Reversible proton coupled electron transfer in a peptide-incorporated naphthoquinone amino acid

Article abstract of DOI:10.1039/b815915g

The synthesis of a naphthoquinone amino acid and its electrochemical characterization in a peptide is presented. The Royal Society of Chemistry.

Full text of DOI:10.1039/b815915g

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Reversible proton coupled electron transfer in a peptide-incorporated
naphthoquinone amino acidw
´
Bruce R. Lichtenstein, Jose F. Cerda,z Ronald L. Kodery and P. Leslie Dutton*
Received (in Cambridge, UK) 11th September 2008, Accepted 5th November 2008
First published as an Advance Article on the web 25th November 2008
DOI: 10.1039/b815915g
The synthesis of a naphthoquinone amino acid and its electro-
chemical characterization in a peptide is presented.
ation provides chemically rigorous protection of the hydro-
quinone hydroxyls throughout the synthesis and during
peptide assembly, we were able to make use of a highly
specific oxidative deprotection step, selective enough to avoid
concomitant oxidative modification of tryptophan and
tyrosine within the same peptide.
Quinones are critical cofactors in the generation of trans-
membrane electrochemical proton gradients that drive the
production of ATP in photosynthesis and respiration.1–5
Relatively weak binding, poorly resolved structural details,
and indistinct spectroscopic characteristics of quinones in
catalytic sites of membrane protein complexes have long
challenged progress toward understanding their mechanistic
roles in energy conversion. In contrast, work on the catalytic
mechanisms of soluble quinoproteins has advanced in large
part because their amino-acid derived quinonoid cofactors are
covalently secured.6–9 The development of simple, water-
soluble, artificial proteins (maquettes) that bring the advan-
tages of covalently secured bioenergetic quinone cofactors to
the protein backbone provides both a platform to study
membrane quinone catalysis and immediate access to novel
biosynthetic devices. The key first step in producing such
maquettes is the development of a quinone amino acid,
reported here. We have chosen a naphthoquinone amino acid
(Naq, z) based upon menadione, one of the quinone head
groups common to energy conversion in the membranes of
photosynthetic and anaerobic organisms.
Synthesis begins with the protective acidic methylation of the
natural product 1,4-dihydroxy-2-naphthoic acid to methyl
1-hydroxy-4-methoxy-2-naphthoate (1)16 (Scheme 1). Further
methylation of 1 under basic conditions affords methyl
1,4-dimethoxy-2-naphthoate (2)17 in 84% yield after two steps.
Arylbromide, derived from carboxylate 2, combined with the
third-generation cinchonidinium18,19 asymmetric phase transfer
catalyst (PTC) alkylated tert-butyl N-(diphenylmethylene)-
glycinate to produce protected Naq (3) in 86% yield with
93% enantiomeric excess (ee). Standard acidic aqueous
deprotection conditions on 3 result in a black tar.20 While the
tar is avoided in acidic methanol,8 the yield is improved with
deprotection of 3 in TFA with ethanedithiol (EDT) as a
scavenger, which produces Naq(OMe/OMe) amino acid
hydrochloride 4 nearly quantitatively. Protection of the amine
Naq proves amenable to solid phase peptide synthesis
(SPPS) and incorporation into synthetic proteins. It is an
a-amino acid with a steric bulk similar to that of tryptophan,
which promises incorporation into natural secondary struc-
tural elements of proteins with little of the perturbation
encountered with cysteine ligation of quinones,10–12 and hence
facilitating catalytic site design. In addition, with substitution
of the highly reactive Michael centers around the para-quinone
ring, Naq is chemically robust, thereby allowing its study
and use under a wide variety of conditions; this contrasts with
non-substituted ortho- or para-quinone amino acids.13,14
The high yielding and readily scalable Naq synthesis centers
around the asymmetric phase transfer based synthesis of
amino acids developed by O’Donnell et al.15 While methyl-
The Johnson Research Foundation and Department of Biochemistry
and Biophysics, University of Pennsylvania, Philadelphia,
PA 19104-6059, USA. E-mail: Dutton@mail.med.upenn.edu;
Fax: +1 (1) 215-898-0465; Tel: +1 (1) 215-898-0991
w Electronic supplementary information (ESI) available: Synthetic
methodologies, compound characterization and spectroelectro-
chemical experimental setup. See DOI: 10.1039/b815915g
z Current address: Department of Chemistry, St. Joseph’s University,
Philadelphia, PA 19131, USA. Tel: +1 (1) 610-660-1787.
y Current address: Department of Physics, The City College of New
York, New York, NY 10031, USA. Fax: +1 (1) 212-650-6940;
Tel: +1 (1) 212-650-5502.
Scheme
1
Synthesis of Fmoc–Naq(OMe/OMe)–OH. (a) H2SO4,
MeOH, reflux, 93.4%; (b) CH3I, K2CO3, DMF, D, 90%; (c) LiAlH4,
THF, 0 1C, 99%; (d) PBr3, CCl4, 0 1C, 95.8%; (e) CsOH, N-(diphenyl-
methylene)glycine tert-butyl ester, cinchonidinium PTC, Tol, ꢀ20 1C,
86%, ee 93%; (f) EDT, TFA, 98.2%; (g) Fmoc–OSu, NaHCO3,
DMF, 85%.
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with Fmoc–OSu gives the SPPS-ready derivative Fmoc–
Naq(OMe/OMe)–OH 5.**
Fmoc–Naq(OMe/OMe)–OH (5) readily incorporates into
peptides via SPPS but the standard TFA based cleavage
cocktails fail to remove the methyl protecting groups of
the quinone hydroxyls. We tried a variety of one-step on-resin
deprotection/cleavage of peptides containing Naq using
modifications of Fmoc-SPPS cleavage reagents. Addition of
strong Lewis acids to these cocktails showed promise in
simultaneously deprotecting Naq(OMe/OMe) and natural
amino acids, but undesired side reactions associated with
Naq were unavoidable. Similarly, although the reaction of
Ac–Naq(OMe/OMe)–OMe with [bis(trifluoroacetoxy)iodo]-
benzene (PIFA), one of the mild hypervalent iodine(III)
oxidants used effectively in deprotecting
a number of
methyl-protected quinones,21 quantitatively produced the
electrochemically active quinone, we were unable to eliminate
unwanted reactions of this reagent with other natural amino
acids, notably tyrosine and tryptophan (data not shown).ww
However, reaction in water at pH 1.0 with stoichiometric
quantities of the oxidant cerium ammonium nitrate (CAN)22
led to quantitative and selective oxidative deprotection of the
Naq(OMe/OMe) side chain to yield the desired quinone.
Solution electrochemical characterization of Naq in a
Fig. 2 Oxidation–reduction of heptaNaq. Line drawn is for a two-
electron two-proton coupled redox center (ꢀ58 mV/pH at 20 1C).
Inset: Spectroelectrochemical data fit to the two-electron Nernst
equation from which the values of Em were derived. See ESI for
experimental conditions.w
naphthoquinone at 243 nm, and the isosbestic point at 252 nm
as a function of redox potential (Fig. 2). Naq oxidation–
reduction exhibits a two-electron reversible Nernstian behavior
(n = 2.0) over the pH range 5.2–8.3 at 20 1C. The pH
dependence of the Em (ꢀ58 mV/pH unit) of heptaNaq matches
the theoretical two-electron, two-proton coupled oxidation–
reduction expected for quinones over this pH range.24 The Em
of heptaNaq is ꢀ20 mV at pH 7.5, a value comparable to that
of the Naq analogue 2-methylnaphthoquinone (Em ꢀ47 mV at
pH 7.5). The modest difference can be explained by subtle
environmental effects caused by the peptide or more specific
hydrogen bonding interactions of the Naq side chain polar
groups with the backbone of the peptide.
peptide was performed with
a
synthesized heptamer
(heptaNaq, sequence PDP PDQ–NH2). HeptaNaq was selected
to be proline rich to decrease conformational flexibility while
increasing water solubility and facilitating aqueous Naq
electrochemistry. HeptaNaq(OMe/OMe) was readily syn-
thesized via SPPS and the natural amino acids deprotected
with 95 : 5 : 3 : 2 TFA–thioanisole–EDT–anisole. After HPLC
purification and deprotection, heptaNaq was re-purified to
produce oxidized, electrochemically active heptaNaq.
The reduced and oxidized electronic spectra of heptaNaq are
nearly identical to those of 2-methylnaphthoquinone (Fig. 1).
The oxidation–reduction midpoint potential (Em) of heptaNaq
as a function of pH was determined spectroelectrochemically by
following the difference between the absorption of the reduced
Observation of proton-limited redox potentials was accom-
plished using cyclic voltammetry in the aprotic solvent dimethyl-
formamide. Under slow scan rates, heptaNaq oxidation–reduction
peaks display clear scan-rate dependency likely to originate from
restricted protonation by protons associated with the peptide itself.
Indeed, at 50 mV sꢀ1 a ‘‘protonation peak’’ is observed indicative
of the availability of protons (Fig. 3). Moreover, although in this
scan rate range, the half-wave potential (E1/2 ꢀ328 mV versus
NHE) is significantly depressed relative to the aqueous midpoint, a
complex ece(c) electrochemical mechanism is evident which is
absent from studies of 2-methylnaphthoquinone under identical
conditions (data not shown). However, with increasing scan rates
both anodic and cathodic peaks split into two one-electron steps
and approach scan rate independence consistent with kinetic
bypass of protonation. Thus, at fast scan rates in the range of
2 V sꢀ1 the two proton uncoupled half-wave potentials are
ꢀ362 mV and ꢀ1088 mV (vs. NHE), again modestly positive of
the values of 2-methylnaphthoquinone in anhydrous, aprotic
DMF (ꢀ408 mV and ꢀ1142 mV versus NHE).23 The difference
in electrochemical potentials between heptaNaq and 2-methyl-
naphthoquinone can as before be explained by redox state
dependent interactions of Naq with the peptide or by the possible
presence of minute amounts of residual water in the experimental
setup.25
Fig. 1 Reduced and oxidized electronic spectra of heptaNaq (—) and
2-methylnaphthoquinone (-ꢂ-) in the presence of redox mediators.
Inset: Difference spectra of the two species. See ESI for details.w
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Notes and references
z The Greek character quoppa ( ) was chosen as the one-letter code
for Naq.
8 We suspect that in aqueous acid the methoxy groups are substituted
with hydroxyls which results in oxygen dependent quinone polymer-
ization.
** Interestingly, the 9-methylenefluorene by-product is polymerized if
the crude reaction mixture sits.
ww We believe that the hypervalent iodine species react selectively with
tryptophan and tyrosine due to the lower steric bulk of the covalent
intermediate formed with these amino acids.
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Fig. 3 Cyclic voltammetry of heptaNaq in dimethylformamide. Slow
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The ease with which Naq can be synthesized and incorporated
into robust protein scaffolds offers new ways to examine the
interplay between the protein structure and quinone redox and
protonation states without the immense complexity attendant
with natural quinone proteins. Electrochemically, the two-
electron two-proton couple of Naq is central to the known
range of biological redox cofactors and near the midpoint of the
quinones critical to respiration and photosynthesis. Thus Naq is
well positioned to foster new concepts and constructions of
multicofactor artificial proteins incorporating Naq and designed
to explore mechanistic aspects of natural oxidoreductases, or to
modulate quinone properties for novel specific catalytic pur-
poses. Potentials observed in the aprotic electrochemical studies
of heptaNaq represent an upper limit on the expected values for
these couples within a protein matrix, supporting the utility of
Naq in very low potential electron transfer chains within a
protein core. In addition, the proton-decoupled electron transfer
potentials are favourable for the implementation of constructs
using Naq as a very low potential reductive intermediate for the
generation of stable strongly reducing fuels such as hydrogen.
The authors thank Tammer A. Farid, Anne K. Jones and
Christopher C. Moser for useful discussions. This work was
supported by DOE Grant DF-FG02-05ER46223; USPHS Grant
GM41048 (P.L.D.); NSF-MRSEC Grant DMR05-20020.
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This journal is The Royal Society of Chemistry 2009
170 | Chem. Commun., 2009, 168–170