Spectroscopic characterization of an assembled pair of free-base and zinc porphyrins linked by the cyclic β-sheet peptide Gramicidin S

Article abstract of DOI:10.1039/b402541e

A pair of porphyrins was linked to the Orn side chains of Gramicidin S [cyclo(-D-Phe-Pro-Val-Orn-Leu-)₂] and its derivatives via the amide bonds; the assorted porphyrins were characterized by various spectroscopic methods. The high-field shifts of the porphyrin signals in the ¹H NMR spectrum and the exciton-coupled Cotton effects in the CD spectrum of cyclo[-D-Phe-Pro-Val-Orn(Por)-Leu-]₂ are both intense compared to those of cyclo[-D-Phe-Pro-Val-Dab(Por)-Leu-]₂ or cyclo[-D-Phe-Pro- Val-Lys(Por)-Leu-]₂ (Dab, diaminobutyric acid; Por, the side-chain linked porphyrin). Some ¹H NMR signals of the tolyl protons of the porphyrins coalesce at 353 K in CD₂Cl₂, which reveals dynamic processes of the porphyrins. The intensities of the Cotton effect are: Gramicidin S with a pair of free-base porphyrins ≈ Gramicidin S with a free-base porphyrin and a zinc porphyrin < Gramicidin S with a pair of zinc porphyrins. The ¹H NMR, UV-vis, CD and fluorescence spectra show that the solvent substantially influences the assembly of the porphyrins. These spectroscopic studies suggest that the strength of the intramolecular interactions between a pair of porphyrins is in the order of toluene > CH₂Cl₂ > DMF.

Full text of DOI:10.1039/b402541e

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P A P E R
Spectroscopic characterization of an assembled pair of free-base
and zinc porphyrins linked by the cyclic b-sheet peptide
Gramicidin Sw
Toru Arai,*a Koji Araki,a Naoki Maruo,a Yuko Sumida,a Chie Korosue,a Kengo Fukuma,a
Tamaki Katob and Norikazu Nishino*b
a Faculty of Engineering, Kyushu Institute of Technology, Kitakyushu 804-8550, Japan.
E-mail: arai@che.kyutech.ac.jp
b Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology,
Kitakyushu 808-0196, Japan
Received (in Montpellier, France) 18th February 2004, Accepted 14th April 2004
First published as an Advance Article on the web 21st July 2004
A pair of porphyrins was linked to the Orn side chains of Gramicidin S [cyclo(–D-Phe–Pro–Val–Orn–Leu–)2]
and its derivatives via the amide bonds; the assorted porphyrins were characterized by various spectroscopic
1
methods. The high-field shifts of the porphyrin signals in the H NMR spectrum and the exciton-coupled
Cotton effects in the CD spectrum of cyclo[–D-Phe–Pro–Val–Orn(Por)–Leu–]2 are both intense compared to
those of cyclo[–D-Phe–Pro–Val–Dab(Por)–Leu–]2 or cyclo[–D-Phe–Pro–Val–Lys(Por)–Leu–]2 (Dab,
1
diaminobutyric acid; Por, the side-chain linked porphyrin). Some H NMR signals of the tolyl protons of the
porphyrins coalesce at 353 K in CD2Cl2, which reveals dynamic processes of the porphyrins. The intensities
of the Cotton effect are: Gramicidin S with a pair of free-base porphyrins E Gramicidin S with a free-base
1
porphyrin and a zinc porphyrin o Gramicidin S with a pair of zinc porphyrins. The H NMR, UV-vis, CD
and fluorescence spectra show that the solvent substantially influences the assembly of the porphyrins. These
spectroscopic studies suggest that the strength of the intramolecular interactions between a pair of
porphyrins is in the order of toluene 4 CH2Cl2 4 DMF.
strands of a cyclic decapeptide, Gramicidin S (GS). GS [cyclo
Introduction
(–D-Phe–Pro–Val–Orn–Leu–)2, 2c, Scheme 1]13 is a natural
bacterial polypeptide comprised of a set of antiparallel b-sheets
and two type II’ b-turn units.14 The cyclic framework of this
small polypeptide stabilizes its b-sheet structure. The side
chains of the Orn residues are in a face-to-face orientation,
which can tether two functional molecules. A pair of porphyr-
ins linked to the antiparallel b-sheet may take a face-to-face
orientation, which will be characterized by spectroscopic meth-
ods. Short linkers such as –(CH2)3– between the porphyrin and
peptide will avoid distortion of the peptide framework upon
varying the solvent. Thus, a new tool to examine the solvent
effect on the porphyrin assembly is designed using a spatially
close pair of porphyrins on an antiparallel peptide template.
We have briefly communicated the synthesis and the spectro-
scopic feature of the porphyrins linked to GS.15 Here we report
further investigations on the relative orientation of the por-
phyrins and its solvent dependency as characterized by various
Various functional biomolecules such as hemes, chlorophylls
and vitamin B12 contain porphyrins and related compounds as
the active centers.1 The rational arrangements of the tetrapyr-
roles, cofactors and so on in biosystems are essential for their
excellent functions, for instance, charge separation,2 solar
energy collection3 and redox catalysis.4 Among various bio-
molecules, the planar structure of porphyrins with a hydro-
phobic conjugated system is unusual. A number of model
systems has been constructed with oligomeric,5 polymeric6
and supramolecular porphyrins.7 In particular, various por-
phyrin arrays linked by artificial polypeptides have been
synthesized8,9 by virtue of the recent progress in peptide
synthesis.10 The importance of peptide-linked porphyrins is
increasing because natural proteins are not only the scaffolds of
the porphyrins but also regulate the reactivities of the porphyr-
ins.11 The environments of the natural porphyrins such as
neighboring amino acids, water molecules and lipophilic mo-
lecules substantially effect the function and assembling of
porphyrins. The interactions between porphyrins are largely
hydrophobic and p-p interactions,12 in addition to hydrogen
bonding and metal coordination;2,3 therefore, the solvent may
influence the relative orientation between porphyrins, that is,
the orientation of the porphyrins could be tunable by the
solvent.
1
spectroscopic methods including H NMR.
Results and discussion
Syntheses of Gramicidin S derivatives bearing a pair of
1
porphyrins and the H NMR investigation of the peptide
conformation
Here we wish to introduce a new methodology to investigate
the solvent effect for the interaction between porphyrins. A
pair of porphyrins was linked to the antiparallel b-sheet
A GS derivative bearing a pair of porphyrins at the Orn side
chains, cyclo[–D-Phe–Pro–Val–Orn(PorFb)–Leu–]2 [2d, PorFb
is 4-(10,15,20-tritolylporphyrin-5-yl)benzoyl], and its two
homologs with different spacer lengths between the peptide
and porphyrins, cyclo[–D-Phe–Pro–Val–Dab(PorFb)–Leu–]2
(1d) and cyclo[–D-Phe–Pro–Val–Lys(PorFb)–Leu–]2 (3d), were
prepared (Scheme 1). First, the GS derivatives and homologs
{ Electronic supplementary information (ESI) available: complete
synthetic details and characterization of the GS derivatives. See
http://www.rsc.org/suppdata/nj/b4/b402541e/
T h i s j o u r n a l i s & T h e R o y a l S o c i e t y o f C h e m i s t r y a n d t h e
C e n t r e N a t i o n a l d e l a R e c h e r c h e S c i e n t i f i q u e 2 0 0 4
N e w J . C h e m . , 2 0 0 4 , 2 8 , 1 1 5 1 – 1 1 5 9
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Fig. 1 UV-vis spectra of 2d (trace A), 2dFbZn (trace B, broken line)
and 2dZn2 (trace C) in CH2Cl2.
afforded 2dZn2 bearing a pair of Orn(PorZn) residues. The
UV-vis spectrum of 2dFbZn was almost the average of those of
2d and 2dZn2 in CH2Cl2 (Fig. 1), suggesting that there was
little ground-state interaction between the porphyrins5c in this
solvent (see below). The HPLC confirmed the purity of all the
1
GS derivatives and H NMR (1D and 2D COSY) and FAB
MS/FAB HR-MS spectra characterized their structures.
1H NMR measurements of the GS derivatives bearing a pair
of porphyrins in DMSO-d6 (0.80 mM) show that their cyclic
frameworks take on a conformation similar to that of the
natural GS (2c) in this solvent (Table 1). The temperature
dependencies of the Val–NH (Dd ¼ ꢀ2.0 ppb Kꢀ1
,
ppb ¼ ppm ꢁ 10ꢀ3) and Leu–NH (ꢀ2.6 ppb Kꢀ1) signals of
2d are as small as those of 2c.14c These facts support the
intramolecular hydrogen bonds Val–NHꢂ ꢂ ꢂOC–Leu and
Leu–NHꢂ ꢂ ꢂOC–Val, as shown in the crystal structure of 2c.14a
The large temperature dependencies of the Orn–aNH and
D-Phe–NH signals of both 2c and 2d show that these NHs are
not involved in intramolecular hydrogen bonding and are exp-
osed to the solvent DMSO.20 The coupling constants of
Scheme 1 Synthesis of Gramicidin S derivatives bearing a pair of
porphyrins. (a) TFA, then AcOH/N,N-diisopropylethyamine; (b) H2;
(c) 4Fb, PyBOP, HOBt; (d) TFA/thioanisole/m-cresol; (e) piperidine;
(f) 4Zn, PyBOP, HOBt; (g) Zn(OAc)2.
with the Z protections on the side chains (1a–3a) were synthe-
sized via a cyclization-cleavage method from the decapeptide-
linked oxime resin (p-nitrobenzophenone oxime resin).14c,16
That is, after the removal of the N-terminal Boc group of the
Boc–[D-Phe–Pro–Val–AA(Z)–Leu]2–oxime resin (1a, AA¼ Dab;
2a, Orn; 3a, Lys), the resin was treated with AcOH/N,N-
diisopropylethylamine (2 molar equiv each) for 2 h to yield
Val–NH, Orn–aNH and Leu–NH of both 2c and 2d (3JNHa
¼
8.6–10.4 Hz) show that these residues are involved in the
b-sheet structure, while the D-Phe residues (J ¼ 2.7–3.7 Hz)
are involved in the b-turn.21 In fact, the NOESY spectra of
2d show cross peaks between Val–CaH and Orn–NH, between
Orn–CaH and Leu–NH, between Leu–CaH and D-Phe–NH,
but not between Pro–CaH and Val–NH. These NOESY peaks
support the hypothesis that the –Val–Orn–Leu–D-Phe–
cyclo[–D-Phe–Pro–Val–AA(Z)–Leu–]2
(1b,17
AA ¼ Dab;
2b,17,18 Orn; 3b,14d,17 Lys) in 70, 84 and 91% yields, respec-
tively. The Z groups were then removed and 4-(10,15,20-
tritolylporphyrin-5-yl)benzoic acid (4Fb)5b was condensed onto
each side chain NH2 group to yield 1d, 2d and 3d in 74, 86 and
81% yields, respectively.
Table 1 1H NMR chemical shifts, coupling constants and tempera-
a
ture coefficients of the amide protons of GS derivatives in DMSO-d6
d/ppm (3JHNa/Hz) Dd/ppb Kꢀ1
The GS derivative bearing one Zn porphyrin (PorZn) and a
free-base porphyrin (PorFb) was synthesized from the cyclic
decapeptide with two different protective groups, cyclo[–D-Phe
–Pro–Val–Orn(Fmoc)–Leu–D-Phe–Pro–Val–Orn(Z)–Leu–]
(2f), which was prepared from 2e by the cyclization-cleavage
method. The Z protection was selectively removed by TFA/
thioanisole/m-cresol19 to yield 2g, to which 4Fb was condensed
to yield 2h bearing one Orn(Fmoc) and an Orn(PorFb). The
Fmoc group of 2h was removed by piperidine to yield 2i, and
then 4Zn was condensed to yield 2dFbZn bearing one Orn
(PorZn) and one Orn(PorFb). This method may be applicable
to the synthesis of other GS derivatives bearing different
functional groups. The metallation of 2d with excess Zn(OAc)2
2c (GS)b
2d
2dZn2
3d
Val–NH
Orn–aNH
Leu–NH
D-Phe–NH
a
7.20 (9.5)
ꢀ1.8
7.30 (10.4)
ꢀ2.0
8.66(9.8)
ꢀ4.5
8.50 (8.6)
ꢀ2.6
8.93 (2.7)
ꢀ8.3
7.33 (9.5)
ꢀ2.1
8.73 (9.5)
ꢀ4.5
8.53 (8.5)
ꢀ2.8
8.99 (2.5)
ꢀ7.4
7.29(9.5)
ꢀ2.1
8.59 (9.5)
ꢀ5.3
8.46 (8.9)
ꢀ2.1
8.91 (3.7)
ꢀ8.6
8.65 (9.5)
ꢀ4.8
8.31 (9.2)
ꢀ2.7
9.04 (3.7)
ꢀ7.0
d and 3JHNa were measured at 303 K, while Dd is at 303, 313, 323 and
b
333 K. Ref 14c. At 313 K.
c
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sequence in 2d constructs a b-sheet structure22 similar to that
of 2c. The 1H NMR measurements of the other GS derivatives
bearing porphyrins, 1d, 3d, 2dFbZn and 2dZn2, show essentially
the same results. The derivation of GS at the Orn (or Dab/Lys)
side chain did not distort the conformation of the peptide
framework comprised of a set of antiparallel b-sheets and
b-turns. 1H NMR spectra in DMSO-d6 and D2O–H2O have
already been employed to examine the relationship between
peptide conformation and the NMR data (d, Dd and J).20–22
Therefore, we do not discuss the peptide conformation of these
GS derivatives in a solvent other than DMSO, such as in
toluene or dichloromethane (GS derivatives are insoluble in
water).
1H NMR investigation of the assembled porphyrins and the
solvent effect
1
The H NMR spectra of 2d also show a spatially close pair of
porphyrins on the cyclic polypeptide. In DMF-d7, the reference
monomeric porphyrin, 5-p-methoxycarbonylphenyl-10,15,20-
tritolylporphyrin (5), has a simple 1H NMR spectrum (data
not shown). The pyrrole-b protons of 5 appear at d 8.95 (s, 8H)
and three tolyl groups appear at d 8.20 (d, 6H), 7.70 (d, 6H)
and 2.72 (s, 9H). That is, the tolyl groups cis to the methox-
ycarbonylphenyl group and the trans tolyl group appear at the
same chemical shifts. In contrast, the 1H NMR spectrum of 2d
in DMF-d7 (2.0 mM, 500 MHz) is more complex. The pyrrole-
b protons of 2d appear at d 8.89 (m, 12H) and 8.76 (d, 4H,
overlapped with an NH signal) at 303 K [Fig. 2(a), for the
discussion of the pyrrole-b signals, see below]. The tolyl signals
of 2d appear as three sets, namely tolyl-A [d 8.18 (d, 4H), 7.69
(d, 4H) and 2.71 (s, 6H)], tolyl-B (d 8.01, 7.45 and 2.52) and
tolyl-C (d 7.98, 7.38 and 2.52). The aromatic signals of tolyl-B
and C overlap with each other and also with the solvent signal
(DMF), Val–NH and the side chain of Phe. However, a careful
inspection of the spectrum, including the signal intensity,
measurement at elevated temperature (see below) and the
2D1H–1H COSY spectrum [Fig. 2(b)] unambiguously indi-
cated the existence of three magnetically non-equivalent tolyl
groups for 2d. In the 2D COSY spectrum, three cross peaks
appear between the tolyl ortho (to porphyrin) and meta
protons. Moreover, long-range COSY signals (not shown)
appear between the tolyl meta protons and tolyl-Me protons,
that is, between the signals at d 8.18 and 2.71, between d 7.45
and 2.52 and between d 7.38 and 2.52. Thus, the three tolyl
groups of a porphyrin in 2d are non-equivalent at 303 K, which
attests to the unsymmetrical character of the porphyrins. Fig.
2(d) depicts a speculative molecular profile of 2d in DMF,
having an edge-to-edge orientation of the porphyrins with an
offset. The chemical shifts of tolyl-A of 2d are similar to those
of the tolyl groups of 5, which means that these protons are not
affected by the ring current of the other porphyrin. The tolyl-B
and C are high-field shifted compared to tolyl-A, probably
affected by the ring current of the other porphyrin existing
nearby.z At 353 K, the pyrrole-b protons of 2d appear as four
doublet peaks at d 8.87 (4H), 8.84 (4H), 8.82 (4H) and 8.72
(4H) [Fig. 2(c)]. Tolyl-A is shifted slightly (d 8.14, 7.66 and
2.71); however, tolyl-B and C show coalescence phenomena at
this temperature, with their signals at d 7.94, 7.42 and 2.53.
These facts suggest a dynamic process within the time scale of
NMR.23 The coalescence of tolyl-B and C suggests a symme-
trical character of the porphyrin at 353 K. The appearance of
four doublet signals for the pyrrole-b protons supports the
symmetrical character of the porphyrin. This interpretation
may account for the complex signals appearing for the pyrrole-
b protons at 303 K [Fig. 2(a)]. The coalescence at 353 K
Fig. 2 1H NMR spectra (in DMF-d7, aromatic region) of 2d: (a) 1D,
(b) 2D COSY at 303 K; (c) 1D at 353 K and (d) an illustration
depicting three non-equivalent tolyl groups.
suggests that the spacer between the porphyrin and peptide is
slightly flexible or that the rotation of the porphyrin ring along
the porphyrin–Orn side-chain axis is slightly restricted; how-
ever, further examination in this solvent failed because of the
poor peak separation for tolyl-B and C.
In CD2Cl2 (2.0 mM), the 1H NMR signals of 2d are
broadened compared to those in DMF-d7. The pyrrole-b
protons of 2d (2.0 mM) appear as four doublet peaks, for
instance at 303 K, at d 8.75, 8.61, 8.56 and 8.33 [Fig. 3(a)].
Their couplings with each other were confirmed by the 2D
COSY spectra [Fig. 3(b)] and the obstructive NH signals could
be eliminated when the solution of 2d was treated with CD3OD
(data not shown). In CD2Cl2, the three magnetically non-
equivalent tolyl signals are fairly well separated from each
other. The signals of tolyl-A at d 8.02 and 7.53 [at 303 K, Fig.
3(a)] are sharp and their chemical shifts are close to those of the
tolyl groups of 5 (d 8.10 and 7.59 in CD2Cl2). The signals of
tolyl-B (d 7.46 and 6.91) and tolyl-C (d 7.29 and 6.73) are
significantly broadened at 303 K, which suggests the involve-
ment of some dynamic process. These well-separated signals of
tolyl-B and C [compare the COSY cross peaks of Figs. 2(b) and
{ The reason why the signals of tolyl-B are high-field shifted is not yet
clear. However, the dynamic exchange between tolyl-B and C described
below may account for this.
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the rate constant k for the chemical exchange process including
the tolyl-B and C was estimated to be 0.92 sꢀ1. M0B and MB are
the signal intensities of signal B before and after the spin-
saturation, T1B is the spin-lattice relaxation time for signal B
(2.22 s in this case). Fig. 3(e) depicts a hypothetical illustration
of the dynamic process involved in 2d. In this figure, the
rotation of the porphyrin along the porphyrin–Orn side-chain
axis interchanges the tolyl C and tolyl B groups. Three tolyl
groups of a porphyrin are non-equivalent at 303 K and some
signals coalescence at the elevated temperature. The sharp four
doublet signals of pyrrole-b protons at 353 K suggest the
symmetrical character of the porphyrin.
It is interesting that the tolyl-B and C signals are still
broadened at 353 K but that the pyrrole-b signals are sharp
at that temperature [Fig. 3(d)]. As one of the reviewers pointed
out, four AB systems (eight doublets) are expected for the
pyrrole-b signals of the slow exchange system of 2d. In fact,
sharp four doublets appear in DMF-d7, CD2Cl2 and toluene-d8
(described below) at 353 K and broadened signals or complex
1
multiplet signals appear at 303 K. Low-temperature H NMR
measurements (303 to 208 K, 2.0 to 0.5 mM) were tried in
CD2Cl2, in the expectation of spectral changes; however, the
signals were broadened without any change of chemical shifts,
intensity and couplings for the porphyrin signals. Probably
intermolecular aggregation occurs at low temperature (poly-
peptides are hardly soluble in dichloromethane).
In toluene-d8 (2.0 mM), the 1H NMR signals of 2d are
extremely broadened at 303 K [Fig. 4(a)] and, as a matter of
fact, no information could be obtained from this spectrum. In
this solvent, 2d shows sharpened 1H NMR signals at 353 K
[Fig. 4(b)] in which the spectral profile resembles that in
CD2Cl2 at 353 K [Fig. 3(d)], with four sets of pyrrole protons
and two sets of tolyl signals. The extremely broadened signals
of 2d at 303 K are probably due to aggregation of the
porphyrins in toluene-d8. No obvious spectrum change was
observed when the concentration of 2d was varied from 1.0 to
4.0 mM at 303 K. However, no direct and clear evidence has
been obtained yet on whether the porphyrins assemble in
toluene intramolecularly or intermolecularly.
In any case, these 1H NMR spectra of 2d show that in DMF-
d7, CD2Cl2 and toluene-d8 some of the tolyl groups are affected
by the ring current of another porphyrin, which means that the
porphyrins are located close to each other on the polypeptide
Fig. 3 1H NMR spectra (in CD2Cl2, aromatic region) of 2d: (a) 1D,
(b) 2D COSY, (c) 2D NOESY at 303 K; (d) 1D at 353 K and (e) an
illustration depicting the dynamic behavior of a pair of porphyrins.
3(b)] help further investigations on the dynamic processes. (1)
The dynamic process including the tolyl-B and C groups is
observed in the 2D NOESY spectra [Fig. 3(c)]. It is known that
the proton signals involved in the chemical exchange process
often show pseudo-NOESY cross peaks.23b Fig. 2(c) clearly
shows the cross peaks between tolyl-B (ortho) and tolyl-C
(ortho) and between tolyl-B (meta) and tolyl-C (meta) protons.
(2) The CD2Cl2 solution of 5b was heated in an NMR sample
tube fitted with a pressure/vacuum valve to observe the coales-
cence of the peaks of tolyl-B and C at 353 K [Fig. 3(d)] At this
temperature, the signals of pyrrole are sharpened compared to
those at 303 K; however, the signals of tolyl-B and C are still
broadened. (3) Finally, the spin-saturation transfer technique
was applied to the chemical exchange process of tolyl-B and
C.23c Irradiating the tolyl-C signal (ortho, d 7.29) at 303 K
caused spin-saturation transfer to the signal of tolyl-B (ortho, d
7.46), and the intensity of the latter signal became 33% of the
intensity before the saturation of tolyl-C (data not shown).
From the equation23c
Fig. 4 1H NMR spectra (in toluene-d8, aromatic region) of 2d: at (a)
303 K and (b) 353 K.
k ¼ (M0B/MB ꢀ 1)/T1B
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template. The magnetic non-equivalence between tolyl-B and C
suggests that the porphyrins are unsymmetrically arranged.
That is, the porphyrins are not in a symmetric face-to-face
orientation but probably in a side-by-side orientation with an
offset as depicted in Figs. 2(d) and 3(e), although the detailed
structure of 2d is not yet clear because no single crystal has
been obtained (for UV-vis and CD spectral results, see below).
Comparing the 1H NMR spectra of 2d in DMF-d7, CD2Cl2
and toluene-d8 (Figs. 2–4), the signal width becomes broader in
the order DMF-d7 o CD2Cl2 o toluene-d8 (at both 303 and
353 K). This order may reflect the kinetics of the chemical
exchange process involved in 2d or the stability of the aggre-
gated porphyrins in the solvent examined.
It is interesting that the high-field shift of the tolyl groups of
the porphyrins is in the order of 1d o 3d o 2d; for instance, the
high-field-shifted tolyl protons (meta to porphyrin) of 1d, 2d
and 3d appear at 7.35, 7.23 and 7.33 ppm (in DMSO-d6),
respectively. These facts might mean that the porphyrins in 2d
are assembled more closely than those in 1d and 3d, which is an
unexpected result because the spacer length between the por-
phyrin and peptide follows the order: 1d o 2d o 3d. This result
is concerned with the CD results of these GS derivatives
described below.
It may be noted here that the chemical shifts of PorFb and
PorZn were too close to distinguish; therefore, the NOESY
experiment of 2dFbZn to examine the orientations of PorFb
and PorZn failed.
CD investigation on the orientation of porphyrins bound to the
GS framework
The GS derivatives bearing a pair of PorFb, 1d, 2d and 3d,
show similar UV-vis spectra in CH2Cl2, with lmax at around
420, 516, 552, 592 and 646 nm (see Fig. 1). In the same solvent,
1d, 2d and 3d show different CD spectra [Fig. 5(a)]. Split
Cotton effects appear for 1d in the porphyrin Soret band
region at 421 nm [Fig. 5(a), trace A, De ¼ ꢀ25.9 Mꢀ1 cmꢀ1
]
and 414 nm (De ¼ 11.1 Mꢀ1 cmꢀ1), which are slightly unsym-
metrical. Split Cotton effects also appear for 2d at 424 nm [Fig.
5(a), line B, De ¼ ꢀ52.9 Mꢀ1 cmꢀ1] and 416 nm (De ¼ 27.9
M
ꢀ1 cmꢀ1), in which the mid-point of two symmetric CD
bands (420 nm) corresponds to the UV absorption. These split
Cotton effects are assigned to the exciton-coupled CD that
arises from two porphyrin chromophores. It is known that the
two porphyrin rings located near each other in the chiral
orientation show exciton-coupled Cotton effects in the por-
phyrin Soret band region, which are characterized by two
symmetrically split intense bands with inverse signs.24 The pair
of porphyrins of 2d located near each other on the cyclic
peptide and their chiral orientations are determined by the
peptide framework. In contrast, 3d shows a weak single CD
band at 420 nm (De ¼ –17.0 Mꢀ1 cmꢀ1), which is assigned to
the CD induced by the chiral amino acids or the side-chain
phenyl groups of the D-Phe residue,25 not the exciton-coupled
CD. The longer –(CH2)4– spacers between the porphyrin and
peptide in 3d would account for its weak CD, although the
reason why 2d shows an intense CD compared to 1d is yet
unclear. It is interesting that the high-field shifts of the tolyl
proton signals in 1H NMR are in the order of 1d o 3d o 2d as
described above, which may be associated with the order of the
CD intensities of 3d o 1d o 2d. It should be noted here that the
CD spectra were taken at 1.6 mM peptide concentration, in
which the peptide concentrations were determined by the
amino acid analysis (see Experimental). However, no concen-
tration dependency was observed in the 0.4 to 16 mM concen-
tration range.
Fig. 5 CD spectra of (a) 1d (trace A), 2d (trace B) and 3d (trace C) in
CH2Cl2; (b) 2d (trace A), 2dFbZn (trace B, broken line) and 2dZn2
(trace C) in CH2Cl2 and (c) 2d (trace A), 2dFbZn (trace B, broken line)
and 2dZn2 (trace C) in MeOH ([peptide] ¼ 1.6 mM).
spectra in the Soret band region of 2dFbZn and 2d suggest that
Zn porphyrin and the free-base porphyrin behave similarly in
the CD event. In contrast, intense Cotton effects appear for
2dZn2at 425 nm (De ¼ ꢀ114.2 Mꢀ1 cmꢀ1) and at 415 nm
(De ¼ 101.3 Mꢀ1 cmꢀ1). Zn porphyrins are usually of low
solubility compared to the free-base porphyrins, which possi-
bly accounts for the intramolecular assembling of PorZn in
2dZn2 to show the intense Cotton effects. The same tendency
for Cotton effects was observed in MeOH [Fig. 5(c)]; 2d and
2dFbZn show similar CD spectra except for their wavelength.
The different wavelengths of the Cotton effects reflect the
different UV absorptions, lmax in MeOH is 415 nm for 2d,
420 nm for 2dZn2 and two lmax (416 and 421 nm) are found for
2dFbZn. Again, 2dZn2 shows an intense CD compared to 2d
and 2dFbZn in MeOH. The Cotton effects of these three
compounds in MeOH are more intense than those in CH2Cl2
(for the solvent effect on the CD spectra, see below).
Next, the effect of Zn metallation of the porphyrin on the
exciton-coupled CD was examined using 2d, 2dFbZn and
2dZn2. In CH2Cl2, 2dFbZn shows a CD spectrum similar to
that of 2d [Fig. 5(b)]. The similar CD and the absorption
N e w J . C h e m . , 2 0 0 4 , 2 8 , 1 1 5 1 – 1 1 5 9
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Further solvent effects for the intramolecular assembling of
two PorZn in 2dZn2 were examined in toluene, triethyl phos-
phate, TFE, pyridine and DMF (Table 2), besides CH2Cl2 and
MeOH described above. The Cotton effects are solvent-depen-
dent, varying in their intensities, wavelengths and profiles. As
for the intensities of the Cotton effects of 2dZn2, strong Cotton
effects appeared in toluene at 435 nm (De ¼ –288.3 Mꢀ1 cmꢀ1),
427 nm (De ¼ 386.3 Mꢀ1 cmꢀ1) and 418 nm (De ¼ –70.8
M
ꢀ1 cmꢀ1). It is not yet clear why 2dZn2 shows such an
unsymmetrical CD spectra in toluene.24g The CD spectra of
2dZn2 in the other solvents are all weaker than that in toluene
and the order of intensities, A, is: toluene 4 MeOH 4 triethyl
phosphate 4 TFE 4 CH2Cl2 4 pyridine 4 DMF. This order
is not associated with the dielectric constant, dipole moment
or the acceptor/donor character of the solvents.26 The coordi-
nation of the solvent to Zn or the solubility of porphyrins is not
related to this observed order of the solvent effect. However,
the order of the Cotton effect intensity is in line with the
p* scale, which is an index of solvent dipolarity/polarizability
and reflects the ability of the solvent to stabilize a charge
or a dipole.26,27 The reported values of the p* parameter are
0.54 (toluene), 0.60 (MeOH), 0.72 (triethyl phosphate), 0.73
(TFE), 0.82 (CH2Cl2), 0.87 (pyridine) and 0.88 (DMF).27b In a
solvent with a lesser ability to stabilize a dipole, such as toluene
with a small p* parameter, the porphyrins may prefer the
assembled structure. This interpretation agrees with the
electrostatic model for porphyrin-porphyrin stacking in non-
aqueous media.12
Fig. 6 UV-vis spectra of 2dZn2 in CH2Cl2 (trace A) showing the sharp
Soret band and in toluene (trace B) showing a shoulder peak (1.0 mM).
disappears upon the addition of 4,40-dipyridyl, to show a weak
induced CD at 424 nm [De ¼ 23.7 Mꢀ1 cmꢀ1, Fig. 7(b)]. A
hypothetical interpretation of this result is that the pair of
porphyrins in 2dZn2 takes a side-by-side orientation without
4,40-dipyridyl as described above and the chiral orientation
between the porphyrins is determined by the peptide frame-
work (see below). However, when an excess of 4,40-dipyridyl is
added, this diamine ligand forms a host-guest complex with
2dZn2. The intramolecular bridging by 4,40-dipyridyl may
bring the pair of porphyrins to a face-to-face orientation
[Fig. 7(c), right]; therefore, merely a weak Cotton effect
appears for the mixture of 2dZn2 and 4,40-dipyridyl. Thus,
the addition of 4,40-dipyridyl might change the side-by-side
orientation of porphyrins in 2dZn2.
The exciton-coupled Cotton effects of 2d, 2dFbZn and 2dZn2
in all the solvents examined have (ꢀþ) sign [from longer to
shorter wavelengths; see Figs. 5(b), 5(c)], although the Cotton
effect is sometimes distorted. From the exciton chirality meth-
od, the (ꢀþ) sign of the Cotton effect means a negative
chirality, which arises from two excitons in a left-twisted
arrangement.24c Because the b-strands of the natural polypep-
tide including GS are generally left-twisted,31 the exciton
The Cotton effects of 2dZn2 appear at different wavelengths
depending on the solvents but essentially appear around the
lmax of the absorption spectra. The lmax value of 2dZn2 differs
in various solvents but is the same as the lmax value of Zn
tetramesitylporphyrin (ZnTMP) in the same solvent, except in
toluene. In toluene only, the lmax of 2dZn2 (424 nm) is red-
shifted and shows a shoulder peak at around 435 nm (trace B in
Fig. 6), in contrast to the sharp peak of ZnTMP at 421 nm
(data not shown). In the other solvents examined, for instance
in CH2Cl2 (trace A in Fig. 6), the Soret absorption of 2dZn2 is
not red-shifted compared to ZnTMP and no shoulder peak is
observed. This fact supports the interpretation that the two
porphyrins in 2dZn2 favor an associated structure in toluene,
probably in the side-by-side fashion. Based on Kasha’s theory
about exciton coupling and the orientation of the transition
moment in porphyrins, the long-wave shift in the absorption
spectra arises from the side-by-side interaction of the chromo-
phores,28 which is often observed in water-soluble porphyrins29
and dimeric porphyrins linked in a side-by-side fashion.5f
In order to support the side-by-side orientation of the pair of
porphyrins in 2dZn2, 4,40-dipyridyl was added to intramolecu-
larly bridge the zinc porphyrins.30 When the solution of 2dZn2
in CH2Cl2 (20 mM) was titrated with 4,40-dipyridyl, the absorp-
tion of 2dZn2 at 421, 550 and 589 nm shifted to 426, 562 and
603 nm with clear isosbestic points [Fig. 7(a)].15b Interestingly,
the exciton-coupled Cotton effect of 2dZn2 [Fig. 5(b), trace C]
Table 2 The exciton-coupled Cotton effects in the CD spectra of
a
2dZn2
l )
ext/nm (De/Mꢀ1 cmꢀ1
A
Solvent
Toluene
MeOH
435 (ꢀ288.3) 427 (386.3) 418 (ꢀ70.8) ꢀ675
425 (ꢀ243.5) 415 (180.2)
–424
ꢀ301
ꢀ259
ꢀ216
ꢀ201
ꢀ176
Triethyl phosphate 429 (ꢀ175.8) 421 (124.7)
TFE
423 (ꢀ152.8) 414 (106.6)
425 (ꢀ114.2) 415 (101.3)
CH2Cl2
Pyridine
DMF
a
434 (ꢀ133.8)
428 (67.1)
424 (60.7)
Fig. 7 (a) UV-vis spectra of the titration of 2d (20 mM in CH2Cl2) by
4,40-dipyridyl (0, 0.2, 0.4. . .2.0 equiv). (b) CD spectra of 2d with 3.0
equiv of 4,40-dipyridyl in CH2Cl2. (c) An illustration depicting the
orientational change of the porphyrins from ‘‘left-twisted’’ to ‘‘face-to-
face’’ induced by 4,40-dipyridyl.
432 (ꢀ115.0)
1.6 mM at 298 K. lext, peak of trough wavelength; A, couplet
amplitude.
1156
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coupling between the porphyrins attached to the b-sheet may
reflect the left-twisted arrangement of the polypeptide strands
[Fig. 7(c), left].
608 nm is 80% quenched, the emission of PorFb at 655 nm is
85% enhanced (after subtracting the residual emission from
PorZn) and the emission of the PorFb at 723 nm is 99%
enhanced. The decrease in the emission of PorZn corresponds
to an increase in the emission of PorFb at 655 and 723 nm,
supporting the mechanism of excited state energy transfer. The
excitation spectrum of 2dFbZn, monitoring the emission of
PorFb (lem ¼ 723 nm, data not shown), is different from the
absorption spectra of 2dFbZn, 2d or 2dZn2. However, the
excitation spectrum fits very well to the calculated spectra
assuming 82% energy transfer, that is, the sum of the UV-vis
spectra of 2d þ 0.82 ꢁ 2dZn2. Thus, we assume the apparent
energy-transfer efficiency of 2dFbZn in toluene to be (at least)
82%.5c The other mechanisms for the quenching of the excited
state of PorZn, such as excited state intersystem crossing to
afford the triplet state and electron transfer from PorZn to
PorFb, have been considered to be negligible.5c Of course, the
exact energy-transfer efficiency should be obtained from the
measurement of the excited state lifetime t of PorZn. However,
the lifetime of PorZn in 2dFbZn seems to be below a nanose-
cond, because, for instance, the reported lifetime of ZnTPP is
2.0 ns (in toluene) and the lifetime of Zn porphyrin in
phenylene–ethyne–phenylene linked dimers is as short as 24–
90 ps.5c Because of the inaccessibility of ps-order time-resolved
fluorescence or absorption spectroscopies, we used a semi-
quantitative evaluation of the energy-transfer efficiency by
steady-state fluorescence spectroscopy.
Steady-state fluorescence spectra were used to evaluate the
energy-transfer efficiencies from PorZn to PorFb in 2dFbZn
not only in toluene as described above but also in MeOH,
CH2Cl2 and DMF. Upon exciting the lmax of PorZn at 549–
557 nm of 2dFbZn, three emissions appear at 603–609 nm
(from PorZn), 653–655 nm (overlap of the emissions from the
PorZn and PorFb) and 716–723 nm (from PorFb) in all the
solvents examined. In all the solvents examined, the emission
from PorZn at 603–609 nm decreases significantly compared
with the calculated spectra assuming 0% energy transfer while
the emission from PorFb is much larger than calculated. By
averaging the quenching efficiency of the emission from PorZn
at 603–609 nm and the enhancement of the emission of PorFb
at 653–655 nm, the energy-transfer efficiencies in MeOH,
CH2Cl2 and DMF were estimated to be 75, 65 and 60%,
respectively. Trace C in Fig. 8 shows the emission spectra of
2dFbZn in DMF, where the emission of PorZn at 609 nm
decreases to 61%, the emission of PorFb at 654 nm is 58%
enhanced (after subtracting the residual emission from the
PorZn) and the emission of PorFb at 720 nm is 69% enhanced.
Thus, the energy-transfer efficiency is in the order of tolue-
ne 4 MeOH 4 CH2Cl2 4 DMF, which suggests that the dis-
tance between the PorZn and the PorFb is in the order of
toluene o MeOH o CH2Cl2 o DMF. However, the possibility
that the different energy-transfer efficiencies might be due to
other solvent effects can not be neglected.
Fluorescence energy transfer between porphyrins bound to GS
The NMR, UV-vis and CD spectra described above strongly
suggest that the orientation of porphyrins bound to GS differs
in various solvents. We next examined the solvent effect on the
fluorescence emission spectra, because (1) the emission of
PorZn overlaps the absorption of PorFb, (2) PorFb and PorZn
of 2dFbZn are located in close proximity, (3) the energy
transfer from the excited state PorZn to the ground state
PorFb is expected to occur intramolecularly5,6 and (4) the
energy-transfer efficiency depends on the orientation of the
porphyrins.5 The fluorescence emission spectra of 2d (lex ¼ 516
nm, lem ¼ 655 and 719 nm, fluorescence quantum yield
Ff ¼ 0.10) and 2dZn2 (lex ¼ 552 nm, lem ¼ 608 and 652 nm,
Ff ¼ 0.025) in toluene are similar to those of FbTPP and
ZnTPP, respectively.32 These facts show that the two porphyr-
ins in 2d or 2dZn2 are independent in the absorption and
emission events.
Upon exciting 2dFbZn in toluene at 552 nm, the emission
appears at 608, 655 and 723 nm with intensities of 5.0, 70 and
24 (arbitrary units; trace A in Fig. 8, the fluorescence spectra in
Fig. 8 are normalized for the absorption at lex). PorFb absorbs
40% of the excitation incident light and PorZn 60% in 2dFbZn
[calculated from the ratio of e552 of 2d (28 600 Mꢀ1 cmꢀ1) and
2dZn2 (42 200 Mꢀ1 cmꢀ1)]. If no energy transfer occurs be-
tween the porphyrins of 2dFbZn in the fluorescence event, the
emission of 2dFbZn should be equal to 0.40 ꢁ 2d þ 0.60 ꢁ
2dZn2 (Fig. 8, trace B). However, the emission spectra of
2dFbZn (Fig. 8, trace A) differed substantially from this
calculated spectrum. Upon exciting 2dFbZn in toluene at
516 nm, where PorFb absorbs 87% of the incident light, the
emission is observed exclusively from PorFb (655 and 723 nm)
and no emission from PorZn (608 nm, data not shown) is seem.
These facts indicate that the excited state energy transfer
occurs intramolecularly from the excited state of PorZn in
2dFbZn to the ground state of PorFb.
From a comparison of the emission spectra of 2dFbZn (lex
552 nm; trace A in Fig. 8) and the calculated one assuming
¼
0% energy transfer (trace B in Fig. 8), the emission of PorZn at
Conclusion
The interaction between a pair of porphyrins bound to a
1
natural cyclic peptide, Gramicidin S, was investigated by H
1
NMR, CD, UV-vis and fluorescence spectroscopy. H NMR
analyses indicate that two tolyl groups among the three tolyl
groups of a porphyrin are high-field shifted by the ring current
effect of the other porphyrin, which suggests a side-by-side
arrangement of the porphyrins. The high-field shift and the
signal line broadening are in the order toluene-d8 4 CD2Cl2 4
DMF-d7. The variable temperature NMR measurements in
CD2Cl2 indicate a dynamic process involving the porphyrins.
The high-field shifts of the 1H NMR signals depend on the
length of the spacer between the porphyrin and peptide, being
in the order of 1d o 3d o 2d. The exciton-coupled Cotton
effects at the porphyrin Soret band region also characterize
Fig. 8 Fluorescence emission spectra of 2dFbZn (lex ¼ 552 nm) in
toluene (trace A), a calculated spectrum assuming 0% energy transfer
in toluene (trace B, broken line) and 2dFbZn (lex ¼ 557 nm) in DMF
(trace C).
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the assorted structures of the porphyrins. The intensities are in
the order: between two zinc porphyrins 4 between a zinc
porphyrin and a free-base porphyrin E between two free-
base porphyrins. The Cotton effects are also solvent-
dependent, with toluene 4 MeOH 4 triethyl phosphate 4
TFE 4 CH2Cl2 4 pyridine 4 DMF, in which this order is in
line with the solvent p* parameter. The efficiency of the
intramolecular energy transfer from the excited state PorZn
to the ground state PorFb are also solvent-dependent. The
energy-transfer efficiencies are in the order of toluene 4
MeOH 4 CH2Cl2 4 DMF, which is similar to the order
observed in the 1H NMR and CD spectra. In the solvents
examined in this study, toluene is the most favorable
solvent for the assembling of porphyrins. These findings hope-
fully will be utilized in a future study to build a more
sophisticated porphyrin assembly on the polypeptide frame-
work, and furthermore, possibly to control the structure and
the function of the b-sheet polypeptide by conjugation with
porphyrins.
Acknowledgements
We thank Professor Hideo Akisada and Ms. Junko Nishida at
Kyushu Kyoritsu University for the CD measurements, Pro-
fessor Masahiko Sisido at Okayama University for the fluor-
escence measurements, Dr. Masako Fujiwara at JEOL, Ltd.,
Japan, for the molecular modeling study, Ms. Keiko Yama-
guchi at Kyushu Institute of Technology for the FAB MS
measurements and also Dr. Yuji Tanaka and Mr. Takaaki
Nakashima at Kyushu Institute of Technology for technical
assistance. We also thank the referees for their constructive
comments. This work was supported in part by Grants-in-aid
for Scientific Research (Nos. 12650 869 and 15550 149) from
the Ministry of Education, Culture, Sports, Science and Tech-
nology, Japan.
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ZnTMP (tetramesitylporphyrinato zinc) was synthesized as
reported.33
HPLC was carried out with a Hitachi L-7100 pump and L-
7420 UV-vis detectors (220 and 420 nm) using (1, normal
phase) a Chemcosorb 100 A silica gel column (4.6 ꢁ 150 mm,
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(10 ꢁ 250 mm, preparative) eluted with a linear gradient of
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eluted with a linear gradient of 68–95% CH3CN–0.1% TFA
over 10 min and then with 95% CH3CN–0.1% TFA with a
1.0 mL minꢀ1 flow rate.
5
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spectrometer (500 MHz), in which the chemical shifts were
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curve fitting. FAB MS, including FAB HR-MS, were measured
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benzyl alcohol as a matrix unless otherwise noted. UV-vis
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6
M
ꢀ1 cmꢀ1 for 2d, e421 ¼ 772 000 Mꢀ1 cmꢀ1 for 2dFbZn and
e424 ¼ 795 000 Mꢀ1 cmꢀ1 for 2dZn2 in toluene, in which the
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acid analyses (Peptide Institute, Osaka, Japan). Fluorescence
spectra were recorded on a Hitachi F-2500 fluorescence
7
spectrophotometer equipped with
photomultiplier at 298 K, with a scan speed of 300 nm minꢀ1
a Hamamatsu R928F
,
2.5 nm slit widths for both excitation and emission, a photo-
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excitation wavelength was kept around 0.08, which means a ca.
1.5 mM peptide concentration. The fluorescence quantum yield
Ff was obtained relative to the known values for FbTPP and
TPPZn.32
8
9
Syntheses of the GS derivatives
See Electronic supplementary information (ESI).
1158
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