Tyrosine analogues for probing proton-coupled electron transfer processes in peptides and proteins

Article abstract of DOI:10.1021/ja907921w

A series of amino acids analogous to tyrosine, but differing in the physicochemical properties of the aryl alcohol side chain, have been prepared and characterized. These compounds are expected to be useful in understanding the relationships between structure, thermodynamics, and kinetics in long-range proton-coupled electron transfer processes in peptides and proteins. Systematic changes in the acidity, redox potential, and O-H bond strength of the tyrosine side chain could be induced upon substituting the phenol for pyridinol and pyrimidinol moieties. Further modulation was possible by introducing methyl and t-butyl substitution in the position ortho to the phenolic hydroxyl. The unnatural amino acids were prepared by Pd-catalyzed cross-coupling of the corresponding halogenated aryl alcohol protected as their benzyl ethers with an organozinc reagent derived from N-Boc L-serine carboxymethyl ester. Subsequent debenzylation by catalytic hydrogenation yielded the tyrosine analogues in good yield. Spectrophotometric titrations revealed a decrease in tyrosine pK _a of ca. 1.5 log units per included nitrogen atom, along with a corresponding increase in the oxidation (peak) potentials of ca. 200 mV, respectively. All told, the six novel amino acids described here have phenol-like side chains with pK_a's that span a range of 7.0 to greater than 10, and an oxidation (peak) potential range of greater than 600 mV at and around physiological pH. Radical equilibration EPR experiments were carried out to reveal that the O-H bond strengths increase systematically upon nitrogen incorporation (by ca. 0.5-1.0 kcal/mol), and radical stability and persistence increase systematically upon introduction of alkyl substitution in the ortho positions. The EPR spectra of the aryloxyl radicals derived from tyrosine and each of the analogues could be determined at room temperature, and each featured distinct spectral properties. The uniqueness of their spectra will be helpful in discerning one type of aryloxyl in the presence of other possible aryloxyl radicals in peptides and proteins with multiple tyrosine residues between which electrons and protons can be transferred.

Full text of DOI:10.1021/ja907921w

Published on Web 12/15/2009
Tyrosine Analogues for Probing Proton-Coupled Electron
Transfer Processes in Peptides and Proteins
Susheel J. Nara,† Luca Valgimigli,*,‡ Gian Franco Pedulli,‡ and Derek A. Pratt*,†
Department of Chemistry, Queen’s UniVersity, 90 Bader Lane, Ontario K7L 3N6, Canada, and
Dipartimento di Chimica Organica “A. Mangini” Via San Giacomo 11, UniVersita` di Bologna,
40126, Bologna, Italy
Received September 17, 2009; E-mail: luca.valgimigli@unibo.it; pratt@chem.queensu.ca
Abstract: A series of amino acids analogous to tyrosine, but differing in the physicochemical properties of
the aryl alcohol side chain, have been prepared and characterized. These compounds are expected to be
useful in understanding the relationships between structure, thermodynamics, and kinetics in long-range
proton-coupled electron transfer processes in peptides and proteins. Systematic changes in the acidity,
redox potential, and O-H bond strength of the tyrosine side chain could be induced upon substituting the
phenol for pyridinol and pyrimidinol moieties. Further modulation was possible by introducing methyl and
t-butyl substitution in the position ortho to the phenolic hydroxyl. The unnatural amino acids were prepared
by Pd-catalyzed cross-coupling of the corresponding halogenated aryl alcohol protected as their benzyl
ethers with an organozinc reagent derived from N-Boc L-serine carboxymethyl ester. Subsequent
debenzylation by catalytic hydrogenation yielded the tyrosine analogues in good yield. Spectrophotometric
titrations revealed a decrease in tyrosine pKa of ca. 1.5 log units per included nitrogen atom, along with a
corresponding increase in the oxidation (peak) potentials of ca. 200 mV, respectively. All told, the six novel
amino acids described here have phenol-like side chains with pKa’s that span a range of 7.0 to greater
than 10, and an oxidation (peak) potential range of greater than 600 mV at and around physiological pH.
Radical equilibration EPR experiments were carried out to reveal that the O-H bond strengths increase
systematically upon nitrogen incorporation (by ca. 0.5-1.0 kcal/mol), and radical stability and persistence
increase systematically upon introduction of alkyl substitution in the ortho positions. The EPR spectra of
the aryloxyl radicals derived from tyrosine and each of the analogues could be determined at room
temperature, and each featured distinct spectral properties. The uniqueness of their spectra will be helpful
in discerning one type of aryloxyl in the presence of other possible aryloxyl radicals in peptides and proteins
with multiple tyrosine residues between which electrons and protons can be transferred.
Introduction
between the electron donor and acceptor moieties and that this
would make long-range ET in peptides and proteins too slow
Long-range electron transfer (ET) in peptides and proteins
is now recognized as a common phenomenon, which plays a
central role in processes as diverse as energy conversion (e.g.,
photosynthesis and respiration) and catalysis (e.g., ribonucleotide
reduction and prostaglandin biosynthesis).1 Early efforts to
understand how the movement of charge between redox-active
protein cofactors, or from a cofactor to a catalytic amino acid-
derived radical, focused on characterizing the ability of the
protein structure to act as a medium to facilitate tunneling.2 From
these studies, it was clear that ET through peptides and proteins
decreases in rate in a logarithmic way with increasing distance
to be useful for either energy conversion or catalysis. This
necessitates that long-range ET through peptides and proteins
be rationalized on the basis of multiple ET steps via intervening
redox-active amino acids, each one serving as an effective
electron relay. Among the naturally occurring amino acids,
tryptophan and tyrosine are most relevant as participants in the
electron relay, due to their readily oxidizable indole and phenol
side chains, respectively, and, of these two, tyrosine following
its oxidation to the tyrosyl radical figures most prominently in
both catalysis and energy conversion.3
The archetypical example of long-range ET mediated by
intervening tyrosine (and tryptophan) residues occurs in the
class I ribonucleotide reductase (RNR), which catalyzes the
rate-limiting step of DNA biosynthesis in E. coli and other
prokaryotes, reduction of ribonucleotides to deoxyribonucle-
† Queen’s University.
‡ Universita` di Bologna.
(1) See, for example: Cordes, M.; Giese, B. Chem. Soc. ReV. 2009, 38,
892–901. Giese, B.; Graber, M.; Cordes, M. Curr. Opin. Chem. Biol.
2008, 12, 755–759. Gray, H. B.; Winkler, J. R. Proc. Natl. Acad. Sci.
U.S.A. 2005, 102, 3534–3539. Mose, C. C.; Page, C. C.; Dutton, P. L.
Curr. Opin. Chem. Biol. 2003, 7, 551–556. Stubbe, J.; Nocera, D. G.;
Yess, C. S.; Chang, M. C. Y. Chem. ReV. 2003, 103, 2167–2201, and
references therein.
(3) Stubbe, J.; van der Donk, W. A. Chem. ReV. 1998, 98, 705. Stubbe,
J. Chem. Commun. 2003, 20, 2511. Pesavento, R. P.; van der Donk,
W. A. AdV. Protein Chem. 2001, 58, 317.
(4) Sjo¨berg, B.-M.; Reichardt, P.; Gra¨slund, A.; Ehrenberg, A. A. J. Biol.
Chem. 1978, 253, 6863–6865. Stubbe, J. J. Biol. Chem. 1990, 265,
5329–5332. Nordlund, P.; Sjo¨berg, B.-M.; Eklund, H. Nature 1990,
345, 593–598.
(2) See, for example: Gray, H. B.; Winkler, J. R. Q. ReV. Biophys. 2003,
36, 341–372, and references therein. Prytkova, T. R.; Kumikov, I. V.;
Beratan, D. N. Science 2007, 315, 622–625.
9
10.1021/ja907921w  2010 American Chemical Society
J. AM. CHEM. SOC. 2010, 132, 863–872 863

A R T I C L E S
Nara et al.
otides.4 The catalytic cycle is initiated when the di-iron tyrosine
(Y122) cofactor of one of the subunits of the enzyme (R2)
oxidizes a cysteine residue on the other subunit (R1) located
35 Å away, prompting the suggestion that ET occurs in a
stepwise fashion via the side chains of a tryptophan residue
(W48) and three tyrosine residues (Y356, Y731, and Y730),
which lie between the cofactor and the catalytic cysteine.1,5 By
studying the kinetic competence of RNR’s wherein each of the
tyrosine residues that form the putative ET relay were replaced
with unnatural amino acids, which differed in their oxidation
potentials (i.e., electron-poor such as 1-66 and electron-rich
such as 7,788), Stubbe and co-workers were able to demonstrate
that the redox chemistry of these intervening amino acid side
chains is indeed involved in the radical propagation necessary
for the initiation of catalysis.
other amino acids, cofactors) that are important in maintaining
effective long-range ET kinetics remain to be clearly elucidated.
While the work of Stubbe and co-workers has provided some
examples of unnatural tyrosine analogues that could be useful
in this regard (vide supra),6 we sought to develop a series of
unnatural amino acids whose acid-base, redox, and radical
chemistry changed in a well-defined and systematic way, which
could be relatively easily accessed by conventional synthetic
methods and readily characterizable by EPR as well as UV
spectroscopy. We conceptualized the incorporation of nitrogen
atoms into the aromatic ring of tyrosine to construct pyridyl
and pyrimidinyl analogues of tyrosine. The idea stemmed from
our previous studies concerning the efficacy of 3-pyridinols and
5-pyrimidinols as radical-trapping antioxidants.11 These previous
investigations revealed that upon incorporation of a N-atom at
the 3-position of phenol in lieu of the usual C-atom, the O-H
bond dissociation enthalpy (BDE) increases by ∼1 kcal/mol,
the ionization (oxidation) potential (IP) increases by a few 100
mVs, and the acidity also increases by ∼1.5 log units. With
the second substitution of a N-atom, this time for the C-atom
at the 5-position, the trend progressed in the same direction,
increasing the bond strength, oxidation potential, and acidity
even further. Thus, we expected that the O-H BDE, Eo, and
pKa of tyrosine could be varied in an isosteric manner by N-atom
incorporation in the phenolic ring and that we could further
substitute tyrosine and the pyridine and pyrimidine analogues
in a systematic way to afford a series of unnatural amino acids,
which may be exploited to help address some of the yet
unanswered questions concerning long-range ET in peptides and
proteins and the roles of tyrosyl radicals in energy conversion
and catalysis.
Recent work by Gray and Winkler9 as well as Giese and co-
workers10 has shone further light on the abilities of intermediate
amino acid side chains to act as charge transfer relays. Of
particular interest is the polypeptide model system of Giese and
co-workers, which allows one to monitor charge migration
between a 2,4-dialkoxyphenylalanine radical cation and a
tyrosine residue via an intervening polyprolyl polypeptide of
well-defined structure. By monitoring the change in charge
migration as a function of intervening amino acid in the
polyprolyl sequence, one can probe the role of the identity of
the amino acid side chain on the kinetics of ET between donor
and acceptor. Because the key thermodynamic parameter
governing the rate of each of the intermediate ET steps in long-
range ET is the redox potential of the amino acid side chain,
which is intimately linked to its ability to give up a proton to
the surrounding medium (which may be coupled to a proton
transfer event involving another side chain presenting a basic
or H-bond accepting site), it is highly desirable to use a series
of amino acids that differ systematically in both their electron-
and proton-donating abilities. However, due to the limited
variability in the structural, acid-base, and redox properties of
natural amino acids, the characteristics of the amino acids and
their interactions with the surrounding medium (i.e., solvent,
(5) Uhlin, U.; Eklund, H. Nature 1994, 370, 533–539. Bennati, M.;
Robblee, J. H.; Mugnaini, V.; Stubbe, J.; Freed, J. H.; Borbat, P. J. Am.
Chem. Soc. 2005, 127, 15014–15015. Uppsten, M.; Fa¨rnegårdh, M.;
Domkin, V.; Uhlin, U. J. Mol. Biol. 2006, 359, 365–377.
(6) (a) Seyedsayamdost, M. R.; Yee, C. S.; Reece, S. Y.; Nocera, D. G.;
Stubbe, J. J. Am. Chem. Soc. 2006, 128, 1562–1568. (b) Seyedsayam-
dost, M. R.; Reece, S. Y.; Nocera, D. G.; Stubbe, J. J. Am. Chem.
Soc. 2006, 128, 1569–1579. (c) Reece, S. Y.; Seyedsayamdost, M. R.;
Stubbe, J.; Nocera, D. G. J. Am. Chem. Soc. 2006, 128, 13654–13655.
(d) Reece, S. Y.; Seyedsayamdost, M. R.; Stubbe, J.; Nocera, D. G.
J. Am. Chem. Soc. 2007, 129, 13828–13830.
(7) Seyedsayamdost, M. R.; Nocera, D. G. J. Am. Chem. Soc. 2006, 128,
2522–2523. Seyedsayamdost, M. R.; Chan, C. T. Y.; Mugnaini, V.;
Stubbe, J.; Bennati, M. J. Am. Chem. Soc. 2007, 129, 15748–15749.
Seyedsayamdost, M. R.; Stubbe, J. J. Am. Chem. Soc. 2007, 129, 2226–
2227.
(8) Seyedsayamdost, M. R.; Xie, J.; Chan, C. T. Y.; Schultz, P. G.; Stubbe,
J. J. Am. Chem. Soc. 2006, 128, 15060–15071.
Results
(9) Shih, C.; Museth, A. K.; Abrahamsson, M.; Blanco-Rodriguez, A. M.;
Di Billo, A. J.; Sudhamsu, J.; Crane, B. R.; Ronayne, K. L.; Towrie,
M.; Vlcek, A.; Richards, J. H.; Winkler, J. R.; Gray, H. B. Science
2008, 320, 1760–1762.
A. Synthesis. The preparation of the pyridyl (10) and pyrim-
idyl (11) analogues of tyrosine was carried out using an
organozinc reagent derived from N-Boc protected L-serine
methyl ester (16)12 and 2-bromo-5-benzyloxy pyridine (17) and
pyrimidine (18), respectively, as shown in Scheme 1. The
pyridyl bromide 17 and the pyrimidyl bromide 18 were obtained
from the corresponding pyridyl and pyrimidyl amines, both
reported in a recent paper from our group,11g by nonaqueous
halo-dediazoniation.13 The Negeishi-type Pd-catalyzed cross-
(10) (a) Wang, M.; Gao, J.; Muller, P.; Giese, B. Angew. Chem., Int. Ed.
2009, 48, 4232–4234. (b) Giese, B.; Wang, M.; Gao, J.; Stoltz, M.;
Muller, P.; Graber, M. J. Org. Chem. 2009, 74, 3621–3625. (c) Cordes,
M.; Jacques, O.; Kottgen, A.; Jasper, C.; Boudebous, H.; Giese, B.
AdV. Synth. Catal. 2008, 350, 1053–1062. (d) Cordes, M.; Kottgen,
A.; Jasper, C.; Jacques, O.; Bodebous, H.; Giese, B. Angew. Chem.,
Int. Ed. 2008, 47, 3461–3463. (e) Giese, B.; Napp, M.; Jacques, O.;
Boudebous, H.; Taylor, A. M.; Wirz, J. Angew. Chem., Int. Ed. 2005,
44, 4073–4075.
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864 J. AM. CHEM. SOC. VOL. 132, NO. 2, 2010

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A R T I C L E S
Scheme 1a
a Reagents and conditions: for 19 and 20, (a) PdCl2(PPh3)2, DMF, room temperature, 12 h; for 23 and 24, (b) Pd2(dba)3, P(o-tolyl)3, DMF, 50 °C, 12 h;
(c) Pd-C, H2, MeOH, room temperature, 12 h (abbreviations: pyr ) pyridine; pym ) pyrimidine).
Scheme 2a
B. Acid-Base Chemistry. The pKa’s of the tyrosine analogues
(10-15) were determined using UV-vis spectroscopy and
compared to that measured for tyrosine itself (9). To avoid
complications arising from the free carboxylic acid and amino
groups of the amino acid, they were left protected as the methyl
ester and tert-butoxy carbamate groups, respectively. The UV
spectra, which were recorded as a function of pH to obtain
characteristic sigmoidal titration curves, revealed a slight red
shift upon incorporation of N-atoms in the aromatic ring of
tyrosine. Upon deprotonation, all of the derivatives exhibited a
bathochromic shift, and from the changes in absorbance the
fraction of the amino acid in its aryloxide ion form (faryloxide
)
was determined at different pH values. The pKa values were
then determined by plotting faryloxide as a function of pH. The
data are shown in Figure 1 along with the calculated pKa values,
which span a range of 7.0-10.7 for 9-14. The pyridyl
analogues 10 and 13 exhibited double sigmoidal curves due to
protonation of the ring nitrogen at acidic pH. Introduction of
nitrogen in tyrosine’s phenolic ring lead to a decrease in the
phenolic O-H pKa of 1.9 on going from 9 to 10 and another
1.3 upon introduction of a second nitrogen on going from 10
to 11 (left panel of Figure 1). Methyl substitution raised the
pKa’s of all of these analogues, by 0.5 and 0.3 for the phenol
and pyrimidinol, respectively, and 1.3 for the pyridinol (right
panel of Figure 1). The pKa of the tert-butylated tyrosine
analogue (15) was determined to be ∼13 in the same way (see
Supporting Information).
C. Redox Chemistry. The oxidation (anodic) peak potentials
(Epa) of the tyrosine analogues (10-15) were determined as a
function of pH in the physiologically relevant range of ∼6.0 to
∼9.0 using differential pulse voltammetry and, again, compared
to the profile for tyrosine (9). The data are shown in Figure 2
and display a potential range from ∼250 to ∼950 mV for 9-14
a Reagents and conditions: (a) NBS, MeCN, 12 h, reflux, 91% (R )
Me), 85% (R ) t-Bu), (b) BnBr, NaH, THF, room temperature, 12 h, 81%,
(c) CuI, NaI, N,N-dimethylethylene diamine, 1,4-dioxane, reflux, 48 h,
quant., (d) for 31, 30, Pd2(dba)3, P(o-tolyl)3, DMF, 50 °C, 12 h, 48%; for
15, 28, PdCl2(PPh3)2, 80 °C, 12 h, 22%, (e) Pd-C, H2, MeOH, room
temperature, 12 h, 85%.
coupling proceeded best with PdCl2(PPh3)2 at room temperature
in DMF. The next step, cleavage of the benzyl ethers of 19 and
20 to yield the aryl alcohols 10 and 11, was easily accomplished
by catalytic hydrogenation in MeOH and purified by flash
column chromatrography in good yield. This sequence was also
employed in the preparation of the analogous pyridinol- and
pyrimidinol-based amino acids featuring methyl substitution in
the ortho positions relative to the phenolic hydroxyl. The cross-
couplings of the more electron-rich methylated aryl bromides
21 and 22, also prepared from the corresponding amines,
proceeded best at slightly higher temperature (50 °C) with
Pd2(dba)3/P(o-tolyl)3 as the catalyst to give the benzyl-protected
alcohols 23 and 24, which were liberated to yield 13 and 14.
The preparation of analogues of tyrosine with methyl and
tert-butyl substitution in the ortho positions relative to the
phenolic hydroxyl involved the same two-step Pd-catalyzed
cross-coupling/catalytic hydrogenation sequence, following
preparation of the appropriately substituted aryl halides (Scheme
2). Bromination of the commercially available 2,6-dialkylated
phenols yielded the corresponding 4-bromophenols 25 and 26,
but while benzyl protection of the former was facile, benzylation
of the latter proved cumbersome. Hence, the cross-coupling of
26 and 16 was carried out directly to yield 15, albeit in low
yield, while the methylated substrate 25 was coupled as the
benzyl ether following a Cu-catalyzed Finkelstein reaction on
2714 to generate the more reactive aryl iodide coupling partner
28. Subsequent removal of the benzyl group from 29 by catalytic
hydrogenation yielded the ortho methylated tyrosine 12.
(11) (a) Pratt, D. A.; DiLabio, G. A.; Brigati, G.; Pedulli, G. F.; Valgimigli,
L. J. Am. Chem. Soc. 2001, 123, 4625–4626. (b) Valgimigli, L.;
Brigati, G.; Pedulli, G. F.; DiLabio, G. A.; Mastragostino, M.;
Arbizzani, C.; Pratt, D. A. Chem.-Eur. J. 2003, 9, 4997–5010. (c)
Wijtmans, M.; Pratt, D. A.; Valgimigli, L.; DiLabio, G. A.; Pedulli,
G. F.; Porter, N. A. Angew. Chem., Int. Ed. 2003, 42, 4370–4373. (d)
Wijtmans, M.; Pratt, D. A.; Brinkhorst, J.; Serwa, R.; Valgimigli, L.;
Pedulli, G. F.; Porter, N. A. J. Org. Chem. 2004, 69, 9215–9223. (e)
Kim, H.-Y.; Pratt, D. A.; Seal, J. R.; Wijtmans, M.; Porter, N. A.
J. Med. Chem. 2005, 48, 6787–6789. (f) Nam, T.-G.; Rector, C. L.;
Kim, H.-Y.; Sonnen, A. F.-P.; Meyer, R.; Werner, W. M.; Atkinson,
J.; Rintoul, J.; Pratt, D. A.; Porter, N. A. J. Am. Chem. Soc. 2007,
129, 10211–10219. (g) Nara, S. J.; Jha, M.; Brinkhorst, J.; Zemanek,
T. J.; Pratt, D. A. J. Org. Chem. 2008, 73, 9326–9333.
(12) Tabanella, S.; Valancogne, I.; Jackson, R. F. W. Org. Biomol. Chem.
2003, 1, 4254–4261.
(13) Francom, P.; Janeba, Z.; Shibuya, S.; Robins, M. J. J. Org. Chem.
2002, 67, 6788–6796.
(14) Klapars, A.; Buchwald, S. L. J. Am. Chem. Soc. 2002, 124, 14844–
14845.
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A R T I C L E S
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Figure 1. Fraction of aryloxide ion as a function of pH for tyrosine (b) and its analogues: 10 (9), 11 (2), 12 (O), 13 (0), and 14 (4).
Figure 2. Oxidation (peak) potentials as a function of pH for tyrosine (b) and its analogues: 10 (9), 11 (2), 12 (O), 13 (0), and 14 (4).
Table 1. EPR Spectral Parameters (HSC ) Hyperfine Splitting Constants) for the Aryloxyl Radicals Derived From Tyrosine (9) and Its
Analogues (10-15)
HSCs ortho (G)
HSCs meta (G)
HSCs methylene (G)
g-factor
9
a(2H) 6.61
a(2H) 1.84
a(H) 7.29; a(H) 6.77
a(H) 11.41; a(H) 7.7
a(H) 10.72; a(H) 9.1
a(H) 6.30; a(H) 6.35
a(H) 10.32; a(H) 10.02
a(H) 9.14; a(H) 9.12
a(H) 7.10; a(H) 6.53
2.0048
2.0048
2.0049
2.0047
2.0048
2.0049
2.0046
10
11
12
13
14
15
a(H) 8.15; a(H) 5.5
a(H) 8.65; a(H) 8.0
a(6H) 6.50
a(3H) 8.95; a(3H) 6.52
a(6H) 6.84
a(N) 2.17; a(H) 1.40
a(N) 2.75; a(N) 0.95
a(2H) 1.77
a(N) 2.20; a(H) 1.15
a(2N) 1.86
a(18H) ∼0.1
a(2H) 1.79
over this pH range. Introduction of nitrogen in tyrosine’s
phenolic ring lead to an increase in Epa of ca. 200 mV on going
from 9 to 10 and another ca. 200 mV on going from 10 to 11
(left panel of Figure 2) and resulted in a leveling of the Epa-pH
profile for 10 and 11 once the pH reached the values of their
phenolic pKa’s (vide supra). Methyl substitution lead to a
systematic drop in Epa values by roughly 150-200 mV on
comparing 12-14 to 9-11 (right panel of Figure 2 to right
panel of Figure 2). The corresponding potential range through
the same pH range for the tert-butylated tyrosine analogue (15)
was determined to be roughly 230-370 mV (see Supporting
Information).
D. EPR Spectra. The EPR spectra of the aryloxyl radicals
derived from the tyrosine analogues (10-15) were recorded
upon exposure to a 10% v/v solution of di-tert-butyl peroxide
in benzene, and compared to the spectrum derived from tyrosine
(9) obtained under the same conditions. The spectral parameters
are collected in Table 1. The g-factors, determined to be in the
range of 2.0046-2.0049, were corrected with respect to 2,6-
di-tert-butyl-4-methylphenol (or butylated hydroxytoluene, BHT)
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866 J. AM. CHEM. SOC. VOL. 132, NO. 2, 2010

Tyrosine Analogues for Probing PCET Processes
A R T I C L E S
Figure 3. EPR spectra of the phenoxyl radicals derived from tyrosine (9), 3,5-dimethylated tyrosine (12), and 3,5-di-tert-butylated tyrosine (15) in benzene
at 298 K and the corresponding simulated spectra.
Table 2. O-H Bond Dissociation Enthalpies in kcal/mol of
and 3,5-di-tert-butylphenol, for which g-values of 2.0046 and
Tyrosine (9) and Its Analogues (10-15) Determined by the
2.0048 are known, respectively.15 The spectra for the series of
Radical Equilibration EPR Technique with the Indicated Reference
Compound
aryloxyl radicals derived from 9, 12 and 15 are shown as
examples in Figure 3 (other representative spectra are included
as Supporting Information). The increase in the persistence of
the aryloxyl radicals upon introduction of methyl and tert-butyl
groups in the ortho positions relative to the phenoxyl oxygen
is clear from these spectra.
reference
measured Keq ((SD) O-H BDE/kcal/mola
9
3,5-di-tert-butylphenol
10 3,5-di-tert-butylphenol
11 3,5-di-tert-butylphenol
12 2,6-di-tert-butyl-4-methylphenol
13 2,6-di-tert-butyl-4-methylphenol
14 2,6-di-tert-butyl-4-methylphenol
15 2,6-di-tert-butyl-4-methylphenol
0.6 ( 0.1
5.2 ( 3.1
9.0 ( 6.7
12.6 ( 9.5
29.3 ( 3.9
57.6 ( 14.0
1.7 ( 0.3
85.2 ( 0.1
86.5 ( 0.6
86.8 ( 0.8
81.4 ( 0.8
81.9 ( 0.1
82.3 ( 0.2
80.2 ( 0.1
E. Radical Stability. The O-H bond dissociation enthalpies
(BDEs) of the tyrosine analogues (10-15) were determined
using the radical equilibration EPR (REqEPR) technique15 and
compared to that measured for tyrosine itself (9). The REqEPR
technique relies on the generation of the aryloxyl radical derived
from 9-15 in the presence of a reference compound of known
O-H BDE close to that of the compound under investigation
(within 2.5 kcal/mol), such that an equilibrium is established
that reflects the difference in the strengths of the O-H bonds.
The reference compounds used in these experiments were 2,6-
di-tert-butyl-4-methylphenol (or butylated hydroxytoluene, BHT)
and 3,5-di-tert-butylphenol, whose O-H BDEs are 79.9 ( 0.1
and 85.52 ( 0.3 kcal/mol,15 respectively.16 The EPR spectra
recorded under these conditions are thus superpositions of the
spectra of the two equilibrating aryloxyls, from which we can
obtain the equilibrium constants. Assuming a negligible entropy
change in the H-atom transfer reaction, the O-H BDEs can be
calculated directly from the equilibrium constants. The O-H
BDEs of 9-15, the equilibrium constants from which they were
derived, and the reference compounds used to establish the
equilibria are given in Table 2.
a Errors account for the SD in the measurement of Keq and do not
include the error in the reference BDE.
folding,17 ion channel function,18 translation,19 and signaling.20
The adaptation of native chemical ligation to protein semisyn-
thesis21 has resulted in a veritable surge in the application of
this approach to a variety of problems, one of the most
interesting of which is radical transport in proteins,1 a topic that
has captured the fascination of many.
Our previous work on pyridinol and pyrimidinol antioxi-
dants11 led us to believe that pyridinol and pyrimidinol analogues
of tyrosine could serve as useful alternatives to the fluorinated
tyrosine analogues (1-6)6 and 3-hydroxytyrosine (7)7 and
3-aminotyrosine (8)8 used by Stubbe, Nocera, and co-workers
to study charge transport in peptides and proteins. From our
previous work, we expected that the incorporation of nitrogen
in the phenolic side chain of tyrosine would substantially lower
the pKa and elevate E° and O-H BDE in a systematic way.
The capability to modulate the electronic properties of the
phenolic ring without the introduction of substituents onto the
ring that may change the steric requirements of the side chain
Discussion
The preparation and site-specific incorporation of unnatural
amino acids into proteins is a very powerful approach to the
study of a variety of protein-mediated biological processes,
including catalysis and energy conversion, as well as protein
(17) For example: Arnold, U.; Hinderaker, M. P.; Koditz, J.; Golbik, R.;
Ulbrich-Hofmann, R.; Raines, R. T. J. Am. Chem. Soc. 2003, 125,
7500–7501.
(18) For example: Valiyaveetil, F. I.; Sekedat, M.; MacKinnon, R.; Muir,
T. W. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 17045–17049.
(19) Maag, D.; Fekete, C. A.; Gryczynski, Z.; Lorsch, J. R. Mol. Cell 2005,
17, 265–275.
(15) Lucarini, M.; Pedrielli, P.; Pedulli, G. F.; Cabiddu, S.; Fattuoni, C. J.
Org. Chem. 1996, 61, 9259–9263. Lucarini, M.; Pedulli, G. F.;
Cipollone, M. J. Org. Chem. 1994, 59, 5063–5070.
(20) For example: Lu, W.; Gong, D.; Bar-Sagi, D.; Cole, P. A. Mol. Cell
2001, 8, 759–769.
(16) The original values included in reference 15 have been given as 1.1
kcal/mol lower here according to the revised value of the O-H BDE
in 2,4,6-tri-tert-butylphenol, see: Mulder, P.; Korth, H. G.; Pratt, D. A.;
DiLabio, G. A.; Valgimigli, L.; Pedulli, G. F.; Ingold, K. U. J. Phys.
Chem. A 2005, 109, 2647–2655.
(21) Muir, T. W. Annu. ReV. Biochem. 2003, 72, 249–289. David, R.;
Richter, M. P.; Beck-Sickinger, A. G. Eur. J. Biochem. 2004, 271,
663–677. Schwarzer, D.; Cole, P. A. Curr. Opin. Chem. Biol. 2005,
9, 561–569.
9
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A R T I C L E S
Nara et al.
seemed ideal for the purpose of making unnatural tyrosine
analogues for probing proton-coupled electron transfer reactions
in peptides and proteins.
slightly for the tyrosine and pyrimidine analogues (the pKa’s of
Tyr and mTyr are 10.2 and 10.7, respectively; Pym and mPym
are 7.0 and 7.3, respectively), but more so for the pyridine
analogue (the pKa’s of Pyr and mPyr are 8.3 and 9.6,
respectively). The origin of the greater susceptibility of the
pyridine analogue of tyrosine to changes in the acidity of the
O-H bond as a function of ortho methyl substitution is unclear,
but, most importantly, the trends are maintained. The increase
in pKa upon methyl substitution can be rationalized by the
destabilization of the aryloxide anion by the electron-donating
methyl substituent groups. This also leads to lower oxidation
potentials, roughly 200 mV lower for each of mTyr, mPyr, and
mPym relative to Tyr, Pyr, and Pym, respecitvely, allowing a
range of driving force of ca. 600 mV to be examined by the
full set of analogues. The O-H BDEs also drop as a result of
the electron donation of the methyl substituents, which stabilize
the electron-poor aryloxyl radicals derived from the aryl
alcohols. The difference is roughly 4 kcal/mol for each of the
analogues.
The preparation of the analogues was straightforward due to
the work of Jackson and co-workers on the Pd-catalyzed
arylation of an organozinc reagent derived from serine,12 which
was demonstrated to work well to attach various aryl moieties
to the methylene adjacent the R carbon of the amino acid. While
the isolated yields of the cross-coupling reactions are only
moderate (ca. 60%), the sequence is scaleable, allowing gram
quantities of the unnatural amino acids to be prepared, an
important feature that allows facile preparation of the quantities
required for introduction into model peptides by solid-phase
peptide synthesis, and eventually for incorporation into proteins
via expressed protein ligation.
The trends in physicochemical properties of tyrosine (Tyr)
and its pyridine and pyrimidine analogues (hereafter Pyr and
Pym) fit the expectations based on our earlier work. Upon
introduction of nitrogen into the phenolic ring of Tyr in lieu of
carbon at the 2- or 2- and 6-positions, the pKa dropped by 1.9
and 3.2 log units, respectively. These effects are slightly larger
than those obtained upon introduction of fluorine at either the
3- or 3- and 5-positions of tyrosine,6b which yield decreases of
∼1.6 and ∼2.8 log units, respectively, despite being further
removed from the reactive hydroxyl moiety. The oxidation
(peak) potentials also increase systematically along the Tyr/
Pyr/Pym series by ca. 200 mV for each nitrogen at physiological
pH. These differences are also greater than those seen in the
fluorinated tyrosines,6b which are ca. 60 mV for the 3-F and
then another 50 mV for the 3,5-F2.
The EPR spectra of the aryloxyl radicals derived from Tyr
and its pyridine and pyrimidine analogues could be determined
at room temperature in benzene, and the resulting spectra were
then used to determine the O-H BDEs of Tyr, Pyr, and Pym
by the radical equilibration EPR technique. These experiments
yielded an O-H BDE in tyrosine (protected as the N-Boc and
carboxymethyl ester) of 85.2 ( 0.2 kcal/mol, 2 kcal/mol lower
than the O-H BDE of phenol determined by the same method.16
This value compares well with the only other value in the
literature of 86.5 ( 0.5 kcal/mol determined from the pKa of
tyrosine and the one-electron reduction potential of the tyrosyl
radical in water (the O-H BDE of phenol by this method is
88.2 ( 0.3 kcal/mol).22 The pyridine and pyrimidine analogues
have O-H BDEs that are higher by 1.2 and 1.6 kcal/mol,
respectively. The increase in O-H BDE upon incorporation of
the nitrogen atom(s) is consistent with our previous observations
and is the result of their imparting greater electron poorness to
any already electron-deficient aryloxyl radical.11
The aryloxyl radicals derived from tyrosine and the analogues
each have characteristic EPR spectra. The pyrimidinoxyl radicals
derived from Pym and mPym are distinguished from the
phenoxyl radicals derived from Tyr and mTyr due to their
particular hyperfine splitting patterns, which include small
couplings due to the ring nitrogens (aN ) 1.0-2.8 G).
Interestingly, the pyrimidinoxyl radical derived from Pym is
asymmetric on the EPR time scale, with the two ring nitrogens
possessing distinctly different HSCs (and likewise the two ring
protons). This may indicate either the formation of an intramo-
lecularly-H-bonded structure, wherein one of the pyrimidine
nitrogen atoms is H-bonded to the amide N-H proton in a six-
membered ring conformation, or the restricted rotation of the
pyrimidine ring about the bond connecting C2 of the pyrimidine
ring and the benzyl-like carbon. The former explanation is
supported by the different magnitudes of the ring nitrogen
couplings, which are consistent with increased (negative) spin
density in the H-bonded position, while decreasing the spin
density at the counterlateral ring position. Of course, while this
interaction may be important in an isolated molecule in a non-
H-bond donating solvent, it is unclear if it would be observed
in a peptide or protein, providing nonetheless a potential source
of information (vide infra). Perhaps more significantly, the
benzyl-like diastereotopic hydrogens have different HSCs (this
is clearly supported by the corresponding second derivative
spectra; see Supporting Information for an example), and these
are 2-4 G larger for the pyrimidinoxyl radicals derived from
Pym (9.1, 10.72 G) and mPym (9.14, 9.12 G) than for the
phenoxyls derived from Tyr (7.29, 6.77 G) and mTyr (6.30,
6.35 G), reflecting the greater electron deficiency of the
pyrimidine ring. These couplings will allow for the unambiguous
assignment of a tyrosyl radical containing these heteroatoms.
The vacant ortho positions of the phenolic rings of Tyr, Pyr,
and Pym permit their further modification to systematically
extend the range of structure-activity space that can be accessed
by studies employing these analogues. The introduction of
methyl substituents in these positions leads to unnatural amino
acids (hereafter referred to as mTyr, mPyr, and mPym) with
elevated pKa’s as compared to the unsubstituted analogues,
The spectra of the pyridinoxyl radicals derived from Pyr and
mPyr are more complicated than either the phenoxyls or the
pyrimidinoxyls due to their asymmetry. Nevertheless, their EPR
spectra could be deconvoluted and the HSCs determined by
comparison with simulated spectra (see Supporting Information).
As for the pyrimidinoxyl radicals, the HSCs of the diastereotopic
benzylic H-atoms were characteristically larger for the pyridi-
noxyl radicals when compared to the phenoxyl radicals, with
values of 7.7 and 11.4 G for Pyr, and 10.32 and 10.02 G for
mPyr, respectively. Of course, intramolecular H-bonding be-
tween the ring nitrogen and amide N-H is expected to be more
prominent in pyridinoxyl radicals, which contain a more basic
(22) Using pulse radiolytic techniques to obtain a reversible potential as
in: Lind, J.; Shen, X.; Eriksen, T. E.; Mere´nyi, G. J. Am. Chem. Soc.
1990, 112, 479–482. It is important to note that the determination
of accurate O-H BDEs for these analogues allows for a better
rationalization of the effect of changes in driving force on changes
in the kinetics of PCET processes. Because the electrochemistry
of the tyrosyl/tyrosinate redox couples measured here, and also of
the fluorinated tyrosine analogues,6b is irreversible, thermodynamic
data cannot be cleanly extracted.
9
868 J. AM. CHEM. SOC. VOL. 132, NO. 2, 2010

Tyrosine Analogues for Probing PCET Processes
A R T I C L E S
ring nitrogen, than in the case of the pyrimidinoxyl radicals,
thereby leading to larger HSCs upon H-bond formation. Indeed,
the aN values of 2.17 and 2.20 G measured for pyridinoxyl
radicals from 10 and 13, respectively, are in agreement with
this expectation.
in mechanistic studies where systematic changes in the side
chain acidity, oxidizability, radical stability, nucleophilicity,
and nucleofugality are desired.
Experimental Section
Rationalization of the complex and distinctive hyperfine
pattern of the aryloxyl radicals derived from tyrosine and its
pyridine and pyrimidine analogues is an important step toward
the use of these unnatural amino acids to probe proton-coupled
electron transfer processes along peptide chains. Indeed, the
relative persistence of these aryloxyl radicals, particularly those
bearing either methyl or tert-butyl substituents, will allow room
temperature EPR investigations of the peptides into which they
will be inserted, enabling one to unambiguously assign the
position of the unpaired electron on the basis of the relevant
spectral differences among the aryloxyl radicals. The larger
coupling constants of the benzyl-like methylenic hydrogens for
the pyridinoxyl and pyrimidinoxyl radicals allow for their clear
distinction from phenoxyl radicals; furthermore, the hyperfine
pattern due to paramagnetic nuclei in meta-positions (1H S )
1/2, or 14N S ) 1) is distinctive even when similar HSC are
displayed (e.g., 9 a(2Hm) 1.84 G vs 14 a(2N) 1.86 G). As
discussed, intramolecular H-bonding in aryloxyl radicals derived
from compounds 10, 11, and 13 is clearly detectable in the EPR
spectra from variation of the spin density at the corresponding
positions. While this results in nonequivalence of the two meta
(and consequently ortho) positions, complicating the spectra and
reducing their intensity, it will also provide an excellent tool to
probe the cybotactic region and the occurrence of intermolecular
H-bonding in the corresponding sites of a peptide incorporating
such unnatural amino acids. Even the disappearance of such
intramolecular interaction upon incorporation of the amino acid
in the peptide would turn into a valuable source of information.
Materials and Methods. All reagents were purchased from
commercial sources and used without further purification, unless
otherwise indicated. Column chromatography was carried out using
Silica-P Flash silica gel (60 Å 40-63 µm, 500 m2/g) from Silicycle.
1H NMR and 13C NMR spectra were recorded at 25 °C on a Bruker
Avance spectrometer at 400 and 100 MHz, respectively. Mass
spectra were obtained on an Applied Biosystems/MDS Sciex
QSTAR XL QqTOF mass spectrometer. EPR spectra were recorded
on a Bruker Elexsys 500 equipped with a Super X-Band ER049
microwave bridge, FF-lock, and Gauss-meter for accurate field
calibration and g-factor measurement.
General Procedure for Bromo-dediazoniation. To a solution
of 5-benzyloxy-2-aminopyri(mi)dine (2.27 mmol) in 20 mL of dry
dibromomethane was added benzyltrimethylammonium bromide
(10.22 mmol, 4.5 equiv) followed by dropwise addition of tert-
butylnitrite (22.73 mmol, 10 equiv). The solution was stirred at
room temperature under nitrogen atmosphere and was monitored
on TLC. The reaction times ranged from 1 to 12 h depending on
the nucleophilicity of the amine. Upon completion, the reaction
was quenched by addition of aqueous NaHCO3 solution, and the
product was extracted with dichloromethane. The combined organic
extracts were washed with brine, dried over Na2SO4, and concen-
trated in vacuo. The residue was subjected to flash chromatography
(eluent: ethyl acetate/hexanes). The purified product was character-
1
ized by H NMR, 13C NMR, and mass spectrometry.
5-Benzyloxy-2-bromopyridine (17). Yield: 45%. 1H NMR (400
MHz, CDCl3): δ 8.15 (d, J ) 3.2 Hz, 1H, arom.), 7.43-7.37 (m,
6H, arom.), 7.17 (dd, J ) 8.8, 3.2 Hz, 1H, arom.), 5.11 (s, 2H,
-CH2) ppm. 13C NMR (100 MHz, CDCl3): δ 154.61, 137.90,
135.62, 132.42, 128.81, 128.51, 128.19, 127.54, 125.32, 70.77 ppm.
HRMS (ES+) calculated (M) 262.9946, observed 262.9946.
1
5-Benzyloxy-2-bromopyrimidine (18). Yield: 36%. H NMR
Conclusions
(400 MHz, CDCl3): δ 8.31 (s, 2H, arom.), 7.43-7.40 (m, 5H,
arom.), 5.16 (s, 2H, -CH2) ppm. 13C NMR (100 MHz, CDCl3): δ
152.44, 146.74, 142.97, 134.68, 128.97, 128.90, 127.65, 71.23 ppm.
HRMS (ES+) calculated (M) 265.9878, observed 265.9877.
Unnatural amino acids containing phenol, 3-pyridinol, and
5-pyrimidinol side chains have been synthesized by Pd-catalyzed
cross-couplings of appropriately substituted haloaryl benzyl
ethers and an organozinc reagent derived from serine followed
by catalytic hydrogenation. The pKa’s and oxidation (peak)
potentials of the analogues reveal that a wide range in both
proton- and electron-donor ability can be accessed by these
unnatural amino acids as a function of both incorporation of
nitrogen in the phenolic ring and introduction of methyl
substituents on the phenolic ring. The EPR spectra of the
aryloxyl radicals generated from these analogues are character-
ized by different signals, which could be useful in distinguishing
between tyrosyl radicals and the aryloxyl radicals derived from
its analogues, some of which differ considerably in their
stability, as revealed by radical equilibration EPR experiments
used to determine the O-H BDEs in tyrosine as well as the
newly synthesized analogues.
While our motivation for carrying out this work is to help
clarify the role of coupling of proton and electron transfer
in charge transport processes in peptides and proteins, it
should be pointed out that the unnatural amino acids reported
here are likely to be helpful in exploring other phenomena.
For example, Kim and Cole23 employed the fluorinated
tyrosine analogues 1-6 in mechanistic studies of protein
tyrosine kinase-catalyzed phosphoryl transfer reactions. One
can envision still other uses for our newly prepared analogues
(2S)-(N-tert-Butoxycarbonylamino)-3-(5′-benzyloxypyrid-2′-yl)-
propionic Acid Methyl Ester (19). To a flame-dried Schlenk flask
was weighed zinc powder (3.46 mmol, 0.226 g), and then it was
dried by heating it with a heat gun under vacuum. Iodine (0.076
mmol, 0.02 g) was then added to the dried zinc powder under
nitrogen. The flask was then heated with heat gun while under high
vacuum for 10 min for activation. The flask containing zinc slurry
was then cooled to 0 °C in an ice bath, and to it a solution of
iodoserine (1.153 mmol, 0.380 g) in DMF (3 mL) was added
dropwise and stirred at room temperature for 90 min for Zn
insertion. To this solution of organozinc compound were then
added 2-bromo-5-benzyloxypyridine (1.534 mmol, 0.406 g) and
PdCl2(PPh3)2 (0.062 mmol, 0.044 g), and they were stirred at room
temperature overnight. The reaction mixture was diluted with ethyl
acetate and washed with water and brine. The combined organic
extracts were dried over Na2SO4 and concentrated in vacuo. The
crude oil was subjected to flash chromatography (eluent: ethyl
acetate/hexanes) to yield 58%. 1H NMR (400 MHz, CDCl3): δ 8.30
(d, J ) 2.8 Hz,1H, arom.), 7.44-7.36 (m, 5H, arom.), 7.21-7.18
(dd, J ) 8.8, 3.2 Hz, 1H, arom.), 7.06 (d, J ) 8.4 Hz, 1H, arom.),
5.82 (d, 1H, J ) 7.2 Hz, 1H, -NH), 5.10 (s, 2H, -CH2), 4.67-4.64
(m, 1H, -CH), 3.70 (s, 3H, -OCH3), 3.27 (dd, J ) 14.8, 6.0 Hz,
1H, HCH), 3.21 (dd, J ) 14.8, 4.8 Hz, 1H, HCH), 1.44 (s, 9H,
-C(CH3)3) ppm. 13C NMR (100 MHz, CDCl3): δ 172.51, 155.50,
153.71, 149.36, 137.42, 136.19, 128.72, 128.30, 127.52, 123.87,
122.37, 79.69, 70.44, 53.28, 52.22, 38.45, 28.33 ppm. HRMS (ES+)
calculated (M) 386.1842, observed 386.1852.
(23) Kim, K.; Cole, P. A. J. Am. Chem. Soc. 1998, 120, 6851–6858.
9
J. AM. CHEM. SOC. VOL. 132, NO. 2, 2010 869

A R T I C L E S
Nara et al.
(2S)-(N-tert-Butoxycarbonylamino)-3-(5′-benzyloxypyrimid-2′-
yl)-propionic Acid Methyl Ester (20). The organozinc reagent (1.153
mmol) was prepared in the same way as described for pyridyl
analogue. To the solution of organozinc compound were then
added 2-bromo-5-benzyloxypyrimidine (1.534 mmol, 0.408 g)
and PdCl2(PPh3)2 (0.062 mmol, 0.044 g), and the mixture was
stirred at 50 °C overnight. The reaction mixture was diluted with
ethyl acetate and washed with water and brine. The combined
organic extracts were dried over Na2SO4 and concentrated in vacuo.
The crude oil was subjected to flash chromatography (eluent: ethyl
acetate/hexanes) to yield 55% and was characterized by 1H NMR,
13C NMR, and mass spectrometry. 1H NMR (400 MHz, CDCl3): δ
8.38 (s, 2H, arom.), 7.42-7.35 (m, 5H, arom.), 5.84 (d, 1H, J )
8.4 Hz, 1H, -NH), 5.13 (s, 2H, -CH2), 4.80-4.76 (m, 1H, -CH),
3.69 (s, 3H, -OCH3), 3.50 (dd, J ) 15.4, 5.4 Hz, 1H, HCH), 3.37
(dd, J ) 15.4, 4.6 Hz, 1H, HCH), 1.43 (s, 9H, -C(CH3)3) ppm.
13C NMR (100 MHz, CDCl3): δ 172.45, 159.46, 155.51, 151.16,
144.16, 135.31, 128.85, 128.65, 127.59, 79.74, 70.80, 52.30, 52.12,
39.79, 28.32 ppm. HRMS (ES+) calculated (M) 387.1794, observed
387.1791.
subjected to flash chromatography (eluent: ethyl acetate/hexanes)
1
to yield 57% and was characterized by H NMR, 13C NMR, and
mass spectrometry. 1H NMR (400 MHz, CDCl3): δ 7.45-7.34 (m,
5H, arom.), 6.81 (s, 1H, arom.), 5.89 (d, 1H, J ) 7.6 Hz, 1H, -NH),
4.81 (s, 2H, -CH2), 4.64 (m, 1H, -CH), 3.71 (s, 3H, -OCH3),
3.22 (dd, J ) 14.4, 5.6 Hz, 1H, HCH), 3.14 (dd, J ) 14.6, 4.6 Hz,
1H, HCH), 2.47 (s, 3H, -CH3), 2.23 (s, 3H, -CH3), 1.44 (s, 9H,
-C(CH3)3) ppm. 13C NMR (100 MHz, CDCl3): δ 172.55, 155.52,
151.68, 151.24, 150.92, 140.49, 136.92, 128.61, 128.27, 127.91,
124.03, 79.58, 74.55, 53.19, 52.15, 38.43, 28.34, 19.55, 15.95 ppm.
HRMS (ES+) calculated (M) 414.2155, observed 414.2153.
(2S)-(N-tert-Butoxycarbonylamino)-3-(5′-benzyloxy-4′,6′-dimeth-
ylpyrimidin-2′-yl)-propionic Acid Methyl Ester (24). The orga-
nozinc reagent (1.153 mmol) was prepared in the same way as
described for pyri(mi)dyl analogues. To the solution of organozinc
compound were then added 2-bromo-5-benzyloxy-4,6-dimethylpy-
rimidine (1.153 mmol, 0.339 g), P(o-tolyl)3 (0.125 mmol, 0.038
g), and Pd2dba3 (0.062 mmol, 0.057 g), and the mixture was stirred
at 50 °C overnight. The reaction mixture was diluted with ethyl
acetate and washed with water and brine. The combined organic
extracts were dried over Na2SO4 and concentrated in vacuo. The
crude oil was subjected to flash chromatography (eluent: ethyl
acetate/hexanes) to yield 57% and was characterized by 1H NMR,
13C NMR, and mass spectrometry. 1H NMR (400 MHz, CDCl3): δ
7.38-7.34 (m, 5H, arom.), 5.82 (d, 1H, J ) 8.4 Hz, 1H, -NH),
4.81 (s, 2H, -CH2), 4.77-4.74 (m, 1H, -CH), 3.68 (s, 3H,
-OCH3), 3.43 (dd, J ) 15.8, 5.4 Hz, 1H, HCH), 3.28 (dd, J )
15.6, 4.4 Hz, 1H, HCH), 2.38 (s, 6H, 2 × -CH3), 1.42 (s, 9H,
-C(CH3)3) ppm. 13C NMR (100 MHz, CDCl3): δ 172.49, 160.82,
160.19, 155.50, 148.57, 136.14, 128.71, 128.58, 128.09, 79.65,
75.19, 52.21, 52.01, 39.97, 28.34, 19.07 ppm. HRMS (ES+)
calculated (M) 415.2107, observed 415.2121.
General Procedure for Hydrogenolysis of 5-Benzyloxy Deriva-
tives of Pyri(mi)dines. A solution of benzyloxy protected amino
acid (0.545 mmol, 0.211 g) in 10 mL of MeOH was treated with
5% Pd on C (0.04 g), and the resulting black suspension was stirred
at room temperature under H2 atmosphere (1 atm) overnight. The
catalyst was removed by filtration through a pad of Celite, and
filtrate was concentrated under reduced pressure. The residue was
subjected to flash chromatography (eluent: ethyl acetate/hexanes).
1
The purified product was characterized by H NMR, 13C NMR,
and mass spectrometry. Although the conversion was quantitative
in all of the cases, the purification and isolation of the polar
derivatives led to slightly reduced yields.
1
1
Compound 10. Yield: 81%. H NMR (400 MHz, CDCl3): δ
Compound 13. Yield: 75%. H NMR (400 MHz, CDCl3): δ
8.17 (s, 1H), 7.19 (d, J ) 8 Hz, 1H), 7.08 (d, J ) 8 Hz, 1H), 5.75
(d, J ) 8 Hz, 1H), 4.67 (bs, 1H), 3.72 (s, 3H), 3.22 (m, 2H), 1.41
(s, 9H) ppm. 13C NMR (100 MHz, CDCl3): δ 172.73, 155.58,
152.42, 147.67, 136.74, 124.63, 124.46, 80.08, 53.51, 52.50, 38.36,
28.29 ppm. HRMS (ES+) calculated (M) 296.1372, observed
296.1377.
6.84 (s, 1H), 5.95 (d, J ) 8 Hz, 1H), 4.61 (d, J ) 4 Hz, 1H), 3.73
(s, 3H), 3.19 (m, 2H), 2.45 (s, 3H), 2.25 (s, 3H), 1.43 (s, 9H) ppm.
13C NMR (100 MHz, CDCl3): δ 172.53, 155.63, 148.80, 146.30,
143.66, 134.65, 124.27, 79.79, 53.55, 52.31, 37.60, 28.33, 18.39,
15.92 ppm. HRMS (ES+) calculated (M) 324.1685, observed
324.1686.
1
1
Compound 11. Yield: 82%. H NMR (400 MHz, CDCl3): δ
Compound 14. Yield: 88%. H NMR (400 MHz, CDCl3): δ
9.34 (bs, 1H), 8.30 (s, 2H), 5.95 (d, J ) 8.4 Hz, 1H), 4.79-4.81
(m, 1H), 3.71 (s, 3H), 3.44 (dd, J ) 15.4, 5.8 Hz, 1H), 3.36 (dd,
J ) 15.0, 4.2 Hz, 1H), 1.42 (s, 9H) ppm. 13C NMR (100 MHz,
CDCl3): δ 172.93, 157.53, 155.93, 150.26, 144.69, 80.51, 52.67,
52.35, 39.55, 28.31 ppm. HRMS (ES+) calculated (M) 297.1325,
observed 297.1323.
5.95 (d, 1H, J ) 8.0 Hz, 1H), 4.74-4.72 (m, 1H), 3.70 (s, 3H),
3.40 (dd, J ) 15.4, 5.4 Hz, 1H), 3.26 (dd, J ) 15.4, 4.6 Hz, 1H),
2.40 (s, 6H), 1.42 (s, 9H) ppm. 13C NMR (100 MHz, CDCl3): δ
172.77, 156.55, 155.73, 153.32, 146.22, 80.01, 52.38, 52.24, 39.23,
28.31, 18.64 ppm. HRMS (ES+) calculated (M) 325.1638, observed
325.1647.
5-Benzyloxy-2-bromo-4,6-dimethylpyridine (21). Yield: 33%. 1H
NMR (400 MHz, CDCl3): δ 7.43-7.36 (m, 5H, arom.) 7.14 (s,
1H, arom.), 4.81 (s, 2H, -CH2), 2.48 (s, 3H, -CH3), 2.22 (s, 3H,
-CH3) ppm. 13C NMR (100 MHz, CDCl3): δ 153.69, 151.89,
143.55, 136.46, 134.93, 128.70, 128.49, 128.05, 127.66, 74.85,
19.38, 15.95 ppm. HRMS (ES+) calculated (M) 291.0259, observed
291.0249.
4-Bromo-2,6-dimethylphenol (25). To the solution of 2,6-
dimethylphenol (16.37 mmol, 2.00 g) in acetonitrile (20 mL) was
added NBS (17.189 mmol, 3.06 g), and it was refluxed overnight.
After the completion of reaction as indicated by TLC, the reaction
mixture was concentrated under reduced pressure and residue
reconstituted in ethyl acetate. The organic solution was washed with
water and brine. The combined organic extracts were dried over
Na2SO4 and concentrated in vacuo. The crude residue was subjected
to flash chromatography (eluent: ethyl acetate/hexanes) to yield
5-Benzyloxy-2-bromo-4,6-dimethylpyrimidine (22). Yield: 33%.
1H NMR (400 MHz, CDCl3): δ 7.43-7.41 (m, 5H, arom.), 4.88
(s, 2H, -CH2), 2.44 (s, 6H, 2 × -CH3) ppm. 13C NMR (100 MHz,
CDCl3): δ 163.90, 149.87, 145.21, 135.61, 128.89, 128.86, 128.29,
75.60, 19.14 ppm. HRMS (ES+) calculated (M) 292.0211, observed
292.0208.
1
91%. H NMR (400 MHz, CDCl3): δ 7.13 (s, 2H), 4.69 (s, 1H),
2.24 (s, 6H) ppm. 13C NMR (100 MHz, CDCl3): δ 151.32, 131.02,
125.27, 112.04, 15.78 ppm. HRMS (ES+) calculated (M) 199.9837,
observed 199.9831.
(2S)-(N-tert-Butoxycarbonylamino)-3-(5′-benzyloxy-4′,6′-dimeth-
ylpyrid-2′-yl)-propionic Acid Methyl Ester (23). The organozinc
reagent (1.153 mmol) was prepared in the same way as described
for pyri(mi)dyl analogues. To the solution of organozinc compound
were then added 2-bromo-5-benzyloxy-4,6-dimethylpyridine (1.534
mmol, 0.450 g), P(o-tolyl)3 (0.125 mmol, 0.038 g), and Pd2dba3
(0.062 mmol, 0.057 g), and the mixture was stirred at 50 °C
overnight. The reaction mixture was diluted with ethyl acetate and
washed with water and brine. The combined organic extracts were
dried over Na2SO4 and concentrated in vacuo. The crude oil was
4-Bromo-2,6-di-tert-butylphenol (26). To the solution of 2,6-di-
tert-butylphenol (9.709 mmol, 2.00 g) in acetonitrile (20 mL) was
added NBS (10.194 mmol, 1.814 g), and it was refluxed overnight.
After the completion of reaction as indicated by TLC, the reaction
mixture was concentrated under reduced pressure and residue
reconstituted in ethyl acetate. The organic solution was washed with
water and brine. The combined organic extracts were dried over
Na2SO4 and concentrated in vacuo. The crude residue was subjected
to flash chromatography (eluent: ethyl acetate/hexanes) to yield
1
85%. H NMR (400 MHz, CDCl3): δ 7.28 (s, 2H), 5.19 (s, 1H),
9
870 J. AM. CHEM. SOC. VOL. 132, NO. 2, 2010

Tyrosine Analogues for Probing PCET Processes
A R T I C L E S
1.44 (s, 18H) ppm. 13C NMR (100 MHz, CDCl3): δ 152.92, 138.18,
127.85, 112.62, 34.50, 30.07 ppm. HRMS (ES+) calculated (M)
284.0776, observed 284.0782.
Determination of pKa’s of the Tyrosine Analogues by UV
Spectroscopy. A buffer solution of 10 mM potassium phosphate
and 200 mM KCl was adjusted to the desired pH using 1 M KOH
or 1 M HCl in the range of pH 2-14. The buffer solution (2 mL)
was transferred to a cuvette, and a blank UV spectrum was recorded.
To this solution was added the solution of amino acid in absolute
ethanol (25 mM, 4 µL), and the UV spectrum was recorded and
the pH remeasured. The absorption at the wavelength of maximum
absorption for the aryloxide ion was used to determine the fraction
of amino acid in its aryloxide ion form, faryloxide, using eq 1, where
2-Benzyloxy-5-bromo-1,3-dimethylbenzene (27). To the solution
of 4-bromo-2,6-dimethylphenol (8.409 mmol, 1.7 g) in dry tet-
rahydrofuran (20 mL) was added sodium hydride (9.249 mmol,
0.37 g), and the mixture was stirred for 5 min. The vigorous
evolution of hydrogen gas was observed. To the resulting suspension
was added benzyl bromide (9.249 mmol, 1.124 mL) dropwise, and
the mixture was stirred at room temperature overnight. After the
completion of reaction as indicated by TLC, the reaction was
quenched by addition of aqueous saturated NH4Cl solution. The
solvent was removed under reduced pressure, and residual oil was
diluted with ethyl acetate. The organic layer was washed with 1%
HCl solution, water, and brine. The combined organic extracts were
dried over Na2SO4 and concentrated in vacuo. The crude residue
was subjected to flash chromatography (eluent: ethyl acetate/
hexanes) to yield 81%. 1H NMR (400 MHz, CDCl3): δ 7.49-7.38
(m, 5H, arom.), 7.19 (s, 2H, arom.), 4.81 (s, 2H, -CH2), 2.29 (s,
6H, 2 × -CH3) ppm.13C NMR (100 MHz, CDCl3): δ 154.90,
137.26, 133.39, 131.49, 128.58, 128.15, 127.85, 116.53, 74.18,
16.27 ppm. HRMS (ES+) calculated (M) 290.0306, observed
290.0302.
A is the absorbance at λmax
.
A(pH) - A(pHlow
A(pHhigh) - A(pHlow
)
faryloxide
)
(1)
)
Plotting the values of faryloxide as a function of pH resulted in the
familiar titration curve, which was fit to eq 2 to obtain the pKa.
10(pH-pKa)
faryloxide
)
(2)
(
)
1 + 10(pH-pKa)
Anodic Peak Potentials of Tyrosine and Its Analogues by
Differential Pulse Voltammetry. Voltammagrams were obtained
using an Electrochemical Analyzer from CH Instruments. The
experiments were performed in a glass cell with three electrodes.
The working electrode was glassy carbon and was polished using
0.05 µm alumina powder before each measurement. The reference
electrode was an Ag/AgCl reference electrode, and the counter
electrode was a platinum wire. The buffer solution, 10 mM
potassium phosphate containing 3 M KCl as supporting elec-
trolyte, was adjusted to the desired pH using 1 M KOH or 1 M
HCl in the range of pH 6-8.5. To 2 mL of buffer solution was
added the solution of amino acid in absolute ethanol (25 mM,
10 µL), and the pH was remeasured. The solutions were
continuously sparged to remove oxygen. Under these conditions,
in buffered solutions of pH 7.0 (25 mM NaH2PO4/Na2HPO4),
the reversible Fe(CN)63-/4- couple was +203 mV versus the Ag/
AgCl, which allows potentials to be calculated versus NHE24
by adding 204 mV to the peak potentials in Figure 2. Voltam-
metry parameters: initial potential ) 0 V, final potential ) 1 V,
pulse width ) 0.05 s, pulse period ) 0.2 s.
1
2-Benzyloxy-5-iodo-1,3-dimethylbenzene (28). Yield: 79%. H
NMR (400 MHz, CDCl3): δ 7.47-7.39 (m, 7H), 4.80 (s, 2H), 2.27
(s, 6H) ppm. 13C NMR (100 MHz, CDCl3): δ 155.77, 137.59,
137.23, 133.80, 128.59, 128.16, 127.85, 87.88, 74.16, 16.08 ppm.
HRMS (ES+) calculated (M) 338.0168, observed 338.0164.
(2S)-(N-tert-Butoxycarbonylamino)-3-(2′-benzyloxy-1′,3′-dimeth-
ylphen-5′-yl)-propionic Acid Methyl Ester (29). The organozinc
reagent (1.153 mmol) was prepared in the same way as described
for pyri(mi)dyl analogues. To the solution of organozinc compound
were then added 5-benzyloxy-2-iodo-4,6-dimethylbenzene (1.153
mmol, 0.390 g), P(o-tolyl)3 (0.125 mmol, 0.038 g), and PdCl2 (0.062
mmol, 0.011 g), and the mixture was stirred at 50 °C for 1 h. The
reaction mixture was diluted with ethyl acetate and washed with
water and brine. The combined organic extracts were dried over
Na2SO4 and concentrated in vacuo. The crude oil was subjected to
flash chromatography (eluent: ethyl acetate/hexanes) to yield 48%.
1H NMR (400 MHz, CDCl3): δ 7.40-7.33 (m, 2H), 7.31-7.26
(m, 3H), 6.70 (s, 2H), 4.91 (d, J ) 8 Hz, 1H), 4.71 (s, 2H),
4.45-4.42 (m, 1H), 3.64 (s, 3H), 2.93 (dd, J ) 16.0, 8.0 Hz, 1H),
2.85 (dd, J ) 16.0, 8.0 Hz, 1H), 2.18 (s, 6H), 1.35 (s, 9H) ppm.
13C NMR (100 MHz, CDCl3): δ 171.50, 154.10, 153.76, 136.63,
130.39, 130.06, 128.68, 127.48, 126.93, 126.72, 78.83, 72.93, 53.49,
51.09, 36.61, 27.29, 15.38 ppm. HRMS (ES+) calculated (M)
413.2202, observed 413.2184.
EPR Spectra of the Tyrosyl Radical and Its Analogues. Radicals
were generated at 298 K by irradiation of the sample with a 500
W high-pressure Hg-lamp directly in the spectrometer cavity,
equipped with a quartz Dewar, controlled by a Bruker B-VT100
variable temperature unit. The actual temperature inside the cavity
was monitored before and after each experiment with a Delta OHM
HD9218 type K thermocouple and was stable within (0.1 °C.
Samples, prepared in 4.0 mm i.d. suprasil quartz tubes and
consisting of doxygenated benzene solutions of di-tert-butylperoxide
(10% v/v M) containing the protected aminoacids 9-15 at various
concentration, were photolyzed inside the cavity (vide infa), and
spectra were collected. Digitized spectra were subjected to cepstral
analysis and autocorrelation numerical approaches to obtain initial
values of HSC, which were subsequently used in interactive fitting
with computer simulated spectra, using Monte Carlo method. The
software used both for numerical analysis and for simulation was
SIMESR developed at the University of Bologna by Prof Marco
Lucarini. HSC values were assigned by analogy with know
phenoxyl,15 pyridinoxyl,11c and pyrimidinoxyl11c radicals.
1
Compound 12. Yield: 85%. H NMR (400 MHz, CDCl3): δ
6.73 (s, 2H), 5.00 (d, J ) 8.0 Hz, 1H), 4.52 (d, J ) 8.0 Hz, 1H),
3.74 (s, 3H), 2.98-2.92 (m, 2H), 2.21 (s, 6H), 1.44 (s, 9H) ppm.
13C NMR (100 MHz, CDCl3): δ 172.67, 155.14, 151.30, 129.39,
127.32, 123.09, 79.86, 54.63, 52.12, 37.39, 28.32, 15.91 ppm.
HRMS (ES+) calculated (M) 323.1733, observed 323.1732.
Compound 15. The organozinc reagent (1.029 mmol) was
prepared in the same way as described for pyri(mi)dyl analogues.
To the solution of organozinc compound were then added 4-bromo-
2,6-di-tert-butylphenol (0.776 mmol, 0.222 g) and PdCl2(PPh3)2
(0.055 mmol, 0.039 g), and the mixture was stirred at 80 °C for
12 h. The reaction mixture was diluted with ethyl acetate and
washed with water and brine. The combined organic extracts were
dried over Na2SO4 and concentrated in vacuo. The crude oil was
subjected to flash chromatography (eluent: ethyl acetate/hexanes)
to yield 22%. 1H NMR (400 MHz, CDCl3): δ 6.90 (s, 2H, arom.),
5.14 (s, 1H, -OH), 4.98 (d, 1H, J ) 8.0 Hz, 1H, -NH), 4.60-4.55
(m, 1H, -CH), 3.74 (s, 3H, -OCH3), 3.03 (d, J ) 5.6 Hz, 2H,
CH2), 1.44 (s, 27H, 3 × -C(CH3)3) ppm. 13C NMR (100 MHz,
CDCl3): δ 172.54, 155.03, 152.82, 135.88, 126.32, 125.89, 79.74,
54.43, 52.15, 37.96, 34.25, 30.31, 28.36 ppm. HRMS (ES+)
calculated (M) 408.2749, observed 408.2737.
Determination of O-H BDEs of Tyrosine and Its Ana-
logues. A deoxygenated benzene solution containing the compound
under investigation (0.01-1 M), the appropriate reference phenol
(0.01-0.2 M), and di-tert-butyl peroxide (0.1M) was sealed under
nitrogen in a suprasil quartz EPR tube sitting inside the thermo-
statted cavity of an EPR spectrometer. Photolysis was carried out
by focusing the unfiltered light from a 500 W high-pressure mercury
(24) O’Reilly, J. E. Biochim. Biophys. Acta 1973, 292, 509.
9
J. AM. CHEM. SOC. VOL. 132, NO. 2, 2010 871

A R T I C L E S
Nara et al.
lamp on the EPR cavity. With compounds originating relatively
persistent radical species (i.e., aryloxyl radicals showing no decay
during an EPR fieldsweep), a short (1-5 s) UV pulse was used,
while with the less persistent radicals continuous photolysis was
necessary to establish “radical buffer”conditions.15 The temperature
was controlled with a standard variable temperature accessory
and was monitored before and after each run. Relative radical
concentrations were determined by comparison of the digitized
experimental spectra with computer simulated ones. An iterative
least-squares fitting procedure based on the systematic applica-
tion of the Monte Carlo method was performed constraining the
spectral parameters to a limited variation, while varying their
relative intensities. At least three different initial concentration
ratios of the two equilibrating species were investigated for each
compound, and at least three determinations were obtained for
each concentration. To ensure that equilibrium was established,
different irradiation intensities were obtained by intercepting the
UV-light with calibrated metal sectors.
Acknowledgment. We are grateful for the support of the Natural
Sciences and Engineering Research Council of Canada, Ontario
Ministry of Innovation and Queen’s University. D.A.P. also
acknowledges the support of the Canada Research Chairs program.
L.V. and G.F.P. acknowledge financial support from MURST
(Rome, Italy) and from the University of Bologna (Italy). We are
grateful to Prof M. Lucarini (Bologna) for access to the EPR
analysis/simulation software.
Supporting Information Available: Further synthetic details
1
and associated characterization, H and 13C NMR spectra of
key compounds, titration curve and pH-dependent potentials for
15, sample EPR spectra, and associated simulations. This
material is available free of charge via the Internet at http://
pubs.acs.org.
JA907921W
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872 J. AM. CHEM. SOC. VOL. 132, NO. 2, 2010