Synthesis of redox-active fluorinated 5-hydroxytryptophans as molecular reporters for biological electron transfer

Article abstract of DOI:10.1039/d1cc00187f

Fluorinated 5-hydroxytryptophans (F_n-5HOWs) were synthesized in gram scale quantities and incorporated into a β-hairpin peptide and the protein azurin. The redox-active F_n-5HOWs exhibit unique radical spectroscopic signatures that expand the function of 5HOW as probes for biological electron transfer.

Full text of DOI:10.1039/d1cc00187f

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Synthesis of redox-active fluorinated
5-hydroxytryptophans as molecular reporters for
biological electron transfer†
Cite this: Chem. Commun., 2021,
57, 3107
Received 11th January 2021,
Accepted 10th February 2021
Amanda Ohler,‡a Hanna Long,‡a Kei Ohgo,a Kristin Tyson,a David Murray,a
Amanda Davis,a Chris Whittington,a Eli G. Hvastkovs,a Liam Duffy,b Alice Haddy,b
a
Andrew L. Sargent,a William E. Allena and Adam R. Offenbacher
*
DOI: 10.1039/d1cc00187f
rsc.li/chemcomm
Fluorinated 5-hydroxytryptophans (Fn-5HOWs) were synthesized in and spectroscopic properties, could have many potential
gram scale quantities and incorporated into a b-hairpin peptide and applications, ranging from biophysical probes to protein
the protein azurin. The redox-active Fn-5HOWs exhibit unique radical engineering and peptide-based pharmaceuticals.7
spectroscopic signatures that expand the function of 5HOW as probes
for biological electron transfer.
Among available indole rings with subtle ring modifications,
5-hydroxytryptophan (5HOW) was selected for its desirable
redox properties (Table S1, ESI†). In this report, we show
Proton-coupled electron transfer (PCET) is a ubiquitous and that oxidation of 5HOW occurs at the hydroxy group, which
fundamental process in biological energy conversion and enables manipulation of its indoxyl pKa and the spectral
catalysis. In addition to metals, proteins exploit their natural features of its radical state through introducing a flanking,
amino acid building blocks, primarily tyrosine (Y) and trypto- nuclear spin active fluorine atom, similar to that reported for
phan (W), to shuttle electrons, sometimes reversibly, over long fluorotyrosines.9,10 Further, we present a facile synthetic
distances.1,2 The mechanistic exploration of tyrosine-mediated approach that leads to high yields (80–90% conversion from
PCET has been advanced by the advent of unnatural amino the indole) of enantiomerically pure, monofluorinated L-5HOWs
acids (UAAs), most notably fluoro- and nitro-tyrosine, that have and their Fmoc derivatives using a one-pot reaction (Scheme 1).
altered redox properties to enable the ‘trapping’ of organic The strategy, which exploits an evolved TrpB synthase,11 marks
radicals associated with PCET reactions.3 Despite its low a significant improvement over conventional synthetic meth-
abundance in nature, tryptophan is particularly appealing ods for unnatural L-Trp derivatives that require multiple steps
because it has a chemically complex and electron rich ring and are associated with low yields.12,13 While 5HOW is expected
structure that is expected to serve as a vital intermediary in to have different electrostatic properties than Trp (see Fig. S1,
long-range electron hopping pathways, including ribonucleo- ESI†), incorporation of 5HOW into different proteins has
tide reductase, cytochrome c oxidase, DNA photolyase, and been performed using human and E. coli expression systems
peroxidases.2 Tryptophan wires, found abundantly in oxidor- without significant deleterious effects on protein structure or
eductases, have been implicated in mitigating oxidative
damage.4 Mechanistic details of Trp-mediated electron transfer
in select systems are not well resolved, in part due to a lack of
tractable UUAs. Further, quantum coherent electron spins
between a flavin and tryptophan radical pair in cryptochromes
might even be responsible for macroscopic properties including
avian migration and plant circadian clocks.5,6 Thus, development
of novel tryptophan-based UAAs, with distinct thermodynamic
a Department of Chemistry, East Carolina University, Greenville, NC, USA.
E-mail: offenbachera17@ecu.edu
b Department of Chemistry and Biochemistry, University of North Carolina,
Scheme 1 One-pot chemoenzymatic synthetic strategy to prepare
L-Fn-5HOW UAAs and their Fmoc-protected derivatives. The model of
the b-hairpin peptide was generated from PepFold.8 The site of 5HOW is
shown by cyan sticks. For the peptide sequence, X represents the Fn-5HOWs
and O refers to L-ornithine. The azurin structure is from the PDB: 1E67.
Greensboro, NC, USA
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d1cc00187f
‡ Contributed equally.
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function.14,15 In former applications, 5HOW has been utilized
for its red-shifted electronic and fluorescence spectral features
as well as its ability to perform bioconjugation. Results,
presented herein, reveal unique thermodynamic and radical
spectroscopic properties of Fn-5HOWs. These data implicate,
for the first time, that 5HOW can be used as a biophysical
probe for electron transfer.
We began assessment of the spectroscopic and electrochemical
properties of the 5-hydroxyindole (5HOI) functional group.
The reduction potential of the 5HOIꢀ/5HOI couple from square
wave voltammetry (SWV) was found to be 565 Æ 5 mV at pH 7
(Fig. S2 and Table S2, ESI†). The value is ca. 300 mV lower than
Y (928 Æ 4 mV) or W (983 Æ 5 mV) at the same pH.
Mono-fluorination of the 5HOI ring at either the 4 or 6 position
(Scheme 1) produces E0 values of 579 Æ 6 and 595 Æ 4 mV,
respectively. The trends, though not the magnitude, of these poten-
tials are consistent with observations from fluorotyrosine9 and track
the DFT-calculated ionization potentials (IPs) of Fn-5HOI derivatives
(Table S2, ESI†). To explain these trends, the HOMOs, calculated
from the geometry optimized structure of 5HOI (Fig. 1), reveal that
the positions 4 and 6 in the 5-hydroxyindole ring have unequal p
contributions. This potentially serves as a framework to understand
the different IPs emerging for the 4F- and 6F-substituted 5HOI
species, since the impact from the halide’s inductive withdrawing
and resonance donating character will be communicated to the p
system to differing degrees depending on position.
Fig. 2 UV-vis (A–E) and EPR (F–J) spectra of Fn-5HOI (A–C and F–H),
tryptophan (D and I), and tyrosine (E and J). EPR spectra were collected at
77 K post UV photolysis from a pulsed Nd-YAG laser. EPR parameters are
presented in the ESI.† For UV, the solutions were prepared at 200 mM in
50 mM potassium phosphate, pH 7. For EPR, the solutions were prepared
at 50 mM in 50 mM sodium borate, pH 11. A pre-flash spectrum is shown
as a dashed line in (F).
Within the pH dependent range, E0 values for Fn-5HOI show
the expected pH dependence of B59 mV per pH unit,
consistent with loss of a proton coupled with side chain
oxidation (Fig. S2, ESI†). At elevated pH values (Z10), the
potentials are no longer dependent on pH, consistent with
the pKa values of the ground state species. To corroborate this
behaviour, the pKa values were determined using UV-visible
spectroscopy. The UV-visible spectra of aqueous solutions of
Fn-5HOI are shown in Fig. 2A–C. Their electronic absorbance
spectra are significantly red-shifted relative to and distinct from
Y and W (Fig. 2D and E). The features are dependent upon the
presence and positioning of the fluorine atom. Deprotonation
of the indoxyl proton causes a ca. 30 nm red shift of the 290 nm
feature in the electronic absorption spectrum (Fig. S3, ESI†).
From this shifted band, the pKa values of the Fn-5HOI species
were then estimated by pH titrations. The pKa of 5HOI was
determined as 10.90 Æ 0.02, comparable to the literature value
(11.1).16 As expected, mono-fluorination of 5HOI led to one-unit
decreases in the pKa (Table S2, ESI†).
Fig. 2F–J presents X-band EPR spectra at 77 K of photolysis-
generated radical species of Y, W, 5HOI, 4F-5HOI, and 6F-5HOI.
The 5HOIꢀ EPR spectrum (Fig. 2F) exhibits a typical line shape
for a g = 2 organic radical with little-to-no hyperfine interaction.
Importantly, the EPR spectra of 4F-5HOIꢀ (Fig. 2G) and 6F-5HOIꢀ
(Fig. 2H) are quite distinct from 5HOIꢀ and the characteristic
features of natural Y and W radicals (Fig. 2I and J). These
distinguishing EPR features establish that Fn-5HOIs could serve
Fig. 1 HOMO of the ground state for indole and 5HOI (A and B) and
Mulliken spin densities for Fn-5HOW radicals (D–F). The spin densities
were calculated for (C) Wꢀ, (D) 5HOWꢀ, (E) 4F-5HOWꢀ, and (F) 6F-5HOWꢀ
and the positive Mulliken spin density charges are displayed.
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as useful reporters of Trp radicals. Note that the hyperfine an off-white powder and was confirmed by MS and NMR.
interaction from the 19F nucleus at position 4 (Fig. 2G) with Similar yields were determined for the fluorinated derivatives.
the delocalized electron spin for the 5HOI radical is more
As proof-of-principle, L-5HOW and L-4F-5HOW were inserted
prominent than from the fluorine at the 6 position (Fig. 2H). into a designed b-hairpin peptide18 (cf. Scheme 1) using Fmoc
To provide a physical basis for these spectra, DFT calculated spin solid-state chemistry. One notable exception was the substitution
densities for the 5HOW derivatives (Fig. 1D–F) reveal larger spin of leucine at position 11 for phenylalanine to model the cross-
density character at the four position of the 5-hydroxyindole ring strand, staggered Trp-Phe p–p stacking interaction in the protein,
which, when coupled to the nuclear moment of a fluorine atom azurin (see below). The HPLC-purified 5HOW2, 4F-5HOW2,
substituted at this position, provides a rationale for the observed and 6F-5HOW2 peptides are highly soluble in aqueous solutions
hyperfine structure in the EPR spectrum for the 4F-5HOI radical (Z10 mM) and confer b-hairpin structure at room temperature
system relative to that of 6F-5HOI.
based on characteristic CD spectra (Fig. S11, ESI†). These peptides
Oxidation of tryptophan occurs by loss of a hydrogen atom exhibit reversible folding as indicated by the reproducible room
(Hꢀ) from the indole N–H bond in a PCET reaction. The spin temperature CD spectra before and after thermal denaturation.
density of the resulting unpaired electron is primarily spread The incorporations of Fn-5HOWs are confirmed by HRMS and the
across the indole ring with some spin density delocalized into UV-visible spectra (Fig. S12A–C, ESI†). It is important to note that
the backbone (Fig. 1C). The EPR detected hyperfine inter- the electronic spectra are slightly influenced by the secondary
actions and DFT calculations support that oxidation of Fn-5HOIs structure, consistent with the observation seen with tryptophan
by photolysis occurs through loss of Hꢀ at the indole OH bond. and tyrosine at position 2 in the peptide (Fig. S12D and E, ESI†).
Unlike Wꢀ, there is no predicted delocalization of spin density The light-generated neutral radical species were characterized by
into the backbone for 5HOWꢀ (Fig. 1D–F and Fig. S4, ESI†). EPR spectroscopy (cf. Fig. S12F–J, ESI†). Qualitatively, the EPR
Consistent with this, EPR spectra of 5HOI and L-5HOW neutral signals of Fn-5HOW radicals in the b-hairpin peptides are compar-
radicals are identical (Fig. S5, ESI†). Therefore, the EPR spectra of able to their sidechains (cf. Fig. 2, 3A and B and Fig. S12, ESI†),
Fn-5HOIꢀ in Fig. 2H–J are reasonable model spectra.
though the hyperfine interactions are less defined. Similar obser-
The Fn-5HOW amino acids were synthesized from Fn-5HOI vations were reported for tyrosyl radicals in other b-hairpins, and
and L-serine using an engineered, pyridoxal phosphate (PLP)- have been attributed to the dynamic interactions within the
dependent tryptophan B synthase (TrpB) variant from Thermo- peptide.19,20
toga maritima, Tm9D8*. This TrpB variant was previously found
To further demonstrate the functionality of Fn-5HOWs as
to be highly efficient for the conversion of substituted, non- amino acids, these UAAs were incorporated into the robust
canonical indole rings and was functionally optimized for model protein, azurin. Azurin was chosen because it contains
37 1C,17 which provides the ability to prepare redox-active side only a single tryptophan residue and only two tyrosines; the
chains in bacterial cultures. Reversed-phase HPLC analysis latter were mutated to phenylalanine to generate a tyrosine-
was used to determine the conversion of 5HOI (RT, 11 min) deficient variant (Y72F and Y108F) to enable direct spectro-
to L-5HOW (RT, 5.7 min) (Fig. S6A–F, ESI†). Previous reactions scopic and electrochemical characterization of the UAAs.
with engineered TrpB variants were carried out for extended Further, the tryptophan (W48) is buried and completely solvent
times (Z24 h) and with excess L-serine. Herein, we show excluded,21 enabling us to test the impact that these more polar
that with sub-stoichiometric L-serine (0.9 eq.), near complete 5HOW sidechains have on protein stability. 5HOW was
conversion of 5HOI to 5HOW can be attained within 4 h. At incorporated into azurin using AMBER stop codon suppression
longer reaction times, unwanted biproducts emerged. Using at position 48 with an engineered tryptophanyl–tRNA synthe-
these conditions, high conversions of 4F- and 6F-5HOW were tase (TrpRS)/tRNA pair. This strategy resulted in protein yields
achieved (cf. Fig. S7A–E, ESI†). Thus, we demonstrate an expanded of 2–5 mg LÀ1 of pure azurin (Fig. S13, ESI†) with L-5HOW
scope of this TrpB variant to these di-substituted indoles.
selectively incorporated at position 48 (80–90% incorporation
From the optimized Tm9D8* enzymatic reaction, the amino assessed by LC-MS/MS; Table S3, ESI†). Exclusion of either
groups of Fn-5HOW were Fmoc protected in the same reaction 5HOW or the TrpRS/tRNA pair to the expression system
vessel through the addition of dioxane as a co-solvent (see SI for resulted in truncated protein as assessed by SDS-PAGE
more details). Prior to adding Fmoc-succinimide (1 eq.), (not shown). CD spectra of 5HOW48 azurin is nearly identical
Tm9D8* was removed by Ni-NTA resin and filtration. The as- to the wild-type protein (Fig. S14, ESI†); the CD-derived
isolated, Fmoc-protected Fn-5HOW products (RT, 13 min) were melting temperature (Tm) of 5HOW48 is 71.9 Æ 0.2 1C versus
obtained in good yield (Z 90% crude mass) and at ca. 80% 74.0 Æ 0.7 1C for WT azurin (Table S4, ESI†). Contrary to 5HOW
purity as judged by HPLC integrations (Fig. S6H–J, ESI†). The solutions, the UV-visible spectrum of 5HOW48 shows some fine
final products of newly synthesized Fmoc-4F-5HOW and structure (cf. Fig. S15, ESI†), consistent with a low dielectric
Fmoc-6F-5HOW were confirmed by 1H and 13C NMR (Fig. S8 environment (cf. Fig. S16, ESI†).21
and S9, ESI†) and HRMS (Fig. S10, ESI†). To demonstrate the
Extending this biosynthetic method for the insertion of
scalability of the two-step synthesis, near quantitative yield of 4F- and 6F-5HOW in azurin resulted in low yields (1 mg mLÀ1).
Fmoc-L-5HOW (0.85 g crude; 96%) was isolated from 0.27 g For EPR characterization, the Fn-5HOW derivatives were incor-
of 5HOI starting material. The crude reaction product was porated using a tryptophan auxotroph (20 mg mLÀ1; see
sticky until purified. After purification, the product became Methods for details); this has been described for 5HOW
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analysed data. H. L. and A. S. performed DFT calculations.
A. O., H. L., and A. R. O. wrote the text.
The authors thank Prof. Frances Arnold for the plasmids
encoding evolved TrpB, Prof. Peter Schultz for plasmids encod-
ing the orthogonal TrpRS/tRNA pair, and Prof. John Latham for
help with HRMS data collection. This work was supported by
the National Science Foundation (REU 1851844) for A. Ohler
and (20-03956) to A. Offenbacher.
Conflicts of interest
There are no conflicts to declare.
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