Ergothioneine in a peptide: Substitution of histidine with 2-thiohistidine in bioactive peptides

Article abstract of DOI:10.1002/psc.3339

Ergothioneine (EGT) is the betaine of 2-thiohistidine (2-thioHis) and may be the last undiscovered vitamin. EGT cannot be incorporated into a peptide because the α-nitrogen is trimethylated, although this would be advantageous as an EGT-like moiety in a peptide would impart unique antioxidant and metal chelation properties. The amino acid 2-thioHis is an analogue of EGT and can be incorporated into a peptide, although there is only one reported occurrence of this in the literature. A likely reason is the harsh conditions reported for protection of the thione, with similarly harsh conditions used in order to achieve deprotection after synthesis. Here, we report a novel strategy for the incorporation of 2-thioHis into peptides in which we decided to leave the thione unprotected. This decision was based upon the reported low reactivity of EGT with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), a very electrophilic disulfide. This strategy was successful, and we report here the synthesis of 2-thioHis analogues of carnosine (βAH), GHK-tripeptide, and HGPLGPL. Each of these peptides contain a histidine (His) residue and possesses biological activity. Our results show that substitution of His with 2-thioHis imparts strong antioxidant, radical scavenging, and copper binding properties to the peptide. Notably, we found that the 2-thioHis analogue of GHK-tripeptide was able to completely quench the hydroxyl and ABTS radicals in our assays, and its antioxidant capacity was significantly greater than would be expected based on the antioxidant capacity of free 2-thioHis. Our work makes possible greater future use of 2-thioHis in peptides.

Full text of DOI:10.1002/psc.3339

Received: 2 March 2021
DOI: 10.1002/psc.3339
Revised: 27 April 2021
Accepted: 30 April 2021
R E S E A R C H A R T I C L E
Ergothioneine in a peptide: Substitution of histidine with
2-thiohistidine in bioactive peptides
Kaelyn A. Jenny1 | Erik L. Ruggles1,2 | Matthew D. Liptak2
Douglas S. Masterson3 | Robert J. Hondal1,2
|
1Department of Biochemistry, College of
Medicine, University of Vermont, Burlington,
Vermont, USA
Ergothioneine (EGT) is the betaine of 2-thiohistidine (2-thioHis) and may be the last
undiscovered vitamin. EGT cannot be incorporated into a peptide because the
α-nitrogen is trimethylated, although this would be advantageous as an EGT-like
moiety in a peptide would impart unique antioxidant and metal chelation properties.
The amino acid 2-thioHis is an analogue of EGT and can be incorporated into a
peptide, although there is only one reported occurrence of this in the literature. A
likely reason is the harsh conditions reported for protection of the thione, with
similarly harsh conditions used in order to achieve deprotection after synthesis. Here,
we report a novel strategy for the incorporation of 2-thioHis into peptides in which
we decided to leave the thione unprotected. This decision was based upon the
reported low reactivity of EGT with 5,50-dithiobis(2-nitrobenzoic acid) (DTNB), a very
electrophilic disulfide. This strategy was successful, and we report here the synthesis
of 2-thioHis analogues of carnosine (βAH), GHK-tripeptide, and HGPLGPL. Each of
these peptides contain a histidine (His) residue and possesses biological activity.
Our results show that substitution of His with 2-thioHis imparts strong antioxidant,
radical scavenging, and copper binding properties to the peptide. Notably, we found
that the 2-thioHis analogue of GHK-tripeptide was able to completely quench the
hydroxyl and ABTS radicals in our assays, and its antioxidant capacity was signifi-
cantly greater than would be expected based on the antioxidant capacity of free
2-thioHis. Our work makes possible greater future use of 2-thioHis in peptides.
2Department of Chemistry, University of
Vermont, Burlington, Vermont, USA
3School of Mathematics and Natural Sciences,
Chemistry and Biochemistry, University of
Southern Mississippi, Hattiesburg, Mississippi,
USA
Correspondence
Robert J. Hondal, Department of Biochemistry,
College of Medicine, University of Vermont,
89 Beaumont Ave, Given Laboratory, Room
B413, Burlington, VT 05405, USA.
Email: robert.hondal@uvm.edu
Funding information
National Heart, Lung, and Blood Institute,
Grant/Award Number: HL141146
K E Y W O R D S
2-thio, histidine, antioxidant, antioxidant peptide, carnosine, copper binding, ergothioneine,
GHK-tripeptide, histidine
Abbreviations: 2-thioHis, 2-thiohistidine; ABTS, 2,20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt; Cu (II), copper (II); Cys, cysteine; DCM, dichloromethane; DIC,
diisopropylcarbodiimide; DMF, dimethylformamide; DMPO, 5,5-dimethyl-1-pyrroline-N-oxide; DPPH, 2,2-diphenyl-1-picrylhydrazyl; DTNB, 5,50-dithiobis(2-nitrobenzoic acid); EGT,
Ergothioneine; EPR, electron paramagnetic resonance; EtCA, ethyl (hydroxyimino)cyanoacetate; Fmoc, fluorenylmethoxycarbonyl; Fmoc-OSu, Fmoc N-hydroxysuccinimide ester; HATU, 1-[bis
(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate; His, histidine; HPLC, high-pressure liquid chromatography; KA, equilibrium association constant; KD
equilibrium dissociation constant; Meb, 4-methylbenzyl; MS, mass spectrometry or mass spectrometric; NMM, N-methylmorpholine; OH, hydroxyl radical; ROS, reactive oxygen species; RSA,
radical scavenging activity; SPPS, solid-phase peptide synthesis; TEA, triethylamine; TEAC, Trolox equivalent antioxidant capacity; TFA, trifluoroacetic acid; TIS, triisopropylsilane; TLC, thin-layer
chromatography; Trolox, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; UV–Vis, ultraviolet–visible; Αsc, ascorbate; βA, β-alanine; Η-βAH-OH or βAH, carnosine.
,
J Pep Sci. 2021;e3339.
wileyonlinelibrary.com/journal/psc
© 2021 European Peptide Society and John Wiley & Sons, Ltd.
1 of 12
https://doi.org/10.1002/psc.3339

2 of 12
JENNY ET AL.
1
|
INTRODUCTION
Ergothioneine (EGT) is the trimethylated betaine of 2-thiohistidine
(2-thioHis) and may be the last undiscovered vitamin.[1–7] EGT is pro-
duced by fungi and mycobacteria and has been shown to be a potent
antioxidant compound in vitro, but its precise biological function is
unknown.[1–7] Humans and other animals obtain EGT by ingesting
food and accumulate it in target tissues such as red blood cells, by the
use of a specific cation transporter.[1–8] EGT also chelates metals,
especially Cu (II), and prevents the metal from undergoing redox
cycling reactions that generate reactive oxygen species (ROS) that
damage biological macromolecules.[1,2,6,9]
FIGURE 2 Naturally occurring His-containing peptides. Carnosine
is a His-containing dipeptide found in the muscle of mammals where
it has important antioxidant, metal chelation, and buffering properties.
GHK-tripeptide is a matrikine from the extracellular matrix. It has a
high affinity for Cu (II) and helps to prevent oxidative stress and is
important for wound healing. Each has a His that can be replaced with
2-thioHis
EGT cannot be incorporated into
a peptide because the
α-nitrogen is trimethylated (Figure 1), although incorporation of an
EGT-like moiety into a peptide would impart unique antioxidant and
metal chelation properties to the peptide. This goal could be achieved
by incorporating 2-thioHis, an EGT analogue, into a peptide and is
possible because 2-thioHis has both α-amino and α-carboxylic acid
groups that enable coupling to other amino acids. An advantage of
such an approach is that peptides enable precise targeting to specific
tissues and organelles and have defined hydrophobicity.
and ultraviolet–visible (UV–Vis) spectroscopy. Our results support our
contention that the thione of 2-thioHis does not need to be protected
in solid-phase peptide synthesis (SPPS) and that the resulting
2-thioHis-containing peptides have enhanced antioxidant and metal
chelation properties.
We are aware of only one other reported instance of incorporat-
ing 2-thioHis into
a
peptide.[10] The previous work used
a
2
|
MATERIALS AND METHODS
| Materials
4-methylbenzyl (Meb) protecting group to protect the thione using
harsh conditions and then similarly harsh conditions to affect
deprotection.[10] Due to the reported low reactivity of the thione of
EGT towards thiol/disulfide exchange, we decided to take the novel
approach of not using any protecting group for the thione.[3,11] Such
an approach easily enables the advantageous incorporation of
2-thioHis in place of His. Examples of bioactive His-containing pep-
tides include carnosine, GHK-tripeptide, GHTD-amide, histatins, and
collagen-like peptides, some of which are depicted in Figure 2.[12–22]
In this report, we synthesized the 2-thioHis analogs of bioactive
peptides H-HGPLGPL-OH, H-GHK-OH, and H-βAH-OH (carnosine).
These His-containing peptides were chosen as targets because it has
been reported that they each have antioxidant and/or metal chelat-
ing properties that could be enhanced by substitution with
2-thioHis.[14–22]
2.1
Solvents for peptide synthesis were purchased from Fisher Scientific
(Pittsburgh, PA). 2,20-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
diammonium salt (ABTS) was purchased from TCI America (Portland,
Oregon). N-Fmoc amino acids were purchased from RSsynthesis
(Louisville, KY, USA). 2-chlorotritylchloride resin (100–200 mesh, 1%
DVB) was purchased from Novabiochem (St. Louis, MO, USA).
L-Carnosine was purchased from Acros Organics (Pittsburgh, PA, USA).
1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-oxide hexafluorophosphate (HATU) was purchased from Oakwood
Chemical (Estill, SC, USA). All other chemicals were purchased from
either Sigma-Aldrich (Milwaukee, WI), Acros Organics (Pittsburgh, PA,
USA), or Fisher Scientific (Pittsburgh, PA, USA). Samples were ionized
by electrospray ionization (ESI) on a Thermo Q Exactive mass spec-
trometer (Thermo Scientific, Waltham, MA, USA). 1H-nuclear mag-
netic resonance (NMR) and 13C-NMR spectra were recorded with a
Bruker Advance III HD 500 MHz NMR spectrometer. All UV–Vis
assays were performed on a Cary 50 UV–Vis spectrophotometer
(Varian, Walnut Creek, CA, USA). All EPR experiments were per-
formed on a Bruker EMXplus EPR spectrometer (Billerica, MA). High-
pressure liquid chromatography (HPLC) analysis of all samples was
performed using a Shimadzu HPLC system with a Symmetry® C18
5-μm column from Waters Corp (Milford, MA, USA) (4.6 ꢀ 150 mm).
For HPLC analysis, peptides were dissolved in water/HPLC-grade ace-
tonitrile (5:1) to a concentration of 0.5 mM following lyophilization.
The aqueous and organic phases were 0.1% trifluoroacetic acid (TFA)
in distilled, deionized water (Buffer A) and 0.1% TFA in HPLC-grade
acetonitrile (Buffer B), respectively. Beginning with 100% Buffer A,
We measured their antioxidant capacity by measuring their ability
to scavenge hydroxyl radicals and various organic radicals. We also
qualitatively determined their ability to bind Cu (II) by using low-
temperature electron paramagnetic resonance (EPR) spectroscopy
FIGURE 1 Structure of ergothioneine and 2-thiohistidine.
Ergothioneine (EGT) is the betaine (contains a trimethylated amine) of
2-thioHis

JENNY ET AL.
3 of 12
Buffer B was increased by 1% up to 50% over 50 min with a 1.4 ml/
min gradient elution. Buffer B was then increased from 50% to 100%
over 10 min. This method was used for analysis of each sample. Pep-
tide elution was monitored via absorbance at both 214 and 254 nm.
acidifying with 20 ml of 1 N HCl. The reaction was extracted three
times with ethyl acetate followed by a back extraction of the ethyl ace-
tate layer with water, 1 N HCl, and brine (1:1:1). The ethyl acetate solu-
tion was then dried with MgSO4, filtered with a Büchner funnel, and
roto-evaporated to dryness. The oil was dissolved in 10–20 ml of ethyl
acetate with 1–2 ml of methanol. The addition of hexanes precipitated
the N-Fmoc-2-thioHis derivative as a cream colored solid. The solid
was purified by redissolving it in 10–20 ml of warm ethyl acetate and
1–2 ml of methanol, filtering the solution through a Büchner funnel,
and then reprecipitating the product with cold hexanes. The product
was dried under high vacuum and used without further purification. MS
analysis showed a dominant peak at 410 m/z for the product as well as
smaller peaks for the Na+ adduct (M + 23) at m/z 432 and the K+
adduct (M + 39) at m/z 448. 1H-NMR (MeOD): δ 2.88 (dd, 1H), 3.08
(dd, 1H), 4.22 (t, 1H), 4.35 (d, 2H), 4.43 (dd, 1H), 6.60 (s, 1H), 7.32 (t,
2H), 7.40 (t, 2H), 7.65 (d, 2H), 7.80 (d, 2H); 13C-NMR (MeOD) δ 26.77,
53.06, 66.61, 119.49, 124.81, 126.78, 127.38, 141.16, 143.76, 143.83,
157.00, and 172.87. An average yield was 82.5%. We note that that the
MS of one of the reactions showed either a mixed disulfide between
Fmoc-2-thioHis and 2-thioHis or a symmetrical disulfide with Fmoc-
2-thioHis (Figure S1). This disulfide could be eliminated by redissolving
the product in ethyl acetate and extracting with 10-mM ascorbate
(Asc), pH 4.25 (Figure S1). If this step was necessary, the yield was
reduced significantly. The disulfide was only observed when addition of
the Fmoc group to 2-thioHis did not go to completion.
2.2
|
Synthesis of L-2-thio-histidine
L-2-Thiohistidine was synthesized according to the procedure from
Erdelmeier et al.[23] This reaction works best when performed on 10 g
or higher scale. Histidine (14 g, 66.8 mmol, 1.0 eq.) was dissolved in
134 ml of deionized water. After the His was fully dissolved, this solu-
tion was cooled in an ice bath at 0ꢁC. Once the reaction was cooled,
bromine (4.45 ml, 86.8 mmol, 1.3 eq.) was added resulting in a bright
orange solution. After 6 min, Cys (24.3 g, 200.4 mmol, 3.0 eq.) was
added to the reaction. The solution was stirred at 0ꢁC for 1 h. An oil
bath was preheated to 95ꢁC. After 1 h, 3-mercaptopropionic acid
(34.9 ml, 400.7 mmol, 6.0 eq.) was added to the reaction, and the
reaction was transferred to the oil bath at 95ꢁC. A condenser was
attached to the reaction, and the reaction was stirred for 18 h at
95ꢁC, after which the reaction had turned dark brown. The reaction
was removed from the oil bath and condenser and allowed to cool to
room temperature. The aqueous solution was then extracted with
ethyl acetate. The aqueous layer remained dark brown after extrac-
tion. The aqueous layer was transferred to a clean flask and placed in
an oil bath preheated to 40ꢁC. The pH of the solution was adjusted to
6.5 with 30% ammonia hydroxide to precipitate 2-thioHis. The reac-
tion was chilled to allow complete precipitation. The off-white precipi-
tate was filtered out of the reaction and washed with cold deionized
water and ethanol. The precipitate was dried under high vacuum to
give 5.04 g (26.9 mmol) of an off-white powder. The percent yield of
this reaction was 40% which is consistent with the findings
of Erdelmeier et al.[23] Mass spectrometric (MS) analysis revealed a
peak at 188.1 m/z. 1H-NMR (D2O/DCl): δ 3.06–3.20 (2H, (3.06 dd),
(3.20 dd)), 4.21 (1H, dd), 6.79 (1H, s); 13C-NMR (D2O/DCl): δ 25.32,
51.82, 115.96, 123.23, 156.49, 170.38.
2.4
|
Peptide synthesis
All His-containing peptides (H-βAH-OH, H-HGPLGPL-OH, and H-
GHK-OH) were synthesized according to standard SPPS on
a
0.1-mmol scale using a glass vessel shaken with a model 75 Burrell
wrist action shaker. We used 300 mg of 2-chlorotrityl chloride resin
(100–200 mesh, Chem-Impex) for each peptide. This resin was
swelled in dichloromethane (DCM) for 20 min. The first amino acid
was directly coupled to the resin using N-methylmorpholine (NMM)/
DCM (2:98), shaking for 1 h. The resin was then capped using
DCM/methanol/NMM (8:1:1). Subsequent amino acids were coupled
using 0.2 mmol of Fmoc-protected amino acid, 0.2 mmol of HATU,
and 1.8 mmol NMM in dimethylformamide (DMF), shaking for 1 h.
Between amino acid couplings, the Fmoc-protecting group was
removed by two 10-min washes with piperidine/DMF (2:8). The suc-
cess of Fmoc removal and amino acid couplings were monitored quali-
tatively using a ninhydrin test.[24] Peptides were cleaved from the
resin with a cleavage cocktail consisting of TFA/triisopropylsilane
(TIS)/water (96:2:2) for 1.5 h. Following cleavage, the resin was
washed with DCM, and the volume of the cleavage solution
was reduced by evaporation with argon gas. Each peptide was precipi-
tated with cold, anhydrous ether. Centrifugation at 3000 rpm on a
clinical centrifuge (International Equipment Co, Boston, MA, USA) for
5 min pelleted the peptide. Peptides were dried under argon gas, then
dissolved in a minimal amount of water/HPLC-grade acetonitrile (5:1),
lyophilized, and used without further purification.
2.3
|
Addition of Fmoc protecting group to L-
2-thioHis
N-Fmoc-L-2-thioHis was prepared using a standard procedure for the
addition of fluorenylmethoxycarbonyl (Fmoc) protecting groups to
amino acids. In a 250-ml round-bottom flask, 2-thioHis (1.0 g) was
added to 5–10 ml of deionized water to create a slurry. Triethylamine
(TEA) (750 μl, 5.35 mmol, 1.0 eq.) was added to the amino acid slurry,
and the reaction was stirred at room temperature. Fmoc N-
hydroxysuccinimide ester (Fmoc-OSu) (1.99 g, 5.89 mmol, 1.1 eq.) was
dissolved in 20–30 ml of acetonitrile and added to the amino acid
slurry. A second eq. of TEA (750 μl) was added to the reaction along
with acetonitrile and water to completely dissolve the 2-thioHis. The
reaction was stirred for 2 h at room temperature and monitored by
thin-layer chromatography (TLC). The reaction was quenched by

4 of 12
JENNY ET AL.
The
2-thioHis-containing
peptides
(H-βAthioH-OH
the peptides (Figure S2). These curves were used to calculate Trolox
equivalent antioxidant capacity (TEAC) values for each of the peptides
by calculating the ratio of peptide to Trolox to achieve the same per-
cent reduction of DPPH.
[thioHcarnosine], H-thioHGPLGPL-OH, and H-GthioHK-OH) were
synthesized with a similar protocol prior to coupling of 2-thioHis. For
the coupling of 2-thioHis, 0.12 mmol of Fmoc-2-thioHis, 0.12 mmol
of ethyl (hydroxyimino)cyanoacetate (EtCA), and 0.36 mmol of
diisopropylcarbodiimide (DIC) was dissolved in DMF, added to the
resin and shaken for 2 h. Double couplings were typically performed.
When 2-thioHis was the first amino acid coupled to the resin, as was
the case for H-βAthioH-OH, coupling was performed with 0.11 mmol
of Fmoc-2-thioHis and 1.8 mmol NMM in DCM for 1 h. All subse-
quent couplings after 2-thioHis were performed with 0.2 mmol of
Fmoc-protected amino acid, 0.2 mmol ETCA, and 0.6 mmol DIC in
DMF for 1 h. The rest of the procedure is identical to the one used
for the His-containing peptides. We note that the peptides containing
2-thioHis had a yellow color upon precipitation in ether and after
lyophilization, while the His-containing peptides were white.
2.6
|
Measurement of 2,20-azino-di-
[3-ethylbenzthiazoline sulfonate(6)] radical scavenging
activity
2,20-Azino-di-[3-ethylbenzthiazoline sulfonate(6)] (ABTS) scavenging
assays were performed according to previously published protocols
with some modifications.[25,27,28] A 10-mM stock solution of potas-
sium persulfate was prepared in deionized water. A stock solution of
ABTS was prepared by dissolving 7-mM ABTS and 2.45-mM potas-
sium persulfate in deionized water. This solution was allowed to incu-
bate at room temperature in the dark for 12–16 h to allow potassium
persulfate to oxidize the ABTS. Following oxidation, the solution was
a dark blue/green color. This solution was stable in the dark at room
temperature for at least 48 h. This solution was diluted with 100-mM
phosphate buffered saline, pH 7.2 so that the final absorbance was
0.70 0.02 at 734 nm. Stock solutions for all peptides and for Trolox
were prepared in a range from 0.1 mM to 3.2 mM in 100-mM phos-
phate buffered saline, pH 7.2. To test the ability of Trolox and pep-
tides to reduce the ABTS radical cation, samples were prepared with
990 μl of the diluted ABTS solution and 10 μl of each of the peptide
or Trolox stock solutions. For every trial, one sample was prepared
with 990-μl ABTS and 10-μl of buffer. The samples were allowed to
incubate at room temperature in the dark for 10 min. The spectropho-
tometer was blanked with 1 ml of 100-mM phosphate buffered saline,
pH 7.2 from 200–800 nm. Scans were taken for each of the samples
and the change in absorbance at 734 nm was monitored. Every sam-
ple was run in triplicate and the results were averaged. The percent-
age of inhibition of ABTS oxidation was calculated using Equation 2.
2.5
|
Measurement of 2,2-diphenyl-
1-picrylhydrazyl radical scavenging activity
2,2-Diphenyl-1-picrylhydrazyl
(DPPH)
activity
assays
were
conducted according to the procedures outlined by Liu et al. and You
et al. with some modifications.[25,26] A 1-mM stock solution of DPPH
was prepared by dissolving DPPH in 95% ethanol. Peptide stock
solutions were prepared at a concentration of 5 mM in 100 mM
potassium phosphate buffer, pH 7.0 for each of the His- and
2-thioHis-containing peptides. 6-hydroxy-2,5,7,8-tetramethylchromane-
2-carboxylic acid (Trolox) was used as a standard for antioxidant activ-
ity and a positive control. A 5-mM solution of Trolox in 100-mM
potassium phosphate, pH 7.0 was prepared. A blank was prepared by
mixing 600 μl of 95% ethanol with 400 μl of 100-mM potassium
phosphate, pH 7.0 in a cuvette. This solution was used to blank the
spectrophotometer from 200–800 nm. In each sample, a 60:40 ratio
of ethanol:buffer was maintained. To test the ability of Trolox and the
different peptides to reduce DPPH, 50-μM DPPH was combined with
different concentrations of peptide or Trolox ranging from 5 to
500 μM and the total volume of each sample was adjusted to 1 ml
with buffer and ethanol. The samples were allowed to incubate at
room temperature in the dark for 15 min, after which each sample
was scanned and changes in the absorbance at 517 nm were moni-
tored. Trials were run in triplicate for each of the peptides and Trolox.
For every trial, one sample was prepared with only 50-μM DPPH
(no peptide or Trolox) to serve as a control. The percentage of inhibi-
tion of DPPH oxidation was calculated using Equation 1:
Acontrol ꢂAsample
ABTS scavenging ð%Þ ¼
ꢀ100
ð2Þ
Acontrol
where Acontrol is the absorbance of the control (ABTS and buffer only)
and Asample is the absorbance at 734 nm of the test peptide/standard.
A Trolox calibration curve was prepared with concentrations ranging
from 1–32 μM (Figure S3). Similar curves were prepared for each of
the peptides (Figure S3). These curves were used to calculate TEAC
values for each of the peptides by calculating the ratio of peptide to
Trolox to achieve the same percent reduction of ABTS.
Acontrol ꢂAsample
DPPH scavenging ð%Þ ¼
ꢀ100
ð1Þ
Acontrol
2.7
|
Measurement of hydroxyl radical scavenging
activity
where Acontrol is the absorbance of the control (DPPH and buffer only)
and Asample is the absorbance at 517 nm of the test peptide/standard.
A Trolox calibration curve was prepared with concentrations ranging
from 5–50 μM (Figure S2). Similar curves were prepared for each of
Hydroxyl radical scavenging activity (RSA) of peptides was measured
with room temperature EPR spectroscopy according to the procedure
of Nazeer et al. with some modifications.[29] Hydroxyl radicals were

JENNY ET AL.
5 of 12
generated by the iron-catalyzed Fenton Haber–Weiss reaction, and
the generated hydroxyl radicals were reacted with the nitrone spin
trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO).[30] The resultant
DMPO-OH adducts were detectable with EPR spectroscopy. The
peptide (1-mM final concentration) was added to a solution con-
taining: 24mM DMPO, 0.8-mM FeSO4, and 0.8-mM H2O2 in 100-mM
potassium phosphate buffer, pH 7.4 in a final reaction volume of
250 μl. This solution was immediately transferred into a 50-μl quartz
capillary tube. After 5 min, the EPR spectrum was recorded using a
Bruker X-band EPR spectrometer operating at 9.42 GHz. The experi-
mental conditions employed were as follows: magnetic field, 336.5
10 mT; power, 2 mW; modulation amplitude, 2.00 G; time constant,
327.68 ms, modulation frequency, 100 kHz; receiver gain, 30-dB
sweep time, 60 s. Trials were performed in triplicate for each peptide.
The double integral of the EPR spectra was obtained and used to
determine the signal intensity for each sample. The hydroxyl radical
scavenging ability was calculated using Equation 3, in which H and H0
are relative peak height of radical signals with and without sample,
respectively.
samples were frozen in liquid nitrogen and stored in liquid nitrogen
prior to use. Control samples were prepared with only buffer and with
200-μM CuCl2 in potassium phosphate buffer. Low temperature EPR
spectra were gathered with a Bruker EPR spectrometer operating at
9.42 GHz with the following parameters: scan range, 6000 G; field
set, 3200 G; time constant, 164 ms; scan time, 60 s; modulation
amplitude, 7.000 G; modulation frequency, 100 kHz; receiver gain,
30 dB; microwave power, 20 mW; and number of scans, 5.
3
|
RESULTS AND DISCUSSION
| Past use of 2-thioHis in peptides
3.1
As mentioned in the Introduction, 2-thioHis has only been incorpo-
rated into a peptide one time in the entire history of peptide science.
While lack of commercial availability is certainly one cause for its
dearth of use in peptides, it can be readily synthesized from histidine
(His) and cysteine (Cys) via a labile His-bromolactone intermediate on
a gram scale.[23] We believe the problem of protection/deprotection
of the thione reported by Maggiora et al. has created a barrier to entry
for many peptide chemists such that 2-thioHis has not been incorpo-
rated into a peptide since their initial report (31 years).[10]
1ꢂH
Hydroxyl radical scavenging ð%Þ ¼
ꢀ100
ð3Þ
H0
In order to achieve protection of the thione, Maggiora et al. used
harsh conditions consisting of sodium dissolved in liquid ammonia
followed by addition of α-bromo-p-xylene to install the Meb group on
the sulfur.[10] The Boc-2-thioHis (Meb) derivative was then incorpo-
rated into a peptide using Merrifield SPPS. After synthesis of the pep-
tide was complete, deprotection was achieved using an excess of
monohalogenated alkanes such as bromomethane or excess bis-
halogenated alkanes in sodium/liquid ammonia to yield various mono-
alkylated and bisalkylated products (Figure 3).[10] Of note, they were
able to create a unique disulfide mimic by connecting two 2-thioHis
residues with an alkyl linker by addition of 1,6-dibromohexane in the
sodium/liquid ammonia reaction.[10]
2.8
|
Spectrophotometric determination of Cu
(II) binding constants (KD)
We attempted to quantify the Cu (II) binding constants for carnosine
and thioHcarnosine by titrating each peptide with Cu (II) and monitor-
ing changes in the absorbance spectra. To detect formation of the Cu
(II) complex, 10-mM carnosine in 50-mM MOPS buffer, pH 7.4 was
titrated with CuCl2 at concentrations ranging from 1–25mM, whereas
100-μM thioHcarnosine in 50-mM MOPS buffer, pH 7.4 was titrated
with CuCl2 at concentrations ranging from 5–250 μM. The
thioHcarnosine/Cu (II) solution was allowed to incubate at room tem-
perature for 40 min before absorbance spectra were recorded to
ensure complete formation of the Cu (II) complex. Both peptides had
a unique wavelength of maximum absorbance for the Cu (II) complex:
thioHcarnosine at 260 nm and carnosine at 630 nm. The KA values
were calculated by fitting the resulting titration data to the appropri-
ate equation describing the association constant.[31] The KD values
were calculated from the reciprocal of the KA value for each peptide.
We decided to attempt incorporation of 2-thioHis into a peptide
without protecting the thione due to the reported low reactivity of
EGT with 5,50-dithiobis(2-nitrobenzoic acid) (DTNB), a highly reactive
disulfide.[3,11] The thione of 2-thioHis (like EGT) is in equilibrium with
the thiol form, with the thione being the dominant form in solution.[1–
7]
We reasoned that the thione form is even more favored in DMF
compared to water and that the low 2-electron nucleophilicity of the
thione would render it unreactive with the carbodiimide of DIC given
its lack of reactivity with DTNB. If true, this would remove a large bar-
rier to using 2-thioHis in peptide synthesis.
2.9
|
Low-temperature EPR studies
The ability of each peptide to bind Cu (II) was determined by low-
temperature (95ꢁK) EPR spectroscopy using the methods from Zhu
et al. and Motohashi et al. with some modifications.[9,32] Samples of
200- and 400-μM peptide were incubated for 10 min at room temper-
ature with 200-μM CuCl2 in 100-mM potassium phosphate, pH 7.4.
Following incubation, aliquots of each sample were transferred into
4-mm inner diameter quartz EPR tubes (Wilmad-LabGlass). The
3.2
|
Synthesis and analysis of 2-thioHis-
containing peptides
Our results show that we were able to successfully insert 2-thioHis
into the three target peptides in place of His using standard Fmoc
SPPS techniques with carbodiimide-mediated coupling. The three

6 of 12
JENNY ET AL.
FIGURE 3 Inserting 2-thioHis into a peptide. (A) Method used by Maggiora et al.[10] to protect 2-thioHis before insertion into a peptide,
followed by deprotection in the presence of linker. (B) Our simplified method eliminates protection of 2-thioHis prior to insertion into a peptide
peptides we chose place 2-thioHis as the first amino acid coupled
(thioHcarnosine), the middle amino acid coupled (GthioHK), or the last
amino acid coupled (thioHGPLGPL). The yield of each crude peptide
after precipitation into ether and lyophilization was 90% or greater.
HPLC analysis of the 2-thioHis-containing peptides shows that
the purity of the GthioHK and thioHcarnosine peptides were nearly
identical to their His-containing counterparts (Figure 4A–D). We
note that 2-thioHis-containing peptides are readily identified in
the analytical HPLC due to their strong absorbance at 254 nm. The
imidazothione of 2-thioHis has a λmax at 257 nm with an extinction
coefficient 14,000 Mꢂ1 cmꢂ1.[5] In the case of peptide thioHGPLGPL,
we observed an unidentified peak that eluted very late in the gradient.
The main peak corresponding to thioHGPLGPL is clearly identified in
the chromatogram due to the very strong absorbance at 254 nm
(Figure 4F). The unidentified peak has a much less intense absorbance
at 254 nm. It is possible that this unidentified peak is a significant
impurity due to a side-reaction or incomplete coupling. However,
another possibility is that this peak is the disulfide form of the pep-
tide. A disulfide can form between two 2-thioHis residues as we
observed during the synthesis of Fmoc-2-thioHis as we indicated in
the Methods section. We synthesized this peptide several times, and
this peak was always present. To investigate the possibility that this
was the disulfide, we incubated the sample with 1-mM Asc at pH 4.5
for 1 h and then injected the reaction onto the column. The result
showed that the unidentified peak disappeared (Figure S4). This is
strong evidence that this peptide formed a disulfide between two
monomers following precipitation into ether and dissolution into
water prior to lyophilization. In fact, the MS analysis of each peptide
shows evidence for disulfide formation. The MS analysis of each
peptide in Figure 4 is given in Figure S5. The disulfide bond between
two 2-thioHis residues must be very weak because Asc was able to
reduce it. We did not utilize the fact that disulfide bond formation is
possible between 2-thioHis residues, but this should make for an
interesting future application.
3.3
|
Antioxidant activity of 2-thioHis-containing
peptides
The His- and 2-thioHis containing peptides, as well as 2-thioHis, were
tested for their ability to scavenge DPPH and ABTS. Trolox, a water-
soluble vitamin E analogue, was used as a standard for the ABTS and
DPPH assays. The reduction of DPPH and ABTS was determined by
monitoring the decrease in absorbance at 517 and 734 nm, respec-
tively, after the addition of peptide or standard. The His-containing
peptides all showed little or no ability to scavenge DPPH or ABTS
radicals at the concentration of peptides chosen here. We note that
carnosine and HGPLGPL have been reported to scavenge the DPPH
radical, but at higher concentrations than we tested.[14,33] Peptide
GHK was the strongest antioxidant peptide tested of the His-
containing peptides and caused only a slight decrease in the DPPH
absorbance peak at 517 nm and no decrease in the ABTS absorbance
peak at 734 nm (Figure 5). In contrast to the His-containing peptides,
all of the 2-thioHis-containing peptides showed strong RSA with both
DPPH and ABTS radicals. Peptide GthioHK was the strongest antioxi-
dant peptide tested overall with the ability to completely reduce the
DPPH and ABTS radicals (Figure 5). The same absorbance spectra for
the other peptides in this study are given in Figures S6 and S7.

JENNY ET AL.
7 of 12
FIGURE 4 Analytical high-pressure liquid chromatography (HPLC) analysis of His- and thioHis-containing peptides in this study. (A) GHK.
(B) GthioHK. (C) Carnosine. (D) ThioHcarnosine. (E) HGPLGPL. (F) ThioHGPLGPL
Next, the His- and 2-thioHis containing peptides, as well as
2-thioHis, were tested for their ability to scavenge •OH. Asc was used
as a standard for •OH scavenging activity, with 1-mM Asc completely
reducing the hydroxyl radical. The reduction of •OH was measured
using EPR spectroscopy. The Fenton Haber–Weiss reaction was used
to produce •OH in the presence of peptide and the spin-trapping
reagent DMPO.[30] The RSA was calculated by measuring the
decrease in peak height upon addition of 1-mM peptide (final
concentration) compared with the control with no peptide.[29] All RSA
values represent the average of three or more trials. Each of the
peptides tested showed ability to scavenge •OH, although the
2-thioHis-containing peptides showed improved activity over their
His-containing counterparts. GthioHK had the highest RSA of all
compounds tested. (Figure 6, Table 1).
2-thioHis for His in various peptides. However, we found that this
substitution produced a synergistic effect for all of the peptides stud-
ied here, with the increase in •OH scavenging ability ranging from a
12% to 13% increase for peptides thioHGPLGPL and thioHcarnosine,
respectively, to a dramatic 31.6% increase for GthioHK (Table 1). It is
interesting to note that in the case of thioHGPLGPL, the •OH
scavenging ability is equivalent to 2-thioHis itself, while for
thioHcarnosine, the •OH scavenging ability is less than 2-thioHis.
Carnosine itself was not a good •OH scavenger in comparison to the
other peptides tested.
Substitution of His with 2-thioHis in peptide GHK produced the
most dramatic and interesting result as is clearly evidenced by
the EPR spectra shown in Figure 6. The GthioHK peptide showed a
similar RSA as Asc, with 1-mM GthioHK nearly completely abolishing
the signal. Clearly, the 2-thioHis residue, in the context of the amino
acid sequence of the peptide, produces an antioxidant that is much
better than expected in comparison with the difference between the
In comparing the •OH scavenging ability of His with 2-thioHis,
we found an increase of 7.5% (Table 1). We could therefore expect a
similar increasing in •OH scavenging ability when substituting

8 of 12
JENNY ET AL.
FIGURE 5 Radical scavenging ability of peptides against DPPH and ABTS. (A) Absorbance spectrum for 50-μM DPPH with different
concentrations of GHK peptide. (B) Absorbance spectrum for 50-μM DPPH with different concentrations of GthioHK peptide. (C) Absorbance
spectrum for ABTS solution with different concentrations of GHK peptide. (D) Absorbance spectrum for ABTS solution with different
concentrations of GthioHK peptide
ability of His and 2-thioHis to scavenge •OH. Our data demonstrate
that GHK itself is a good antioxidant, which agrees with other
studies.[18,20,21] The naturally occurring matrikine peptide GHK and its
Cu (II) complex are commonly used in skin care products and
cosmeceuticals. The GHK tripeptide has well-studied wound healing
and anti-aging properties stemming in part from its ability to block the
formation of reactive oxygen and carbonyl species as well as its ability
to readily chelate redox active metals such as Cu (II) and Fe (II).[17–21]
Studies of the antioxidant capabilities of GHK have demonstrated its
strong ability to scavenge •OH in vitro suggesting it may play a role in
managing oxidative stress in cells.[21] Our GthioHK analogue could
also be potentially used as a therapeutic or in cosmetics due to its
enhanced antioxidant properties.
GthioHK having a slightly higher TEAC value than the other 2-thioHis
peptides. All three 2-thioHis-containing peptides were stronger scav-
engers of the ABTS radical compared to both Trolox and 2-thioHis on
its own (Table 1). 2-thioHis had the same ability as Trolox to scavenge
the DPPH radical. The His-containing peptides showed no significant
ability to scavenge the DPPH radical even at 10:1 peptide: DPPH
(Table 1 and Figure S6). In contrast, the 2-thioHis-containing peptides
showed significant ability to scavenge the DPPH radical; however,
they were less efficient than Trolox and took longer to react with the
radical, likely due to steric hindrance (Table 1).[34] Although we did not
study the kinetics of the reaction specifically, we noticed an immedi-
ate color change of the DPPH solution upon addition of Trolox
(change from purple to orange color), while the 2-thioHis-containing
peptides took several minutes to achieve the same color change. The
ability of the 2-thioHis-containing peptides to scavenge the DPPH
radical and ABTS radical indicates that the presence of 2-thioHis
imparts strong antioxidant activity to these peptides. In comparison,
the His-containing peptides had very weak antioxidant activity in the
same assays.
3.4
|
Summary of antioxidant activities of
2-thioHis-containing peptides
The antioxidant capacities of all peptides tested were compared by
calculating TEAC values for both the ABTS and DPPH assays
(Table 1). All TEAC values represent the average from three or more
trials. The curves used to determine TEAC values for 2-thioHis pep-
tides are shown in Figures S2 and S3. The His-containing peptides all
had TEAC values of approximately 0 and showed no ability to scav-
enge the ABTS radical at the concentrations tested (Table 1). The
2-thioHis-containing peptides possessed high ABTS RSA, with
3.5
|
Copper binding ability of 2-thioHis-
containing peptides
Next, we investigated the stoichiometry and affinity of copper
binding of the 2-thioHis-containing peptides in comparison to the

JENNY ET AL.
9 of 12
His-containing peptides. The ability of the test peptides to chelate Cu
(II) was determined with low temperature (95ꢁK) EPR spectroscopy.
The EPR experiments showed that the 2-thioHis-containing peptides
could all bind Cu (II) as evidenced by the disappearance of the Cu
(II) signature in the EPR spectra and the change in peak shape and
position (Figure 7). Comparing the EPR spectra of the 2-thioHis-
containing peptides to that of 2-thioHis indicates that the 2-thioHis
containing peptides bind Cu (II) in a similar configuration as 2-thioHis
(Figure 7). The 2-thioHis-Cu (II) complex peak is almost identical to
the peaks for the three 2-thioHis containing peptides indicating that
these peptides are binding Cu (II) through the sulfur (Figure 7).[35] In
contrast, all of the His-containing peptides have unique EPR spectra
compared to each other, which indicates differences in Cu (II) binding
geometry between the three of them or differences in Cu
(II) binding affinity. (Figure 7).
In addition to Cu (II) binding ability, the EPR data in Figure 8
offers some insight into the equilibrium dissociation constants (KD)
and binding stoichiometry for some of the peptides. For peptide GHK,
the KD for Cu (II) has previously been determined to be
7.0 ꢀ 10ꢂ14 M using isothermal titration calorimetry with competitive
chelators (such as glycine) in different buffer systems.[36] Because
GHK has a very high affinity for Cu (II), it can be concluded that in the
conditions used for EPR, all of the Cu (II) in solution would be bound
to GHK. Based on this information, we can conclude from the EPR
spectra that GHK binds to Cu (II) in a 2:1 ratio because at 1:1 GHK:Cu
(II) the Cu (II) trace is still visible in the EPR spectrum, but with 2:1
GHK:Cu (II) the Cu (II) trace is almost completely gone (Figure 8). The
EPR spectra for GthioHK shows a similar trend indicating that
GthioHK, like 2-thioHis and GHK, likely also binds Cu (II) in a 2:1 ratio
(Figure 8).
However, the affinity for Cu (II) and binding stoichiometry
appears to be significantly different for carnosine in comparison to
thioHcarnosine upon comparison of their EPR spectra (Figure 8). The
KA value reported in the literature for carnosine is 1.1 M, which indi-
cates a weak affinity for Cu (II).[37] Carnosine has been reported to
bind Cu (II) in a 1:1 ratio which is confirmed in the EPR spectra as the
Cu (II) trace is still highly visible at both 1:1 and 2:1 peptide: Cu
(II) (Figure 8).[37] Although we cannot make any quantitative determi-
nations of KA for thioHcarnosine from the EPR spectra, we can
FIGURE 6 Hydroxyl radical scavenging activity of peptides
measured by electron paramagnetic resonance (EPR). Hydroxyl radical
was generated by a reaction of 0.8-mM FeSO4, with 0.8-mM H2O2,
pH 7.0 (250 μl). The hydroxyl radicals were trapped with
5,5-dimethyl-1-pyrroline-N-oxide (DMPO) prior to obtaining spectra
TABLE 1 Antioxidant capacity of
His- and 2-thioHis-containing peptides
Analyte or peptide
ABTS radical TEAC
1.00
DPPH radical TEAC
1.00
Hydroxyl radical RSAa (%)
NAb
Trolox
compared with Trolox or ascorbate and
NAb
NAb
100
2-thioHis
Asc
His
NAb
NAb
48.5 7.3
56.0 2.1
46.8 1.3
59.7 3.0
65.4 0.008
97.0 0.3
31.4 4.2
43.5 1.1
2-thioHis
HGPLGPL
thioHGPLGPL
GHK
1.50 0.03
NDc
1.00 0.02
NDc
2.14 0.02
NDc
0.95 0.01
NDc
GthioHK
Carnosine
thioHCarnosine
2.61 0.01
NDc
0.95 0.02
NDc
2.25 0.05
0.80 0.01
aAll analytes were tested at a concentration of 1 mM.
bNot applicable because it was not used in the assay.
cThe activity was too low to be determined, these peptides have TEAC values close to zero and are not
comparable to Trolox.

10 of 12
JENNY ET AL.
reasonably conclude that it has a higher affinity for Cu (II) than
unlike carnosine, thioHcarnosine binds Cu (II) with a 2:1 stoichiometry
as indicated by the change in the EPR signal as the peptide concentra-
tion is increased from 200 to 400 μM while keeping the Cu
(II) concentration constant at 200 μM (Figure 8).
carnosine due to the lack of
a strong Cu (II) signal at 1:1
thioHcarnosine:Cu (II) (Figure 8). In addition, we can conclude that
Based on the comparison of EPR spectra of the His and
2-thioHis-containing peptides, we can conclude that substitution of
2-thioHis for His in peptides can change the Cu (II) binding geometry,
binding affinity, and binding stoichiometry. The comparison of the
2-thioHis-containing peptides to their His counterparts and free
2-thioHis indicates that these peptides are binding Cu (II) through the
sulfur with a geometry similar to 2-thioHis.[35] Using known KA values
for GHK and carnosine, it can also be concluded that insertion of
2-thioHis in place of His resulted in a 2:1 peptide: Cu (II) binding
geometry, and in the case of thioHcarnosine, also increased the Cu
(II) binding affinity.
We then attempted to quantify KD for Cu (II) for carnosine and
thioHcarnosine via titration of Cu (II) using UV–Vis spectroscopy. As
has been reported in the literature, carnosine forms a blue complex
when binding Cu (II), which can be detected at 630 nm.[37] We also
detected this blue complex upon addition of Cu (II) to carnosine as is
shown by the plot of absorbance versus wavelength at various Cu
(II) concentrations as shown in Figure 9A. We then made a plot of the
change in absorbance at 630 nm versus Cu (II) concentration
(Figure S8). The resulting curve was fit to the appropriate equation for
a 1:1 association constant.[31] This yields an association constant (KA)
FIGURE 7 Low temperature electron paramagnetic resonance
(EPR) spectra of peptide-Cu (II) complexes in potassium phosphate
buffer, pH 7.4. All peptides and 2-thioHis are at a concentration of
400- with 200-μM CuCl2
FIGURE 8 Low temperature electron
paramagnetic resonance (EPR) spectra of
peptide-Cu (II) complexes in potassium
phosphate buffer, pH 7.4. (A) 200-μM CuCl2,
(B) GHK with 200-μM CuCl2, (C) GthioHK
with 200-μM CuCl2, (D) carnosine with
200-μM CuCl2, and (E) thioHcarnosine with
200-μM CuCl2

JENNY ET AL.
11 of 12
FIGURE 9 Absorbance spectral scan of carnosine and thioHcarnosine as a function of CuCl2 concentration. (A) Absorbance scan of carnosine
(10 mM) as CuCl2 is titrated from 0 mM (black line) to 30 mM (light grey line). The absorbance at 610 nm increases as CuCl2 is added to the
peptide and then levels off. (B) Absorbance scan of thioHcarnosine (100 μM) as CuCl2 is titrated from 0 μM (black line) to 250 μM (light beige
line). The absorbance at 255 nm decreases as CuCl2 is added to the peptide, whereas the absorbance at 220 nm increases. The tailing absorbance
at 300 nm also increases as CuCl2 is titrated
of 0.228 M (KD = 4.39 M). This compares to a reported value of
1.1 M.[37] We note that we made our measurement in MOPS buffer
at pH 7.4, while the literature value was determined in pure water.[37]
We then performed the same analysis for thioHcarnosine as
shown by the absorbance scan shown in Figure 9B. However, we
were unable to do the same analysis because of the strong absor-
bance of the 2-thioHis residue at 255 nm, which limited the concen-
tration of peptide we could analyze by UV–Vis spectroscopy. This led
us to perform the experiment with thioHcarnosine on a different scale
from carnosine, rendering comparison impossible using this method.
Department of Chemistry. These studies were supported by National
Heart, Lung, and Blood Institute grant HL141146 to Robert J. Hondal.
ORCID
Robert J. Hondal
https://orcid.org/0000-0002-1466-2852
REFERENCES
1. Melville DB. Ergothioneine. Vitam. Horm. 1959;17:155-204.
2. Akanmu D, Cecchini R, Aruoma OI, Halliwell B. The antioxidant action
of ergothioneine. Arch. Biochem. Biophys. 1991;288(1):10-16.
3. Hartman PE. Ergothioneine as an antioxidant. Methods Enzymol.
1990;186:310-318.
However, the EPR data in Figure
8 lead us to believe that
thioHcarnosine binds Cu (II) more tightly than carnosine, but we have
not quantified this parameter.
4. Ey J, Schomig E, Taubert D. Dietary sources and antioxidant effects
of ergothioneine. J. Agric. Food Chem. 2007;55(16):6466-6474.
5. Paul BD, Snyder SH. The unusual amino acid L-ergothioneine is a
physiologic cytoprotectant. Cell Death Differ. 2010;17(7):1134-1140.
6. Cheah IK, Halliwell B. Ergothioneine; antioxidant potential,
physioplogical function and role in disease. Biochim. Biophys. Acta.
2012;1822(5):784-793.
4
|
CONCLUSIONS
7. Borodina I, Kenny LC, McCarthy CM, et al. The biology of
ergothioneine, an antioxidant nutraceutical. Nutr. Res. Rev. 2020;
33(2):190-217.
Our decision to leave the thione of 2-thioHis unprotected during
SPPS while seemingly unconventional proved to be sound and
enabled the facile synthesis of multiple biologically relevant peptides.
Our study has shown that the substitution of 2-thioHis for His in bio-
active peptides greatly enhances their existing antioxidant properties,
equal to that of 2-thioHis, or in some cases much more. An example
of this greater enhancement is substitution of 2-thioHis for His in the
matrikine GHK-tripeptide, which very strongly quenched both
the ABTS and hydroxyl radicals. The therapeutic potential for the
GthioHK analogue is high and not yet fully explored. Substitution of
His with 2-thioHis also confers the ability to bind Cu (II) to the pep-
tide. Overall, the newfound ability to easily insert 2-thioHis into a
peptide opens up a new and unexplored realm of possibilities for
peptide chemistry and therapeutics.
8. Gründemann D, Harlfinger S, Golz S, et al. Discovery of the
ergothioneine transporter. Proc. Natl. Acad. Sci. 2005;102(14):
5256-5261.
9. Zhu B, Mao L, Fan R, et al. Ergothioneine prevents copper-induced
oxidative damage to DNA and protein by forming a redox-inactive
ergothioneine-copper complex. Chem. Res. Toxicol. 2011;24(1):30-34.
10. Maggiora LL, Smith CW, Hsi A. L-2-thiol-histidine: Introduction of
conformational constraints into peptides via thioether linkage. Tetra-
hedron Lett. 1990;31(20):2837-2840.
11. Turner E, Klevit R, Hopkins PB, Shapiro BM. Ovothiol: a novel
thiohistidine compound from sea urchin eggs that confers NAD(P)H-
O2 oxidoreductase activity on ovoperoxidase. J. Biol. Chem. 1986;
261(28):13056-13063.
12. Komatsu T, Kobayashi K, Helmerhorst E, Oppenheim F, Chang-il LM.
Direct assessment of the antioxidant property of salivary histatin.
J. Clin. Biochem. Nutr. 2019;65(3):217-222.
ACKNOWLEDGEMENTS
13. Paule SG, Nikolovski B, Gray RE, et al. GHTD-amide: a naturally
occurring beta cell-derived peptide with hypoglycemic activity.
Peptides. 2009;30(5):955-961.
The authors are grateful for help with EPR spectroscopy from Dr
Monika Ivancic and Aarzoo Grover of the University of Vermont

12 of 12
JENNY ET AL.
14. Mendis E, Rajapakse N, Kim S. Antioxidant properties of a radical-
scavenging peptide purified from enzymatically prepared fish skin
gelatin hydrolysate. J. Agric. Food Chem. 2005;53(3):581-587.
15. Boldyrev AA, Aldini G, Derave W. Physiology and pathophysiology of
carnosine. Physiol. Rev. 2013;93(4):1803-1845.
weight bioactive subfractions isolated from cultures of wood
degrading fungus Cerrena unicolor. PLoS ONE. 2018;13(6):e0197944.
29. Nazeer RA, Sampath Kumar NS, Ganesh RJ. In vitro and in vivo studies
on the antioxidant activity of fish peptide isolated from the croaker
(Otolithes ruber) muscle protein hydrolysate. Peptides. 2012;35(2):
261-268.
16. Tanaka K, Kawahara M. Carnosine and lung disease. Curr. Med. Chem.
2020;27(11):1714-1725.
30. Rosen GM, Rauckman EJ. Spin trapping of superoxide and hydroxyl
radicals. Methods Enzymol. 1984;105:198-209.
17. Pickart L, Freedman JH, Loker WJ, et al. Growth modulating plasma
tripeptide may function by facilitating copper uptake into cells.
Nature. 1980;288(5792):715-717.
31. Thordarson P. Determining association constants from titration
experiments in supramolecular chemistry. Chem. Soc. Rev. 2011;40(3):
1305-1323.
18. Pickart L, Vasquez-Soltero JM, Margolina A. GHK-Cu may prevent
oxidative stress in skin by regulating copper and modifying expression
of numerous antioxidant genes. Cosmetics. 2015;2:236-247.
32. Motohashi N, Mori I, Sugiura Y. Complexing of copper ion by
ergothioneine. Chem. Pharm. Bull. 1976;24(10):2364-2368.
33. Abdelkader H, Longman M, Alany RG, Pierscioinek BG. On the
anticataractogenic effects of L-carnosine: Is it best described as an
antioxidant, metal-chelating agent or glycation inhibitor? Oxidative
Med. Cell. Longev. 2016;2016:e3240261.
ꢀ
19. Bossak-Ahmad K, Wisniewska MD, Bal W, Drew SC, Frączyk T.
Ternary Cu (II) complex with GHK peptide and cis-urocanic acid as
potential physiologically functional copper chelate. Int. J. Mol. Sci.
2020;21(17):6190-6207.
20. Pickart L, Vasquez-Soltero JM, Margolina A. The human tripeptide
GHK-Cu in prevention of oxidative stress and degenerative condi-
tions of aging: implications for cognitive health. Oxidative Med. Cell.
Longev. 2012;2012:1-8.
34. Holtz RW. In: Dayan N, ed. Skin Aging Handbook: An Integrated
Approach to Biochemistry and Product Development. Norwich, NY:
William Andrew Publishing; 2009:13.
35. De Luna P, Bushnell EAC, Gauld JW. A density functional theory
investigation into the binding of antioxidants ergothioneine and
ovothiol to copper. J. Phys. Chem. 2013;117(19):4057-4065.
36. Trapaidze A, Hureau C, Bal W, Winterhalter M, Faller P. Thermody-
namic study of Cu2+-binding to the DAHK and GHK peptides by iso-
thermal titration calorimetry (ITC) with the weaker competitor
glycine. J. Biol. Inorg. Chem. 2012;17(1):37-47.
21. Sakuma S, Ishimura M, Yuba Y, Itoh Y, Fujimoto Y. The peptide glycyl-
L-histidyl-L-lysine is an endogenous antioxidant in living organisms
possibly by diminishing hydroxyl and peroxyl radicals. Int J Physiol
Pathophysiol Pharmacol. 2018;10(3):132-138.
22. Kohen R, Yamamoto Y, Cundy KC, Ames BN. Antioxidant activity of
carnosine, homocarnosine, and anserine present in muscle and brain.
Proc. Natl. Acad. Sci. U. S. A. 1988;85(9):3175-3179.
37. Velez S, Nair NG, Reddy VP. Transition metal ion binding studies of
carnosine and histidine: biologically relevant antioxidants. Colloids
Surf. B: Biointerfaces. 2008;15(2):291-294.
23. Erdelmeier I, Duanay S, Lebel R, Farescour L, Yadan JC. Cysteine
as
a sustainable sulfur reagent for the protecting-group-free
synthesis of sulfur-containing amino acids: biomimetic synthesis of
L-ergothioneine in water. Green Chem. 2012;14(8):2256-2266.
24. Kaiser E, Colescott RL, Bossinger CD, Cook PI. Color test for detec-
tion of free terminal amino groups in the solid-phase synthesis of
peptides. Anal. Biochem. 1970;34(2):595-598.
SUPPORTING INFORMATION
Additional supporting information may be found online in the
Supporting Information section at the end of this article.
25. Liu J, Jin Y, Lin S, Jones GS, Chen F. Purification and identification of
novel antioxidant peptides from egg white protein and their antioxi-
dant activities. Food Chem. 2015;175:258-266.
How to cite this article: Jenny KA, Ruggles EL, Liptak MD,
Masterson DS, Hondal RJ. Ergothioneine in a peptide:
Substitution of histidine with 2-thiohistidine in bioactive
peptides. J Pep Sci. 2021;e3339. https://doi.org/10.1002/psc.
3339
26. You L, Zhao M, Cui C, Zhao H, Yang B. Effect of degree of hydrolysis
on the antioxidant activity of loach (Misgurnus anguillicaudatus)
protein hydrolysates. Innov. Food Sci. Emerg. 2009;10(2):235-240.
27. Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C.
Antioxidant activity applying an improved ABTS radical cation
decolorization assay. Free Radic. Biol. Med. 1999;26:1231-1237.
28. Matuszewska A, Jaszek M, Stefaniuk D, Ciszewski T, Matuszewski L.
Anticancer, antioxidant, and antibacterial activities of low molecular