Visible Light-Mediated Synthesis of Se?S Bond-Containing Peptides

Article abstract of DOI:10.1002/adsc.202100751

A visible light-initiated method has been developed for preparation of Se?S bond-containing peptides. The method is based on generation of sulfur-centered radical employing organic dye. The protocol is tolerant to unprotected peptides with “sensitive” amino acids. The stability of Se?S bond is evaluated in buffers at different pH (3.0–10.0) and also in the presence of oxidants and reducing agents. Additionally, the ability of Se?S bond to serve as an oxidation sensitive linker in biocompatible materials has been confirmed. (Figure presented.).

Full text of DOI:10.1002/adsc.202100751

COMMUNICATIONS
doi.org/10.1002/adsc.202100751
Visible Light-Mediated Synthesis of SeÀ S Bond-Containing
Peptides
Sindija Lapcinska,a Pavels Dimitrijevs,a and Pavel Arsenyana,*
a
Latvian Institute of Organic Synthesis, Aizkraukles 21, LV-1006, Riga, Latvia
E-mail: pavel@osi.lv
Manuscript received: June 18, 2021; Revised manuscript received: July 13, 2021;
Version of record online: ■■■, ■■■■
Supporting information for this article is available on the WWW under https://doi.org/10.1002/adsc.202100751
diphenyl diselenide with silver trifluoromethylsulfide.
Abstract: A visible light-initiated method has been
This exchange reaction is favored due to the selenolate
developed for preparation of SeÀ S bond-containing
stabilization with silver atom.[9] Typically, SeÀ S bond
peptides. The method is based on generation of
is prepared by reaction between thiol and electrophilic
sulfur-centered radical employing organic dye. The
protocol is tolerant to unprotected peptides with
selenyl species – selenyl halides[10,11] and organyl
seleninic acids.[12–14] Benzeneselenol can also be
“sensitive” amino acids. The stability of SeÀ S bond
utilized for the synthesis of related phenylselenyl
is evaluated in buffers at different pH (3.0–10.0)
sulfide by employing aryl or alkyl thiols and a catalytic
and also in the presence of oxidants and reducing
amount of tBuOK,[15] whereas reaction of benzenesele-
agents. Additionally, the ability of SeÀ S bond to
nol with electrophilic N-phenyl-trifluoromethane-
serve as an oxidation sensitive linker in biocompat-
sulfenamide[16] occurs in acidic conditions. Notably,
ible materials has been confirmed.
various sugar-selenyl sulfides have been synthesized
directly from sugar diselenides and glutathione in a
phosphate buffer. Moreover, this efficient method has
Keywords: glutathione; peptide; photocatalysis; se-
been extended from using glutathione as a thiol group-
lenocysteine
containing substrate to a protein – a single-cysteine
mutant of subtilisin.[17] Another example of synthesis
of SeÀ S bond-containing substrate obtained by direct
The biological importance of SeÀ S bond is based on reaction between diselenide (selenocystine) and thiol
the fact that this bond is found in the active center of (penicillamine) occurs in the presence of Et3N.[18] SeÀ S
redox regulating enzyme, namely, thioredoxin reduc- bond-containing cyclic dipeptides are obtained by
tase – one of the major components of the antioxidant treating À Se-benzhydryl and À S-trityl dipeptide with
system in mammalian cells.[1,2] SeÀ S bond- containing iodine.[19] Significantly, UV light has been employed
intermediate is formed in the catalytic cycle of the for the exchange reaction between diaryl disulfide and
glutathione peroxidase–selenoenzyme that is responsi- dialkyl diselenide. The authors have also stated that
ble for reduction of H2O2 and other peroxides by SeÀ S bond is formed under UV light, while longer
glutathione.[3] Recently, low-molecular weight com- wavelength (>410 nm, visible light) reverses the
pounds with SeÀ S bond have been used as fluorescent reaction.[20]
probes for detection of reactive sulfur species (RSS) –
Recently, we have reported an efficient method for
H2S and H2S2.[4,5] Selenosulfides have also been used the functionalization of SeÀ Se bond-containing pep-
as prodrugs for inhibition of protein tyrosine tides, based on the generation of a selenium radical via
phosphatases.[6]
visible light-initiated reaction.[21] As a continuation of
Compounds with SeÀ S bond are considered un- our research related to development of methods for
stable thus the synthesis can be challenging.[7] The modification of selenocysteine[22–23] (Sec) and
exchange reaction between diselenide and thiol, cysteine[24] peptides, here we report a simple protocol
although theoretically possible, is unfavorable because for preparation of SeÀ S bond-containing peptides using
the selenolate byproduct is a stronger nucleophile than visible light-initiated reaction.
thiol.[8] However, the reaction can be performed under
The optimization of reaction conditions was per-
suitable conditions.[7] For example, the SeÀ S bond- formed using dipeptide dimer Boc-Sec-Gly-OBn 1a
containing compound has been obtained by reacting and glutathione (GSH) (2) as model substrates. The
Adv. Synth. Catal. 2021, 363, 1–6
1
© 2021 Wiley-VCH GmbH
��
These are not the final page numbers!

COMMUNICATIONS
asc.wiley-vch.de
search for the most suitable photocatalyst was per-
Next, the tolerance of amino acids with “sensitive”
formed using 1 equiv. 1a, 10 equiv. 2, 0.02 equiv. groups (Arg, Glu, Lys, His, Trp, Tyr, Met) was
transition metal catalyst or 0.05 equiv. organic dye in established (Scheme 1). Gratifyingly, selenocysteine
the mixture of acetonitrile and water (1:1), while and selenocystamine derivatives with Glu, Tyr, Arg,
irradiating the reaction mixture with blue LED light His, Lys showed excellent tolerance and provided the
(max 460 nm, bright blue, x=01440, y=0.0395, >50 desired SeÀ S bond-containing products. Even Trp-
000 lx) for 1 hour. The reaction performed without the containing product 3c was isolated, albeit the yield
catalyst showed only traces of the product 3a. The was lower due to formation of side products that arise
highest selectivity and conversion of the starting from Trp oxidation. A rapid oxidation of methionine
materials was achieved employing Rose Bengal (RB). has been reported under visible light irradiation in the
Other catalysts were: (i) less effective (bis[2-(4,6- presence of RB.[25] Thus, not surprisingly, Met
difluorophenyl)pyridinato-C2,N](picolinato)iridium(III) sulfoxide-containing product 3f was selectively ob-
(FIrPic)); (ii) induced formation of the respective alkyl tained in good yield starting from Met-containing
seleninic acid which subsequently led to deselenylation substrate.
by elimination of H2SeO3 providing dehydroalanine
Obviously, selenocystamine fragment can serve as a
(Dha) peptide[21] (Ru(bpy)3Cl2, 2,4,5,6-tetrakis(9H-car- convenient linker. Furthermore, it has been demon-
bazol-9-yl) isophthalonitrile (4-CzIPN), 9-mesityl-10- strated that in certain structures the selenoethyl moiety
methylacridinium tetrafluoroborate, ethyl eosin, fluo- can be eliminated under reductive conditions.[26]
rescein), or (iii) failed to initiate the reaction (5-
Notably, 5-nitropyridine-2-thiol (4) was also suc-
carboxytetramethylrhodamine). Performing the reac- cessfully employed in the visible light-mediated reac-
tion in methanol and ethanol resulted in lower tion providing the respective SeÀ S bond-containing
conversion of 1a in the given time. The reaction products 5a,b in good yields (Scheme 2). Therefore, a
performed in the day-light or in the dark did not lead convenient and straightforward method is demon-
to formation of 3a, thus, confirming the necessity of strated for the preparation of 5-nitropyridin-2-yl)thio-
LED460. Other tested light sources (compact fluorescent Sec peptides as an alternative to the existing method.[27]
lamp (CFL) – warm white (2291 K, x=0.4915, y= This type of compounds has been reported to be used
0.4105), red LED (max 660 nm, deep red, x=0.5939, as electrophilic Se sources.[28] Furthermore, analogous
y=0.2488)) were less efficient. The decrease of GSH SÀ S bond-containing products 5c,d were synthesized
amount (5 equiv.) did not result in full conversion of employing
BocÀ CysÀ GlyÀ OBn
and
BocÀ Glu
1a.
(OtBu)À CysÀ GlyÀ OBn. Although the preparation of
The presence of TEMPO did not prevent the asymmetrical disulfide may not be an easy task due to
formation of 3a. Presumably, the generation of the formation of a mixture of symmetrical disulfides,
*
glutathionyl (GS ) radical under visible light irradi- the products 5c,d were obtained in moderate yields
ation, followed by the formation of SeÀ S bond or and only negligible amounts of symmetrical disulfides
homocoupling reaction, proceeds much faster, than were detected.
radical quenching with TEMPO.
Scheme 1. Scope and limitation studies for the preparation of SeÀ S bond-containing compounds. Reaction conditions: diselenide
(1 equiv.), GSH (10 equiv.), RB (0.1 equiv.), MeCN/H2O, LED460
.
Adv. Synth. Catal. 2021, 363, 1–6
2
© 2021 Wiley-VCH GmbH
��
These are not the final page numbers!

COMMUNICATIONS
asc.wiley-vch.de
ure 1). The photoluminescence quenching of RB was
performed in a degassed mixture of acetonitrile/water
(1:1). The quenching rate constant of RB with GSH
was significantly higher (8.8·10À 3 l/mol) than with 1a
(0.12·10À 3 mol/l). Therefore, it allows the assumption
that S-centered radical is rapidly formed under LED460
irradiation. Next, the GS radical reacts with the
diselenide forming the desired SeÀ S bond-containing
peptide 3a or it undergoes the homocoupling reaction
forming oxidized glutahione (GS-SG), therefore an
excess of 2 is required for full consumption of 1a
(Scheme 3). Notably, both parts of the diselenide are
utilized in the reaction.
Next, the stability of SeÀ S bond-containing gluta-
thione derivatives was evaluated. First, we examined
the stability of 3a under visible light irradiation.
Prolonged irradiation (>3 h) of 3a and RB solution in
MeCN/water with LED460 resulted in homolytic cleav-
age of SeÀ S bond, forming respective selenyl radical
Scheme 2. Synthesis of (5-nitropyridin-2-yl)thio-Sec and Cys
peptides. Reaction conditions: a) 1a or 1b (1 equiv.), 4
(5 equiv.), RB (0.1 equiv.), MeCN, LED460; b) Cys peptide
(1 equiv.), 4 (1 equiv.), RB (0.05 equiv.), MeCN, LED460
.
With the purpose to determine whether Se- or S- and GS radical. Selenyl radical is quickly oxidized to
centered radical is formed via visible light-initiated selenyl electrophile that reacts with H2O2 forming alkyl
reaction, Stern-Volmer analysis was performed (Fig- seleninic acid
6
and BocÀ DhaÀ GlyÀ OBn
7
(Scheme 4).[21] H2O2 is produced from singlet oxygen
reaction with water.
The reaction of 3a with H2O2 led to fast oxidation
to alkyl seleninic acid. Intermediate 6a was detected
by HRMS ([M+Na]=471.0633) and 77Se NMR (δ
1219.8 ppm) spectroscopy. Besides, a small signal of
selenonic acid 6b (δ 1050.8 ppm) was also detected in
the 77Se NMR spectra (Figure S1). The alkyl seleninic
acid 6a is too unstable to be isolable, it delivers 7 via
deselenylation.[21] The use of tBuOOH led to the
formation of 7 in 2 h as well.
Reduction of 3a with 3 equiv. 1,4-dithiothreitol
(DTT) or tris(2-carboxyethyl)phosphine hydrochloride
(TCEP) provided diselenide 1a and GSH in a short
reaction time (30 min). Furthermore, increasing the
amount of TCEP to 10 equiv. led to deselenylation and
provided Boc-Ala-Gly-OBn 9 (Scheme 4). The reduc-
tion proceeds via formation of the respective selenol 8
Figure 1. Photoluminescence quenching of RB with 1a and
GSH.
Scheme 4. Stability of 3a. Reaction conditions: a) H2O2 or
tBuOOH, MeCN/H2O; b) DTT, MeCN/H2O; c) TCEP, MeCN/
H2O.
Scheme 3. Proposed mechanism for the formation of SeÀ S
bond-containing substrates.
Adv. Synth. Catal. 2021, 363, 1–6
3
© 2021 Wiley-VCH GmbH
��
These are not the final page numbers!

COMMUNICATIONS
asc.wiley-vch.de
that was detected by LC-MS ([M+Na]=439.03) and compound was stable in pH range 3.0–8.0 for 24 h,
77Se NMR spectroscopy (δ -72.5 ppm). The deselenyla- whereas a considerable amount of diselenide 11 was
tion proceeded smoothly yielding 9 and TCEP=Se formed at pH 10.0 after 24 h (Table S1). Inspired by
([MÀ H]=328.9704).[29,30]
the good stability of a linker at physiological pH,
Further, derivative 12 was prepared starting with 7- absorption and emission spectra were collected for the
hydroxy-2-oxo-2H-chromene-3-carboxylic acid 10 solutions of 11, 12 and 13a in PBS (pH=7.4, c=
(Scheme 5) to evaluate the possibility to utilize 10 μM) (Figures S2–S4). The studied compounds
alkylselenylsulfide moiety as a cleavable linker for the showed absorbance maximum at 396–404 nm that is
introduction of fluorescent probe to thiol group- typical for the coumarin ring. Upon excitation at
containing peptide,. Diselenide 11 was successfully 350 nm the compounds‘ solutions emit light in blue
employed in the visible light-initiated reaction with region (Figure 2). Diselenide’s 11 photoluminescence
glutathione, thus, a mixture of DMSO/H2O was used to quantum yield is low (Φ=1.6%), however, it is
ensure that the starting materials are fully dissolved. considerably higher in the case of 12 (SeÀ S, Φ=
Luckily, the desired SeÀ S bond-containing product 12 29.5%). The highest emission quantum yield was
was easily obtained in 37% yield. Next, the stability of observed after oxidation of 12 with hydrogen peroxide
12 was established. A quick oxidation and formation – the seleninic acid‘s 13a Φ is equal to 52.4%. The
of the respective seleninic acid 13a was observed in emission was characterized by CIE coordinates of pure
the presence of H2O2. Another oxidant – NaClO blue light (x:0.1585, y:0.0160).
provided the respective chlorinated seleninic acid 13b
In conclusion, a simple protocol was developed for
([MÀ H]=377.9290) The use of DTT (10 equiv.) led to the synthesis of SeÀ S bond-containing peptides. A
formation of diselenide 11. Recently, it was demon- visible light-initiated reaction was employed for gen-
strated that a cyclic selenosulfide subjected to reduc- eration of sulfur-centered radical from unprotected
tive conditions (TCEP or DDT), spontaneously elimi- glutathione that further reacted with protected and
nates selenoethyl moiety releasing ethylene molecule, unprotected selenocysteine or selenocystamine pepti-
besides, all reactions were very slow (up to 6 days).[26] des. Amino acids with sensitive groups (Arg, Lys, Trp,
We did not observe such transformation in the case of Tyr, His, Glu) showed tolerance under reaction
12 – interaction with TCEP (10 equiv.) was very conditions, although the products were obtained only
efficient providing ethyl amide 14 in 30 min.
in moderate yields. The use of Met-containing sub-
Considering a possible utilization of SeÀ S bond as strate provided the respective Met-sulfoxide. Notably,
a sensitive linker in biocompatible materials, the asymmetrical disulfides can be synthesized from 5-
stability of 12 in phosphate-buffered saline (PBS, nitropyridine-2-thiol and Cys peptides. Fluorescent
20 mM) was established under various pH. The coumarin-selenocystamine conjugate with SeÀ S bond
is stable under physiological conditions allowing to
propose the SeÀ S bond as a valuable linker in
biocompatible materials under oxidative stress condi-
tions.
Experimental Section
Representative procedure for visible light-mediated Se-S bond
formation. Rose Bengal (0.1 equiv.) was added to a solution of
Sec-peptide 1 (1 equiv.) and l-glutathione 2 (10 equiv.) in
Scheme 5. Synthesis and stability of 12. Reaction conditions: a)
selenocystamine×2HCl (1 equiv.), 10 (2.5 equiv.), N-meth-
ylmorpholine (NMM) (3 equiv.), 1-hydroxybenzotriazole
(HOBt)
(1 equiv.),
1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide (EDC) (3 equiv.), DMF; b) GSH (10 equiv.), RB
(0.1 equiv.), DMSO/H2O, LED460; c) DTT, PBS; d) H2O2; e)
NaClO, PBS; f) TCEP, PBS.
Figure 2. Emission spectra of 11, 12, 13a upon excitation at
350 nm (PBS, pH=7.4).
Adv. Synth. Catal. 2021, 363, 1–6
4
© 2021 Wiley-VCH GmbH
��
These are not the final page numbers!

COMMUNICATIONS
asc.wiley-vch.de
mixture of MeCN/H2O (1:1) and the mixture was irradiated by
36 W blue LEDs for 1 hour. After evaporation the residue was
purified by reverse phase flash chromatography (C-18, MeCN/
H2O, 10–85%) to give the product 3.
[13] M. Abdo, S. Knapp, J. Org. Chem. 2012, 77, 3433–3438.
[14] M. Abdo, S. Knapp, J. Am. Chem. Soc. 2008, 130, 9234–
9235.
[15] Y. Xu, X. Shi, L. Wu, RSC Adv. 2019, 9, 24025–24029.
[16] M. Jereb, D. Dolenc, RSC Adv. 2015, 5, 58292–58306.
[17] O. Boutureira, G. J. L. Bernardes, M. Fernández-Gonzá-
lez, D. C. Anthony, B. G. Davis, Angew. Chem. Int. Ed.
2012, 51, 1432–1436; Angew. Chem. 2012, 124, 1461–
1465.
[18] M. Haratake, Y. Tachibana, Y. Emaya, S. Yoshida, T.
Fuchigami, M. Nakayama, ACS Omega. 2016, 1, 58–65.
[19] S. Shimodaira, M. Iwaoka, Phosphorus Sulfur Silicon
Relat. Elem. 2019, 194, 750–752.
Acknowledgements
Financial support from Latvian Institute of Organic Synthesis is
gratefully acknowledged (internal grant: IG-2021-01).
References
[20] F. Fan, S. Ji, C. Sun, C. Liu, Y. Yu, Y. Fu, H. Xu, Angew.
Chem. Int. Ed. 2018, 57, 16426–16430; Angew. Chem.
2018, 130, 16664–16668.
[1] A. Holmgren, C. Johansson, C. Berndt, M. E. Lönn, C.
Hudemann, C. H. Lillig, Biochem. Soc. Trans. 2005, 33,
1375–1377.
[21] S. Lapcinska, P. Dimitrijevs, L. Lapcinskis, P. Arsenyan,
Adv. Synth. Catal. 2021, 363, 3318–3328.
[2] A. Canal-Martín, R. Pérez-Fernández, Nat. Commun.
2021, 12, 163.
[22] P. Arsenyan, S. Lapcinska, A. Ivanova, J. Vasiljeva, Eur.
J. Org. Chem. 2019, 4951–4961.
[23] S. Lapcinska, P. Arsenyan, Eur. J. Org. Chem. 2020,
784–795.
[24] S. Lapcinska, P. Arsenyan, Synthesis 2021, 53, 1805–
1820.
[3] N. Metanis, J. Beld, D. Hilvert, in Patai’s chemistry of
functional groups. Wiley, New York, 2011.
[4] S. I. Suarez, R. Ambrose, M. A. Kalk, J. C. Lukesh,
Chem. A Eur. J. 2019, 25, 15736–15740.
[5] Y. Wang, C. T. Yang, S. Xu, W. Chen, M. Xian, Org.
Lett. 2019, 21, 7573–7576.
[25] N. Emmanuel, C. Mendoza, M. Winter, C. Horn, A.
Vizza, L. Dreesen, B. Heinrichs, J.-C. M. Monbaliu, Org.
Process Res. Dev. 2017, 21, 1435–1438.
[6] C. C. Tjin, K. D. Otley, T. D. Baguley, P. Kurup, J. Xu,
A. C. Nairn, P. J. Lombroso, J. A. Ellman, ACS Cent. Sci.
2017, 3, 1322–1328.
[26] V. Diemer, N. Ollivier, B. Leclercq, H. Drobecq, J.
Vicogne, V. Agouridas, O. Melnyk, Nat. Commun. 2020,
11, 2558.
[27] K. M. Harris, S. Flemer, R. J. Hondal, J. Pept. Sci. 2007,
13, 81–93.
[28] D. T. Cohen, C. Zhang, C. M. Fadzen, A. J. Mijalis, L.
Hie, K. D. Johnson, Z. Shriver, O. Plante, S. J. Miller,
S. L. Buchwald, B. L. Pentelute, Nat. Chem. 2019, 11,
78–85.
[29] S. Dery, P. S. Reddy, L. Dery, R. Mousa, R. N. Dardashti,
N. Metanis, Chem. Sci. 2015, 6, 6207–6212.
[30] N. J. Mitchell, J. Sayers, S. S. Kulkarni, D. Clayton,
A. M. Goldys, J. Ripoll-Rozada, P. J. B. Pereira, B.
Chan, L. Radom, R. J. Payne, Chem. 2017, 2, 703–715.
[7] A. Hamsath, M. Xian, Antioxid. Redox Signal. 2020, 33,
1143–1157.
[8] R. J. Hondal, S. M. Marino, V. N. Gladyshev, Antioxid.
Redox Signal. 2013, 18, 1675–1689.
[9] P. Saravanan, P. Anbarasan, Chem. Commun. 2019, 55,
4639–4642.
[10] A. Hamsath, Y. Wang, C. Yang, S. Xu, D. Cañedo, W.
Chen, M. Xian, Org. Lett. 2019, 21, 5685–5688.
[11] D. P. Gamblin, P. Garnier, P. S. Van Kasteren, N. J.
Oldham, A. J. Fairbanks, B. G. Davis, Angew. Chem. Int.
Ed. 2004, 43, 828–833; Angew. Chem. 2004, 116, 846–
851.
[12] M. Abdo, Z. Sun, S. Knapp, Molecules. 2013, 18, 1963–
1972.
Adv. Synth. Catal. 2021, 363, 1–6
5
© 2021 Wiley-VCH GmbH
��
These are not the final page numbers!

COMMUNICATIONS
Visible Light-Mediated Synthesis of SeÀ S Bond-Containing
Peptides
Adv. Synth. Catal. 2021, 363, 1–6
S. Lapcinska, P. Dimitrijevs, P. Arsenyan*