechanism of
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Basu et al.
3960 The Journal of Physical Chemistry, Vol. 97, No. 16, 1993 10000
-
3.5
/
2.5 n
5
s
from alkyl bromide data
e"
I?
1000;
(slop -0.63)
\
-5
Br-D,L-cdh 1.5
0
2
100
t
-
Br-3SQct
0.5 0.0
0
I
I
I
50
100
150
Time (ns)
10000
9
2.0
Figure4. Plot of the "Meding;-Wi1kinson"relation ( 4 6 ) . Each peptide is depicted in the figure as a point calculated from their measured value of (C$F
