Synthesis of dityrosine cross-linked peptide dimers using the Miyaura-Suzuki reaction

Article abstract of DOI:10.1021/ol034801t

(Matrix presented) Since peroxidase-catalyzed dityrosine formation is inefficient for peptides, we have developed alternative conditions for intermolecular dityrosine formation using the Miyaura-Suzuki reaction. A one-pot reaction is effective for cross-l

Full text of DOI:10.1021/ol034801t

ORGANIC
LETTERS
2003
Vol. 5, No. 16
2817-2820
Synthesis of Dityrosine Cross-Linked
Peptide Dimers Using the
Miyaura−Suzuki Reaction
Joshua C. Yoburn and David L. Van Vranken*
Department of Chemistry, UniVersity of California,
IrVine, California 92697-2025
dlVanVra@uci.edu
Received May 9, 2003
ABSTRACT
Since peroxidase-catalyzed dityrosine formation is inefficient for peptides, we have developed alternative conditions for intermolecular dityrosine
formation using the Miyaura−Suzuki reaction. A one-pot reaction is effective for cross-linking short peptides, but longer peptides inhibit the
Suzuki step, mandating a traditional two-step procedure using potassium acetate for the Miyaura reaction and potassium carbonate for the
Suzuki coupling. These palladium-based methods are complementary to the well-established peroxidase-catalyzed oxidative phenolic coupling
of full-length proteins.
Long-lived human proteins undergo a variety of posttrans-
lational modifications. Among the most interesting protein
modifications are those that lead to the formation of carbon-
carbon bonds. Dityrosines are biaryl cross-links that result
from the ortho coupling of two tyrosyl radicals (Figure 1).
protein.1 Unlike cystine disulfides, dityrosine cross-links are
not susceptible to reductive cleavage.
While there are numerous ways to efficiently make the
dimer of the amino acid tyrosine, the synthesis of dityrosine
cross-linked peptide dimers is considerably more difficult.
Peroxidase-catalysis is an effective method for forming
dityrosine cross-links between the free amino acid tyrosine
or between larger peptides and proteins.2,3 Unfortunately, it
is notoriously inefficient when carried out in small- to
medium-sized peptides, particularly those suited for detailed
spectroscopic studies.4 Eickhoff and co-workers recently
reported a 6.5% yield for the preparation of a dityrosine
cross-linked peptide dimer. In this case, 2.5 mg of enzyme
generated 3 mg of product.3 While peroxidase-catalyzed
Figure 1. Three types of symmetrical cross-links.
(1) (a) Amado, R.; Aeschbach, R.; Neukom, H. Methods Enzymol. 1984,
107, 377-388. (b) Giulivi, C.; Davies, K. J. Methods Enzymol. 1994, 233,
363-371. (c) Tilley, K. A.; Benjamin, R. E.; Bagorogoza, K. E.; Okot-
Kotber, B. M.; Prakash, O.; Kwen, H. Amyloid 1999, 6, 7-13.
(2) Malencik, D. A.; Sprouse, J. F.; Swanson, C. A.; Anderson, S. R.
Anal. Biochem. 1996, 242, 202-213.
(3) Eickhoff, H.; Jung, G.; Rieker, A. Tetrahedron 2001, 57, 353-364.
(4) Michon, T.; Chenu, M.; Kellershon, N.; Desmadril, M.; Gueguen,
J.; Biochemistry 1997, 36, 8504-8513.
The robust dityrosine linkage is structurally analogous to the
carbon-carbon bond that secures a ditryptophan cross-link.
Dityrosines occur naturally in collagens, elastins, proteo-
glycans, lens proteins, wheat gluten, and Alzheimer’s Aâ-
10.1021/ol034801t CCC: $25.00 © 2003 American Chemical Society
Published on Web 07/04/2003

reactions are rapid, they are troubled by overoxidation,
oligomerization, and low mass balance. In addition, the
dimeric product must be separated from the voluminous
excess of buffer salts. Clearly, a complementary synthetic
approach devoid of these problems is of paramount interest.
Scheme 1. One-Pot Homocoupling of Peptidesa
Palladium-catalyzed coupling reactions have revolutionized
the synthesis of biaryls.5,6 There are numerous examples of
palladium-catalyzed Ullmann cross-coupling methods to
make unsymmetrical dimers using ditin7 and diboron8
reagents. Recently, Carbonelle and Zhu reported the applica-
tion of the Miyaura-Suzuki coupling to the synthesis of a
15-membered ring biphenomycin analogue containing an
intramolecular O,O′-dimethyl-dityrosine cross-link.9 Unfor-
tunately, mass balance was generally poor and deiodination
was a predominant side reaction. Despite the modest yields
(10% at 5 mM and 45% 20 mM) that were reported, the
one-step Miyaura-Suzuki reaction procedure appeared to
be a promising method for formation of intermolecular
dityrosine cross-links, particularly for intermolecular reac-
tions that are not complicated by ring strain or competitive
oligomerization.
a Conditions: aryl iodide (100 mol %), diboron (60 mol %),
PdCl2dppf (10 mol %), K2CO3 (600 mol %), DMSO, 90 °C. An
asterisk (*) denotes a 2,2′-biaryl cross-link.
Biaryls are common byproducts in the palladium-catalyzed
Miyaura reaction of aryl halides with bispinacolatodiboron,10
and a palladium-catalyzed Ullmann homocoupling with
bispinacolatodiboron has been used in the synthesis of
dimeric pyranonaphthoquinones.11 We hypothesized that the
Miyaura-Suzuki reaction might be efficiently applied to the
formation of intermolecular dityrosine cross-links as opposed
to intramolecular macrocyclic ring closure reactions. In
addition, it was expected that O-benzyl would be more easily
removed from dityrosines than O-methyl protecting groups.
potassium carbonate, which was harmful in the biphenomycin
study, led to efficient formation of dimer 1b. However,
potassium phosphate, which is more basic (and presumably
more nucleophilic) than potassium carbonate, gave a mul-
titude of products. 1,2-Dimethoxyethane, a common solvent
for Suzuki reactions of aryl boronate esters, generated
dityrosine 1b in 42% yield. Efforts to enhance reactivity by
using alternative Pd(0) sources were not effective; for
example, when the reaction was carried out with 5 mol %
Pd2dba3 and 10 mol % dppf, the aryl iodide was not
consumed. Elevated temperatures (up to 150 °C) generated
dimer in lower yield. The use of biscatecholdiboron led to
the formation of dimer 1b in only 10% yield from 1a,
confirming the superiority of bispinacolatodiboron. A tran-
sient intermediate is observed to build up prior to the
formation of dityrosine product; the Rf and mass of this
intermediate is consistent with the aryl boronate ester.12 This
observation suggests that the Miyaura reaction proceeds faster
than the Suzuki coupling.
We next applied the optimized one-pot conditions13
(K2CO3, PdCl2dppf, DMSO) to the coupling of a dipeptide
substrate 2a. The yield for dimer 2b was slightly lower
(74%), and some unreacted aryl iodide remained after product
formation had ceased. Only trace amounts of product
resulting from either deiodination or protodeboronylation
were observed. Homocoupling of tripeptide 3a to form
dityrosine 3b proceeded in similar yield despite the position-
The Miyaura reaction of aryl halides provides a simple
method for the synthesis of aryl boronate esters from aryl
halides. When only 50 mol % bispinacolatodiboron is used,
50 mol % of the aryl halide can be boronylated (in situ) to
the aryl boronate, which then undergoes Suzuki coupling with
the remaining 50 mol % unreacted aryl halide (Scheme 1).
The resulting transformation, equivalent to an Ullmann
coupling, proceeds under milder conditions than the tradi-
tional copper-promoted reaction. Beginning with 10 mol %
of a Pd(II) source such as PdCl2dppf, we used an additional
10 mol % bispinacolatodiboron for the reduction of Pd(II)
to the active Pd(0) species.
Potassium acetate, deemed necessary for the intramolecular
Miyaura-Suzuki reaction, led to boronylation of iodotyrosine
1a with no detectable formation of biaryl 1b. In contrast,
(5) (a) Yokoyama, Y.; Ito, S.; Takahashi, Y.; Murakami, Y. Tetrahedron
Lett. 1985, 2, 6457-6460.
(6) Miyaura, N.; Suzuki, A. Chem. ReV. 1995, 95, 2457-2483.
(7) Kelly, T. R.; Li, Q.; Bhushan, V. Tetrahedron Lett. 1990, 31, 161-
164.
(8) (a) Elder, A. M.; Rich, D. H. Org. Lett. 1999, 1, 1443-1446. (b)
For an example of two-step, one-pot cross-couplings, see: Giroux, A.; Han,
Y.; Prasit, P. Tetrahedron Lett. 1997, 38, 3841-3844.
(9) (a) Carbonnelle, A. C.; Zhu, J. P. Org. Lett. 2000, 2, 3477-3480.
(b) Boisnard, S.; Carbonnelle, A.-C.; Zhu, J. P. Org. Lett. 2001, 3, 2061-
2064.
(12) ESI-MS of the transient TLC spot is consistent with an aryl boronate
intermediate. Ironically, the aryl iodide (m/z 453.04) and the aryl boronate
(m/z 453.23) have similar m/z and were therefore difficult to distinguish by
low-resolution mass spectrometry.
(13) General One-Pot Procedure for the Homocoupling of Aryl
Iodides. A flame-dried flask was charged with aryl iodide (100 mol %),
K2CO3 (600 mol %), PdCl2dppf (10 mol %), and bispinacolatodiboron (60
mol %). The solids were flushed with nitrogen for 5 min prior to the addition
of DMSO. The reaction was heated to 90 °C for 6-72 h. Concentration in
vacuo followed by silica gel chromatography provided the dityrosine cross-
linked peptide dimer.
(10) Ishiyama, T.; Murata, M.; Miyaura, N. J. Org. Chem. 1995, 60,
7508-7510.
(11) Brimble, M. A.; Neville, D.; Duncalf, L. J. Tetrahedron Lett. 1998,
39, 5647-5650.
2818
Org. Lett., Vol. 5, No. 16, 2003

ing of the iodotyrosine in the middle of the peptide strand.
Overall, reaction times required for homocoupling increased
as the substrate length was increased. Unfortunately, cyclic
hexapeptide 4a gave disappointing results. Here, consumption
of aryl iodide was sluggish even after 3 days. With this and
other longer substrates, a substantial amount of unreacted
aryl iodide remained.
In theory, the amide bonds of peptidic substrates could
chelate to and deactivate the palladium catalyst or other
necessary intermediates. To assess the potential inhibitory
effect of oligopeptide chains on the reaction, the homo-
coupling of aryl iodide 1a was carried out in the presence
of 100 mol % tetrapeptide 5 for 6 h, at which point no further
reaction was observed (Scheme 2). Therefore, it seemed that
the presence of additional peptides lengthens the reaction
half-lives. Under these conditions the desired dityrosine 1b
was formed in only 58% yield, while starting material and
aryl boronic ester accounted for the remainder of the material
and were present in a 1:1 ratio.
In an attempt to overcome potential catalyst depletion,
homocoupling of 1a was performed with a solid mixture of
10 mol % PdCl2dppf and 10 mol % diboron reagent being
added 6 h into the ongoing reaction in order to generate more
active catalyst in situ. Unfortunately, the reaction did not
appear to progress further to a noticeable extent. Thus, while
the one-pot homocoupling procedure is both direct and useful
for the construction of small dityrosine cross-linked dimers,
it is slow and impractical for the synthesis of larger ones.
An alternative approach to the homocoupling of difficult
substrates is to initially prepare the aryl boronic ester using
Miyaura conditions and then cross-couple this intermediate
with additional aryl iodide using Suzuki conditions (Scheme
3). The utility of this two-step protocol relies first upon the
efficient preparation of aryl boronic ester. In the presence
of tetrapeptide 5 (100 mol %), aryl iodide 1a could be
boronylated in excellent yield in 3 h using 110 mol %
diboron, 10 mol % PdCl2dppf, and 600 mol % KOAc
(Scheme 3). In the absence of added tetrapeptide 5, these
conditions led to a shorter half-life for boronylation. Impor-
tantly, tetrapeptide 5 does not appear to have a major
deleterious effect in the overall efficiency of the Miyaura
boronylation reaction under these conditions.
Scheme 2a
Scheme 3. Two-Step Homocoupling of Peptidesa
a Conditions: additive + aryl iodide (100 mol %), diboron (60
mol %), PdCl2dppf (10 mol %), K2CO3 (600 mol %), DMSO, 90
b
°C. Approximate time required for the reaction to reach comple-
tion.)
Since aryl iodide and aryl boronate remained unreacted,
it seemed likely that the active catalyst had been depleted.
However, the palladium did not black out, as is typical for
reactions that generate unligated Pd(0). Pinacol (100 mol %)
had virtually no effect on the homocoupling reaction of aryl
iodide 1a, confirming its nonparticipation in reaction inhibi-
tion (Scheme 2).
a Reaction conditions: (a) aryl iodide (100 mol %), diboron (110
mol %), PdCl2dppf (10 mol %), KOAc (600 mol %), DMSO, 90
°C; (b) aryl iodide (100 mol %), diboron (120 mol %), PdCl2dppf
(20 mol %), K2CO3 (600 mol %), DMSO, 90 °C. An asterisk (*)
denotes a 2,2′-biaryl cross-link.
When aryl iodide 1a was subjected to a homocoupling in
the presence of tetrapeptide 5 (100 mol %) and 300 mol %
diboron, dityrosine 1b and aryl boronic ester 1c were formed
in a 1:1 mixture in excellent yield with no detectable starting
material. Under these conditions, the use of less diboron (120
mol %) led to a 1:1:1 mixture of unreacted aryl iodide, biaryl,
and aryl boronic ester. Thus, while the use of excess diboron
reagent facilitates the consumption of aryl iodide, it also leads
to the formation of aryl boronic ester at the expense of biaryl
formation.
With quantitative conversion of 1a to aryl boronate 1c,
the intermediate reaction was filtered through a pad of Celite
and silica to remove palladium black and any insoluble salts.
The reaction was immediately recharged with additional aryl
iodide (100 mol %), catalyst (10 mol %), diboron (10 mol
%), and K2CO3 (600 mol %). After a total of 8 h, the Suzuki
reaction had progressed minimally (<20% biaryl) and aryl
iodide 1a and aryl boronate were still present in roughly
Org. Lett., Vol. 5, No. 16, 2003
2819

equal amounts, again suggesting catalyst deactivation. Re-
peating the Suzuki step again but with higher catalyst loading
(20 mol % PdCl2dppf + 20 mol % diboron) provided biaryl
1b in 84% overall yield.
Scheme 4
Optimized two-step conditions14 were applied to larger
substrates. For example, the N-terminal tyrosine substrate
6a was dimerized in good yield, confirming that the reaction
tolerates tyrosine at various positions along the peptide
strand. Tripeptide 3a afforded dimer 3b in overall 87% as
compared to the one-step protocol, which required an
additional 20 h and resulted in a lower yield. As a final
demonstration of the viability and dependability of this two-
step approach for large peptidic substrates, heptapeptide 7a
was dimerized in 59% yield over an 11 h time period. Thus,
substrates that are reluctant to form dityrosine cross-links
using the one-step protocol are likely to benefit from the
two-step procedure, which while less direct, is ultimately
faster and more efficient.
The benzyl protecting groups are easily removable through
catalytic hydrogenolysis of the benzyl ethers. For example,
the benzyl ethers and Cbz groups of dimer 3b were removed
using catalytic Pd-C in methanolic acetic acid providing
dityrosine cross-linked peptide 3c in 94% yield (Scheme 4).
In conclusion, we have shown that the Miyaura-Suzuki
reaction is an effective approach to the synthesis of dityrosine
cross-linked peptide dimers. In the first examples of this
reaction carried out by Zhu and co-workers, the yield may
have been artificially low due to a challenging substrate. For
the one-step intermolecular cross-linking reaction of short
peptides, it is best to use potassium carbonate as the
nucleophile. In the two-step transformation, the initial
Miyaura step should be performed with potassium acetate
followed by a traditional Suzuki coupling using potassium
carbonate. To date, these methods offer the highest reported
yields for the preparation of dityrosine cross-linked peptide
dimers. The strategy is best suited for peptides of moderate
length and is complementary to the peroxidase-catalyzed
method. However, unlike peroxidase-catalysis, the Miyaura-
Suzuki approach to dityrosine cross-linked peptides is devoid
of overoxidation, oligomerization, or the need for tedious
separation of product from buffer salts.
Acknowledgment. The authors would like to thank the
National Institutes of Health (R01 GM54523) for its generous
financial support, as well as Dr. John Greaves for assistance
with mass spectrometry.
(14) General Two-Step Proceedure for the Homocoupling of Aryl
Iodides. A flame-dried flask was charged with aryl iodide (100 mol %),
KOAc (600 mol %), PdCl2dppf (10 mol %), and bispinacolatodiboron (110
mol %). The solids were flushed with nitrogen for 5 min prior to the addition
of DMSO (0.25 M). The reaction was heated at 90 °C for 3-5 h. The
reaction mixture was passed through a pad of silica and then a pad of Celite.
The filtrate was supplemented with aryl iodide (100 mol %), K2CO3 (600
mol %), PdCl2dppf (20 mol %), and bispinacolatodiboron (20 mol %) and
sufficient DMSO to solubilize the reagents (about 0.125 M). The reaction
was again heated at 90° C for 3-6 h. Concentration in vacuo followed by
silica gel chromatography provided the dityrosine cross-linked peptide dimer.
Supporting Information Available: Details describing
the synthesis and characterization of all starting materials
and products. This material is available free of charge via
the Internet at http://pubs.acs.org.
OL034801T
2820
Org. Lett., Vol. 5, No. 16, 2003