Controlling multiple fluorescent signal output in cyclic peptide-based supramolecular systems

Article abstract of DOI:10.1021/ja066885h

A multicomponent equilibrium network based on self-assembling α,γ-cyclic peptides with controlled fluorescence output is described. The network takes advantage of the large association constant of α,γ-cyclic peptides and the controlled formation homo- and

Full text of DOI:10.1021/ja066885h

Published on Web 01/23/2007
Controlling Multiple Fluorescent Signal Output in Cyclic
Peptide-Based Supramolecular Systems
Roberto J. Brea,† M. Eugenio Va´zquez,† Manuel Mosquera,‡ Luis Castedo,†
and Juan R. Granja*,†
Contribution from Departamento de Qu´ımica Orga´nica and Departamento de Qu´ımica F´ısica,
UniVersidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain
Received September 25, 2006; E-mail: qojuangg@usc.es
Abstract: A multicomponent equilibrium network based on self-assembling R,γ-cyclic peptides with controlled
fluorescence output is described. The network takes advantage of the large association constant of R,γ-
cyclic peptides and the controlled formation homo- and heterodimers, making use for the first time of excimer/
FRET effects in conjunction for studying complex interaction networks. In addition, we study the Dapoxyl/
pyrene FRET pair for the first time.
Introduction
assembly. However, their work was aimed at the development
of molecular electronics applications and was not concerned
One of the most fundamental and pressing problems in the
field of supramolecular chemistry is the control of self-assembly
processes through the design of the molecular components1 and
the practical application at the macromolecular level of such
supramolecular associations.2 Self-assembling peptide nanotubes
(SPN) have been widely studied as supramolecular model
systems and as tools in materials and biological sciences due
to their ease of synthesis and modification.3 In addition,
fluorescence techniques have been extensively used in the study
of self-assembly processes in macromolecular chemistry,4 but
their application to the study of SPN is still scarce. For example,
Ghadiri et al. have recently reported that D,L-R-peptides bearing
1,4,5,8-naphthalenetetracarboxylic acid diimide (NDI) side
chains show intermolecular NDI excimer formation upon self-
with the detailed study of the equilibria involved or the control
of the signal output.5
Design and Synthesis
In an approach to novel supramolecular systems, we have
recently reported that high-affinity homo- and heterodimerization
can be controlled in the context of R,γ-cyclic peptides (R,γ-
CPs). This could be done by appropriate design of the peptide
sequences involving (1S,3R)-3-aminocyclohexanecarboxylic
acids (γ-Ach), (1S,3R)-3-aminocyclopentanecarboxylic acids
(γ-Acp), and L-R-amino acids.6,7 We also found that hetero-
dimeric complexes of these cyclic peptides are more stable than
the corresponding homodimers. In this report, we have applied
this differential R,γ-CPs homo/heterodimer complex formation
in the design of a novel multicomponent equilibrium network
of fluorescently derivatized self-assembling R,γ-cyclic peptides
that can be brought into different association states, with
distinguishable fluorescent output signals, by controlling the
concentration of the different R,γ-CPs.8 This approach might
be relevant in the development of molecular systems for
information processing based on photoinduced electron or
energy transfer.9 In addition, we also report for the first time
the use of pyrene and Dapoxyl as a fluorescence resonance
energy transfer (FRET) pair that, due to the unique spectroscopic
† Departamento de Qu´ımica Orga´nica.
‡ Departamento de Qu´ımica F´ısica.
(1) (a) Lehn, J.-M. Supramolecular Chemistry: Concepts and PerspectiVes;
Wiley-VCH: Weinheim, Germany, 1995. (b) Philp, D.; Stoddart, J. F.
Angew. Chem., Int. Ed. Engl. 1996, 35, 1155-1196. (c) ComprehensiVe
Supramolecular Chemistry; Atwood, J. L., Davies, E. D., Lehn, J.-M.,
MacNicol, D. D., Vogtle, F., Eds.; Pergamon Press: Oxford, 1996.
(d) Bermejo, M. R.; Gonza´lez-Noya, A. M.; Pedrido, R. M.; Romero, M.
J.; Va´zquez, M. Angew. Chem., Int. Ed. 2005, 44, 4182-4157. (e)
Amendola, V.; Fabbrizzi, L.; Mangano, C.; Pallavicini, P. Acc. Chem. Res.
2001, 34, 488-493.
(2) (a) Balzani, V.; Venturi, M.; Credi, A. Molecular DeVices and Machines:
A Journey into the Nano World; Wiley-VCH: Weinheim, Germany, 2003.
(b) Bushey, M. L.; Nguyen, T. Q.; Zhang, W.; Horoszewski, D.; Nuckolls,
C. Angew. Chem., Int. Ed. 2004, 43, 5446-5453. (c) Rothemund,
P. W. K. Nature 2006, 440, 297-302.
(3) (a) Brea, R. J.; Granja, J. R. Self-Assembly of Cyclic Peptides in Hydrogen-
Bonded Nanotubes. In Dekker Encyclopedia of Nanoscience and Nano-
technology; Schwarz, J. A., Contescu, C. I., Putyera, K., Eds.; Marcel
Dekker: New York, 2004; pp 3439-3457. (b) Martin, C. R.; Kohli,
P. Nat. ReV. 2003, 2, 29-37. (c) Ferna´ndez-Lo´pez, S.; Kim, H.-S.; Choi,
E. C.; Delgado, M.; Granja, J. R.; Khasanov, A.; Kraehenbuehl, K.; Long,
G.; Weinberger, D. A.; Wilcoxen, K. H.; Ghadiri, M. R. Nature 2001, 412,
452-455. (d) Motesharei, K.; Ghadiri, M. R. J. Am. Chem. Soc. 1997,
119, 11306-11312.
(4) (a) Martin, R. B.; Fu, K.; Li, H.; Cole, D.; Sun, Y.-P. Chem. Commun.
2003, 2368-2369. (b) Kanekiyo, Y.; Naganawa, R.; Tao, H. Chem.
Commun. 2004, 1006-1007. (c) Winnik, F. M. Chem. ReV. 1993, 93, 587-
614. (d) Amendola, V.; Fabbrizzi, L.; Foti, F.; Licchelli, M.; Mangano,
C.; Pallavicini, P.; Poggi, A.; Sacchi, D.; Taglietti, A. Coord. Chem. ReV.
2006, 250, 273-299.
(5) (a) Horne, W. S.; Ashkenasy, N.; Ghadiri, M. R. Chem.sEur. J. 2005, 11,
1137-1144. (b) Ashkenasy, N.; Horne, W. S.; Ghadiri, M. R. Small 2006,
2, 99-102.
(6) For four amino acid R,γ-CPs, see: (a) Amor´ın, M.; Brea, R. J.; Castedo,
L.; Granja, J. R. Org. Lett. 2005, 7, 4681-4684. (b) Amor´ın, M.; Brea,
R. J.; Castedo, L.; Granja, J. R. Heterocycles 2006, 67, 575-583. For six
amino acid R,γ-CPs, see: (c) Amor´ın, M.; Castedo, L.; Granja, J. R.
J. Am. Chem. Soc. 2003, 125, 2844-2845. (d) Amor´ın, M.; Villaverde,
V.; Castedo, L.; Granja, J. R. J. Drug DeliVery Sci. Technol. 2005, 15,
87-92. For eight amino acid R,γ-CPs, see: (e) Amor´ın, M.; Castedo, L.;
Granja, J. R. Chem.sEur. J. 2005, 11, 6539-6457.
(7) For R,γ-CP heterodimers, see: Brea, R. J.; Amor´ın, M.; Castedo, L.; Granja,
J. R. Angew. Chem., Int. Ed. 2005, 44, 5710-5713.
(8) For earlier studies involving DL-R-peptides by fluorescence, see: Karlstro¨m,
A.; Unde´n, A. Biopolymers 1997, 41, 1-4.
9
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J. AM. CHEM. SOC. 2007, 129, 1653-1657
1653

A R T I C L E S
Brea et al.
Scheme 1. Cyclopeptides Used in This Studya
Ach-based CPs were synthesized as follows. The linear hexa-
peptides were prepared by a solution-phase method following
the strategy previously reported6,7 and cyclized in dichloro-
methane in the presence of TBTU. The resulting cyclic peptides
were hydrogenated (i.e., balloon pressure) with 10% Pd/C
catalyst to provide unprotected cyclic hexapeptides R,γ-CPAchH
and R,γ-CPAcpH. The resulting cyclo-[L-Ser-D-MeN-γ-Ach-(L-
Phe-D-MeN-γ-Ach)2-] (R,γ-CPAchH) was subsequently coupled
with 1-pyreneacetic acid in the presence of DIC/DMAP to give
R,γ-CPAchpyr, while peptide R,γ-CPAcpdap was prepared by
reaction of cyclo-[L-Lys-D-MeN-γ-Acp-(L-Leu-D-MeN-γ-Acp)2-
] (R,γ-CPAcpH) with Dapoxyl 3-sulfonamido propionic acid
succinimidyl ester in a 1:4 DMF/CHCl3 solution. The NMR
spectra of the resulting cyclic peptides in CHCl3 exhibited clear
â-sheet signals, characteristic of R,γ-CP dimeric structures, in
which the three nonequivalent dimers (rotamers) are slowly
exchanging on the NMR time scale (Scheme 1, bottom).
Steady State Fluorescence Studies
The first equilibrium studied was the homodimerization
process of R,γ-CPAchpyr (Scheme 2a). Irradiation of dilute
solutions of R,γ-CPAchpyr (50 nM in CHCl3) generated the
typical emission spectrum of pyrene, with two maxima at 377
and 397 nm. Increasing the concentration of R,γ-CPAchpyr by
addition of successive aliquots of a concentrated stock solution
of R,γ-CPAchpyr resulted in the appearance of a new band at
472 nm, corresponding to the pyrene excimer emission
(Figure 1).12
Considering that the formation of excimers is geometrically
allowed in only one of the three possible dimers, we used the
observed excimer band for the calculation of the homodimer-
ization binding constant of R,γ-CPAchpyr, resulting in KA
)
5.3 × 105 M-1 (Scheme 2a and Figure 1).13 From previous
studies, we expected that the heterodimer formation of R,γ-
CPAchpyr and any complementary Acp-based CP would take
place with much higher affinity than homodimerization of either
cyclic peptide.7 Thus, addition of R,γ-CPAcpZ competed with
the R,γ-CPAchpyr/R,γ-CPAchpyr homodimer formation by
efficiently sequestering R,γ-CPAchpyr as a heterodimeric R,γ-
CPAchpyr/R,γ-CPAcpZ complex, thus abolishing the excimer
emission band (Figure S1 in Supporting Information). This
observation supports the model of homodimeric species as
responsible for excimer emission.
a Ach and Acp peptide series and perspective representation of cyclo-
peptide dimers showing the hydrogen bonds between both cyclopeptides.
Schematic representation of the three nonequivalent dimeric forms that are
produced by successive shifts of register between both cyclopeptide rings.
Light gray represents nonfluorescent side chains, while dark gray represents
fluorescent side chains (either pyrene or Dapoxyl in Ach or Acp cyclic
peptides, respectively).
properties of pyrene (i.e., excimer emission), is particularly
suited for studying systems involving homo- and heterodimeric
species in equilibrium, yielding distinctive fluorescent signals.10
Although there are examples of pyrene in FRET pairs with other
fluorophores such as coumarins or fluorescein,11 to our knowl-
edge, this is the first time that pyrene excimer formation and
FRET processes are combined in the context of complex
intermolecular association studies.
This preference for heterodimer formation between Acp-based
and Ach-based CPs rather than homodimerization was also
exploited for the fluorescence resonance energy transfer process
between cyclic peptides R,γ-CAchpyr and R,γ-CAcpdap. Irradia-
tion at 340 nm of a 0.1 µM solution of R,γ-CPAcpdap resulted
in a large fluorescence emission band at 500 nm. Addition of
successive aliquots of R,γ-CPAchpyr over that solution increased
the fluorescence emission intensity at 500 nm (Figure S2 in
Supporting Information). Given that R,γ-CPAchpyr does not emit
at 500 nm, and the concentration of the cyclic peptide R,γ-
CPAcpdap is kept constant throughout the titration, the increased
The network presented in this paper was formed by different
equilibria involving two R,γ-CPs (Scheme 1): cyclo-[L-Ser(1-
pyreneacetyl)-D-MeN-γ-Ach-(L-Phe-D-MeN-γ-Ach)2-] (R,γ-
CPAchpyr) and cyclo-[L-Lys(Dapoxyl)-D-MeN-γ-Acp-(L-Leu-D-
MeN-γ-Acp)2-] (R,γ-CPAcpdap). Both the Acp-based and
(9) (a) Jang, S. S.; Jang, Y. H.; Kim, Y.-H.; Goddard, W. A., III; Flood,
A. H.; Laursen, B. W.; Tseng, H.-R.; Stoddart, J. F.; Jeppesen, J. O.; Choi,
J. W.; Steuerman, D. W.; DeIonno, E.; Heath, J. R. J. Am. Chem. Soc.
2005, 127, 1563-1575. (b) Li, X.; Sinks, L. E.; Rybtchinski, B.;
Wasielewski, M. R. J. Am. Chem. Soc. 2004, 126, 10810-10811.
(10) Giepmans, B. N. G.; Adams, S. R.; Ellisman, M. H.; Tsien, R. Y. Science
2006, 312, 217-224.
(11) (a) Margulies, D.; Melman, G.; Felder, C.; Arad-Yelin, R.; Shanzer, A.
J. Am. Chem. Soc. 2004, 126, 15400-15401. (b) Hossain, M. A.; Mihara,
H.; Ueno, A. J. Am. Chem. Soc. 2003, 125, 11178-11179.
(12) (a) Garc´ıa-Echeverr´ıa, C. J. Am. Chem. Soc. 1994, 116, 6031-6032. (b)
Ueno, A.; Suzuki, I.; Osa, T. J. Am. Chem. Soc. 1989, 111, 6391-6397.
(13) KA was determined by least-square analysis fitting to appropriate equations
using Mathematica 4.1 (Wolfram Research, Champaign, IL). For model
and equation used, see: (a) Park, J. W.; Song, H. E.; Lee, S. Y. J. Org.
Chem. 2003, 68, 7071-7076. (b) Martin, R. B. Chem. ReV. 1996, 96, 3043-
3064.
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1654 J. AM. CHEM. SOC. VOL. 129, NO. 6, 2007

Controlling Multiple Fluorescent Signal Output
A R T I C L E S
Figure 1. (a) Pyrene fluorescence emission of R,γ-CPAchpyr in CHCl3 (340 nm excitation wavelength) from 0.3 µM (brown) to 7.4 µM (dark blue),
denoting homodimer formation; 1-pyreneacetic acid (7.4 µM) is also shown (black). Inset shows titration for KA calculation. (b) Absorption spectrum of
Dapoxyl (solid line) showing the overlap with pyrene emission (dashed line). All spectra were normalized for representation.
Scheme 2. Equilibria between R,γ-CPAchpyr and R,γ-CPAcpdap and Signal Outputs Arising from the Different Complexesa
a Insert: the two other possible R,γ-CPAchpyr/R,γ-CPAcpdap heterodimeric rotamers responsible for the observed FRET.
emission intensity must arise from intramolecular FRET between
pyrene and Dapoxyl in the heterodimeric R,γ-CPAcpdap/R,γ-
CPAchpyr complex (Scheme 2b and Figure 2). This energy
transfer is possible because the spectral overlap between
Dapoxyl absorption and pyrene emission is almost complete,
which ensures efficient transfer between the two fluorophores
(Figure 1). Considering the quantum yield of pyrene under these
conditions to be 0.03,14 we calculated from these spectra the
Fo¨rster radius as 23 Å.
resulted in an increase in the natural emission of pyrene at 377
and 397 nm and a reduction of the 500 nm signal of Dapoxyl.
This observation is consistent with formation of the het-
erodimeric R,γ-CPAcpdap/R,γ-CPAchBn and disruption of the
supramolecular dimeric species R,γ-CPAcpdap/R,γ-CPAchpyr
(Scheme 2c and Figure 2). As expected, addition of a 100-fold
excess of 1-pyreneacetic acid did not induce any change in the
R,γ-CAcpdap emission intensity, consistent with the absence of
the cyclic peptide dimerization unit.
Addition of competitive cyclopeptide R,γ-CPAchBn to the
heterodimeric R,γ-CPAcpdap/R,γ-CPAchpyr complex solution
Time-Resolved Fluorescence Studies
These results demanded a full characterization of the dynamic
processes taking place using time-resolved fluorescence tech-
(14) Masuko, M.; Ohuchi, S.; Sode, K.; Ohtani, H.; Shimadzu, A. Nucleic Acids
Res. 2000, 28, e34.
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A R T I C L E S
Brea et al.
efficiency in the transfer process using eq 1.
R0
r )
(1)
(τ0D/τte)1/6
where τ0D is the excited-state lifetime of pyrene in the absence
of transfer (24 ns) and τte is the excited-state lifetime of energy
transfer process (1 ns).15,16 This distance corresponds to the
rotamers where pyrene and Dapoxyl are not in proximity. The
third rotamer that places both fluorophores in close contact must
have an even faster transfer that is not detectable in the
nanosecond time scale.
Conclusions
Figure 2. Formation of R,γ-CPAcpdap/R,γ-CPAchpyr heterodimer. Fluo-
rescence emission of (a) b monomeric R,γ-CPAcpdap; (b) [ heterodimeric
R,γ-CPAcpdap/R,γ-CPAchpyr; and (c) O heterodimeric R,γ-CPAcpdap/R,γ-
In summary, these studies show the design and characteriza-
tion of a novel multicomponent network of pyrene and Dapoxyl-
derivatized R,γ-CPs that display controlled fluorescent signal
output. These studies might be particularly relevant for the
development of new biosensor systems, specifically tailored for
studying networks involving homo- and heterodimerization
processes. In addition, to our knowledge, this is the first time
that pyrene excimer formation is used together with FRET to
achieve distinct fluorescent signals associated with diverse
association states. Finally, we also report for the first time the
use of a new FRET pair between pyrene and Dapoxyl character-
ized by a high transfer efficiency and convenient excitation and
emission wavelengths for biological imaging studies.
CPAchpyr with excess of R,γ-CPAch
.
Experimental Section
Peptide Synthesis. Linear peptides Boc-[L-Phe-D-MeN-γ-Ach]2-L-
Ser(Bn)-D-MeN-γ-Ach-OFm and Boc-[L-Leu-D-MeN-γ-Acp]2-L-Lys(Z)-
D-MeN-γ-Acp-OFm were prepared following the synthetic strategy
previously described.6,7
Cyclo-[L-Lys(Z)-D-MeN-γ-Acp-(L-Leu-D-MeN-γ-Acp)2-]
(R,γ-
CPAcpZ). A solution of Boc-[L-Leu-D-MeN-γ-Acp]2-L-Lys(Z)-D-MeN-
γ-Acp-OFm (400 mg, 0.35 mmol) in 20% piperidine in DCM (5 mL)
was stirred at room temperature for 20 min. After removal of the
solvent, the residue was dissolved in DCM (10 mL), and this solution
was washed with HCl (5%), dried over Na2SO4, filtered, and concen-
trated. The resulting residue was dissolved in 4 mL of TFA/DCM
(1:1) and stirred at room temperature for 15 min. After removal of the
solvent, the residue was dried under high vacuum for 3 h and used
without further purification. The linear peptide was dissolved in DCM
(345 mL) and treated with TBTU (133 mg, 0.41 mmol), followed
(dropwise) by DIEA (241 µL, 1.38 mmol) [an additional 1 equiv of
TBTU (111 mg, 0.35 mmol) and 4 equiv of DIEA (241 µL, 1.38 mmol)
were added when the starting material was detected by HPLC, and the
resulting mixture was stirred for 3 h at room temperature to complete
the reaction]. After 12 h, the solvent was removed under reduced
pressure, and the crude was purified by HPLC, affording 160 mg of
R,γ-CPAcpZ as a white foam [54%, Rt ) 16 min (Phenomenex Maxsil-
Figure 3. (a) 380 nm emission of 5 µM monomeric R,γ-CPAchpyr; (b)
470 nm emission of homodimeric R,γ-CPAchpyr; (c) 380 nm emission of
heterodimeric R,γ-CPAcpdap/R,γ-CPAchpyr (5 µM R,γ-CPAchpyr and 1
µM R,γ-CPAcpdap). Dashed line represents the laser pulse. Gray lines are
the best fit to the experimental data.
niques. Irradiation of homodimeric R,γ-CPAchpyr/R,γ-CPAchpyr
resulted in a fluorescence decay that could be fitted to a
biexponential model with two different times of 24 and 7 ns
(Figure 3). The longer 24 ns time corresponds to the normal
pyrene emission decay from the two rotamers where excimer
formation is geometrically impossible (Scheme 1); the shorter
7 ns lifetime is assigned to the rotamer with stacked pyrene
side chains, which can give rise to excimer emission. Measure-
ments of fluorescence decays at the excimer emission wave-
length (470 nm) also showed a biexponential decay (19 and 7
ns). The longer time corresponds to the excimer decay, while
the shorter 7 ns decay, having a negative preexponential factor,
is assigned to the process of conformational change that gives
the required geometrical arrangement for excimer formation
(Figure 3).
1
10 semipreparative column, 5-15% MeOH in CH2Cl2, 30 min)]. H
NMR (CDCl3, 500.13 MHz, δ): 8.31-8.11 (overlapping doublets, 3H,
NH), 7.41-7.27 (m, 5H, Ar Z), 5.25-4.98 (m, 3.4H, 2 × HR Leu +
HR Lys + 0.4 × NH), 5.07 (s, 2H, CH2 Z), 4.96-4.64 (m, 3.6H, 3 ×
Hγ γ-Acp + 0.6 × NH), 3.25-3.10 (m, 2H, CH2NH), 3.09-3.00
(overlapping singlets, 9H, NMe γ-Acp), 2.99-2.87 (m, 3H, HR γ-Acp),
2.30 (m, 3H, CH γ-Acp), 2.09 (m, 3H, CH γ-Acp), 1.96-1.18 (m,
24H, 12 × CH γ-Acp + 2 × CH2 Leu + 2 × CH Leu + 3 × CH2
When the heterodimeric complex R,γ-CPAcpdap/R,γ-CPAch
-
pyr was studied, we were able to observe the expected pyrene
decay (24 ns), together with a new fast process with an
exponential decay of 1 ns. This lifetime was assigned to the
energy transfer process which, together with the previously
calculated Fo¨rster radius (R0), allowed us to determine an
average pyrene-Dapoxyl distance of 14 Å, with a 96%
(15) Valeur, B. Molecular Fluorescence: Principles and Applications; Wiley-
VCH: Weinheim, Germany, 2002.
(16) The estimated error in the average pyrene-Dapoxyl distance determination
is about 10%, derived mainly form the Fo¨rster radius calculation: Stryer,
L. Annu. ReV. Biochem. 1978, 47, 819- 846. See also ref 15.
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Controlling Multiple Fluorescent Signal Output
A R T I C L E S
Lys), 1.04-0.85 (m, 12H, CH3 Leu). 13C NMR (CDCl3, 125.77 MHz,
δ): 175.3 (CO), 173.5 (CO), 156.3 (CO), 136.6 (C), 128.5 (CH), 128.1
(CH), 128.0 (CH), 66.7 (CH2), 54.7 (CH), 48.2 (CH), 46.9 (CH), 42.4
(CH2), 42.3 (CH), 40.9 (CH2), 35.8 (CH2), 33.1 (CH2), 29.8 (NCH3),
29.3 (CH2), 27.2 (CH2), 24.8 (CH), 23.1 (CH3), 22.8 (CH2), 22.6 (CH2),
22.4 (CH3). FTIR (293 K, CHCl3): 3303 (amide A), 3005, 2960, 1662,
1626 (amide I), 1529 (amide IIII) cm-1. MS (MALDI-TOF) [m/z (%)]:
902 ([M + K]+, 10), 886 ([M + Na]+, 40), 864 ([MH]+, 100). HRMS
(MALDI-TOF) calcd for C47H74N7O8 ([MH]+) 864.5593, found 864.5578.
Cyclo-[L-Ser(Bn)-D-MeN-γ-Ach-(L-Phe-D-MeN-γ-Ach)2-] (R,γ-
CPAchBn).4 Prepared in the same way as R,γ-CPAcpZ from Boc-{[L-
Phe-D-MeN-γ-Ach]2-L-Ser(Bn)-D-MeN-γ-Ach-}OFm (188 mg, 0.21
mmol). Yield after HPLC purification was 125 mg (68%); white solid.
1H NMR (CDCl3, 250.13 MHz, δ): 8.79-8.24 (overlapping doublets,
J ) 9.2 Hz, 3H, NH), 7.20 (m, 15H, 2 × Ar Phe + Bn), 5.48-5.21
(m, 3H, 2 × HR Phe + HR Ser), 4.70-4.24 (m, 3H, Hγ γ-Ach), 3.77-
3.63 (m, 2H, CH2âSer), 3.15-3.01 (m, 6H, 2 × CH2âPhe + CH2Βn),
2.88-2.78 (m, 3H, HR γ-Ach), 2.51 (s, 9H, NMe γ-Ach), 2.06-1.03
(m, 24H, CH2 γ-Ach). FTIR (293 K, CHCl3): 3303 (amide A), 2934,
2863, 1664, 1627 (amide I), 1529 (amide IIII) cm-1. MS (FAB+) [m/z
(%)]: 1778 ([2MH]+, 12), 889 ([MH]+, 100). HRMS [MH]+ calcd for
C52H69N6O7 889.5228, found 889.5219.
[78%, Rf ) 0.15 (10% MeOH in CH2Cl2)]. 1H NMR (CDCl3, 750 MHz,
δ): 8.22-8.07 (overlapping doublets, 3H, NH), 7.89 (d, J ) 8.2 Hz,
2H, Ar Dap), 7.64 (m, 1H, Ar Dap), 7.53 (d, J ) 8.6 Hz, 2H, Ar Dap),
7.46 (m, 1H, Ar Dap), 7.05 (s, 1H, Ar Dap), 6.70 (d, J ) 8.6 Hz, 2H,
Ar Dap), 5.34-5.23 (m, 7H, 2 × HR Leu + HR Lys + CH2 + 2 ×
NH), 5.16-4.97 (m, 3H, Hγ γ-Acp), 4.74 (m, 2H, CH2NH), 2.99-
2.95 (overlapping singlets, 9H, NMe γ-Acp), 2.99 (overlapping singlets,
6H, NMe Dap), 3.25-3.13 (m, 3H, HR γ-Acp), 2.54 (m, 2H, CH2),
2.03-1.02 (m, 30H, 9 × CH2 γ-Acp + 2 × CH2 Leu + 2 × CH Leu
+ 3 × CH2 Lys), 0.90-0.69 (m, 12H, CH3 Leu). MS (MALDI-TOF)
[m/z (%)]: 1165 ([M + K]+, 39), 1149 ([M + Na]+, 16), 1127 ([MH]+,
31), 1012 (100). HRMS (MALDI-TOF) calcd for C59H87N10O10S
([MH]+) 1127.6322, found 1127.6331.
Cyclo-[L-Ser(1-pyreneacetyl)-D-MeN-γ-Ach-(L-Phe-D-MeN-
γAch)2-] (R,γ-CPAchpyr). A solution of 1-pyreneacetic acid (1.6
mg, 6.3 µmol) in CDCl3 (250 µL) was stirred and sonicated at room
temperature for 10 min, and then DIC (1.5 µL, 9.4 µmol), R,γ-CPAchH
(5.0 mg, 6.3 µmol), and DMAP (1.2 mg, 9.4 µmol) were successively
added. After 1 h stirring at room temperature, the solution was washed
with HCl (5%) and NaHCO3(sat.), dried over Na2SO4, and concentrated
under reduced pressure. The residue was purified by HPLC, giving
5.2 mg of R,γ-CPAchpyr as a yellow solid [80%, Rt ) 27 min
(Phenomenex Maxsil-10 semipreparative column, 0-10% MeOH in
CH2Cl2, 30 min)]. 1H NMR (CDCl3, 500.13 MHz, δ): 8.63-8.51
(overlapping doublets, 2H, NH), 8.30-7.75 (m, 10H, Pyr + NH), 7.24-
7.07 (m, 10H, Ar Phe), 5.45-5.14 (m, 3H, 2 × HR Phe + HR Ser),
4.62-3.93 (m, 7H, 3×x Hγ γ-Ach + CH2 Pyr + CH2âSer), 3.21-
2.78 (m, 7H, 2 × CH2 âPhe + 3 × HR γ-Ach), 2.65-2.38 (overlapping
singlets, 9H, NMe γ-Ach), 1.92-0.68 (m, 24H, CH2 γ-Ach). MS
(MALDI-TOF) [m/z (%)]: 1079 ([M + K]+, 31), 1063 ([M + Na]+,
100), 1041 ([MH]+, 9). HRMS (MALDI-TOF) calcd for C63H72N6NaO8
([M + Na]+) 1063.5304, found 1063.5277.
Cyclo-[L-Lys-D-MeN-γ-Acp-(L-Leu-D-MeN-γ-Acp)2-] (R,γ-CPAcpH).
A solution of R,γ-CPAcpZ (80 mg, 93 µmol) in EtOH (1 mL) was
treated with 10% Pd/C (20 mg, 19 µmol) and stirred at room
temperature under hydrogen overnight. The resulting mixture was
filtered through a celite pad; the residue was washed with ethanol, and
the pooled filtrate and washings were concentrated under reduced
1
pressure, affording 62 mg of R,γ-CPAcpH as a white solid (91%). H
NMR (DMSO-d6, 500.13 MHz, δ): 8.28-8.12 (overlapping doublets,
3H, NH), 7.61 (br s, 2H, NH2), 4.91-4.52 (m, 6H, 2 × HR Leu + HR
Lys + 3 × Hγ γ-Acp), 2.92-2.82 (overlapping singlets, 9H, NMe
γ-Acp), 2.78-2.69 (m, 2H, CH2 NH2), 2.67-2.57 (m, 3H, HR γ-Acp),
1.98-1.11 (m, 30H, 9 × CH2 γ-Acp + 2 × CH2 Leu + 2 × CH Leu
+ 3 × CH2 Lys), 0.91-0.81 (m, 12H, CH3 Leu). MS (MALDI-TOF)
[m/z (%)]: 751 ([M + Na]+, 45), 729 ([MH]+, 100). HRMS (MALDI-
TOF) calcd for C39H67N7O6 ([MH]+) 729.5147, found 729.5100.
Cyclo-[L-Ser-D-MeN-γ-Ach-(L-Phe-D-MeN-γ-Ach)2-] (R,γ-CPAchH).6d
Prepared in the same way as R,γ-CPAcpH from R,γ-CPAcpBn (40 mg,
45 µmol). Yield after HPLC purification was 36 mg (99%); white solid.
1H NMR (CDCl3, 750 MHz, δ): 8.81-8.59 (m, 2H, NH), 8.36 (d, J )
8.5 Hz, 0.25H, NH Phe), 8.31 (d, J ) 8.7 Hz, 0.25H, NH Phe), 7.92
(d, J ) 8.1 Hz, 0.5H, NH Ser), 7.20 (m, 10H, Ar Phe), 5.33 (m, 2.5H,
2 × HR Phe + 0.5 × HR Ser), 5.12 (s, 0.5H, HR Ser-HR Ser), 4.57 (m,
3H, Hγ γ-Ach), 3.90 and 3.75 (s, 2H, CH2âSer), 3.11-2.92 (m, 7H,
2 × CH2 âPhe + 3 × HR γ-Ach), 2.52 and 2.48 (s, 9H, NMe γ-Ach),
2.09-0.98 (m, 24H, CH2 γ-Ach). FTIR (293 K, CHCl3): 3303 (amide
A), 2928, 2857, 1661, 1624 (amide I), 1525 (amide IIII) cm-1. MS
(FAB+) [m/z (%)]: 799 ([MH]+, 100). HRMS [MH]+ calcd for
C45H63N6O7 799.4758, found 799.4765.
Cyclo-[L-Lys(Dapoxyl)-D-MeN-γ-Acp-(L-Leu-D-MeN-γ-Acp)2-] (R,γ-
CPAchdap). A solution of R,γ-CPAcpH (5.0 mg, 6.9 µmol) in DMF
(100 µL) was stirred and sonicated at room temperature for 10 min,
and then DIEA (4.8 µL, 27.4 µmol) was added. After 5 min stirring at
room temperature, a solution of Dapoxil 3-sulfonamido propionic acid
succinimidyl ester (3.5 mg, 6.9 µmol) in CDCl3 (400 µL) was added.
After 1 h stirring at room temperature, the resulting mixture was
concentrated under reduced pressure. The residue was dissolved in
CHCl3 (2 mL), washed with HCl (5%) and NaHCO3(sat.), dried over
Na2SO4, and concentrated under reduced pressure. The resulting crude
was purified by preparative thin-layer chromatography (5% MeOH in
CH2Cl2; two times), giving 6.0 mg of R,γ-CPAcpdap as a yellow solid
Time-Resolved Fluorescence. Fluorescence lifetimes were deter-
mined by time-correlated single-photon counting on an Edinburgh
Instruments CD-900 spectrometer equipped with a hydrogen-filled
nanosecond flash lamp. The instrumental response width of the system
is 1.0 ns. We measured usually until 10 000 counts were reached in
(2 × 103 channels). The emission band-pass for the lifetime measure-
ments was usually 20 nm. The experiments were performed at room
temperature, and samples were purged with argon prior to measurement.
Acknowledgment. This work was supported by the Spanish
Ministry of Education and Science and the ERDF (SAF2004-
01044). R.J.B. thanks the Spanish M.E.C. for his FPU fellow-
ship, and M.E.V. thanks the Human Frontier Science Program
Organization for the Career Development Award (CDA0032/
2005-C) and the Spanish M.E.C. for his Ramo´n y Cajal contract.
We also wish to thank Dr. Miguel Va´zquez (University of
Santiago) and Dr. Timm Knoerzer (Nazareth College, Rochester,
NY) for their useful advice in the preparation of the manuscript.
Supporting Information Available: Figures S1 (elimination
of the excimer emission band of dimeric R,γ-CPAchpyr by
addition of CPAcpZ); S2 (addition of R,γ-CPAchpyr over a
solution of R,γ-CPAcpdap). Characterization of peptides R,γ-
CPAcpZ, R,γ-CPAcpH, R,γ-CPAcpdap, and R,γ-CPAchpyr, in-
cluding NMR (1H and 13C, NOESY, and/or ROESY), FTIR,
and UV spectra. This material is available free of charge via
the Internet at http://pubs.acs.org.
JA066885H
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J. AM. CHEM. SOC. VOL. 129, NO. 6, 2007 1657