Development of a model system for the study of long distance electron transfer in peptides

Article abstract of DOI:10.1002/adsc.200700605

We have designed and synthesized a peptide model in which stepwise electron transfer (ET) through amino acid side chains could be observed. An injection system, which generates an electron hole upon laser irradiation, was connected directly to the aromati

Full text of DOI:10.1002/adsc.200700605

FULL PAPERS
DOI: 10.1002/adsc.200700605
Development of a Model System for the Study of Long Distance
Electron Transfer in Peptides
Meike Cordes,a Olivier Jacques,a Agnieszka Kçttgen,a Christian Jasper,a
Hassen Boudebous,a and Bernd Giesea,*
a
Department of Chemistry, University of Basel, St. Johanns-Ring 19, 4056 Basel, Switzerland
Fax : (+41)-61-267-1105; e-mail: bernd.giese@unibas.ch
Received: December 21, 2007; Published online: March 20, 2008
Dedicated to Andreas Pfaltz, a great colleague and scientist, on the occasion of his 60th birthday.
Supporting information for this article is available on the WWW under http://asc.wiley-vch.de/home/.
Abstract: We have designed and synthesized a pep- a different signal in the transient absorption spec-
tide model in which stepwise electron transfer (ET) trum. Additional non-natural oxidizable aromatic
through amino acid side chains could be observed. amino acids were synthesized as spectroscopic sen-
An injection system, which generates an electron sors to detect oxidized amino acid side chains as
hole upon laser irradiation, was connected directly to chemical intermediates in long range ET.
the aromatic side chain of a modified C-terminal
amino acid. This electron acceptor could be observed
by transient absorption spectroscopy. The N-terminal Keywords: aromatic amino acids; electron transfer;
amino acid tyrosine acts as an electron donor, giving laser flash photolysis; peptides
Introduction
today.[9,10] Oligopeptidic model systems have provided
additional insight regarding the role of H-bonding.[11]
An alternative model for long range ET is the hop-
Long range electron transfer (ET) plays an important
role in biological systems. While ET through DNA is ping mechanism, which requires the occurence of oxi-
known to occur accidentally, causing damage to the dized chemical intermediates in the ET process.[12]
biopolymer, ET through proteins is a controlled pro- Electron hopping through peptides with contribution
cess which is of basic significance to such important of the peptidic bonds[13] has been proposed, but
biological processes as photosynthesis and respira- though there have been some experimental hints for
tion.[1,2] The mechanism of ET through proteins has amide participation in ET,[14] the occurrence of oxi-
been investigated intensively during the past years. dized peptide bonds as intermediates seems to be en-
The development of theoretical models for ET ergetically unfavourable.[15] This leads to the question
through proteins started from the Marcus theory for of amino acid side chain participation. The influence
ET.[3,4] In an early approach, the protein was consid- of peptide sequence, that is, which of the 20 natural
ered as a matrix of uniform potential.[5] In contrast to amino acids are present in ET pathways, still remains
this view, the pathway model[6] states the existence of to be investigated in detail. Investigations of ribonu-
ET pathways through different bond types. This cleotide reductase,[16] DNA photolyase[17,18] and photo-
theory has become increasingly detailed, taking into system II,[1] point to the importance of aromatic
account the effects of secondary structure and protein amino acids like tyrosine and tryptophan for ET in
dynamics.[7,8] In this process of refinement, chemical these proteins. Nevertheless, since only 4 aromatic
modifications of proteins with metal cofactors (azurin amino acids occur in nature, the possibilities to detect
and cytochromes) have proven to be a tool to obtain charged intermediates are limited. Moreover, small
experimental data concerning the ET properties of structural changes can lead to complete inhibition of
protein matrices with known and defined structures. ET in enzymes. Therefore, the construction of small
Detailed data, elucidating distance dependences and model systems provides additional possibilities for se-
the effect of secondary structure, is available quence modification. To contribute to the examina-
tion of the role of amino acid side chains in ET we
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time-resolved spectroscopic techniques. d) To keep
the influence of peptide backbone structure constant,
a rigid “innocent” spacer was chosen.
Electron Donor
Figure 1. Schematic design of peptide model 1 for the inves-
tigation of electron hopping through amino acid side chains
We chose tyrosine as an electron donor (Scheme 1).
This natural amino acid is assumed to participate in
ET through proteins by providing a relay for hopping
have designed a model 1, in which the N-terminal steps.[19] In aqueous solution tyrosine is known to de-
amino acid carries an electron donor D, and the C-ter- protonate upon oxidation (2!3), leading to a long-
minal amino acid side chain can be converted into an lived tyrosyl radical, a species with a relatively low
electron acceptor A. The peptide matrix connecting oxidation potential.[20] Therefore tyrosine can function
donor and acceptor contains an additional relay as a thermodynamic sink for the electron hole, sup-
amino acid with an oxidizable side chain R (Figure 1). pressing back ET and reducing intermolecular side re-
The relay amino acid can act as a charge sensor, actions. In addition, the transient absorption spectrum
giving an observable transient upon oxidation and of tyrosine radical shows a sharp band at approx.
thereby allow the simultaneous observation of elec- 410 nm,[21] leaving a broad spectral range for the de-
tron acceptor, electron donor and charged intermedi- tection of other intermediates.
ate.
Electron Acceptor
Results and Discussion
An injection system 4 (Scheme 2) that allows the site
For the design of an appropriate oligopeptidic model selective injection of a positive charge into biomole-
system several prerequisites had to be fulfilled. a) The cules has been developed earlier in our group.[22–24] It
electron donor should provide a sufficient driving- is based on photoinduced cleavage of a tert-butyl
force for ET and allow non-reversible trapping of the ketone functionality attached to a substituted tetrahy-
charge. b) The electron acceptor should be generated drofuran ring. The resulting radical 5 undergoes heter-
selectively at a specific side chain. Generation by olytic b-elimination and yields radical cation 6. The
laser flash photolysis allows time-dependent observa- suitability of this injector for the site-selective oxida-
tion of ET. c) The side chains of the relay amino acids tion of an aromatic amino acid has been shown previ-
should have appropriate redox potentials so that they ously in oligopeptides where the injector was coupled
can function as chemical intermediates in ET. The to an aromatic amino acid at the C-terminus via an
generated oxidized transients should be observable by ester linkage.[24]
In this work, we chose to shorten the linkage be-
tween amino acid and injection unit to ensure fast in-
jection and thereby exclude any intra- or intermolecu-
lar side reactions of radicals 5 and 6. Alcohol 4
should therefore be directly connected to the aromat-
ic ring of an amino acid side chain via an ether link-
age. This was achieved by conversion of primary alco-
hol 4 into the triflate 7, using triflic anhydride and
pyridine (82%), and subsequent coupling of 7 with
the protected 2-methoxy-substituted tyrosine 8, with
caesium carbonate as a mild base to give the desired
Scheme 1. Tyrosine as N-terminal electron donor.
injector-modified aromatic amino acid 9 (Scheme 3).
Scheme 2. Photoinduced radical cation generation.
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Scheme 3. Synthesis of the electron acceptor precursor.
The substitution pattern of the dialkoxyphenylalanine
The synthesis of non-natural amino acid
8
derivative was chosen because of its spectroscopic (Scheme 4) was possible starting from commercially
and electrochemical properties (see below).
available 4-hydroxy-2-methoxybenzaldehyde applying
an Erlenmeyer azlactone procedure.[25,26] Basic open-
Scheme 4. Synthesis of Boc-4-hydroxy-2-methoxyphenylalanine methyl ester 8.
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chromatography yielded free amino acid 13, which
could be converted to the N-Boc protected methyl
ester 8 using standard conditions. However, a migra-
tion of the Boc protection group to the hydroxy func-
tion of the aromatic ring lowered the yield in this re-
action. Due to the instability of compound 8, coupling
with 7 was carried out directly after the isolation.
Irradiation of dialkoxyphenylalanine 9 allows pho-
toinduced fast and direct generation of an oxidized
amino acid side chain, that is, generation of the C-ter-
minal electron acceptor (Scheme 5). The resulting
radical cation 14 shows a transient absorption spec-
trum different from the tyrosyl radical (see below).
Scheme 5. Oxidation of the C-terminal electron acceptor 9.
ing of lactone 10 and asymmetric hydrogenation of
acrylic acid 11 with a commercially available Rhodi-
um Deguphos[27] catalyst that has been used before in Relay Amino Acid
the enantioselective hydrogenation of acrylates[28]
gave the (S)-configured amino acid derivative 12 in It is known from ET through DNA that an electron
96% yield and >95% ee. Deprotection under strongly can hop between monomers of similar redox proper-
acidic conditions and purification via ion exchange ties in a biopolymer and can thereby be translocated
Scheme 6. Synthesis of 2,4-dimethoxyphenylalanine and 2,4,6-trimethoxyphenylalanine.
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over large distances.[23,29–32] To investigate whether
transfer through peptides can occur via a similar
mechanism, using amino acid side chains, an aromatic
amino acid with a redox potential similar to the elec-
tron acceptor was chosen as relay amino acid. For this
reason we synthesized a dimethoxy analogue 21 of di-
alkoxyphenylalanine 9. Furthermore we synthesized
the trimethoxy-substituted phenylalanine 22 as an ad-
ditional relay amino acid, whose radical cation has an
absorption spectrum different from 14 (see below).
The synthesis of enantiopure 2,4-dimethoxyphenylala-
nine 21 and 2,4,6-trimethoxyphenylalanine 22 could
be achieved similarly to the synthesis of compound 13
by Erlenmeyer azlactone synthesis and enantioselec-
tive hydrogenation, yielding the amino acid deriva-
tives 19 and 20 in > 95% ee, as depicted in Scheme 6.
Acidic deprotection followed by ion exchange chro-
matography gave the free amino acids 21 and 22 that
Figure 2. Transient absorption spectra of ac-2,4-dimethoxy-
phenylalanine radical cation (black) and ac-2,4,6-trimeth-
AHCTREoUNG xyphenylalanine radical cation (grey), generated by photo-
could be converted to the fully protected amino acids
23 and 24 needed for cyclovoltammetric measure-
ments and photosensitization experiments (see
below). Boc-protection (not depicted) could be car-
ried out in 80–90% yields analogous to the N-acetyla-
tion described here.
sensitization of 23 or, respectively, 24 with 1,4-dicyanonaph-
thalene as a sensitizer and biphenyl as a codonor.[33]
Since the ET process is induced by a laser flash
with a wavelength of 308 nm, the amino acids used should show defined structural properties. We there-
for the model system have to be transparent at fore chose prolines as spacers since proline oligomers
308 nm. We measured UV spectra of the fully protect- are known to form stable helices starting already
ed amino acids 23 and 24 at millimolar concentrations from 3 units,[34] and decided to set the distances to
in acetonitrile and could not detect any absorption at one repeat unit of the helix, that is, 3 proline mono-
wavelengths above 300 nm.
mers between acceptor or, respectively, donor and
To gain information about the redox properties of relay amino acid (Scheme 7). Intramolecular H-bond-
the dialkoxy- and trimethoxy-substituted amino acids, ing does not occur in our model. We synthesized pep-
we measured cyclovoltamogramms of compounds 23 tide 25 and 26 using a convergent strategy and stan-
and 24 in acetonitrile. Surprisingly, the redox poten- dard Boc methodology in solution phase with 2-(6-
tials of 23 and 24 were almost identical (0.95 V resp. chloro-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroni-
0.94 V vs. the ferrocene redox couple). Both amino um hexafluorophosphate (HCTU) as coupling re-
acids can therefore be regarded as possible hopping agent. The fragments are shown in Scheme 7.
relays analoguous to the heterocyclic bases in DNA.
The CD spectra of 25 and 26 (Figure 3) clearly
The transient absorption spectra of the radical cat- show that the peptides adopt a polyproline II (PPII)
ions of 23 and 24 could be obtained in a photosensiti- conformation[35] in the 3:1 (v/v) acetonitrile/water
zation experiment (Figure 2). They show clearly dis- mixture we used for the laser measurements. The
tinct maxima, with 2,4-dimethoxyphenylalanine radi- helix did not melt upon heating of the solution to
cal cation absorbing around 450 nm, and 2,4,6-trime- 808C, which indicates a high stability of the peptide
thoxyphenylalanine radical cation absorbing around conformation.[35]
550 nm (see also ref.[24]).
Since the lateral translation in a PPII helix is 3.2 Š
The maximum of the tyrosyl radical absorption is per residue, the overall distance between electron ac-
located at 402 nm.[21] This allows spectroscopic differ- ceptor and electron donor in our peptides can be esti-
entiation between the products of oxidation of radical mated to approx. 20 Š. Transfer of an electron across
acceptor 9, radical donor tyrosine and relay amino distances of more than 10 Š is considered to be long
acid 24.
range ET.[12] Therefore our oligopeptides 25 and 26
are suited to gain insight into the participation of spe-
cific amino acid side chains in long range ET. Irradia-
tion of peptides 25 and 26 with a laser flash of 308 nm
wavelength leads to the radical cations 27 and 28,
Peptides
We assembled electron acceptor, electron donor and containing our C-terminal electron acceptor
relay amino acid in a peptide matrix. The matrix (Scheme 8).
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Scheme 7. Assembly of a peptide model for the investigation of long range ET.
We recorded the transient absorption spectra of (2,4-dimethoxyphenylalanine radical cation) cannot
peptides 27 and 28 after a delay of 40 ns.[36] In the be distinguished from the spectrum of the oxidized
transient absorption spectrum of peptide 27 (Figure 4) electron acceptor, we could not decide whether the
one can clearly see the signals of two transients: oxi- relay amino acid became oxidized during ET through
dized electron acceptor (lmax =450 nm) and oxidized the peptide 27. For this reason we synthesized peptide
electron donor (lmax =410 nm). Because the transient 26 in which the relay amino acid gives a characteristic
absorption spectrum of the oxidized relay amino acid signal at 550 nm upon oxidation (Figure 2).
The transient absorption spectrum 40 ns after irra-
diation of peptide 26 shows the simultaneous occur-
ence of all three oxidized species. The oxidized elec-
tron acceptor, generated by our injection system, ab-
sorbs around 450 nm; the N-terminal electron donor
tyrosine has already been oxidized to the tyrosyl radi-
cal, indicated by an absorption peak around 410 nm,
and the oxidized relay amino acid can be observed by
its maximum at 550 nm (marked by an arrow in
Figure 5).
This experiment proves that long distance ET
through peptide 28 occurs in a hopping mechanism,
where the relay amino acid is used as a stepping stone
in the two-step mechanism.
Conclusions
A charge injection system 4 has been optimized for
fast, selective and observable injection of a positive
charge directly into a specific aromatic amino acid
side chain (9!14). In addition, spectroscopic sensors
Figure 3. CD-spectra of 25 (grey) and 26 (black) in acetoni-
trile/water 3:1 (v/v) at 258C. The bands around 206 nm (neg,
s) and 229 nm (pos, w) are characteristic of a PPII helix.
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Figure 4. Transient absorption spectrum recorded 40 ns after
the irradiation of a 5 mM degassed solution of peptide 25 in
acetonitrile/water 3:1 (v/v). The peak around 400 nm can be
assigned to the tyrosyl radical (N-terminal electron donor).
The maximum around 450 nm is characteristic of radical
cation 14 (C-terminal electron acceptor).
Scheme 8. Generation of the C-terminal electron acceptor
by laser flash photolysis.
for the occurrence of oxidized amino acid side chains
in ET reactions through peptides were developed.
Laser flash photolysis of peptide 26 demonstrated the
simultaneous existence of oxidized side chains of all
aromatic amino acids. This proves that the relay
amino acid in 28 acts as a stepping stone for long dis-
tance ET through the peptide. In future experiments,
this relay amino acid will be exchanged by naturally
occurring amino acids, and the question will be asked
which natural amino acids can act as stepping stones
for long distance ET through peptides.[37]
Experimental Section
Figure 5. Transient absorption spectrum recorded 40 ns after
the irradiation of a 5 mM degassed solution of peptide 26 in
acetonitrile/water 3:1. The peak around 400 nm can be as-
signed to the tyrosyl radical (N-terminal electron donor).
The maximum around 450 nm is characteristic of radical
cation 14 (C-terminal electron acceptor), an additional max-
imum at 550 nm (arrow) indicates the oxidation of the relay
amino acid 2,4,6-trimethoxyphenylalanine.
Detailed information, containing complete characterisation
of the compounds, is available as Supporting Information.
Trifluoromethanesulfonic Acid 2-(2,2-Dimethylprop-
ionyl)-3-(diphenoxyphosphoryloxy) tetrahydrofuran-
2-yl Methyl Ester (7)
To a solution of alcohol 4[24] (188 mg, 0.433 mmol) in CH2Cl2
(2 mL), pyridine (0.11 mL, 1.4 mmol) was added at 08C.
After 10 min of stirring, Tf2O (0.14 mL, 0.82 mmol) was
added dropwise and the reaction mixture was warmed to
room temperature. After the addition of saturated aqueous
NH4Cl solution (2 mL) and phase separation, the aqueous
phase was extracted with CH2Cl2 (3”2 mL). The combined
organic phases were dried (MgSO4) and concentrated under
vacuum to give triflate 7 as a white solid; yield: 251 mg
(82%).
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2-tert-Butoxycarbonylamino-3-(4-hydroxy-2-methoxy-
phenyl)-propionic Acid Methyl Ester (8)
2-(S)-Benzoylamino-3-(4-hydroxy-2-methoxyphenyl)-
propionic Acid Methyl Ester (12)
Step 1: To a suspension of amino acid 13 (30 mg, 0.14 mmol)
in MeOH (30 mL), SOCl2 (0.36 mL, 0.71 mmol) was added
dropwise. After 2 days of stirring at room temperature, the
mixture was concentrated under vacuum to give 2-amino-3-
(4-hydroxy-2-methoxy-phenyl)-propionic acid methyl ester
as a yellow solid in quantitative yield.
Acrylic acid methyl ester 10 (411 mg, 1.26 mmol) was sus-
pended in 15 mL of MeOH and after the addition of 3R,4R-
(+)-bis(diphenylphosphino)-1-benzylpyrrolidine(1,5-cyclooc-
tadiene)rhodium(I) (4.0 mg, ca. 0.5 mol%), the mixture was
stirred for 15 min at room temperature. The reaction sus-
pension was then closed in an autoclave and hydrogenated
(50 bar H2, 24 h). The formed brown solution was concen-
trated under reduced pressure and the residue purified by
column chromatography (EtOAc/hexane, 3:1) to afford 12
as a white solid; yield: 400 mg (96%, ee>95%).
Step 2: To a suspension of 2-amino-3-(4-hydroxy-2-me-
thoxyphenyl)-propionic
acid
methyl
ester
(70 mg,
0.31 mmol) in CH2Cl2 (3 mL) was added Et3N (60 mL,
0.47 mmol) at room temperature. After 10 min of stirring,
Boc2O (75 mg, 0.34 mmol) in CH2Cl2 (2 mL) was added
dropwise. 30 min later, saturated aqueous NH4Cl solution
(5 mL) was added and the mixture was extracted with
CH2Cl2 (3”3 mL). The combined organic phases were
dried, concentrated under vacuum and the residue was puri-
fied by column chromatography (EtOAc/hexane 3:1, quick-
ly, as the product slowly decomposes on SiO2!) to give 8 as
a white solid; yield: 40 mg (35%).
2-(S)-Amino-3-(4-hydroxy-2-methoxyphenyl)prop-
ionic Acid (13) (“4-Hydroxy-2-phenylalanine“)
Propionic acid methyl ester 12 (400 mg, 1.21 mmol) was dis-
solved in HClCAHTRE(UNG 32%)/H2O (10 mL, 3:2, v/v) and stirred for
5 h at 1108C. After cooling and concentration under re-
duced pressure, H2O was added (15 mL) and the mixture
was extracted with CH2Cl2 (3”5 mL). The aqueous phase
was concentrated under vacuum, the residue was subjected
to ion exchange chromatography and amino acid 13 was ob-
tained as a white solid; yield: 200 mg (78%).
2-tert-Butoxycarbonylamino-3-{4-[2-(2,2-dimethyl-
propionyl)-3-(diphenoxyphosphoryloxy)tetrahydro-
furan-2-ylmethoxy]-2-methoxyphenyl}propionic Acid
Methyl Ester (9)
4-[1-(2,4-Dimethoxyphenyl)-meth-(Z)-ylidene]-2-
To a solution of 8 (70 mg, 0.22 mmol) in DMF (3 mL) was phenyl-4H-oxazol-5-one (15)
added Cs2CO3 (101 mg, 0.236 mmol) at room temperature.
2,4-Dimethoxybenzaldehyde (10 g, 60 mmol), hippuric acid
After 10 min of stirring, a solution of triflate 7 (218 mg,
0.215 mmol) in DMF (2 mL) was added dropwise and stir-
ring was continued for 3 h. Then, saturated aqueous NH4Cl
solution (2 mL) was added and the mixture was extracted
with EtOAc (3”3 mL). The combined organic phases were
washed with water (3”3 mL), dried (MgSO4) and concen-
trated under vacuum. Column chromatography (hexane/
EtOAc, 2:1) gave 9 as a white solid; yield: 83 mg (52%).
(11 g, 63 mmol) and NaOAc (4.9 g, 60 mmol) were stirred in
acetic anhydride (30 mL) at 858C for 3 h. The mixture was
cooled to 08C and cold EtOH was added. The formed
orange precipitate was filtered and washed with cold EtOH
and hot H2O to afford oxazolone 15 as a yellow solid; yield:
13.3 g (72%).
2-Phenyl-4-[1-(2,4,6-trimethoxyphenyl)-meth-(Z)-
ylidene]-4H-oxazol-5-one (16)
4-(2’-Methoxy-4’-acetoxybenzylidene)-2-phenyloxazol-
5(4H)-one (10)
2,4,6-Trimethoxybenzaldehyde (15 g, 76 mmol), hippuric
acid (14.4, 80 mmol) and sodium acetate (6.3 g, 76 mmol)
were stirred in acetic anhydride (40 mL) at 858C for 2 h.
The mixture was cooled to 08C and cold EtOH was added.
The mixture was left in the fridge for 1 h. The resulting
yellow precipitate was filtered and washed with cold EtOH
and hot H2O to afford oxazolone 16 as a yellow solid; yield:
12.9 g (50%).
4-Hydroxy-2-methoxybenzaldehyd (4.04 g, 26.6 mmol), hip-
puric acid (4.80 g, 26.8 mmol), sodium acetate (2.24 g,
27.3 mmol) and acetic anhydride (6.0 mL, 6.5 g, 64 mmol)
were placed in a 100-mL round-bottom flask, liquefied with
a heat gun and then heated in an oil bath at 1008C for 2 h.
The resulting solid mixture was cooled to room temperature
before H2O (30 mL) was added. The resulting suspension
was filtrated, washed with aqueous Na2CO3 and dried under
vacuum. The desired azlactone 10 was then obtained after
recrystallization from acetone/H2O (2:1) as a yellow solid;
yield: 6.89 g (77%).
(Z)-2-Benzoylamino-3-(2,4-dimethoxyphenyl)-acrylic
acid Methyl Ester (17)
Oxazolone 15 (13.3 g, 43 mmol) and Na2CO3 (13.7 g,
129 mmol) were suspended in MeOH (150 mL) and then re-
fluxed for 2 h. The mixture was filtered hot and the remain-
ing solution was concentrated under reduced pressure to
10% of the original volume. Crystallisation at 48C over
night and subsequent washing with methanol/water (1:1) af-
forded 17 as white crystalline needles; yield: 11.1 g (76%).
2-Benzoylamino-3-(4-hydroxy-2-methoxyphenyl)-
acrylic Acid Methyl Ester (11)
A 100-mL round-bottom flask was charged with 4-acetoxy-
2-methoxyazlactone 10 (6.71 g, 19.9 mmol), a 1:1 mixture of
CH2Cl2 and MeOH (80 mL) and Na2CO3 (1.0 g, 9.4 mmol).
The mixture was stirred for 16 h at room temperature, fil-
tered over celite and the resulting solution was concentrated
under reduced until 11 precipitated as a white solid. yield:
5.53 g (85%).
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change chromatography and amino acid 22 was obtained as
a white solid; yield: 479 mg (70%).
(Z)-2-Benzoylamino-3-(2,4,6-trimethoxyphenyl)-
acrylic Acid Methyl Ester (18)
Oxazolone 11 (14 g, 41 mmol) and sodium carbonate (11.4 g,
107 mmol) were suspended in MeOH (140 mL) and refluxed
for 1 h. The mixture was filtered hot and the remaining solu-
tion was concentrated under reduced pressure to 10% of the
original volume. Recrystallization of the resulting solid from
hot MeOH yielded 18 as large white crystals; yield: 12.9 g
(85%).
2-Acetylamino-(S)-3-(2,4-dimethoxyphenyl)-propionic
Acid Methyl Ester (23)
Amino acid 21 (15 mg, 67 mmol) was dissolved in CH2Cl2
(3 mL). Acetic anhydride (163 mL, 670 mmol) and Hünig-
base (DIPEA) (245 mL, 670 mmol) were added. After stir-
ring for 60 min, aqueous HCl (2M, 6 mL) was added. The
organic phase was dried (MgSO4) and concentrated under
reduced pressure. The residue was dissolved in MeOH
(3 mL) and Cs2CO3 (11 mg, 24 mmol) in water was added.
Coevaporation with toluene to dryness was followed by ad-
dition of DMF (3 mL) and MeI (21 mL, 335 mmol). After
stirring for 30 min, water was added to the reaction mixture.
Extraction with EtOAc/2M aqueous HCl, drying of the or-
ganic phase (MgSO4) and concentration under reduced pres-
sure was followed by column chromatography (4% MeOH
in CH2Cl2) of the crude product to give 23 as a white foam;
yield: 11 mg (60%).
2-Benzoylamino-3-(2,4-dimethoxyphenyl)-propionic
acid Methyl Ester (19)
Acrylic acid methyl ester 17 (1.0 g, 2.9 mmol) was suspended
in MeOH (15 mL). After the addition of 3R,4R-(+)-bis(di-
phenylphosphino)-1-benzylpyrrolidine(1,5-cyclooctadiene)-
rhodium(I) (11 mg, ca. 0.45 mol%), the mixture was stirred
for 15 min at room temperature. The reaction suspension
was then closed in an autoclave and hydrogenated (50 bar
H2, 15 h). The formed brown solution was concentrated
under reduced pressure and the residue purified by column
chromatography (EtOAc/hexane, 2:1) to afford 19 as a
white solid; yield: 970 mg (96%, ee> 95%).
2-Acetylamino-(S)-3-(2,4,6-trimethoxyphenyl)-
propACHTERiUNG onic Acid Methyl Ester (24)
Amino acid 22 (50 mg, 0.19 mmol) was dissolved in CH2Cl2
(5 mL). Acetic anhydride (0.19 mL, 1.9 mmol) and Hünig
base (DIPEA) (0.33 mL, 1.9 mmol) were added. After stir-
ring for 60 min, aqueous HCl (2M, 10 mL) was added. The
organic phase was dried (MgSO4) and concentrated under
reduced pressure. The residue was dissolved in MeOH
(5 mL) and Cs2CO3 (31 mg, 80 mmol) in water was added.
Coevaporation with toluene to dryness was followed by ad-
dition of DMF (5 mL) and MeI (60 mL, 0.95 mmol). After
stirring for 30 min, water was added to the reaction mixture.
Extraction with EtOAc/2M aqueous HCl, drying of the or-
ganic phase (MgSO4) and concentration under reduced pres-
sure was followed by column chromatography (4% MeOH
in CH2Cl2) of the crude product to give 24 as a white foam;
yield: 32 mg (54%).
2-Benzoylamino-3-(2,4,6-trimethoxyphenyl)-propionic
Acid Methyl Ester (20)
Acrylic acid methyl ester 18 (1.0 g, 2.7 mmol) was suspended
in 15 mL of MeOH and after the addition of 3R,4R-(+)-
bis(diphenylphosphino)-1-benzylpyrrolidine(1,5-cyclooctadi-
ene)rhodium(I) (11 mg, ca. 0.5 mol%), the mixture was
stirred for 15 min at room temperature. The reaction sus-
pension was then closed in an autoclave and hydrogenated
(50 bar H2, 15 h). The formed brown solution was concen-
trated under reduced pressure and the residue purified by
column chromatography (EtOAc/hexane 2:1) to afford 20 as
a white solid; yield: 955 mg (95%, ee> 95%).
2-Amino-3-(2,4-dimethoxy-phenyl)-propionic Acid
(21) (“2,4-Dimethoxyphenylalanine“)
Ac-Tyr-(Pro)3-2,4-Dimethoxyphenylalanine-(Pro)3-3-
{4-[2-(2,2-dimethylpropionyl)-3-(diphenoxyphosphor-
yloxy)tetrahydrofuran-2-ylmethoxy]-2-methoxy-
phenyl}propionic Acid Methyl Ester (25)
Propionic acid methyl ester 19 was dissolved in H2O
(15 mL) and acetic anhydride (15 mL). HCl (32%, 30 mL)
was added carefully and the solution was stirred for 2 h at
1108C. After cooling and concentration under reduced pres-
sure, water (15 mL) was added and the mixture was extract-
ed with CH2Cl2 (3”5 mL). The aqueous phase was concen-
trated under vacuum, the residue was subjected to ion ex-
change chromatography and amino acid 21 was obtained as
a white solid; yield: 470 mg (73%).
The peptide was assembled from the fragments Ac-Tyr-
AHCTREU(GN OBn)-(Pro)3-OH and HCl·H2N-2,4-dimethoxyphenylala-
nine-(Pro)3-O-t-Bu, using standard procedures for peptide
coupling in solution (2 equiv. of 2-(6-chloro-1-H-benzotri-
azol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
(HCTU), 4 equiv. ethyldiisopropylamine (DIPEA), DMF/
CH2Cl2, room temperature, 3 h) and t-Bu-ester deprotection
[trifluoroacetic acid (TFA)/CH2Cl2, 2:1, room temperature,
30 min]. Coupling of the resulting free acid to deprotected 9
(Boc-deprotection was carried out with 4M HCl in dioxane,
room temperature, 20 min) was achieved under the same
coupling conditions mentioned above. After reductive cleav-
age of the benzyl protection group (10% Pd/C, H2, room
temperature, 3 h), the resulting peptide 25 was purified by
flash chromatography (4% MeOH in CH2Cl2 to 15%
MeOH in CH2Cl2).
2-Amino-3-(2,4,6-trimethoxyphenyl)-propionic Acid
(22) (“2,4,6-Trimethoxyphenylalanine“)
Propionic acid methyl ester 20 was dissolved in H2O
(15 mL) and acetic anhydride (15 mL). HCl (32%, 30 mL)
was added carefully and the solution was stirred for 2 h at
1108C. After cooling and concentration under reduced pres-
sure, water (15 mL) was added and the mixture was extract-
ed with CH2Cl2 (3”5 mL). The aqueous phase was concen-
trated under vacuum, the residue was subjected to ion ex-
Adv. Synth. Catal. 2008, 350, 1053 – 1062
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
1061

Meike Cordes et al.
FULL PAPERS
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Ac-Tyr-(Pro)3-2,4,6-Trimethoxyphenylalanine-(Pro)3-
3-{4-[2-(2,2-dimethylpropionyl)-3-(diphenoxy-
phosphoryloxy)tetrahydrofuran-2-ylmethoxy]-2-
methoxyphenyl}propionic Acid Methyl Ester (26)
The peptide was assembled using the fragments Ac-Tyr-
ACHTREUNG(OBn)-(Pro)3-OH and HCl·H2N-2,4,6-trimethoxyphenylala-
nine-(Pro)3-O-t-Bu, using standard procedures for peptide
coupling in solution [2 equiv. of 2-(6-chloro-1H-benzotri-
ACHTREUNGazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
(HCTU), 4 equiv. ethyldiisopropylamine (DIPEA), DMF/
CH2Cl2, room temperature, 3 h], and t-Bu-ester deprotection
[trifluoroacetic acid (TFA)/CH2Cl2 2:1, r.t., 30 min]. Subse-
quent coupling of the resulting free acid to deprotected 9
(for Boc-deprotection 4m HCl in dioxane was added at
room temperature and stirred for 20 min) was achieved
under the same conditions described above. After reductive
cleavage of the benzyl-protection group (10% Pd/C, H2,
MeOH, room temoperature, 3 h) the resulting peptide 26
was purified by flash chromatography (4% MeOH in
CH2Cl2 to 15% MeOH in CH2Cl2).
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Acknowledgements
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and biphenyl (BP) is the excitation of DCN. Subse-
quent fast ET from BP generates the BP radical cation,
which is able to oxidize electron-ich aromatic com-
pounds. DCN is regenerated by reaction with oxygen.
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after irradiation, because the half-life time for the gen-
eration of the electron acceptor (9!14) isabout 20 ns.
On the other hand, measurements recorded 80 ns after
the flash have shown that intermolecular ET already
plays a role. Thus, the time window for the detection of
intramolecular ET through peptides 27 and 28 is very
narrow.
This work was supported by the Swiss National Science
Foundation. C. J. thanks the Leopoldina for a grant. We
thank Prof. Wirz/Basel for important discussions.
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ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 2008, 350, 1053 – 1062