Oxidative phenol coupling - Tyrosine dimers and libraries containing tyrosyl peptide dimers

Article abstract of DOI:10.1016/S0040-4020(00)00942-X

The principles of phenol oxidation are outlined and summarized. Various procedures were used to dimerize tyrosine derivatives, especially linear tripeptides and cyclohexapeptides, via cross-linking of their phenolic side-chains. The coupling was performed by potassium hexacyanoferrate(III), phenyliodine(III)-bis(trifluoroacetate), vanadium(V)-oxyfluoride or horseradish peroxidase. The individual dityrosine and isodityrosine derivatives obtained in preparative scale, as well as the peptide-dimer libraries generated, were characterized by HPLC and electrospray ionization mass spectrometry. Further analyses were carried out by NMR spectroscopy, fluorescence spectroscopy and gas chromatography.

Full text of DOI:10.1016/S0040-4020(00)00942-X

TETRAHEDRON
Pergamon
Tetrahedron 57 (2001) 353±364
Oxidative phenol couplingÐtyrosine dimers and libraries
containing tyrosyl peptide dimers
²
Holger Eickhoff, GuÈnther Jung and Anton Rieker
*
È È È È
Universitat Tubingen, Institut fur Organische Chemie, Auf der Morgenstelle 18, 72076 Tubingen, Germany
Received 19 April 2000; revised 30 May 2000; accepted 10 July 2000
AbstractÐThe principles of phenol oxidation are outlined and summarized. Various procedures were used to dimerize tyrosine derivatives,
especially linear tripeptides and cyclohexapeptides, via cross-linking of their phenolic side-chains. The coupling was performed by potas-
sium hexacyanoferrate(III), phenyliodine(III)-bis(tri¯uoroacetate), vanadium(V)-oxy¯uoride or horseradish peroxidase. The individual
dityrosine and isodityrosine derivatives obtained in preparative scale, as well as the peptide-dimer libraries generated, were characterized
by HPLC and electrospray ionization mass spectrometry. Further analyses were carried out by NMR spectroscopy, ¯uorescence spectroscopy
and gas chromatography. q 2000 Elsevier Science Ltd. All rights reserved.
1. Introduction
1.1. General aspects of the oxidative functionalization of
phenols
A wealth of investigations has revealed that phenols are
easily oxidized, both in vivo and in vitro, to give quinolide
systems, which may be rearomatized or transformed into
many other useful classes of compounds.
The primary oxidation process may be described by a
`scheme of squares', in which the individual species are
correlated by electron transfers (`E' processes) and proton
transfers (`C', chemical processes) (Scheme 1).1,2
Neutral phenol 1, cation radical 2, and dication 3 are linked
by two successive one-electron transfers. All three species
1±3 are in equilibrium with their deprotonated forms, anion
Scheme 1. Square scheme of phenol oxidation; only one resonance struc-
ture of the species 1±6 is shown.
4, neutral phenoxyl radical 5, and phenoxenium cation 6,
respectively, which, for their part, are again interrelated by
two consecutive one-electron transfers. The real system is
more complicated, since every species may react with each
of the others via bimolecular electron or proton transfers to
give two other species of the system. Moreover, other side
reactions, such as dimerization of the radicals or electro-
philic attack of the cations on nucleophiles, may take place.
prohibited by means of steric hindrance and that strong
nucleophiles are absent (for the case of sterically less
hindered phenols in water or protic solvents see Refs. 10±
12):
(i) In the absence of a base, phenol 1 itself is oxidized at E1
(ca. 1000±1400 mV ) to give the cation-radical 2 in a one-
³
electron transfer. This species deprotonates to the neutral
phenoxyl 5, which is further oxidized at the applied poten-
tial to the desired cation 6, because E4,E1 (ECE reaction).
Alternatively, 2 and 5 may react in a homogeneous redox
reaction to produce 1 and again 6.
Electroanalytical investigations3±9 disclosed two important
results, provided that dimerization of the radicals 2 and 5 is
Keywords: amino acids and derivatives; biaryls; oxidation; peptide ana-
logues/mimetics.
(ii) In the presence of a base, the phenolate 4 formed from 1
* Corresponding authors. Fax: 149-7071-29-5560;
e-mail: anton.rieker@uni-tuebingen.de
guenther.jung@uni-tuebingen.de
²
³
In the following, all potentials refer to Ag/0.01 M Ag1.
0040±4020/01/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00942-X

354
H. Eickhoff et al. / Tetrahedron 57 (2001) 353±364
as iodobenzene diacetate (PIDA)33±37 or iodobenzene
bis(tri¯uoroacetate) (PIFA)38±41 are often used. In the latter
cases, a complex of the phenol 1 with the periodo
compounds is formed,33,34,38,41 the heterolysis of which
produces an incipient phenoxenium ion 6 (formal two-elec-
tron oxidation). Similar reaction pathways are postulated for
42
VOX3 as oxidant although for vanadium the oxidation
mechanism is rather complex. For Ce41 mediated phenolic
oxidations, the mechanism is equivocal43 (for a more
detailed account on phenol oxidants see Ref. 44).
The formation of the ®nal reaction products will be
controlled by the reactivity of the phenoxyls 5 and phenox-
enium ions 6 (Scheme 2). Both species are stabilized by
resonance (canonical structures a, b, c). In the case of the
phenoxyls 5, this can be proven by ESR measure-
ments.17,18,45 If the substituents R and/or R1 show p-conju-
gation with the aromatic ring, further resonance structures
with a free spin in these substituents also must be con-
sidered.45
Phenoxyls 5 can combine with other radicals Xz, either
added or produced from H±X via dehydrogenation by an
excess of 5 (Eq. (1)):
H±X 1 5 ! H±X1z 1 4 ! Xz 1 1
ꢀ1†
Xz may be halogen atoms,46 oxygen radicals (e.g.
Scheme 2. Reaction paths of phenoxyls 5 and phenoxenium ions 6.
zOOtBu,21,23 zO±Oz,46. zO±NvCR247), nitrogen radicals
z
(e.g. NO,48 zNO2,49 zNHR50), or carbon radicals (e.g.
is anodically oxidized to give the phenoxyl radical 5 at
E3ˆ2600±1300 mV selectively (CE process). In special
cases, the presence of 5 can be proved by electron spin
resonance (ESR)13 or multiple internal re¯ection Fourier
transform infrared spectroscopy (MIRFTIRS).14 At a higher
potential, E4ˆ1700±800 mV, radical 5 is further oxidized
in a one-electron transfer to the phenoxenium ion 6 (CEE
mechanism), although 6 is not generally stable towards
base.
zCH2C6H551). Generally, the cyclohexadienones 8 and/or 9
are formed, while the ethers 7 are only observed with carbon
radicals.51
Products 8 and 9 can also be formed from phenoxenium ions
6(a,b,c) by addition of nucleophiles and deprotonation.
Examples are the addition of H±X ˆ HOH,3,40,52±54
ROH,31,33,35,37,52±54 ROOH,52,53 RCOOH,42,52±55 carbo-
hydrates2, RNH2,33,52,53,56 amides,52,56 imines,56 oximes,56
amino acids (C- and N-terminal or via the side chain; useful
for peptide syntheses).2,57±60 Certain aromatic61 and ole®nic39
nucleophiles react in a similar way.
Thus, phenoxyl 5 or phenoxenium ion 6 can be selectively
generated using the proper potentials and proton concen-
tration.
Scheme 1 is also valid for chemical oxidants. However,
oxidant and substrate now have to come into contact in a
bimolecular step in¯uencing the kinetics. Moreover, the
potential of the oxidant is ®xed and cannot be changed at
will as can that of an electrode; in some cases, a limited
in¯uence is possible via pH adjustment.15,16 One also has to
consider reactions of the oxidant with any of the species
1±6, occurring as non-redox processes.
A certain selectivity in the formation of ortho- and para-
quinolide structures 8 and 9 is ensured by the steric
hindrance of R, R1 and X, and by the electronic nature of
R1. Thus, if R1 is electron-donating, 9 is favored; if R1 is
electron-withdrawing, 8 predominates.51,62
If phenol and nucleophile are combined in the same mole-
cule via a spacer, generally ring closure to spirocyclohex-
adienones occurs upon oxidation.30,33,36,52,63,64 This can be
used for the synthesis of natural products such as alkaloids41
or sponge metabolites.29
Potassium hexacyanoferrate(III), K3Fe(CN)6, in alkaline
solution15,17,18 is generally considered a genuine one-
electron oxidant (oxidizing 4 to 5). Other examples are
metal complexes used in catalyzed oxygenations19±23with
O2 or alkylhydroperoxides, as well as PbO2, Ag2O, NiO2,
or oxygen radicals (HOz, t-BuOz, ArOz).18 Apparently, also
enzymes such as tyrosinase24 and horseradish peroxidase
(HRP)/H2O225±27 belong to this class of oxidants.
Moreover, cyclohexadienones 8 and 9 are synthetic equiva-
lents used for the preparation of phenols, quinones, cyclo-
hexadiene diols, quinol epoxides or cyclopentadienones, to
mention a few applications.65
1.2. Oxidative phenol coupling
As two-electron oxidants, Tl3128±31 or trivalent iodine
compounds (for a recent literature survey see ref.32) such
If Xz in Scheme 2 is another phenoxyl 5, the resulting

H. Eickhoff et al. / Tetrahedron 57 (2001) 353±364
355
Scheme 3. Coupling pattern of two phenoxyl radicals.
cyclohexadienones 8 and 9 are phenoxyl dimers. The
principles of this dimerization are shown in Scheme 3.
phenoxenium ion 6 and phenol 1 with deprotonation
(according to the lower part of Scheme 2). This may
occur if the phenoxenium ion is formed incipiently (e.g.
attack of the phenol on the complex formed from phenol/
oxidant with simultaneous heterolysis at the complex
center; formal two-electron oxidants, vide infra). On the
other hand, a free phenoxenium 6 and phenol 1 would
comproportionate into cation radical 2 and phenoxyl 5,
which would react according to Scheme 3. It is questionable
whether selectivity with respect to CC or CO coupling can
be obtained by selecting formal one- or two-electron
oxidants.15,26,43
Two phenoxyls 5a and 5b combine according to their free
spin density distribution via O, Co and Cp, thus leading to the
coupling pattern shown: O±O (10), C±O (11a,b) and C±C
(12a,b,c). If the phenoxyls 5 are identical (RˆR1), the
process is denoted as `homo-coupling'; for non-identical
phenoxyls (R unequal R1) the term `hetero-coupling' is
used. It should be mentioned that it is not necessary
to prepare 5a and 5b separately. Due to electron and
proton exchange between phenols and phenoxyls according
to Scheme 3, we can start with phenol 1a/phenoxyl 5b,
with phenol 1b/phenoxyl 5a, or even co-oxidize
phenols 1a and 1b. With the exception of the aromatic
Additional diversity in coupling options appears if sub-
stituents R, R1 are p-conjugated (coupling via an atom of
the substituent72a,75,76) or for R, R1ˆH (dienone!phenol
rearrangement and oligomerization43,76a,77).
peroxides 10, all coupled species 11 and 12 have been
1,8,15,26,41,44,46,51,52,62,66±73
found, the type of coupling being
dependent on the steric and electronic effects of the sub-
51,62,70a,74
stituents R and R1.
derived:
Some rough rules can be
A very important case is the oxidation of biphenols, leading
to intramolecular coupling of the resulting biradical. The
ring closure again follows the principles of Scheme 3, the
coupling pattern (C±C, C±O) being determined mainly by
the molecular frame. A series of natural products and
model compounds can be synthesized,32,34,78±80 and also
some natural products might in vivo be formed in this
way,81a e.g. isoquinoline alkaloids,44 ellagitannins,81bor
lignin.25,76,82
(i) homo-coupling: Rortho large, Rpara small!12b
®rst Rortho small, second Rortho large, Rpara large!12a
other cases!11a,11b,12c
(ii) hetero-coupling: Rortho large, Rpara any size, both elec-
tron-donating; R1ortho small, Rp1ara any size !11b
Rortho large, Rpara any size, electron-withdrawing; Ro1rtho
small, R1para any size and electronic effect !11a.
1.3. Oxidation of tyrosine derivatives
It should be emphasized that the coupling products 11 and
12 could, in principle, also result from the combination of
Tyrosine, the only phenol among the essential amino acids,

356
H. Eickhoff et al. / Tetrahedron 57 (2001) 353±364
Scheme 4. Oxidative functionalization pathways of tyrosine derivatives.
and its derivatives 13 should undergo all the oxidative func-
tionalization reactions discussed in the previous sections.
One-electron oxidation to the phenoxyl 14 (Scheme 4),
and dimerization of this species would lead to 16 or 19
(only Co±Co and Co±O coupling is shown for simplicity),
whereas reaction with other radicals Xz would give the
cyclohexadienones 17 and 20. These species could also
arise from the two-electron oxidation product 15 and HX,
with intramolecular COOH addition (for R2ˆH) to give
spiroquinol ethers 18 as a special case. A heterolytic
cleavage of 18 by nucleophiles NuH to the quinol 21
might also be envisaged.
Indeed, many of these reactions have been reported and
were used as crucial steps to synthesize a series of natural
and non-natural products. The electron/proton transfer
equilibria 13O14O15 (see also Scheme 1) were character-
ized by cyclic voltammetry (CV).67 Tyrosinoxyl radicals 14
were identi®ed by ESR (organic solvents,67a enzymes83),
and their conformational behavior was determined. They
react with radicals such as t-BuOOz to give 17 (Xˆ
t-BuOO).84 Reactions via phenoxenium ions 15 to give 17,
spirolactones 18 or the corresponding spirolactams are
known (for earlier work of Wipf, Sharma, McKillop,
Hara, Witkop see Ref. 67).67,84,85a,b Spirolactones 18 react
with nucleophiles like C-terminal-protected amino acids to
give (di)peptides 21 (Nuˆamino acid residue) containing
the tyrosine unit with a masked side chain,67 which can be
reduced to the phenolic structure.67
Scheme 5. Dityrosine and isodityrosine derivatives.
coupling of 14 with other phenoxyls according to Scheme
2).67a,85c
Namely, dityrosine 22a and isodityrosine 23a are structural
elements in various peptidic natural products that may
exhibit high biological activity such as the bastadines,86
the enzyme inhibitor K-13 (24),87 bouvardin (25),88 vanco-
mycin,89 ristocetin,90 or the antitumor agent RA-VII,91
among others.92 Dityrosine occurs as well in cell wall struc-
tural proteins, where the cross-links are responsible for
stability due to restricted conformational ¯exibility (e.g. in
yeasts93). It is also produced by irradiation of proteins and
can be regarded as a marker of oxidative stress.94
The dimerization of phenoxyls 14 is of high practical impor-
tance because the dimers 16 and 19 may rearrange or frag-
ment to the dityrosine (22) or isodityrosine (23) systems
(Scheme 5), especially if RˆH or halogen (for hetero-
Most of the above systems are cyclopeptides with 22a or
23a as ring constituents. Many different approaches through
multi-step syntheses have been explored to achieve ring

H. Eickhoff et al. / Tetrahedron 57 (2001) 353±364
357
Figure 1. Comparison of oxidizing agents in the formation of tyrosine dimers 22 and 23. Substrate oxidized: 30±32 mmol. Solvents: K3FeCN6/TEA, water/
MeOHˆ1:1; K3Fe(CN)6/Na2CO3, water, and CH2Cl2/water/Bu4NBr for Z-Tyr-OMe; CAN, water/MeOH, and MeOH for Z-Tyr-OMe; PIFA, CH2Cl2; VOF3,
CH2Cl2; VOF3/TFA, CH2Cl2. p: Amount not determined.
closure by coupling of two tyrosines in a peptide chain via
non-oxidative methods89±91 (for a non-oxidative synthesis
of 23a itself see Ref. 95). Oxidative coupling (anode,96
produce tyrosine coupling products 22 and/or 23 from the
corresponding tyrosines 13 on a preparative scale. Some of
the results obtained on an analytical scale are presented in
Fig. 1.
Tl31 79,97 enzymes98) of ring mono- or di-halogenated
,
tyrosines was also successful and affords linear or cyclic
systems containing 22a or 23a, whereas the direct chemical
oxidative coupling of unsubstituted tyrosine has not been
the subject of many publications99 and was mainly achieved
enzymatically.99a±d
Potassium hexacyanoferrate(III) in alkaline aqueous solu-
tions led to rapid oligomerization and polymerization,
whereas 13c in a two-phase system, CH2Cl2/Na2CO3/
(Bu)4NBr, gave a good yield of 23c. Application of triethyl-
amine instead of inorganic bases in aqueous methanol
produced mainly dityrosine, isodityrosine and trityrosine,
and only small amounts of polymers from 13d.
It was our aim to develop on a preparative scale oxidative
coupling procedures of peptides and cyclopeptides contain-
ing one tyrosine with an unsubstituted side chain. This
would lead to cross linked peptides in one step, species
which are of importance because the biphenyl or biphenyl
ether cross-link will not be cleaved by proteases. Further-
more, receptor selectivity is usually better achieved by
conformationally restricted cyclic ligands.
For the oxidative coupling of tyrosine derivatives (e.g.
Z-Tyr-OMe 13c, Fmoc-Tyr-OMe) which are poorly soluble
or insoluble in aqueous solutions, PIFA in dichloromethane
proved to be a suitable oxidative agent. The coupling
proceeded under mild conditions and made accessible
compounds 22c and 23c in preparative scale after puri®-
cation by column chromatography on silica. The ratio of
22c and 23c could be in¯uenced by variation of the amount
of PIFA. The highest yield of the dityrosine was achieved
with 2 mol equiv. of PIFA (36% 22c, ,5% 23c). A lower
yield of the dityrosine but the best yield of the isodityrosine
was produced with 0.5 mol equiv. of PIFA (20% 22c, 25%
23c). In contrast to PIFA, VOF3 (in acidic CH2Cl2) gave
only dityrosine derivative 22c. The oxidation with CAN in
methanol/water or methanol was unsatisfactory in all cases.
2. Results and discussion
According to the principles outlined in the previous chap-
ters, oxidative coupling of tyrosines 13 should best proceed
via the phenoxyl state 14. Therefore, at ®rst glance, anodic
oxidation at constant potential in the presence of a base to
ensure one-electron oxidation to 14 would appear as the
method of choice. However, oligomerization or polymeriza-
tion is expected to a great extent, if the phenol unit is unhin-
dered in the ortho-positions, a well-known phenomenon in
phenolic oxidations.43,76a,77 Indeed, the electrode is rapidly
coated, and the yield of dimers is very low.84 Therefore, we
®rst systematically investigated a variety of other oxidants,
already characterized above [HRP/O2, K3Fe(CN)6,
(NH4)2Ce(NO3)6 (CAN), PIFA, VOF3], for their ability to
Horseradish peroxidase (HRP) has been applied for cata-
lytic oxidations of tyrosine derivatives with some
success,99a±d as already mentioned. In most of our own
experiments using a variety of substrates and HRP/H2O2
(the quality of the peroxidase is a crucial point), tyrosines
were coupled selectively to dityrosine, but with 13d only

358
H. Eickhoff et al. / Tetrahedron 57 (2001) 353±364
Table 1. HRP catalyzed dimerization of tyrosine derivatives 13 (378C, 1 h)
The reactions were carried out at 378C, according to Amado
et al.99d Tests at room temperature showed lower yields,
particularly with poorly soluble peptide mixtures. The reac-
tion time was optimized by ¯uorescence measurements;99a
in contrast to isodityrosine, dityrosine emits intensive ¯uor-
escence. Additionally, the samples were analyzed by HPLC
and electrospray mass spectrometry. However, a differentia-
tion of the isobaric dityrosine and isodityrosine derivatives
is not possible by routine electrospray ionization mass spec-
trometry. Therefore, gas chromatography (GC) was used to
assure the exclusive presence of dityrosyl compounds. The
dimeric tyrosine derivatives and peptides (vide supra) were
hydrolyzed and the amino acids transformed into N-tri¯uoro-
acetyl methyl esters whose GC analysis permitted the
comparison with standards of tyrosine dimers.
Solvent
Substrate
Yield of dityrosine (%)
0.2 M borate buffer
0.2 M borate buffer
0.2 M borate buffer
0.2 M borate buffer/20% MeOH Ac-Tyr-NH2
0.2 M borate buffer/33% MeOH Ac-Tyr-NH2
0.2 M borate buffer/50% MeOH Ac-Tyr-NH2
0.2 M borate buffer/66% MeOH Ac-Tyr-NH2
0.2 M borate buffer/50%
dioxane
0.2 M borate buffer/50%
tBuOHa
PBS buffer
Ac-Tyr-NH2
Z-Tyr-OH
Ac-Tyr-OH
.90
60
53
84
69
59
30
38
Ac-Tyr-NH2
Ac-Tyr-NH2
Ac-Tyr-NH2
61
47
a tBuOH:H2Oˆ4:1.
60% Z-Dityr-OH 22d and also 19% Z-Isodityr-OH 23d
were formed. The best yields of dimeric products were
achieved in 0.2 M borate buffer (pH 9.4),99d whereas in
water and phosphate buffers (pH 7.4) only small amounts
of the desired products were formed. In cases of low solu-
bility of the substrates in the aqueous reaction medium, up
to 50% of methanol or dioxane had to be used, which led on
the one hand to a decrease in the yield of dimers (Table 1),
but on the other hand was essential to make the oxidative
coupling possible. In most cases, hydrogen peroxide was
used in only 1.1-fold excess to prevent the production of
oxidized compounds other than tyrosine dimers. Higher
amounts of hydrogen peroxide promoted oligomerization
and formation of various by-products.
After the HRP catalyzed oxidative coupling of tyrosines 13
was optimized, it was also used to couple small tyrosine
peptides. Thus, cyclohexapeptide c(VAYAVa) (aˆd-alanine)
could be oxidized to the dityrosine-bridged product 26. The
1H NMR spectrum of 26 exhibited an ABX pattern of
signals in the aromatic region similar to that of the dityro-
sine derivatives 22b,c although the chemical shifts of indi-
vidual protons varied with the type of groups at the N and C
terminus. The amino acid components were determined by
gas chromatography after total hydrolysis, and the structure
was ®nally con®rmed by HSQC- and HMBC-NMR
measurements. The dimerization yield of 26 was 6.5%.
Similarly, c(VAYASA) and c(VAYALa) could be dimer-
ized in 6 and 5% yield, respectively (Scheme 6).
Scheme 6. HRP/H2O2 Coupling of tyrosine peptides.

H. Eickhoff et al. / Tetrahedron 57 (2001) 353±364
359
Mixtures of cyclohexapeptides were also enzymatically
dimerized. To avoid problems with solubility, peptides
containing Lys, Ser, Arg, Asp and Glu were used. The
cyclopeptide educts were puri®ed by HPLC prior to use
since a test reaction with unpuri®ed peptides gave poor
yields. In the enzymatically oxidized mixture of the three
cyclopeptides c(VAYAVa), c(VAYASA), c(VAYALa), all
possible dimeric coupling products could be detected by
electrospray mass spectrometry; a selective hetero-cross
coupling was not attained.
account of the analysis of single tyrosyl-peptide dimers
obtained by non-combinatorial procedures (vide supra).
The most striking result of the experiments using HRP as
oxidant is that dityrosine formation prevails with low-
molecular tyrosine derivatives and apparently occurs exclu-
sively with tyrosyl peptides. This Co±Co regioselectivity
may be attributed to a complex association of these tyrosyl
peptides with each other, or, more likely, with the peptidic
part of HRP in a way to arrange the phenolic moieties side
by side. Since the spacer linking peptide and phenol moiety
of the substrate extends to just one CH2 group, Co±Co
coupling of the phenoxyls formed by oxidation via the
activated H2O2 should be easier than Co±O coupling. This
explanation is supported by the observation by G. N. Lewis
et al.76b of a regio- and stereo-speci®c coupling of lignin
precursors in the presence of a high-molecular peptide.
These observations could open a new strategy in phenol
coupling.
To obtain a hetero-cross coupled product in satisfactory
yield, the pentapeptide Ac-Tyr-Arg-Gly-Asp-Val-OH was
oxidized in the presence of a large excess of Z-Tyr-OH.
After reaction and acidi®cation, the unreacted Z-Tyr-OH
as well as its dimers and oligomers could be easily extracted
with ethyl acetate, thus leaving the nonreacted pentapeptide
educt and the product 27 in the aqueous phase.
Peptide libraries are valuable tools in the random and
rational screening of small molecule candidates to ®nd
new active compounds.100 With respect to therapeutical
applications, side-chain cross-links are desirable, because
these bonds cannot be cleaved by peptidases. Therefore,
we tested HRP catalyzed coupling in a preliminary feasi-
bility study to demonstrate, in principle, the transformation
of monomeric to dimeric tyrosyl-peptide libraries. Peptides
of the general type Ac-Tyr-X-Gly-NH2 (28,29) were chosen
because N-acetylated peptide amides proved to be ideal
substrates for the dimerization reaction. N-Protecting
groups other than acetyl often caused low solubility of the
peptides and a carboxy group at the C-terminus gave rise to
oligomers via over-oxidation. X stands for all proteinogenic
amino acids, except for tyrosine and cysteine which would
themselves dimerize. Other oxidizable amino acids like
tryptophan, phenylalanine, histidine or arginine did not
cause problems. Additionally, aminobutyric acid (Abu)
and ornithine (Orn) were incorporated in the X-position.
3. Conclusion
In this report, the principal features of oxidation and oxida-
tive coupling of phenols have been compiled in the context
of intra- and intermolecular coupling of tyrosine derivatives
to various natural products. Systematic variation of oxidants
revealed that HRP/H2O2 is the best oxidant to give dityro-
sine derivatives. The enzymatic method was then applied to
the coupling of small linear and cyclic peptides, and
libraries of biphenyl-cross linked peptides were assembled.
These represent novel building blocks which may be used
after deprotection for elongation of peptides or for further
reactions of the cross coupled products.
4. Experimental
4.1. Materials and equipment
To assure that all possible dimers 30 were produced in the
oxidations, test-sublibraries have been generated from
mixtures containing four peptides each. The oxidative
couplings with HRP were performed in ®fty-eight wells of
a microtitre-plate. Each one of the sublibraries consisted of
four different homodimers and twelve heterodimers; but
because of the symmetrical dityrosine bridge, only six of
the latter were different. The same calculation for the whole
library (from twenty peptides) resulted in twenty homo-
dimers and one hundred and ninety heterodimers, a total
of two hundred and ten dimers all together.
Solvents and chemicals for solid phase peptide synthesis
were of p. a. quality (Fluka and Merck). Solid phase peptide
synthesis was performed on a multiple peptide synthesizer
(SMPS 350, Zinsser Analytic; Syro software, MultiSyn-
Tech, Bochum) by Fmoc/tBu strategy. Fmoc-protected
amino acids: Novabiochem (Bad Soden, Germany) and
Bachem Biochemica GmbH (Bubendorf, Switzerland).
Peptide amides were generated on Polystyrene A RAM
resin (200±400 mesh, capacity: 0.57 mmol/g): Rapp
Polymers (TuÈbingen, Germany). Cyclopeptides were
synthesized on 2-chlorotritylchloride resin preloaded with
an Fmoc-protected amino acid (1% DVB, 200±400 mesh,
capacity: 1.3 mmol/g): Senn Chemicals AG (Dielsdorf,
Germany). Horseradish peroxidase (525 U/mg): Boehringer
GmbH (Mannheim, Germany). Microtitre-plates with l mL
PP-wells: Costar GmbH, Bodenheim (Germany).
Characterization of the libraries was achieved by HPLC
coupled to electrospray mass spectrometry. It was not pos-
sible to distinguish every single dimer analytically, neither
in the entire library nor in the sublibraries because some of
the peaks in HPLC chromatograms overlapped. Therefore,
the libraries in total were separated by HPLC from other
components and during this process consecutively subjected
to MS. In this way, the mass peaks of all theoretically pos-
sible dimers could be observed, whereby only the peptides
containing the isobaric amino acids leucine and isoleucine
had identical masses. Although the presence of diisotyro-
sines could not be ruled out de®nitely, it is unlikely on
4.2. Analytical methods
4.2.1. Fluorescence measurements. Samples (10 mL) were
taken after 5, 10, 15, 30, 45 and 90 min, diluted with 0.2 M
borate buffer (3 mL) in a quartz cuvette and measured with a
Luminescence Spectrometer LS5OB (Perkin±Elmer); room

360
H. Eickhoff et al. / Tetrahedron 57 (2001) 353±364
temperature; excitation 300±400 nm (5 nm slit); emission
maximum 385 nm.
4.3.3. Cleavage of the peptide amides from the resin. The
peptide-loaded resins were suspended in a mixture of 82.5%
TFA, 5% water, 5% phenol, 5% thioanisole and 2.5%
ethanedithiol (reagent K) (1 mL) and shaken for 3 h at
room temperature. The cleavage solution was dropped in
an ice-cold mixture of diethyl ether/n-hexane (1:1) to pre-
cipitate the peptides. The precipitates were centrifuged and
washed three times with diethyl ether/n-hexane (1:1). The
pellets were dissolved in tert-butylalcohol/water (4:1),
deep-frozen in liquid nitrogen, lyophilized, and analyzed
with ESI-MS and analytical HPLC and used without further
puri®cation.
4.2.2. Gas chromatography. Samples for routine analysis
were obtained by hydrolysis of the peptides in 6N HC1 at
1108C for 24 h. The hydrolyzate was dried in an air current,
and acetyl chloride in methanol was added. After 15 min at
1108C, the solvent was removed and the methyl esters
dissolved in tri¯uoroacetic anhydride, which was removed
after 10 min in an air stream.
4.2.3. HPLC and TLC. Analytical HPLC was carried out
on a Nucleosil 300 C-18 column (250£2 mm, particle size:
5 mm; Grom, Herrenberg, Germany) using a Beckman
HPLC-system (System-Gold, Beckman, San Ramon,
USA), 214 nm. The ¯ow rate was 0.3 mL/min. Water
(0.12% tri¯uoroacetic acid, TFA) and acetonitrile (0.08%
TFA) (E. Merck, Darmstadt, Germany) were used as
solvents and a gradient from 10 to 100% acetonitrile in
45 min was chosen. Semipreparative HPLC was performed
on a Waters 600 system (Waters, Milford, USA) and a
Nucleosil 100 C-18 column (250£8 mm, particle size:
7 mm, Grom, Herrenberg, Germany), the ¯ow rate was
3.5 mL/min. The libraries were desalted on RP-18 columns
from ICT GmbH (Bad Homburg, Germany). For TLC silica
gel 60/F254 on aluminum foil (E. Merck, Darmstadt,
Germany) was used (solvent system: 80% chloroform,
30% methanol, 5% water). Compounds were detected
with UV-light (254 nm and 366 nm) and by spraying with
TDM-reagent.
4.3.4. Cyclopeptides. The corresponding linear hexa-
peptides (H-VAYAVa-OH, H-VAYALa-OH, H-VAYASA-
OH) were synthesized on Fmoc-amino acid loaded 2-chloro-
trityl-PS/1%DVB resin. The cleavage from the resin was
achieved in a mixture of CH2Cl2 and 1,1,1,3,3,3-hexa¯uoro-
isopropanol (7:3) in 1 h. The cleavage solutions were evapo-
rated and the side-chain protected peptides dissolved in DMF
to form a 1 mmol solution. For cyclization 4 equiv. of DIPEA
and 3 equiv. of a 0.4 mol solution of O-(benzotriazole-1-yl)-
N,N,N0,N0-tetramethyluronium tetra¯uoroborate/HOBt were
added dropwise. After 3±5 h (TLC-monitoring), the solvent
was evaporated. The side-chain protected cyclopeptides were
dissolved in CH2Cl2 and extracted with 5% KHSO4, water,
5% NaHCO3 and water (3 times each). The organic phase
was concentrated in vacuo. Side-chain deprotection was
performed in 95% TFA. After evaporation the products
were lyophilized from tert-butylalcohol/water (4:1), puri®ed
by semipreparative HPLC (214 nm) and characterized by
analytical HPLC and ESI-MS.
4.2.4. Mass spectrometry. Electrospray ionization mass
spectrometry (ESI-MS) was performed on an API III
TAGA 6000 E triple-quadrupole mass spectrometer,
equipped with an IonSpraye interface (Sciex, Thornhill,
Canada).
4.4. Coupling with chemical oxidants
4.4.1. Oxidation of Z-Tyr-OH with K3Fe(CN)6/TEA.
Z-Tyr-OH (100 mg, 0.32 mmol) in a mixture of MeOH
(2 mL), water (3 mL) and triethylamine (TEA) (0.73 g,
7.2 mmol) was stirred for 1 h under Ar. K3Fe(CN)6
(200 mg, 0.61 mmol) was added and stirring continued for
12 h. The reaction was quenched by adding 5% aqueous
KHSO4, the solvents were evaporated in vacuo and the resi-
due exhaustively extracted with MeOH. The combined
methanolic fractions were concentrated to 5 mL. Prepara-
tive HPLC (40!65% MeCN in 30 min) gave 19 mg
(30 mmol, 19%) Z-Dityr-OH 22d, 21 mg (34 mmol, 21 %)
Z-Isodityr-OH 23d, and 12 mg (14 mmol, 13%) Z-Trityr-
OH.
4.2.5. Nuclear magnetic resonance. NMR-Measurements
were performed on Bruker spectrometers AM 250, WM 400
and AMX 600.
4.3. Synthesis of the starting peptides
4.3.1. Multiple peptide synthesis. Parallel reactions were
performed in 2 mL-syringes, which were placed in reaction
blocks with 96 positions, which permitted the synthesis
either of several different peptides or of higher amounts of
one single peptide, using several positions for only one
sequence. The peptide synthesis on the resin (unloaded
Rink-amide resin for peptide amides, preloaded Wang-
resin for peptides with free C-terminus) was carried out in
DMF, using 1-hydroxy-1H-benzotriazole (HOBt) and diiso-
propylcarbodiimide (DIC) for activation and piperidine for
the cleavage of Fmoc-protective groups.
22d. ESI-MS [amu]: 629.5 [M1H]1. 13C NMR (100 MHz,
CD3OD): d 39.3, 58.2, 68.9, 118.8, 128.7, 129.9, 130.2,
130.7, 131.2, 131.5, 131.9, 150.0, 139.5, 155.3, 159.7,
175.4.
23d. ESI-MS [amu]: 629.5 [M1H]1. 13C NMR (100 MHz,
CD3OD): d 38.2, 39.4, 57.9, 58.1, 67.5, 116.8, 118.0,
128.3±134.6, 139.0, 139.8, 154.2, 155.1, 159.8, 160.2,
177.0, 178.3.
4.3.2. N-Acetylation of peptides. The peptide-loaded resin
in the syringes was suspended in a mixture of CH2Cl2
(0.7 mL), ethyldiisopropylamine (DIPEA) (200 mL;
152 mg; 1.2 mmol) and acetic anhydride (100 mL;
108 mg; 1.1 mmol). After 1 h at room temperature in a
shaker, the resin was washed several times with CH2Cl2
and diethyl ether.
1
Z-Trityr-OH. ESI-MS [amu]: 942.5 [M1H]1. H NMR
(100 MHz, CD3OD): d 3.02 (3H, m), 3.23 (3H, m), 4.50
(3H, m), 5.08 (3H, d, Jˆ12.9 Hz), 5.12 (3H, d, Jˆ11.9 Hz),

H. Eickhoff et al. / Tetrahedron 57 (2001) 353±364
361
6.92 (2H, d, Jˆ8.2 Hz), 7.14 (2H, d, Jˆ7.9 Hz), 7.21 (4H,
HRP (1.4 mg; 750 U) in buffer (750 mL) and 3% H2O2
(90 mL) were added. The mixture was maintained at 378C
for 45 min, after acidi®cation diluted with water and
extracted 5 times with ethyl acetate (50 mL). The aqueous
phase was desalted and analyzed by HPLC and ESI-MS.
Semipreparative HPLC: 7.7 mg of cross-coupled 27
(8 mmol; 26%).
m), 7.34 (15H, s).
4.4.2. Oxidation of Z-Tyr-OMe with phenyliodine(III)
A
bis(tri¯uoroacetate).
solution of PIFA (1.29 g,
3.0 mmol) in CH2Cl2 (9 mL) was added dropwise to a solu-
tion of Z-Tyr-OMe (1.0 g; 3.0 mmol) in dry CH2Cl2
(45 mL). After 1.5 h, NaHCO3 (504 mg, 6.0 mmol) was
added, and the mixture stirred for 5 min. After ®ltration
through celite and concentration in a rotary evaporator,
the remaining syrupy raw product was chromatographed
on silica gel (eluent: petroleum ether/ethyl acetate 2:1).
Two fractions were collected: Z-Isodityr-OMe 23c
(Rfˆ0.36, petroleum ether/ethyl acetate 1:1), 106 mg
(162 mmol; 10.6%), ESI-MS [amu]: 657.5 [M1H]1, and
Z-Dityr-OMe 22c (Rfˆ0.25, petroleum ether/ethyl acetate
1:1), 219 mg (334 mmol; 22%), ESI-MS [amu]:
ESI-MS [amu]: 964.5 [M1H]1. 1H NMR (400 MHz,
CD3OD): d 0.94 (3H, d, Jˆ6.8 Hz), 0.95 (3H, d, Jˆ
6.8 Hz), 1.57±1.73 (2H, m), 1.95 (3H, s), 1.82±1.98
(2H, m), 2.13±2.22 (1H, m), 2.69±3.30 (7H, m), 2.74
(1H, d, Jˆ7.5 Hz), 3.76 (1H, d, Jˆ17 Hz), 3.82 (1H, d,
Jˆ17 Hz), 4.23±4.80 (4H, m), 4.50 (1H, t, Jˆ7.6 Hz),
5.01 (1H, d, Jˆ13 Hz), 5.05 (1H, d, Jˆ13 Hz), 6.81 (1H,
d, Jˆ8.0 Hz), 6.84 (1H, d, Jˆ8.0 Hz), 7.08 (2H, dd, Jˆ3.3,
8.0 Hz), 7.13 (1H, d, Jˆ3.3 Hz), 7.16 (1H, d, Jˆ3.3 Hz),
7.26 (5H, m); only shifts of protons bound to carbons are
given.
1
675.5 [M1H]1. Both species were characterized by H-
and 13C NMR (not shown).
In order to simplify the NMR spectra, the Z protecting
groups of 22c and 23c were cleaved with ammonium
formiate and palladium black.101 Semipreparative HPLC
(10!40% acetonitrile/water, 30 min) yielded 22 mg
(57 mmol; 75%) of H-Dityr-OMe 22b and 18 mg
(46 mmol; 61%) of H-Isodityr-OMe 23b.
4.6. Tripeptide dimer libraries by enzymatic oxidative
coupling
4.6.1. General. For optimization test reactions were
performed in microtitre-plates with 250 mL-wells contain-
ing 5 mmol of the peptide mixture in 200 mL buffer. Horse-
radish peroxidase (0.1 mg; 50 U) in 30 mL solvent and 6 mL
3% hydrogen peroxide were added and the microtitre-plate
incubated at 378C for 1.5 h.
1
22b. ESI-MS [amu]: 389.5 [M1H]1. H NMR (250 MHz,
CD3OD): d 3.10 (1H, dd, Jˆ7.6, 14.5 Hz), 3.23 (1H, dd,
Jˆ5.7, 14.4 Hz), 3.81(6H, s), 4.28 (2H, dd, Jˆ5.7,
7.6 Hz), 6.92 (2H, d, Jˆ7.6 Hz), 7.07 (2H, s), 7.10 (2H, d,
Jˆ7.6 Hz). 13C NMR (63 MHz, CD3OD): d 37.7, 54.7,
56.5, 118.7, 127.4, 128.5, 132.0, 134.6, 156.4, 171.6.
4.6.2. Synthesis of dimeric sublibraries in small scale.
Tripeptide Ac-YXG-NH2 mixtures (30 mmol) and 300 U
(0.57 mg) horseradish peroxidase in 0.2 M borate buffer or
borate buffer/dioxane (1:1) (950 mL) were placed in 1 mL
1
23b. ESI-MS [amu]: 389.5 [M1H]1. H NMR (400 MHz,
CD3OD): d 2.92 (4H, m), 3.53 (3H, s), 3.59 (3H, s), 4.05
(2H, m), 6.63 (1H, s), 6.70 (2H, d, Jˆ8.6 Hz), 6.74 (2H, s),
6.98 (2H, d Jˆ8.6 Hz). 13C NMR (400 MHz, CD3OD): d
36.7, 53.5, 55.3, 118.3, 118.8, 123.5, 126.8, 127.5, 129.1,
131.6, 144.7, 150.3, 159.2, 170.4.
wells of
a microtitre-plate. After addition of 3%
hydrogen peroxide (36 mL) the oxidations were performed
at 378C for 1.5 h. The mixtures were carefully acidi®ed
with 5% HCl and samples were taken for analytical
characterization.
4.6.3. Synthesis of the complete tripeptide dimer library
in preparative scale. HRP (6.5 mg; 3400 U) and 3% H2O2
(408 mL) were added to Ac-YXG-NH2 (340 mmol; 17 mmol
of each peptide) in 0.2 M borate buffer (11 mL). The solu-
tion was incubated for 1.5 h at 378C. After careful acidi®ca-
tion with 5% HCl, the work-up was performed as described
below, and the dimeric peptide library was characterized by
ESI-MS.
4.5. Enzymatic oxidative coupling
4.5.1. Enzymatic oxidative coupling of a cyclopeptide.
The cyclopeptide c[VAYAVa] (46 mg; 80 mmol) was
dissolved in 0.2 M borate buffer (45 mL) and HRP
(2.5 mg; 1340 U) and 3% hydrogen peroxide (650 mL;
580 mmol) were added. After 12 h at 378C the reaction
mixture was acidi®ed with dilute HCl and the solvent
removed in a rotary evaporator. Semipreparative HPLC
yielded 3 mg (2.6 mmol; 6.5%) of dimeric cyclopeptide 26.
4.6.4. Puri®cation methods. Horseradish peroxidase was
removed from the samples by centrifugation in Amicon
microconcentrators with a molecular weight cut-off of
3000 Da. The peptide libraries were desalted on RP-18
columns: The columns were equilibrated with 10% aceto-
nitrile/water. The buffer solutions were placed on top of the
column and diluted with water to wash off the salts. Then
the peptides were eluted with 50% acetonitrile/water.
ESI-MS [amu]: 1147.5 [M1H]1, 1169.5 [M1Na]1. H
1
NMR (600 MHz, DMSO-d6): d 0.85 (24H, m), 1.18 (6H,
d, Jˆ6.9 Hz), 1.28 (12H, m), 2.00±2.30 (4H, m), 3.00 (4H,
d, Jˆ7.4 Hz), 3.89±4.37 (12H, m), 6.78 (2H, d, Jˆ8.3 Hz),
6.95 (2H, dd, Jˆ2.2, 8.3 Hz), 6.97 (1H, d, Jˆ2.2 Hz), 7.19
(2H, Jˆ7.6 Hz), 7.84 (2H, d, Jˆ7.4 Hz), 8.03 (6H, m), 8.42
(2H, d, Jˆ5.5 Hz), 9.04 (2H, s).
HPLC-puri®cations were performed on RP-18 columns in
semipreparative scale using acetonitrile (0.1% TFA) and
water (0.1% TFA) as solvents. The gradient was 10 to
50% acetonitrile in 45 min.
4.5.2. Enzymatic cross-coupling. Ac-Tyr-Arg-Gly-Asp-
Val-OH (20 mg; 31 mmol) and Z-Tyr-OH (32 mg;
100 mmol) were dissolved in 0.2 M borate buffer (3 mL).

362
H. Eickhoff et al. / Tetrahedron 57 (2001) 353±364
Fauvet, M.; Gaudemer, A. J. Chem. Soc., Perkin Trans. 1
1984, 1971±1979.
È È
23. Knaudt, J.; Forster, S.; Bartsch, U.; Rieker, A.; Jager, E.-G.
Acknowledgements
We thank R. SuÈûmuth and T. Walk for recording the mass
È
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