Hydrogen-bond promoted intramolecular electron transfer to photogenerated Ru(III): A functional mimic of tyrosine(z) and histidine 190 in photosystem II

Article abstract of DOI:10.1021/ja984048c

As a model for redox components on the donor side of photosystem II (PS II) in green plants, a supramolecular complex 4 has been prepared. In this, a ruthenium(II) tris-bipyridyl complex which mimics the function of P₆₈₀ in PS II, has been covalently linked to a tyrosine unit which bears two hydrogen-bonding substituents, dipicolylamine (dpa) ligands. Our aim is to mimic the interaction between tyrosine(z) and a basic histidine residue, namely His190 in PSII, and also to use the dpa ligands for coordination of manganese. Two different routes for the synthesis of the compound 4 are presented. Its structure was fully characterized by ¹H NMR, COSY, NOESY, ¹³C NMR, IR, and mass spectrometry. ¹H NMR and NOESY gave evidence for the existence of intramolecular hydrogen bonding in 4. The interaction between the ruthenium and the substituted tyrosine unit was probed by steady-state and time-resolved emission measurements as well as by chemical oxidation. Flash photolysis and EPR measurements on 4 in the presence of an electron acceptor (methylviologen, MV²⁺, or cobalt pentaminechloride, Co³⁺) showed that an intermolecular electron transfer from the excited state of Ru(II) in 4 to the electron acceptor took place, forming Ru(III) and the methylviologen radical MV⁺· or Co²⁺. This was followed by intramolecular electron transfer from the substituted tyrosine moiety to the photogenerated Ru(m), regenerating Ru(II) and forming a tyrosyl radical. In water, the radical has a g value of 2.0044, indicative of a deprotonated tyrosyl radical. In acetonitrile, a radical with a g value of 2.0029 was formed, which can be assigned to the tyrosine radical cation. In both solvents the electron transfer is intramolecular with a rate constant κ(ET) > 1 x 10⁷ s^-1. This is 2 orders of magnitude greater than the one for a similar compound 3, in which no dpa arm is attached to the tyrosine unit. Therefore the hydrogen bonding between the substituted tyrosine and the dpa arms in 4 is proposed to be responsible for the fast electron transfer. This interaction mimics the proposed His190 and tyrosine(z) interaction in the donor side of PS II.

Full text of DOI:10.1021/ja984048c

6834
J. Am. Chem. Soc. 1999, 121, 6834-6842
Hydrogen-Bond Promoted Intramolecular Electron Transfer to
Photogenerated Ru(III): A Functional Mimic of TyrosineZ and
Histidine 190 in Photosystem II
Licheng Sun,*,† Mark Burkitt,‡ Markus Tamm,† Mary Katherine Raymond,§
Malin Abrahamsson,§ Denis LeGourrie´rec,§ Yves Frapart,‡ Ann Magnuson,‡
Ping Huang Kene´z,‡ Peter Brandt,† Anh Tran,† Leif Hammarstro1m,*,§
Stenbjo1rn Styring,*,‡ and Bjo1rn A° kermark*,†,|
Contribution from the Department of Chemistry, Organic Chemistry, Royal Institute of Technology (KTH),
S-100 44 Stockholm, Sweden, Department of Physical Chemistry, Uppsala UniVersity, P.O. Box 532,
S-751 21 Uppsala, Sweden, and Department of Biochemistry, Center for Chemistry and Chemical
Engineering, UniVersity of Lund, P.O. Box 124, S-221 00 Lund, Sweden
ReceiVed NoVember 23, 1998
Abstract: As a model for redox components on the donor side of photosystem II (PS II) in green plants, a
supramolecular complex 4 has been prepared. In this, a ruthenium(II) tris-bipyridyl complex which mimics
the function of P680 in PS II, has been covalently linked to a tyrosine unit which bears two hydrogen-bonding
substituents, dipicolylamine (dpa) ligands. Our aim is to mimic the interaction between tyrosineZ and a basic
histidine residue, namely His190 in PSII, and also to use the dpa ligands for coordination of manganese. Two
different routes for the synthesis of the compound 4 are presented. Its structure was fully characterized by 1H
1
NMR, COSY, NOESY, 13C NMR, IR, and mass spectrometry. H NMR and NOESY gave evidence for the
existence of intramolecular hydrogen bonding in 4. The interaction between the ruthenium and the substituted
tyrosine unit was probed by steady-state and time-resolved emission measurements as well as by chemical
oxidation. Flash photolysis and EPR measurements on 4 in the presence of an electron acceptor (methylviologen,
MV2+, or cobalt pentaminechloride, Co3+) showed that an intermolecular electron transfer from the excited
state of Ru(II) in 4 to the electron acceptor took place, forming Ru(III) and the methylviologen radical MV+•
or Co2+. This was followed by intramolecular electron transfer from the substituted tyrosine moiety to the
photogenerated Ru(III), regenerating Ru(II) and forming a tyrosyl radical. In water, the radical has a g value
of 2.0044, indicative of a deprotonated tyrosyl radical. In acetonitrile, a radical with a g value of 2.0029 was
formed, which can be assigned to the tyrosine radical cation. In both solvents the electron transfer is
intramolecular with a rate constant kET > 1 × 107 s-1. This is 2 orders of magnitude greater than the one for
a similar compound 3, in which no dpa arm is attached to the tyrosine unit. Therefore the hydrogen bonding
between the substituted tyrosine and the dpa arms in 4 is proposed to be responsible for the fast electron
transfer. This interaction mimics the proposed His190 and tyrosineZ interaction in the donor side of PS II.
Introduction
quinoneA, and quinoneB.2 The transferred electron is later used
to reduce carbon dioxide, while the chlorophyll unit P680 is
regenerated from its photooxidized form P680+ by electron
transfer (ET) from a nearby manganese cluster.3 This transfer
Sustainable energy systems need to be developed in the future,
and solar light is one very attractive energy source for such
systems. Artificial photosynthesis has therefore attracted con-
siderable attention in the past decades as a potential way of
converting solar energy into fuel or electricity. In nature, solar
light is absorbed by green plants and transformed into bioenergy
by the reduction of carbon dioxide. Simultaneously, water is
oxidized to molecular oxygen. Photosystem II (PSII) in green
plants is the key enzyme in this complex process.1 The
conversion is initiated by the absorption of a photon by the light-
harvesting system of PSII, leading to excitation of the central
chlorophyll dimer P680. An electron is then transferred from the
excited-state P680* to a series of electron acceptors: pheophytin,
is mediated by tyrosineZ,4 located 5-10 Å4b,5 away from the
6
manganese cluster and 10-15 Å from P680
.
After four
(2) (a) Diner, B. A.; Babcock, G. T. In Oxygenic Photosynthesis: The
Light Reactions; Ort, D. R., Yocum, C. F., Eds.; Kluwer Academic
Publishers: Dordrecht, The Netherlands, 1996; pp 213-247, (b) Britt, R.
D. In Oxygenic Photosynthesis: The Light Reactions; Ort, D. R., Yocum,
C. F., Eds.; Kluwer Academic Publishers: Dordrecht, The Netherlands,
1996; pp 137-164.
(3) (a) Hoganson, C. W.; Babcock, G. T. Science 1997, 277, 1953-
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† Royal Institute of Technology.
‡ Lund University.
§ Uppsala University.
| E-mail: bear@orgchem.kth.se.
(6) (a) Svensson, B.; Etchebest, C.; Tuffery, P.; van Kan, P.; Smith, J.;
Styring, S. Biochemistry 1996, 35, 14486-14502. (b) Kodera, Y.; Hara,
H.; Astashkin, A. V.; Kawamori, A.; Ono, T. Biochim. Biophys. Acta 1995,
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(1) (a) Barber, J.; Andersson, B. Nature 1994, 370, 31-34. (b)
Andersson, B.; Styring, S. In Current Topics in Bioenergetics; Lee, C. P.,
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10.1021/ja984048c CCC: $18.00 © 1999 American Chemical Society
Published on Web 07/14/1999

Electron Transfer to Photogenerated Ru(III)
J. Am. Chem. Soc., Vol. 121, No. 29, 1999 6835
consecutive photoinduced ET events, the manganese cluster,
which acts as a reservoir for oxidizing equivalents, recovers all
four electrons by oxidizing two water molecules to molecular
oxygen.2,7 In recent years, model systems8,9 for mimicking the
manganese cluster have been studied extensively. Such systems
have in general been mainly structural models, investigated by
X-ray, EPR spectroscopy, and magnetic measurements. Fewer
examples exist in which the OEC models have undergone
electrochemical or light-induced processes.10 However, to devise
an artificial photochemical system that is capable of producing
oxygen by oxidation of water,11,12 electron transfer from a
manganese complex to a photooxidized sensitizer must first be
demonstrated.
Chart 1
A few years ago, we started to build such model systems
based on tris-bipyridine ruthenium(II) as a substitute for
chlorophyll. In our first studies it was shown that in a dinuclear
Mn(II)-Ru(II) supramolecular model system 1, an intramo-
lecular ET from the coordinated manganese(II) monomer to a
photogenerated ruthenium(III) was possible,13,14 opening up the
way to mimic the ET process in the donor side of PSII. We
were also able to show that the distance between manganese
and ruthenium is crucial. In 1 the intramolecular ET rate is fairly
low, kET ca. 105 s-1. However, when the distance was decreased
as in 2, the lifetime of the excited state was about 2 orders of
magnitude shorter than that of 1 due to efficient quenching by
the manganese (Chart 1). We were therefore unable to observe
the initial ET from the excited Ru(II) of compound 2. In PS II,
the electron transfer is mediated by a phenolic unit, tyrosineZ,
perhaps in order to avoid quenching of the excited state of the
chlorophyll unit by manganese, yet ensure rapid electron transfer
to P680+. The intermediate tyrosineZ may also be essential for
the oxidation of the manganese cluster in PS II by participating
in hydrogen atom transfer from coordinated water.3,4
To build a supramolecular system based on similar principles,
we have synthesized a compound 3, in which L-tyrosine was
covalently linked to a ruthenium tris-bipyridyl complex through
an amide group.14 We could show that a tyrosyl radical was
generated by photoinduced ET from tyrosine to the photoge-
nerated Ru(III). We could also show that this tyrosyl radical
was capable of oxidizing a dinuclear manganese complex that
has recently been prepared by Girerd and co-workers.15,16
Recently, it was found that tyrosineZ most probably is
hydrogen-bonded to a histidine residue, His190 in the D1
polypeptide in PS II.5,6a,17 It is also known that the EPR spectrum
from the oxidized tyrosine represents the neutral, deprotonated
radical. It has been proposed that the histidine mediates proton
transfer from tyrosineZ.18,19 The exact mechanism for the
electron transfer is not clear, but there are three reasonable
alternatives. The first is that an electron is transferred from
tyrosineZ to the photooxidized P680 to give a cation radical,
which is subsequently deprotonated to the tyrosyl radical. The
deprotonation should be very efficient because the radical cation
is a strong acid. In the second alternative, tyrosineZ is first
deprotonated to give a phenolate anion which should be a very
efficient electron donor, giving the tyrosyl radical directly. Since
histidine is a relatively weak base compared to phenolate, the
relative concentration of phenolate should be low, however. The
third alternative is that electron and proton transfers are coupled.
This would be a very plausible mechanism if it is assumed that
electron transfer takes place from a system in which strong
(7) (a) Yachandra, V. K.; Sauer, K.; Klein, M. P. Chem. ReV. 1996, 96,
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A.; Parish, R. V.; Tames, J. J. Chem. Soc., Dalton Trans. 1985, 1391-
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R.; Andersson, M.; Bo¨rje, A.; Korall, P.; Philouze, C.; Almgren, M.; Styring,
S.; A° kermark, B. Chem. Comm. 1997, 607-608. (b) Sun, L.; Berglund,
H.; Davidov, R.; Norrby, T.; Hammarstro¨m, L.; Korall, P.; Bo¨rje, A.;
Philouze, C.; Berg, K.; Tran, A.; Andersson, M.; Stenhagen, G.; Mårtensson,
J.; Almgren, M.; Styring, S.; A° kermark, B. J. Am. Chem. Soc., 1997, 119,
6996-7004. (c) Berglund-Baudin, H.; Sun, L.; Davidov, R.; Sundahl, M.;
Styring, S.; A° kermark, B.; Almgren, M.; Hammarstro¨m, L. J. Phys. Chem.
A 1998, 102, 2512-2518. (d) Hammarstro¨m, L.; Sun, L.; A° kermark, B.;
Styring, S. Biochim. Biophys. Acta 1998, 1365, 193-199.
(16) Magnuson, A.; Frapart, Y.; Abrahamsson, M.; Horner, O.; A° kermark,
B.; Sun, L.; Girerd, J.-J.; Hammarstro¨m, L.; Styring, S. J. Am. Chem. Soc.
1999, 121, 89-96.
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Biochemistry 1998, 37, 11352-11365. (b) Mamedov, F.; Sayre, R. T.;
Styring, S. Biochemistry 1998, 37, 14245-14256.
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A. Chem. ReV. 1998, 98, 705-762.

6836 J. Am. Chem. Soc., Vol. 121, No. 29, 1999
Sun et al.
Chart 2
Scheme 1. Two Different Routes to Synthesize Complex 4
and Its Manganese Complex 5
Figure 1. Schematic presentation of the hydrogen bonding around
redox active tyrosine in photosystem II and in a model system 4.
hydrogen bonding between the phenol and the histidine aids
the electron transfer by increasing electron density on the
tyrosine. The hydrogen bonding should also promote transfer
of the proton because it should very readily move along the
hydrogen bond from a position close to the oxygen to a position
close to the histidine nitrogen.
In an attempt to study these questions we have now
synthesized complex 4, in which two dpa arms are connected
to the o-positions of the phenol group of L-tyrosine. The dpa
arms form a strong hydrogen bond to the phenol hydroxy group
and also act as an internal base. Of course, they also form a
site for coordinating manganese ions to the complex,20 giving
5.
In this paper we present the synthesis and characterization
of complex 4 (Chart 2). We have been able to demonstrate
intramolecular electron transfer from the tyrosine analogue to
the photogenerated Ru(III). We have compared the rate of
electron transfer with that of the unsubstituted tyrosine analogue
in 3, which lacks the hydrogen-bonding substituents, and find
it to be much faster in 4. We have also been able to show that
the photogenerated tyrosyl residue in 4 is a radical cation in
acetonitrile, a moderately polar, aprotic solvent. In contrast, a
neutral tyrosyl radical was observed in aqueous solution, closely
mimicking the photoevents at the donor side of PS II (Figure
1).
routes, shown in Scheme 1. In route A, compound 3, which
has been prepared earlier,14 was subjected to a Mannich reaction
with dipicolylamine(dpa) (see Scheme 1, route A), to give the
substituted tyrosine-ruthenium compound 4, which was sub-
sequently purified by column chromatography. The progress of
1
this reaction could easily be followed by H NMR. The two
aromatic doublet peaks of the tyrosine moiety in the starting
material 3 merged into a singlet at 7 ppm, an area where no
other protons gave a signal in the reaction mixture. The
purification of the crude 4 by column chromatography proved
to be difficult due to the necessary excess of dpa as starting
material. The problem was overcome by purification of the
hydrochloric salt of 4 on Al2O3 using a hydrochloric acid in
methanol as eluent. The product was then obtained by neutral-
izing the product containing fractions with a dilute aqueous
solution of sodium bicarbonate. Compound 4 was characterized
by 1H NMR, COSY, NOESY, 13C NMR, IR, and mass
1
spectrometry. The H NMR shows the phenolic proton at 11
ppm, indicating a strong hydrogen bond to the dpa arms. This
observation is supported by the NOESY spectrum21 in DMSO
which shows an interaction between the phenolic proton, the
benzylic CH2 groups, and two of the pyridine protons from the
dpa arms. Also the absorption of the phenolic OH in the IR at
3405 cm-1 supports the existence of a hydrogen bond. Elec-
trospray ionization mass spectrometry (ESI-MS) spectrum of 4
from acetonitrile gave a mono charged peak at m/z 1386 [M -
Results and Discussion
Synthesis. The compound 4 was prepared by two different
(20) (a) Pessiki, P. S.; Khangulov, S. V.; Ho, D. M.; Dismukes, G. C. J.
Am. Chem. Soc. 1994, 116, 891. (b) Khangulov, S. V.; Pessiki, P. J.; Barynin,
V. V.; Ash, D. E.; Dismukes, G. C. Biochemistry 1995, 34, 2015. (c) Sun,
L.; Raymond, M. K.; Magnuson, A.; LeGourrie´rec, D.; Tamm, M.;
Abrahamsson, M.; Kene´z, P. H.; Ma˚rtensson, J.; Stenhagen, G.; Hammar-
stro¨m, L.; Styring, S.; Åkermark, B. EUCHEM Conference, Artificial
Photosynthesis, May 16-20, 1998, Sigtuna, Sweden.
(21) See Supporting Information.

Electron Transfer to Photogenerated Ru(III)
J. Am. Chem. Soc., Vol. 121, No. 29, 1999 6837
Figure 3. Time-resolved emission decay profile of compound 4 in
N2-purged acetonitrile at room temperature, λex ) 327 nm, λem ) 610
nm.
acetonitrile and water. In acetonitrile, see Figure 2a and 2b,
there is an oxidation with peak potential 1.32 V vs SCE and
three reversible reduction peaks at negative potentials, the first
one with peak potential -1.22 V vs SCE. These are typical for
oxidation of the metal and the reduction of the three ligands in
Ru(bpy)32+ complexes. In Figure 2a the reduction peak of Ru-
(III) looks small compared to the oxidation peak, but scanning
only over the potential range around the Ru peak gave equal
peak hights. The two irreversible oxidation peaks at 0.84 and
1.01 V vs SCE were assigned to the oxidation of the tyrosine
and the dpa arm, respectively. This conclusion is based on the
shape and peak potential obtained in acetonitrile for the free
dpa arm (1.02 V vs SCE) and complex 3 (0.85-0.95 V vs SCE).
These data imply that the tyrosine is easier to oxidize than the
dpa arm and that both of them can be oxidized by the Ru(III)
complex.
Figure 2. Cyclic voltammetry of 1.12 mM 4 in 0.1 M TBABF4/CH3-
CN at a glassy carbon working electrode (L 3 mm): (a) scan from 0.0
to +1.6 V; (b) scan from 0.0 to -2.0 V. A Ag/AgCl electrode (saturated
with LiCl in CH3CN) is used as reference electrode. Scan rate 100
mV/s.
In water solution at pH 7 (not shown) only one irreversible
oxidation peak with a peak potential 0.79 V (0.74 V for 3) vs
SCE was obtained (the redox processes of the Ru(bpy)32+ moiety
are outside the potential window in water). This was assigned
to the oxidation of tyrosine due to similarities in the shape and
peak potential of earlier published results for free tyrosine and
3.16,23 For the free dpa arm in water, we obtained an irreversible
oxidation with the peak potential 0.89 V vs SCE, i.e., a much
lower potential than in acetonitrile. Because oxidized amines
undergo fast secondary reactions, the observed irreversible peak
potentials may lead to poor estimates of the true oxidation
potentials.24,25 The oxidation potential for the dpa arm in 4 may
therefore be even lower than 0.89 V, making it difficult to decide
whether the dpa or the tyrosine unit is the most readily oxidized.
Emission Studies and Flash Photolysis. In acetonitrile,
complex 4 displayed a MLCT absorption band at 456 nm that
- +
-
PF6
]
and doubly charged peaks at m/z 693 [M - PF6
+
H+]2+, and m/z 621 [M - 2PF6
]
- 2+. Fast atom bombardment
mass spectrometry (FAB-MS) using 1-thioglycerol as matrix,
showed the mono charged peaks at m/z 1532 [M + H+]+ and
1386 [M - PF6-]+. These results clearly prove the assigned
structure.
An alternative approach to the synthesis of compound 4 is
described in Scheme 1, route B. In this route, the L-tyrosine
ester 6 was subjected to the Mannich reaction with dpa to give
compound 7. Deprotection of the amino group gave the
compound 8 which was then linked to the ruthenium tris-
22
is typical of Ru(bpy)3 type compounds.26 The emission
2+
bipyridyl carboxylic acid 9 to give 4. Also on this pathway
the complex turned out to be difficult to purify, because the
main side product, the starting material 8, had physical properties
similar to those of the product 4. It was found that the addition
of small amounts of acetic acid to the eluent during column
chromatography made the product 4 eluable while 8 remained
on the column. Unfortunately, the yield of this amide coupling
is still unsatisfactory. Attempts to perform the coupling by other
established methods, for example, via an active ester, gave even
lower yield. Nevertheless, both routes A and B led to the same
compound 4.
maximum (λEM) of 4 in water is 675 nm, and in acetonitrile
656 nm. An emission decay curve for 4 was obtained by single-
photon counting (Figure 3). Complex 4 displayed a single-
exponential decay in acetonitrile and in water with lifetimes τ
) 1350 ns and 470 ns, respectively. In both solvents, 4 had a
similar excited-state lifetime as the compound 3, in which the
(23) Harriman, A. J. Phys. Chem. 1987, 91, 6102.
(24) Clark, C. D.; Hoffman, M. Z. J. Phys. Chem. 1996, 100, 14688.
(25) Chow, Y. L.; Danen, W. C.; Nelsen, S. F.; Rosenblett, D. H. Chem.
ReV. 1978, 78, 243.
(26) For properties of Ru(II)-polypyridyls, especially concerning pho-
toinduced electron transfer, see: (a) Juris, A.; Balzani, V.; Barigelletti, F.;
Campagna, S.; Belser, P.; von Zelewsky, A. Coord. Chem. ReV. 1988, 84,
85. (b) Meyer, T. J. Acc. Chem. Res. 1989, 22, 163. (c) Kalyanasundaram,
K. Photochemistry of Polypyridine and Porphyrin Complexes; Academic
Press: London, 1992, Chapter 6.
Electrochemistry Studies. The electrochemical behavior of
4 (route B) was studied by cyclic voltammetry in both
(22) Peek, B. M.; Ross, G. T.; Edwards, S. W.; Meyer, G. J.; Meyer, T.
J.; Erickson, B. W. Int. J. Pept. Protein Res. 1991, 38, 114.

6838 J. Am. Chem. Soc., Vol. 121, No. 29, 1999
Sun et al.
Figure 4. Transient absorption curves at 600 nm (top) and 450 nm
(bottom) of 4 (4 × 10-5 M) and methylviologen (MV‚2Cl, 1.0 × 10-2
M) in water at room temperature. The Ru(III) to Ru(II) recovery is on
the same time scale as the Ru(II)* excited-state decay (the spike in the
450 nm one).
Figure 5. Transient absorption curves at 600 nm (top) and 450 nm
(bottom) of deoxygenated acetonitrile solution of 4 (4 × 10-5 M) in
the presence of MV‚2PF6 (1.0 × 10-2 M) at room temperature. The
intensity of the slow component of the Ru(II) recovery at 450 nm
increases as the sample is subjected to more light.
ligand contains an unsubstituted tyrosine unit (τ ) 1150 ns in
acetonitrile and 370 ns in water). Neither of the tyrosine moieties
quenched the Ru-excited state, as shown with a reference
compound with alanine instead of tyrosine.14 The shorter
excited-state lifetime in water is a solvent effect on the Ru-
chromophore itself, typical for these Ru-complexes.26
the tyrosine unit which is hydrogen-bonded to it. This conclusion
was supported by direct observation of the photogenerated
phenoxyl radicals in the EPR experiments (see below).
In the flash photolysis experiments, the MV+• signal at 600
nm decayed following second-order kinetics with a rate constant
of ca. 5 × 109 M-1 s-1. The decay was attributed to the
To measure the photoinduced intramolecular ET rate from
the tyrosine moiety to the photogenerated ruthenium(III), flash
photolysis measurements were made. Application of a 460 nm
laser pulse (<30 ns duration) to complex 4 in water in the
presence of the electron acceptor methylviologen (MV2+
)
recombination between MV+• and the tyrosine radical.14
A
resulted in a rapid loss in absorption at 450 nm and an increase
at 600 nm (Figure 4). The bleaching at 450 nm reflects the loss
of Ru(II), due to excitation and the oxidative quenching of the
excited state, Ru(II)* (reaction 1). The absorption observed at
600 nm is caused by the methylviologen radical cation, MV+•,
formed upon reduction by Ru(II)*.
buildup of the methylviologen radical was observed after
repeated flashes to a solution of 4 in acetonitrile (but not in
water). This is probably due to the partial photodecomposition
of the electron donor which would otherwise convert MV+• to
MV2+. Another control experiment, using Ru(bpy)32+ and bis-
(picolyl)ethylamine instead of 4, also gave a buildup of
methylviologen radical, suggesting that the dpa ligand can serve
as an irreversible electron donor in acetonitrile.
Ru(II)* + MV2+ f Ru(III) + MV+•
(1)
The decay of the MV+• absorption occurred much more
slowly than the recovery of the Ru(II) signal, leading to the
significant conclusion that Ru(II) recovery does not simply occur
via the reverse of reaction 1. Recovery of Ru(II) must, therefore,
involve electron transfer from the tyrosine phenolate moiety.
Flash photolysis experiments in acetonitrile gave a similar result
(see Figure 5). The rate of Ru(II) recovery was in fact limited
by the quenching reaction 1, that had a pseudo-first-order rate
constant of 1 × 107 s-1 at the highest concentration of MV2+
used. This gives that the ET reaction that regenerates Ru(II)
has a rate constant kET > 1 × 107 s-1 both in acetonitrile and
in water, which means that the electron transfer must be
intramolecular.
Thermodynamically, both the dpa arm and the tyrosine unit
can work as electron donors. To exclude the possibility of the
dpa arm as a primary electron donor, a control experiment was
performed. Instead of 4, a compound in which the ruthenium-
(II) tris-bipyridyl complex was directly linked to a dpa ligand
via a single carbon bond27 was used in the flash photolysis.
The result showed that the ET rate constant from the dpa ligand
to the photogenerated Ru(III) was 3 orders of magnitude lower,
ca. 104 s-1, despite the short Ru-dpa distance. Therefore, the
primary electron donor in 4 is not likely to be the dpa arm but
Phenoxyl Radicals. To study the light-induced formation of
tyrosyl radicals from 4, a series of EPR experiments were
conducted. Continuous, in situ laser flashing of an aqueous
solution of 4 in the presence of the sacrificial electron acceptor
[Co(NH3)5Cl]2+ resulted in the generation of a neutral phenoxyl
radical, as evidenced by a characteristic EPR signal (Figure 6),
having an isotropic g value of 2.0044 and a peak-to-trough line
width of approximately 8.7 G. This photochemical reaction
proceeds in several steps. First, the Ru(II) moiety transfers an
electron from its MLCT state to the sacrificial electron acceptor,
[Co(NH3)5Cl]2+, resulting in the formation of [Co(H2O)6]2+ and
Ru(III). The Ru(III) moiety retrieves an electron from the dpa-
substituted tyrosine moiety in 4, and a phenoxyl radical is
formed after loss of one proton. The high g value (g ca. 2.0044)
is strong evidence that the observed spectrum represents a
neutral (i.e., deprotonated) phenoxyl radical (see below).
In a similar experiment with 4 in pure acetonitrile, using
MV2+ as an external electron acceptor instead of [Co(NH3)5-
Cl]2+, a protonated phenoxyl radical (PhOH+•) was detected.
Also here, Ru(III) was formed in the first step together with a
methylviologen radical (MV+•) after an intermolecular ET. The
MV+• radical has a large, wide EPR spectrum with many
hyperfine lines. To observe radical species formed from 4 after
a laser pulse, we conducted kinetic EPR experiments which
permit their detection even in the presence of the strong MV+•
signals. To accomplish this, time-mode EPR measurements at
(27) Berg, K. E.; Tran, A.; Raymond, M. K.; Abrahamsson, M.; Wolny,
J.; Redon, S.; Andersson, M.; Sun, L.; Styring, S.; Hammarstro¨m, L.;
Toftlund, H.; A° kermark, B., manuscript in preparation.

Electron Transfer to Photogenerated Ru(III)
J. Am. Chem. Soc., Vol. 121, No. 29, 1999 6839
Figure 6. EPR spectrum recorded during the continuous laser-flashing
of an aqueous solution (10% DMSO) of 4 in the presence of [Co-
(NH3)5Cl]2+. The radical EPR signal has a g value of 2.0044 indicative
of the formation of a neutral phenoxyl radical. The laser flashes were
delivered with 5 Hz flash frequency and the “spikes” in the spectrum
are due to the transient decay of a fraction of the formed radical between
the flashes. For EPR settings see the Experimental Section.
field positions at which the methylviologen radical was deter-
mined to give zero signal intensity were used to construct a
“kinetic” spectrum (see Figure 7A). This revealed the presence
of an underlying signal from a short-lived (t1/2 ≈ 40 ms) species
(see Figure 7B). The signal had a peak-to-trough width of
approximately 16 G and an isotropic g value of 2.0029. This g
value is characteristic of a protonated phenoxyl radical
(PhOH+•),28 which was generated by intramolecular ET from
dpa-substituted tyrosine to the Ru(III). It has been reported that
upon increasing the acidity to below pH 0, phenoxyl radicals
are converted to phenol radical cations. Phenol radical cations
have also been observed in nonaqueous solvents. The phenol
radical cation has a pKa of -2.0 in water and should be rapidly
deprotonated in aqueous solution, as observed. Phenoxyl radicals
are reported to have g values of 2.0044 ( 0.0004, and those
for phenol radical cations have values of 2.0032 ( 0.0004,
strongly supporting our conclusion that the observed radical with
g value of 2.0029 is a protonated phenoxyl radical.28
In summary, these results establish that the tyrosine moiety
is the intramolecular donor to the photogenerated Ru(III), as
observed also for the complex 3, where the tyrosine is
unsubstituted. The electron transfer from the ligand to the
photogenerated Ru(III) in 4 is more than 2 orders of magnitude
faster than in 3.29 We propose that the reason for this is strong
intramolecular hydrogen bonding between the phenolic hydroxyl
group and the dpa arms in 4. Because of the hydrogen bond,
the electron density on the tyrosine is increased, facilitating
electron transfer. Since the electron-transfer rate in the parent
complex 3 goes up as the pH increases,30 partial deprotonation
of the phenol in 4 is also possible.
Figure 7. (A) Flash-induced induction and decay of the radical species
in 4 after a single flash to a mixture of 4 in the presence of
methylviologen (MV‚2PF6) in acetonitrile. The inset shows the spectrum
of the methylviologen radical and the arrowmarked dot indicates the
field position for the transient spectrum shown. The transient signal
represents the average of 250 flash-induced events. (B) Kinetic spectrum
of the radical species formed in A. The spectrum is constructed from
the amplitude of the transient signals (Figure 7A) measured at different
field positions chosen where the amplitude of the methylviologen radical
signal was minimal or zero (A, inset). The approximate g value for the
radical species is 2.0029, indicative of the formation of a protonated
phenoxyl radical. See Experimental Section for details.
sensitizer, here Ru(III), is coupled with a proton-transfer process,
generating a tyrosine centered radical (see Scheme 2). In water
solution, the electron transfer is very fast and the result is a
deprotonated radical. Thus the phenolic proton has been
removed from 4 (Scheme 2, left pathway). In acetonitrile, the
electron transfer is again very fast, but the proton stays in the
close vicinity of the phenolic oxygen in 4 after its oxidation. It
is probable that this is due to very strong hydrogen bonding in
a network such as that indicated in Scheme 2 (pathway on the
right) which retains the phenolic proton despite the very acidic
radical formed. Together, these results suggest that the fast
electron transfer to the photooxidized Ru-center from the
phenolic moiety in 4 is aided by a hydrogen bond network,
involving the nitrogens in the dpa arms, which immediately
accept the proton when the electron leaves the phenolic center.
These results thus show for the first time that a photoinduced
electron-transfer process from a tyrosine unit to a photooxidized
(28) (a) Dixon, W. T.; Murphy, D. J. Chem. Soc., Faraday Trans. 2
1976, 72, 1221-1230. (b) Barry, B. A.; Babcock, G. T. Chem. Scr. 1988,
28A, 117-122.
(29) Molecular mechanics calculations (see Supporting Information)
predict different conformations of 4 in water and in acetonitrile, which may
result in different ET rates. However, that cannot be proven because of the
rate limitation imposed by the diffusion-controlled intermolecular electron
transfer from excited Ru(II) to methylviologen, faster rates than ca. 1 ×
107 s-1 for the intramolecular electron transfer from tyrosine to photoge-
nerated Ru(III) cannot be measured.
This mimics the situation in PS II where the phenolic proton
in tyrosineZ most likely is hydrogen-bonded to Histidine 190
(Figure 1)17-19 which also aids in fast electron transfer from
(30) The electron-transfer rate increases from k ) 5 × 104 s-1 at pH 7
to k ) 7.6 × 104 s-1 at pH 8.2 and k ) 1.3 × 105 s-1 at pH 9.3.

6840 J. Am. Chem. Soc., Vol. 121, No. 29, 1999
Sun et al.
water was Millipore M-Q quality. In the flash photolysis experiments,
an excimer laser pumped a dye laser to produce excitation pulses of
30-ns width at 460 nm. Transient absorption measurements were made
at 450 and 600 nm. All solutions were bubbled with nitrogen.
Scheme 2. Proposed Photoinduced Electron Transfer
Pathways of Model Compound 4 in the Presence of an
Electron Acceptor [Co(NH3)5Cl]2+ in Water or
Methylviologen (MV‚2PF6) in Acetonitrile Solution
Computational Methods. Molecular mechanics modeling was
carried out using MacroModel version 6.0 with the MM3* force field.
The force field was extended to include new parameters for the Ru-
polypyridine moiety.32 A conformational search was carried out using
a Monte Carlo method33 together with a truncated Newton-Raphson
minimization algorithm. The number of Monte Carlo steps taken was
10 000 and a derivative convergent criteria of 0.05 kJÅ-1 mol-1 was
used. Solvation (water and chloroform) was included in the minimiza-
tions using the GB/SA solvation model34 implemented in MacroModel.
Electrochemistry Study. Electrochemical experiments were done
in water and in acetonitrile. The experiments in acetonitrile were
performed in an argon-filled glovebox. The electrolytes used were 0.2
M KCl in water and 0.1 M tetrabutylammonium tetrafluoroborate
(Aldrich, TBABF4) in acetonitrile. The TBABF4 was vacuum-dried at
140 °C for 48 h, and acetonitrile (Aldrich, 99.8%) was used as received.
The glassware was oven-dried. The electrochemical measurements were
carried out by using a three-electrode system connected to an Eco
Chemie model Autolab/GPES electrochemical interface. The working
electrode was a freshly polished glassy carbon disk (diameter 3 mm),
and the counter electrode consisted of a platinum wire. The reference
electrode was commercial Ag/AgCl in 3 M KCl (0.207 V vs NHE) in
water and Ag/AgCl in saturated LiCl (Merck) in acetonitrile. Both the
reference and counter electrodes were separated from the solution by
a salt bridge in contact with the working electrode. A ferrocenium/
ferrocene couple was used as an internal reference in nonaqueous media.
All data obtained in acetonitrile are reported vs SCE. The half potential
2+
of the reversible Ru(III/II) peak for Ru(bpy)3 was assigned to be
the tyrosineZ. In this case the result is the neutral radical as
judged from the spectral properties of tyrosineZox. However, the
phenolic proton still resides in the vicinity and is spectroscopi-
cally observed to participate in a disordered or bifurcated
hydrogen bond.31 It seems clear that the hydrogen bond is
weaker around tyrosineZ than in 4 since the radical which is
formed in PS II is neutral and not cationic.
1.3235 V vs SCE according to the value reported by Bard et al.35 By
using the Ru(III/II) value obtained for Ru(bpy)32+ and the half potential
for the internal reference the data could be reported vs SCE.
Detection of the Neutral Phenoxyl Radical. To a saturated aqueous
solution of [Co(NH3)5Cl]2+ was added a solution of DMSO and water
(10:90, v/v) and 4 (2 mM). The combined solution was transferred by
aspiration to a quartz EPR flat-cell, positioned, and pretuned on a 10%
aqueous solution of DMSO in the EPR cavity. A spectrum was recorded
immediately upon the commencement of continuous laser-flashing (532
nm, 5 Hz, 1.5 W) into the spectrometer cavity from a GCR-200 series
Nd:Yag laser (Spectra-Physics, CA). The following EPR spectrometer
settings were used: modulation amplitude, 5 G; time constant, 20 ms;
sweep time, 84 s; receiver gain, 1 × 105; microwave power, 18.3 mW;
microwave frequency, 9.771 GHz. A solution of diphenylpicrylhydrazyl
(in 10% aqueous solution of DMSO), transferred to the flat-cell without
retuning, was used as a g marker (g ) 2.0036).
Detection of the Protonated Phenoxyl Radical. The reaction
mixture, containing 4 (2 mM) and methylviologen (MV‚2PF6) in
acetonitrile, was transferred by using aspiration to an aqueous flat-cell
positioned in the EPR cavity to obtain the spectrum of the methylvi-
ologen radical. The sample was subjected to continuous laser-flashing
at 5 Hz (as above) for 1 min, after which an EPR spectrum of the
resulting methylviologen radical cation was recorded by using the
following instrument settings: modulation amplitude, 0.71 G; time
constant, 20 ms; sweep time, 84 s; receiver gain, 2 × 104 microwave
power, 17 mW; microwave frequency, 9.770 GHz. The magnetic field
positions at which the multilined spectrum of the methylviologen radical
crosses the baseline (i.e., has no microwave absorption) were noted.
These field positions were used as static field settings in the construction
of a “kinetic EPR” spectrum in the search for underlying, transient
species (i.e., species other than the methylviologen radical). Without
Experimental Section
1
General Methods. H NMR and 13C NMR spectra were measured
using a Bruker-400 MHz spectrometer for 1D or a Bruker-500 MHz
spectrometer for 2D. The electrospray ionization mass spectrometry
(ESI-MS) experiments were performed on a ZacSpec mass spectrometer
(VG Analytical, Fisons Instrument). Electrospray conditions were needle
potential, 3kV; acceleration voltage, 4kV; bath and nebulizing gas;
nitrogen. The liquid flow was 75 µL per min using a syringe pump
(Phoenix 20, Carlo Erba, Fisons instrument). The solvent was pure
acetonitrile. Precise mass measurements were obtained by the use of
poly(ethylene glycol) (PEG) as an internal standard. The fast atom
bombardment mass spectrometry (FAB-MS) experiments were run in
a matrix of 1-thioglycerol. Absorption and steady-state emission spectra
were recorded with Varian Cary 5E UV-vis-NIR and SPEX Fluorolog
II Systems spectrometers. X-Band EPR spectra were recorded on a
Bruker ESP380 spectrometer equipped with an Oxford Instruments
temperature controller. Time-resolved emission measurements were
conducted using single-photon counting equipment, employing a mode-
locked Nd:YAG laser to pump a DCM dye laser. The output from the
dye laser was frequency doubled to 327 nm and used to excite the
samples, and the instrumental response function (fwhm) was 200 ps.
The emission was observed around 610 nm by means of an interference
filter with a 10-nm bandwidth. All solutions used for photophysical
measurements were deoxygenated by purging with nitrogen for fifteen
minutes before measurements and then by being kept under an
atmosphere of nitrogen. Acetonitrile was of spectroscopic grade, and
(32) Brandt, P.; Norrby, T.; A° kermark, B.; Norrby, P.-O. Inorg. Chem.
1998, 37, 4120-4127.
(33) Chang, G.; Guida, W. C.; Still, W. C. J. Am. Chem. Soc. 1989,
111, 4379. Chang, G.; Guida, W. C.; Still, W. C. J. Am. Chem. Soc. 1990,
112, 1419.
(34) Still, W. C.; Tempczyk, A.; Hawley, R. C.; Hendrickson, T. J. Am.
Chem. Soc. 1990, 112, 6127.
(35) Tokel-Takvoryan, N. E.; Hemmingway, R. E.; Bard, A. J.; J. Am.
Chem. Soc. 1973, 95, 6582.
(31) (a) Tommos, C.; McCracken, J.; Styring, S.; Babcock, G. T. J. Am.
Chem. Soc. 1998, 120, 10441-10452. (b) Dorlet, P.; DiValentin, M.;
Babcock, G. T.; McCracken, J. L. J. Phys. Chem. B 1998, 102, 8239-
8247. (c) Force, D. A.; Randall, D. W. and Britt, R. D. Biochemistry 1997,
36, 12062-12070.

Electron Transfer to Photogenerated Ru(III)
J. Am. Chem. Soc., Vol. 121, No. 29, 1999 6841
retuning the instrument, the reaction mixture was replaced with fresh
solution for each magnetic field position employed. Time-mode EPR
spectra were obtained at each field position following the application
of a single laser flash using the following instrument settings:
modulation amplitude, 0.71 G; time constant, 0.16 ms; sweep time,
0.328 s; receiver gain, 1.25 × 105; microwave power, 17 mW;
microwave frequency, 9.7692 GHz. For each static field position used,
traces from 250 sequential laser flashes (523 nm, 5 Hz, 1.5 W) were
accumulated. The EPR recording was triggered to commence 500 µs
after each flash by means of a personal computer. A solution of
diphenylpicrylhydrazyl in acetonitrile was used as a g marker (as above).
An EPR “spectrum” was then constructed by plotting the initial signal
amplitude observed at each static field position.
and extracted with 3 × 50 mL CH2Cl2. The combined extracts were
dried over Na2SO4, and after evaporation of the solvent 510 mg of
(83%) 8 was isolated as a light yellow oil. 1H NMR (400 MHz, CDCl3)
δ in ppm 1.12 (t, J ) 7.0 Hz, 3H, CO2CH2CH3), 1.70 (br s, 2H, NH2),
2.72 (dd, J ) 7.9 and 13.4 Hz, 1H, CHCH2Tyr), 2.93 (dd, J1 ) 4.9
and 13.4 Hz, 1H, CHCH2Tyr), 3.59 (m, 1H, CHCH2Tyr), 3.74 (s, 4 H,
TyrCH2N), 3.81 (s, 8H, PyrCH2N), 4.04 (q, J ) 7.0 Hz, 2H, CO2CH2-
CH3), 6.99 (s, 2H, arom. Tyr-CH), 7.06 (m, 4H, Pyr-CH), 7.43 (m,
4H, Pyr-CH), 7.54 (m, 4H, Pyr-CH), 8.46 (m, 4H, Pyr-CH), 10.95 (s,
1H, TyrOH). 13C NMR (100.6 MHz, CDCl3, DEPT) δ in ppm 14.03
(+), 40.28 (-), 54.55 (-), 55.29 (+), 59.61 (-), 60.54 (-), 121.78
(+), 122.75 (+), 123.97 (Cquart), 126.58 (Cquart), 129.87 (+), 136.33
(+), 148.70 (+), 154.68 (Cquart), 159.05 (Cquart), 174.96 (Cquart). ESI-
MS found: m/z 654 (8 plus Na+ requires 654).
Materials. L-Tyrosine ethyl ester hydrochloride, 4,4′-dimethyl-2,2′-
bipyridine, 2,2′-bipyridine (bpy), 4,4′-bipyridine, rutheniumtrichloride,
silvertriflate, triethylamine, ammonium hexafluorophosphate, p-cresol,
and paraformaldehyde were purchased from Aldrich and used as
received. Silica gel 60 (230-400 mesh, Merck, Darmstadt, Germany)
and aluminum oxide (Aldrich) were used for column chromatography.
cis-Dichloro-bis(bpy)ruthenium (cis-Ru(bpy)2Cl2‚2H2O) was prepared
by the method reported by Meyer et al.36 All solvents were dried by
standard methods. Methylviologen (MV‚2PF6) was made by changing
the counterions from methylviologen (MV‚2Cl, Aldrich) followed by
two times recrystallization (2×) from ethanol/water (90/10). N,N-Di-
(2-picolyl)amine (dpa) was made by reaction of picolyl amine and
picolyl aldehyde followed by reduction with sodium borohydride.37
[Ru(bpy)2[4-Me-4′-CONH-CH(COOEt)(Hbpmp)]].(PF6)2 (4). Route
A. To compound 314 (0.177 g, 0.15 mmol) in EtOH/H2O (1.2 mL/1.5
mL) N,N-di(2-picolyl)amine (0.150 g, 0.75 mmol) and paraformalde-
hyde (0.023 g, 0.75 mmol) were added, followed by addition of 0.05
mL 2 N HCl, and the two-phase reaction mixture was heated under
reflux for 20 h under argon. After the solution was cooled to room
temperature, a solution of NH4PF6 (2 g) in 20 mL of H2O and 20 mL
of CH2Cl2 was added to the reaction mixture, and the organic phase
was isolated. To this organic solution, which contained the product 4
and the excess dpa, a 20 mL solution of 0.2 N HCl and NH4PF6 (1 g)
were added. The organic phase was washed with a solution of 30 mL
of H2O and Na2CO3 (3 g) and then with 30 mL of water. The organic
phase was finally separated and dried over anhydrous sodium sulfate.
After evaporation of the solvent, a red solid was obtained. Further
purification was conducted by three successive flash column chro-
matographies on Al2O3 with eluents of MeOH/2M HCl (95:5, v/v).
The desired fractions were combined and concentrated to about 10 mL
and then neutralized by adding an aqueous solution of Na2CO3.
Extraction with CH2Cl2, drying over Na2SO4, and evaporation of the
2,6-Bis[[N,N-di(2-pyridylmethyl)amino]methyl]-4-methylphe-
nol (Hbpmp). This compound was made by following a modification
of the literature method.37 To N,N-di(2-picolyl)amine (2.50 g, 12.5
mmol) in EtOH/H2O (15 mL/45 mL) was added p-cresol (0.54 g, 5.0
mmol) and paraformaldehyde (0.75 g, 25.0 mmol). To the mixture was
added 0.3 mL of 2 N HCl, and the two-phase reaction mixture was
heated under reflux for 20 h and then allowed to cool to room
temperature. CH2Cl2 (100 mL) and H2O (100 mL) were added to the
reaction mixture, and the organic phase was separated after washing
with another 100 mL of H2O and dried over anhydrous sodium sulfate.
A yellow oil was obtained after evaporation of solvent. Medium-
pressure column chromatography on silica gel with gradient eluents
CH2Cl2 and CH2Cl2/MeOH (94/6) afforded a pale yellow oil (2.25 g,
85% yield) which became a solid upon standing, mp 112 °C (uncor-
1
solvent afforded 4 (0.149 g, 65% yield) as a red solid. H NMR (400
MHz, DMSO-d6) δ in ppm 1.01 (t, J ) 7.0 Hz, 3H, CO2CH2CH3),
2.51 (s, 3H, Me), 3.01-3.12 (m, 2H, CHCH2Tyr), 3.55-3.72 (m, 12H,
CH2N), 3.99 (q, J ) 7.0 Hz, 2H, CO2CH2CH3), 4.62-4.74 (m, 1H,
CHCH2Tyr), 7.09 (s, 2H, Tyr), 7.19 (dd, J ) 4.8 and 7.0 Hz, 4H, Pyr),
7.39 (d, J ) 7.4 Hz, 4H, Pyr), 7.43-7.57 (m, 4H, Bpy), 7.63-7.72
(m, 8H, Bpy, Pyr), 7.72-7.77 (m, 1H, Bpy), 7.80-7.85 (m, 1H, Bpy),
8.09-8.19 (m, 5H, Bpy), 8.39 (dd, J ) 4.8 and 5.4 Hz, 4H, Pyr), 8.69
(d, J ) 19.8 Hz, 1H, Bpy), 8.77-8.86 (m, 5H, Bpy), 9.05 (d, J ) 19.8
Hz, 1H, Bpy), 9.45 (br s, 1H, NH), 10.95 (s, 1H, TyrOH). 13C NMR
(100.6 MHz, DMSO-d6, DEPT) δ in ppm 13.77 (+), 20.61 (+), 35.77
(-), 53.78 (-), 54.84 (+), 58.63 (-), 60.56 (-), 121.55 (+), 122.05
(+, Pyr), 122.49 (+, Pyr), 123.48 (Cquart), 124.34 (+), 125.39 (+),
126.13 (Cquart), 127.80 (+), 128.85 (+), 129.82 (+), 136.51 (+, Pyr),
137.89 (+), 140.80 (Cquart), 148.53 (+, Pyr), 149.79 (Cquart), 149.84
(Cquart), 150.28 (+), 150.97 (+), 151.82 (+), 154.14 (Cquart), 155.48
(Cquart), 156.27 (Cquart), 156.31 (Cquart), 156.36 (Cquart), 156.40 (Cquart),
156.45 (Cquart), 157.31 (Cquart), 157.34 (Cquart), 158.50 (Cquart, Pyr), 162.91
(Cquart), 162.97 (Cquart), 171.02 (Cquart). IR (cm-1) 1669 (ν(CdO)), 1732
(ν(CdO)), 3405 (ν(OH)). Electrospray ionization mass spectrometry
(ESI-MS) spectrum of 4 from acetonitrile gave a mono charged peak
at m/z 1386 (calcd for [M - PF6-]+, 1386) and doubly charged peaks
1
rected). H NMR (400 MHz, CDCl3) δ in ppm 2.23 (s, 3H, -CH3),
3.77 (s, 4H, ph-CH2-N), 3.86 (s, 8H, N-CH2-py), 6.98 (s, 2H, ph-H),
7.11 (m, 4H, py-H), 7.48 (d, J ) 7.8 Hz, 4H, py-H), 7.59 (m, 4H,
py-H), 8.50 (m, 4H, py-H). 13C NMR (100 MHz, CDCl3), δ in ppm
20.6, 54.8, 59.8, 121.9, 122.9, 123.8, 127.3, 129.7, 136.5, 148.9, 153.6,
159.4.
NH2-CH(COOEt)-Hbpmp (8). To a suspension of dpa (500 mg,
2.51 mmol) and paraformaldehyde (100 mg, 3.33 mmol) in 2 mL of
ethanol and 6 mL of water were added HCl (0.5 m, 0.5 mL) and the
tyrosine derivative 6 (300 mg, 0.970 mmol).38 The mixture was heated
under reflux for 4 days, cooled to room temperature and all volatile
fractions were evaporated in vacuo. The remaining highly viscous oil
was dissolved in 5 mL CH2Cl2 at 0 °C, 5 mL of trifluoroacetic acid
were added and the mixture was stirred for additional 12 h while it
warmed to room temperature. The solvents were evaporated in vacuo
and 100 mL of aqueous ammonia, saturated with NaCl, were added.
The mixture was extracted with 5 × 50 mL CH2Cl2, the combined
organic phases were dried over Na2SO4 and again the solvent was
subsequent evaporated in vacuo. Column chromatography was per-
formed on silica gel (30 g) with the following eluents: CH3CN/CH3-
COOH ) 20:1, 100 mL; CH3CN/H2O/CH3COOH ) 100:10:1, 200 mL;
CH3CN/NH3(aqueous) ) 10:1, 200 mL; eluating the desired compound
as the last fraction. This fraction was concentrated in vacuo to 30 mL
at m/z 693 (calcd for [M - PF6- + H+]2+, 693) and m/z 621 (calcd for
-
[M - 2PF6
]
2+, 621). Fast atom bombardment mass spectrometry
(FAB-MS) using 1-thioglycerol as matrix, showed the mono charged
peaks at m/z 1532 (calcd for [M + H+]+, 1532) and 1386 (calcd for
[M - PF6-]+, 1386).
Route B. To Ru-complex-acid 9 (300 mg, 0.33 mmol) was added
freshly distilled SOCl2 (5 mL). The resulting red solution was stirred
for 2 h under reflux and then cooled to room temperature. The SOCl2
was evaporated in vacuo, and CH3CN (10 mL) was added followed by
8 (210 mg, 0.33 mmol) dissolved in CH3CN (5 mL). After 30 min,
K2CO3 (800 mg, 5.79 mmol) was added, and the resulting suspension
was stirred for 24 h at room temperature. The mixture was then diluted
with water (100 mL) and extracted with CH2Cl2 (3 × 80 mL). The
combined organic phases were concentrated by evaporation to 10 mL
and subsequently chromatographed on 30 g silica gel (eluent: CH3-
CN/H2O/KNO3(aq)/CH3COOH ) 100:10:5:2). To the obtained shiny red
(36) Sullivan, B. P.; Salmon, D. J.; Meyer, T. J. Inorg. Chem. 1978, 17,
3334.
(37) Lubben, M.; and Feringa, B. L. J. Org. Chem. 1994, 59, 2227-
2233.
(38) Houlihan, F.; Bouchard, J.; Frechet, J. M.; Willson, C. G. Can. J.
Chem. 1985, 63, 153-162.
(39) Due to the low solubility of the complex 4 in water, it has not been
possible to perform NOESY studies to verify this result.

6842 J. Am. Chem. Soc., Vol. 121, No. 29, 1999
Sun et al.
fraction, was added aqueous K2CO3 (20 mL), followed by evaporation
of the highly volatile parts. After dissolving of NH4PF6 (2.00 g, 12.27
mmol), the mixture was extracted with CH2Cl2 (6 × 50 mL).
Evaporation of the organic solvents and washing of the obtained red
solid with ethyl acetate (50 mL) and hexane (50 mL) gave 4 (266 mg,
53%). The NMR spectra turned out to be identical with the ones from
the other pathway.
Research Program. We thank Drs. Gunnar Stenhagen and Jerker
Mårtensson for performing the ESI-MS and FAB-MS measure-
ment, Dr. Anna Bo¨rje for help with the 2D NMR, Mr. Jiutian
Zheng for synthesis of dpa and Hbpmp, and Drs. Pierre Mialane
and Heimo Schmitt for help with the IR.
Supporting Information Available: A conformational
search of 4 in water and in acetonitrile by molecular mechanics
calculation and a NOESY spectrum of 4 in DMSO-d6 (PDF).
This material is available free of charge via the Internet at
http://pubs.acs.org.
Acknowledgment. This work was financially supported by
grants from the Knut and Alice Wallenberg Foundation, the
Swedish Natural Science Research Council, the Swedish
Research Council for Engineering Sciences, the European TMR
Program (TMR Network CT96-0031), and the Nordic Energy
JA984048C