Application of dehydroalanine as a building block for the synthesis of selenocysteine-containing peptides

Article abstract of DOI:10.1039/C8RA09880H

Selenocysteine (Sec), the 21^st proteinogenic amino acid, is inserted co-translationally into number of natural proteins. It is coded by a dual function stop codon UGA (opal). It is a redox active amino acid found at the active sites of several

Full text of DOI:10.1039/C8RA09880H

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PAPER
Application of dehydroalanine as a building block
for the synthesis of selenocysteine-containing
peptides†
Cite this: RSC Adv., 2019, 9, 34
Kishorkumar M. Reddy and Govindasamy Mugesh
*
st
Selenocysteine (Sec), the 21 proteinogenic amino acid, is inserted co-translationally into number of natural
proteins. It is coded by a dual function stop codon UGA (opal). It is a redox active amino acid found at the
active sites of several enzymes that are involved in oxidation–reduction reactions. These enzymes include
the three major mammalian selenoproteins glutathione peroxidase (GPx), thioredoxin reductase (TrxR), and
iodothyronine deiodinase (Dio). Although Sec is structurally similar to its sulfur analogue cysteine (Cys), the
Received 1st December 2018
Accepted 4th December 2018
a
lower pK of the selenol group in Sec as compared to that of Cys and the interesting redox properties of the
selenium atom in peptides and proteins play crucial roles in redox catalysis. However, the chemical
synthesis of Sec-containing peptides has been a difficult task. In this paper, we report on a new method
for the synthesis of Sec and Sec-containing peptides using dehydroalanine (Dha) as a building block.
DOI: 10.1039/c8ra09880h
rsc.li/rsc-advances
Interestingly, the key enzyme SepSec synthase in eukaryotes
and selenocysteine synthase in prokaryotes are known to
The genetically encoded selenocysteine (Sec, U) is incorpo- convert phosphoserine and serine, respectively, to selenocys-
Introduction
1,2
3
rated into proteins by suppressing the stop codon UGA. The teine on the tRNA using pyridoxal pyrophosphate as a co-factor.
redox active Sec in proteins plays various roles in biological Recent studies showed that the mechanism of this pathway
systems, ranging from maintaining the redox homeostasis to involves the formation of a dehydroalanine (Dha) intermediate,
4
hormone activation and protein folding. Glutathione peroxi- which is further converted to Sec by a nucleophilic attack of
7
dase (GPx), iodothyronine deiodinase (Dio) and thioredoxin selenophosphate (Fig. 1). The formation of Dha through
reductase (TrxR) are some of the important selenoenzymes that a spontaneous non-enzymatic elimination of phosphoserine is
4
8
play key roles in mammalian systems. The lower pK of the detected during protein aging in human cells. Furthermore,
a
selenol group in Sec as compared to the thiol group in cysteine Dha is also produced during lantipeptide natural product
9
(
Cys) and the relatively lower reduction potential of the sele- biosynthesis in prokaryotes.
nium atom in the proteins make Sec a unique moiety for
Dehydroalanine (Dha) is a widely used precursor for the post-
5
catalysis. However, the synthesis of selenoproteins follows translational modications in proteins. The amino acid resi-
a unique pathway. The biosynthesis of Sec involves the use of dues serine (Ser), Cys or Sec are rst inserted at the position of
special mRNA sequence known as selenocysteine inserting interest in proteins. These residues are then activated to form
sequence (SECIS) adjacent to the UGA codon in the prokaryotes good leaving groups, which upon elimination, produces Dha.
0
and at the 3 -untranslation region in the eukaryotes. In Synthetically, Dha can be produced by the oxidative elimination
prokaryotes, it requires a selenophosphate synthetase for the of an alkylated derivatives of Cys and Sec. Recently, biosynthetic
synthesis of selenophosphate, selenocysteine synthase to incorporation of Sec-derivatives followed by an oxidative elim-
convert serine to Sec on tRNA, a specialised tRNA and Sec- ination of the aryl moiety with hydrogen peroxide has been
10
binding protein to bind with tRNA and SECIS. On the other shown to produce Dha moiety on peptides and proteins.
hand, in eukaryotes, the incorporation of Sec into proteins However, only a few reports are available on the incorporation
requires selenophosphate synthetase-2 for the synthesis of of selenium into proteins or peptides by chemical modica-
selenophosphate, SepSec synthase that can convert the phos- tions. It has been shown that the activation of a serine residue
phoserine to Sec on tRNA, a specialised tRNA, a Sec-binding with phenylmethanesulfonyl uoride (PMSF), followed by
6
protein and an elongation factor to bind with tRNA and SECIS.
displacement of the sulfonate with hydrogen selenide at pH 5.5
11
can give Sec-containing proteins. Another method involving
Native Chemical Ligation (NCL) of polypeptides between C-
terminal thioester and N-terminal Sec residue has also been
Department of Inorganic and Physical Chemistry, Indian Institute of Science,
Bangalore 560 012, India. E-mail: mugesh@iisc.ac.in
12
reported. The solid-phase peptide synthesis (SPPS) of Sec-
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/c8ra09880h
containing peptides using Fmoc-Sec(pMob)-OH is widely used,
1
34 | RSC Adv., 2019, 9, 34–43
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Fig. 1 Mechanism of SepSec synthase/selenocysteine synthase, which shows the formation of dehydroalanine before the attack of
7
selenophosphate.
15
but the protected amino acid is known to form Dha via Ca with minor modications. The Dha derivative 1 was synthe-
hydrogen atom abstraction mechanism during the elongation sized starting from L-cysteine. The amine group of cysteine was
step. The Dha residue thus formed can be attacked by piperi- protected with acid labile Boc and the resulting Boc-L-cysteine
13
2 3
dine to form b-piperidine adducts. As Dha plays a key role in was treated with 5 equiv. of K CO in DMF to afford compound
protein modication and SPPS, we have developed a new 1 in 30% yield, along with the methylated cysteine derivative (2)
method to insert Sec into peptides using a synthetic Dha as in about 65% yield (Scheme 1). This observation indicates that it
a building block.
is important to protect the carboxylic group, before treating
with methyl iodide for an alkylation–elimination reaction. Davis
and co-workers showed that the reaction of BocCysOMe with
a series of alkylating agents can produce Dha and the yield of
Dha depends on the nature of alkylating agent. They showed
Results and discussion
Synthesis of peptides having Dha moiety
The early attempts to generate Dha on proteins and peptides that 1,4-dibromo- and 1,4-diiodobutanes are the most efficient
relied on sulfonylation of Ser, followed by elimination under of the reagents for the generation of Dha. 1,4-Dichlorobutane,
experimental conditions that were found to be too harsh for on the other hand, was found to be slow to alkylate, and in this
14
most proteins. Recent studies indicate that dehydroalanine case, they recovered mostly the unreacted BocCysOMe. Other
reagents such as 1,5-diiodopentane, 1,4-bis(methansulfonyl)-
(
Dha) can be generated under mild conditions via post-
0
translational modications of Ser or Cys. When these two butandiol and a,a -dibromo-o-xylene produced BocDhaOMe in
16
amino acids are part of peptides or proteins, the elimination of only 11–12% yield.
water and hydrogen sulde, respectively, from these amino acid
For the synthesis of Dha-based dipeptides 5a–e, we employed
moieties produce the Dha. In our study, the Dha derivatives 1 the Boc-protected amino acids 3a–e. To achieve this,
and 5a–e were synthesized following the literature procedures compounds 3a–e were treated with N-methylmorpholine, ethyl
ꢀ
Scheme 1 Synthetic route to dehydroalanine 1 from the L-cysteine. (a) 1 M aq. NaOH, Boc anhydride, 1,4 dioxane, 27 C, 6 h. (b) K
2 3
CO , MeI, DMF,
ꢀ
27 C, 20 h.
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Scheme 2 Synthetic route to the dipeptides 5a–e from the protected amino acids 3a–e respectively via the formation of the corresponding
ꢀ
thioesters 4a–e. (a) Ethyl chloroformate, N-methylmorpholine, dry THF, molecular sieves, 27 C, 6 h (b) NaBH
4
, MeOH, 5 h. (c) K CO
2 3
, MeI, DMF,
ꢀ
ꢀ
2
7 C, 20 h. (d) K CO , 1,4-diiodobutane, DMF, 27 C, 4 h.
2 3
2 3
chloroformate and thiophenol in dry THF to form the C- dipeptides when treated with 1,4-diiodobutane and K CO in
terminal thioesters 4a–e. Peptide C-terminal thioesters are DMF for 4 h, afforded C-terminal Dha dipeptides in 90–95%
generally used as key intermediates in a variety of applications. without any other side products.
Particularly, they have been used recently for the native chem-
ical ligation methods for the total chemical synthesis of
proteins. Therefore, it was thought worthwhile to explore the
Reaction of Dha with pMob selenol
17
use of C-terminal thioesters for the synthesis of compounds 5a– Synthetically, Dha is a versatile precursor for various natural
18
e. The native chemical ligation was performed between and unnatural amino acid derivatives. The a,b-unsaturated
compounds 4a–e and L-cysteine to produce the dipeptides carbonyl moiety present in Dha can undergo Michael-type
bearing free thiol and carboxylic groups. When these dipeptides conjugate addition reactions with various nucleophiles. These
were treated with K
2
CO
3
in DMF for 24 h, the dipeptides bearing reactions are generally carried out in aqueous media at neutral
C-terminal dehydroalanine centre (5a–e) were obtained or slightly basic pH and temperatures below 40 C, the condi-
ꢀ
(
Scheme 2). Similar to the reaction of Boc-Cys with MeI, the tions that are compatible with proteins. Given the higher
formation of S-methylated peptides was observed as side nucleophilicity of the selenolate as compared to that of a thio-
products in these reactions as well. When we used L-cysteine late, it is expected that the Michael addition of aryl selenolates
methyl ester instead of L-cysteine in the native chemical liga- to Dha amides (selena-Michael addition) should be a highly
tion, we obtained dipeptides with a free thiol group. These favoured process. It has been shown that Dha is suitable for
19
chemically generating Sec in proteins. However, only one
ꢀ
Scheme 3 Synthetic route to selenocysteine derivative 8 and dipeptides 9a–e starting from the dipeptides 5a–e. (a) NaBH
4
, MeOH, 0 C, 10 min,
ꢀ
(
b) 50 mM sodium phosphate buffer pH-8, MeOH, 27 C, 6–8 h.
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Scheme 4 Synthetic route to glutathione analogue 13 starting from the glutathione 10. (a) Trityl alcohol, TFA. (b) Triethylamine, water, Boc
ꢀ
ꢀ
ꢀ
2 3 2 3
anhydride. (c) K CO , MeI, DMF. 27 C, 20 h. (d) Triethylsilane, 4% TFA, DCM, 27 C, 1 h. (e) K CO , 1,4-diiodobutane in DMF, 27 C, 4 h.
example of selena-Michael addition to Dha has been reported in
We have extended this methodology to tripeptides bearing
the literature. This involved the formation of a Se-allyl- Dha at the C-terminal and central positions. For this purpose,
selenocysteine from a selenium nucleophile derived in situ we selected glutathione (GSH, 10) as a model peptide to intro-
19
from the precursor allylselenocyanate. However, this meth- duce Dha between two amino acid residues. It is known that
odology may be useful for broader applications, when suitable GSH is an important antioxidant that prevents damage to
22
selenium nucleophiles are available for the Michael addition. cellular components caused by reactive oxygen species (ROS).
For our study, we selected p-methoxybenzyl (pMob) as the pro- It is a unique tripeptide with a g-peptide linkage between the
tecting group as it is a well-known protecting group for the carboxyl group of the glutamate side chain and the amine group
20
selenium in Sec. For the reactions with Dha, we synthesized of cysteine. The carboxyl group of cysteine is attached by
pMob diselenide (6), which upon reduction with NaBH , in situ a normal peptide linkage to glycine. Therefore, the Dha meth-
can generate pMob selenol (7). The reduction of compound 6 odology may be useful for the synthesis of the selenium
by NaBH was carried out in MeOH, in 50 mM sodium phos- analogue of GSH (GSeSeG). Previously, Tamura et al. synthe-
4
21
4
phate buffer pH 8.0 to obtain the corresponding selenol 7. sized the selenium analogue of glutathione disulphide, g-L-
When the in situ generated pMob selenol 7 was treated with the glutamyl-L-selenocysteinylglycine, by a liquid phase method. In
Dha derivative 1, the pMob-protected selenocysteine derivative 8 this method, the selenol group of Sec was protected by the pMob
was obtained in good yield. Similarly, the reaction of 7 with the group. The overall yield of the nal product was ꢁ9% based on
23
Dha-based dipeptides 5a–e in 50 mM sodium phosphate buffer the starting compound, Se-(pMob)-L-selenocysteine. Later,
at pH 8.0 produced the Sec dipeptides 9a–e in quantitative Iwaoka and co-workers reported the synthesis and antioxidant
yields (Scheme 3).
activity of GSeSeG. They synthesized GSeSeG from the Sec
Scheme 5 Synthetic route to the tripeptide 17 starting from dipeptide 14. (a) Ethyl chloroformate, N-methylmorpholine, dry THF, molecular
ꢀ
ꢀ
4 2 3
sieves, 27 C, 6 h. (b) NaBH , MeOH, 5 h. (c) K CO , 1,4-diiodobutane, DMF, 27 C, 4 h.
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ꢀ
Scheme 6 Synthetic route to seleno-tripeptides 18 and 19 starting from the Dha-peptides 13 and 17 respectively. (a) NaBH
4
, MeOH, 0 C, 10 min,
ꢀ
(
b) 50 mM sodium phosphate buffer pH 8, MeOH, 27 C, 6–8 h.
derivative, Fmoc-Sec(pMob)-OH, using a solid-phase peptide was synthesized by following a conventional solution-phase
24
synthesis method.
peptide synthesis method. The Boc-protected dipeptide amino
To synthesize the GSeSeG via the Dha route, it was necessary acid (Boc-NH-Leu-Ala-COOH, 14) was treated with thiophenol in
to protect the free amino and carboxylic groups. The protected the presence of N-methylmorpholine and ethyl chloroformate
GSH derivative 12 was synthesized by following the literature in dry THF to give the dipeptide bearing C-terminal thioester 15,
25
procedure. Briey, the thiol group of GSH was protected with which was used for the native chemical ligation with L-cysteine
trityl group by treating the thiol with trityl alcohol in TFA. The methyl ester to give the tripeptide 16. Treatment of compound
2 3
resulting trityl-protected GSH was treated with Boc anhydride 16 with 1,4 diiodobutane and K CO in DMF for 4 h afforded the
under basic conditions to afford N-Boc-S-trityl-protected GSH. tripeptide bearing Dha at C-terminal 17 (Scheme 5). We
Esterication of this derivative with MeI in DMF produced N- employed L-cysteine in the chemical ligation and followed by
Boc-S-trityl-glutathione methyl ester 11. The deprotection of the treatment with MeI and K CO in DMF for 24 h afforded the
2
3
trityl group by treating with triethylsilane in TFA afforded N- Dha peptide in 30% yield.
Boc-glutathione methyl ester 12. The Dha analogue of gluta- Although the addition of functionalized thiols to Dha under
thione 13 was obtained by treating 12 with 1,4-diiodobutane in mild conditions is generally a facile process, the nature of
DMF (Scheme 4). amino acid moiety attached to the Dha appears to modulate its
In addition to GSeSeG, several other selenium-containing reactivity. In this study, we observed that the nature of solvents
peptides have been studied as glutathione peroxidase plays an important role in the conversions as well as the reac-
mimetics. Iwaoka and co-workers showed that the change in the tion time. When the coupling of tripeptides bearing Dha (13
amino acid sequence in a series of selenium-containing and 17) with pMob selenol were performed in MeOH and
24
peptides alters the antioxidant activity. For example, they 50 mM sodium phosphate buffer at pH 8, the selenium
showed that the replacement of the g-Glu moiety in GSeSeG analogue of glutathione 18 and selenopeptide 19 were obtained
with a Leu residue signicantly decreased the antioxidant in 80–90% yield. This agrees with an earlier report that the rate
activity. Therefore, we aimed at the synthesis of Sec-containing of Michael addition of amines and thiols to Dha amides was
26
peptides having other amino acids such as Leu and Ala resi- greatly accelerated in the presence of water (Scheme 6).
dues. For this, the tripeptide with C-terminal Dha centre (17)
Scheme 7 Reaction of the selenocysteine dipeptide 9d with iodine in methanol and a subsequent reaction with sodium borohydride and
iodoacetic acid to produce the alkylated derivative 21.
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2 6 4 2
Scheme 8 Coupling of o-nitrobenzyl selenol with the Dha derivative 1 to afford the selenocysteine derivative 22. (a) o-NO –C H –CH SeCN,
ꢀ ꢀ
NaBH , MeOH, 0 C for 30 min; 27 C, 8 h (b) 366 nm UV light, ACN and phosphate buffer pH 7 (1 : 1), 2 h.
4
Formation of epimers
at 366 nm UV light for around 2 h, produced the diselenide 23,
which was characterized by ESI-MS (Fig. S2B and S2C, ESI†). The
UV-visible spectra of compound 23 exhibited a band (lmax) at
around 254, which disappeared upon irradiation (Fig. S2A,
ESI†).
It has been shown earlier that the addition of thiols to Dha lead
to epimeric products, and the formation of epimeric (D-/L-)
mixtures in these reactions can be viewed as a limitation of the
27
synthetic functionalization of Dha. This methodology may not
be useful when enantiomerically pure selenium compounds are
required. However, for the selenocysteine derivatives, the
chirality may not play any key role in certain biological activities
such as peroxynitrite scavenging. To understand the number of
isomers produced in the selenocysteine derivatives, we carried
Conclusions
In this paper, we reported the incorporation of selenocysteine
into peptides via dehydroalanine intermediate (Dha). Using this
methodology, several di- tri- and penta-peptides having Dha
moiety were synthesized. A selenium moiety has been intro-
duced into these peptides using p-methoxybenzyl (pMob) sele-
nol, derived from the corresponding diselenide. The pMob
group can be cleaved easily by treating the peptides with iodine
in methanol. The Dha methodology was found to be useful,
particularly, for the synthesis of the selenium analogue of
glutathione. The Dha route can also be used to synthesize
selenocysteine derivatives having photo-cleavable o-nitrobenzyl
group, which can be readily cleaved under UV irradiation.
Although the Dha methodology yields the selenium compounds
as mixture of epimers, the compounds produced by this method
should be useful for studies that do not depend on the chirality
of the selenocysteine moiety.
7
7
ꢀ
out Se NMR studies in CD OD at 27 C (Fig. S1, ESI†). For
3
compound 9d, two signals at 230.4 and 231.4 ppm were
observed, indicating the formation of two epimers. As the
amino acid attached to the selenocysteine moiety was enantio-
merically pure (R-conguration), the two epimers formed in the
reactions are expected to have R,R and R,S congurations
(Scheme 7).
When compound 9d was treated with I
produced the corresponding diselenide 20. The Se NMR
spectrum obtained for 20 showed four peaks at 295.6, 298.1,
2
in MeOH, it
7
7
298.6, 298.9 ppm, indicating the formation four epimers with
possible congurations of R,R,R,S; R,S,R,R; R,R,R,R and R,S,S,R.
The reduction of the diselenide 20 by sodium borohydride
produced the corresponding selenol, which was trapped by
treating the reaction mixture with iodoacetic acid. The alkylated
compound 21 also showed two peaks in the Se NMR spectrum
at 190.4 and 191.1 ppm, indicating the formation of two
epimers with R,R and R,S congurations.
7
7
Experimental section
General procedure
All the amino acids were purchased from GL Biochem
Selenocysteine derivatives with a photo-cleavable group
(Shanghai) Ltd. Methanol was obtained from Merck. All other
In addition to the use of pMob group, which can be removed chemicals were of the highest purity available. Most reactions
from the peptide by treating with iodine in MeOH, it is known were carried out in a well-ventilated fume hood to avoid the
that a photo labile group such as o-nitrobenzyl group can also unpleasant odour and toxic nature of the reaction mixtures
2
8
be used as a protecting group for selenium in selenocysteine.
For the introduction of o-nitrobenzyl group, we synthesized o- none. Thin-layer chromatography analyses were carried out on
nitrobenzyl selenocyanide (o-NO –C –CH SeCN) as precursor pre-coated silica gel plates (Merck), and spots were visualized
by following the literature procedure. Briey, the procedure under UV radiation. Column chromatography was performed
involved. THF was dried over sodium metal with benzophe-
2
6
H
4
2
2
9
1
13
involves treatment of o-nitrobenzyl bromide or chloride with on glass columns loaded with silica gel. H (400 MHz),
KSeCN in acetone at reux condition. The product must be (100.56 MHz), and Se (76.29 MHz) NMR spectra were obtained
recrystallized using EtOH to remove the impurities. Subse- on a Bruker 400 MHz NMR spectrometer. Chemical shi values
quently, o-nitrobenzyl selenocyanide was treated with NaBH in are cited with respect to SiMe as internal ( H and C) and
MeOH to obtain o-nitrobenzyl selenol, which was added care- Me Se as external ( Se) standard. Mass spectral studies were
C
7
7
1
13
4
4
7
7
2
fully to the Dha derivative 1 in MeOH to obtain compound 22 in carried out on a Bruker Daltonics Esquire 6000 plus mass
around 30% yield (Scheme 8). The irradiation of compound 22 spectrometer with ESI-MS mode analysis.
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Synthesis of 1
(1.0 equiv., 3.74 mmol) was added in MeOH (10 mL), and the
reaction mixture was stirred till the starting materials were fully
consumed (ꢁ4 h, TLC analysis). Aer the removal of MeOH
under reduced pressure, the residue was treated with water (20
mL). The reaction mixture was acidied with 1 N HCl, extracted
with ethylacetate (3 ꢂ 50 mL) and washed with water (2 ꢂ 25
mL), followed by brine (3 ꢂ 25 mL). The organic layer was dried
Step 1. In a 250 mL round bottom ask, L-cysteine (1.0 g, 8.2
mmol) was dissolved in 1 M aq. NaOH (25 mL). To the solution,
Boc anhydride (2.16 g, 9.9 mmol) in 1,4-dioxane (8 mL) was
added and the reaction mixture was stirred for 6 h at 27 C. The
4
reaction mixture was acidied with aq. KHSO (50 mL) and
extracted with ethyl acetate (3 ꢂ 50 mL). The organic layer was
washed with water (3 ꢂ 50 mL) followed by brine (3 ꢂ 50 mL).
The combined organic layer was dried over anhydrous Na SO ,
ꢀ
2 4
over anhydrous Na SO . Aer reducing the volume of the
solvent to ꢁ10 mL, TECP (0.5 equiv., 1.87 mmol) was added to
reduce any disulphide bonds formed during the progress of the
reaction. The reaction mixture was diluted with water (50 mL)
and the aqueous layer was extracted once again with ethyl-
acetate (3 ꢂ 50 mL) and washed with water(2 ꢂ 25 mL), fol-
lowed by brine (3 ꢂ 25 mL). The organic layer was dried over
2
4

ltered, and concentrated under reduced pressure to give thick
oil as product Boc-L-cysteine in good yield (1.7 g, 95%). The
product was used in the next time without purication.
Step 2. To a solution of Boc-L-cysteine (1.5 g, 6.8 mmol) in
DMF (30 mL), 5 equiv. of K
2
CO
3
(4.69 g, 33.9 mmol) was added
ꢀ
2 4
anhydrous Na SO , ltered and concentrated under reduced
at 0 C and the suspension was stirred for 15 min. To this stirred
pressure. The intermediates formed in these reactions were
used directly for the next step without further purication.
These intermediate products were dissolved in DMF (25 mL)
and added MeI (5 equiv., 18.7 mmol) and K CO (5 equiv., 18.7
solution, methyl iodide (5 equiv., 425 mL, 6.8 mmol) was added
and the stirring was continued for 24 h at 27 C. The completion
of reaction was followed by TLC. The reaction mixture was
4
neutralized with aq. KHSO and extracted with ethyl acetate (3
ꢀ
2
3
ꢀ
mmol) at 0 C. Aer 15 min the ice bath was removed, and the
ꢂ 50 mL). The organic layer was washed with water (3 ꢂ 20 mL)
ꢀ
reaction mixture was stirred at 27 C for about 12 h. Aer
and brine (5 ꢂ 20 mL) to remove the DMF. The combined
completion of the reaction, which was followed by TLC, dil. HCl
was added to neutralize the reaction mixture and the compound
was extracted with ethylacetate (3 ꢂ 50 mL). The organic layer
was washed with brine solution (5 ꢂ 25 mL) to remove DMF. It
was dried over anhydrous Na SO , ltered and concentrated
2 4
organic layer was dried over anhydrous Na SO , ltered, and
concentrated under reduced pressure. The crude residue was
puried by column chromatography using 10% Pet. ether and
ethylacetate to afford 1 (30%, 408 mg) as colorless oil. H NMR
1
2
4
(
CDCl ) d (ppm) 6.97 (br, 1H), 6.08 (s, 1H), 5.63–5.64 (d, 1H),
3
1
3
under reduced pressure. The product was puried by column
chromatography using 100–200 mesh silica and 20% petroleum
ether and ethylacetate as eluent to afford the Dha-containing
peptide 5a–e, 17 as viscous liquids which becomes solids on
standing for long time.
3.74 (s, 3H), 1.40 (s, 9H); C NMR (CDCl
3
) d (ppm) 28.15, 52.76,
80.54, 105.05, 131.30, 152.46, 164.35; ESI-MS: m/z calcd for
+
9 4
C H15NO Na [M + Na] : 224.0899, found 224.1136.
General procedure for the synthesis of thioesters 4a–e, 15
Compound 5a. Thick viscous liquid, yield: 40% (386 mg, 1.49
1
mmol). H NMR (D
.88 (s, 3H), 3.79 (s, 3H), 1.47 (s, 9H); C NMR (D -CD OD)
4
-CD
3
OD), d (ppm): 6.43 (s, 1H), 5.88 (s, 1H),
The Boc-protected amino acid (3a–e, 14) (1.0 equiv., 5.71 mmol),
N-methylmorpholine (1.2 equiv., 6.85 mmol) and dry THF (25
1
3
3
4 3
ꢀ
d (ppm) 28.64, 45.43, 49.00, 53.35, 81.00, 110.04, 132.84, 158.51,
mL) were mixed in a dry ask containing 4 A molecular sieves
+
ꢀ
165.38, 171.18; ESI-MS: m/z calcd for C H N O Na [M + Na] :
11 18 2 5
and the solution was cooled to 0 C. To this, ethyl chloroformate
1.5 equiv., 8.56 mmol) was added and the reaction mixture was
2
81.1113, found: 281.0737.
(
Compound 5b. Thick viscous liquid, yield: 30% (305 mg, 1.12
stirred for 15 min. Thiophenol (1.2 equiv., 6.85 mmol) and N-
1
mmol). H NMR (CDCl
3
), d (ppm): 8.45 (br, 1H), 6.55 (s, 1H),
methylmorpholine (1.2 equiv., 6.85 mmol) were added and the
ꢀ
5.86 (s, 1H), 5.16–5.27 (br, 1H), 4.23–4.24 (br, 1H), 3.79 (s, 3H),
reaction mixture was slowly warmed to 27 C. The stirring was
1
3
1
5
.44 (s, 9H), 1.36–1.37 (d, 3H); C NMR (CDCl
3
) d 17.92, 28.26,
continued for 12 h at this temperature. The completion of the
reaction was monitored by TLC. The reaction mixture was
0.98, 52.93, 80.37, 109.26, 130.91, 155.53, 164.25, 171.78; ESI-
+
MS: m/z calcd for C H N O Na [M + Na] : 295.1270, found:

ltered to remove molecular sieves, and the solvent was
12 20 2 5
2
95.1051.
removed under reduced pressure. The resulting residue was
dissolved in dichloromethane (100 mL) and washed sequen-
Compound 5c. Thick viscous liquid, yield: 40% (465 mg, 1.48
1
mmol). H NMR (CDCl
3
), d (ppm): 8.41 (br, 1H), 6.57 (s, 1H),
tially with NaHCO
3
(3 ꢂ 50 mL), H
2
O (3 ꢂ 50 mL), and brine (3
5
(
.86 (s, 1H), 5.05 (br, 1H), 4.19 (br, 1H), 3.79 (s, 3H), 1.66–1.69
ꢂ 50 mL). The organic phase was dried over anhydrous Na
2
SO
4
1
3
m, 2H), 1.47–1.51 (m, 1H), 1.41 (s, 9H), 0.90–0.92 (t, 6H);
C
and concentrated under reduced pressure. The product was
puried by column chromatography to afford the products (4a–
e, 15) (80–90%) as white solids. The spectral values obtained
3
NMR (CDCl ), d (ppm): 21.78, 23.01, 24.81, 28.28, 41.04, 52.93,
5
3.96, 80.27, 109.28, 130.91, 155.74, 164.28, 171.78; ESI-MS: m/z
+
15,30
calcd for C H N O Na [M + Na] : 337.1739, found: 337.1586.
15 26 2 5
were matching with the literature values.
Compound 5d. Thick viscous liquid, yield: 35% (408 mg, 1.30
1
mmol). H NMR (CDCl
.88 (s, 1H), 5.09 (br, 1H), 4.07 (brs, 1H), 3.81 (s, 3H), 1.92–1.93
Cysteine$HCl (1.0 equiv., 3.74 mmol) was added to MeOH (15 (m, 1H), 1.47–1.51 (m, 1H), 1.44–1.51 (m, 1H) 1.42 (s, 9H) 1.09–
3
), d (ppm): 8.22 (br, 1H), 6.59 (s, 1H),
General procedure for the synthesis of Dha peptides 5a–e, 17
5
1
3
mL). NaBH
4
(2.0 equiv., 7.48 mmol) was added slowly, and the 1.16 (m, 1H), 0.87–0.94 (m, 6H); C NMR (CDCl
3
), d (ppm):
mixture was stirred for 1 h under N
2
atm. Thioesters (4a–e, 15) 11.60, 15.73, 24.79, 28.36, 37.40, 53.06, 60.15, 80.27, 109.35,
40 | RSC Adv., 2019, 9, 34–43
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30.71, 155.82, 164.41, 170.78; ESI-MS: m/z calcd for selenocysteine derivatives, seleno dipeptides 8, 9a–e, 18, 19, in
+
C H N O Na [M + Na] : 337.1739, found: 337.2340.
80–90% yields, and 22 in 35% yield.
1
5
26 2 5
Compound 5e. White solid, yield: 35% (457 mg, 1.31 mmol).
Compound 8. Eluent for column chromatography: 20% Pet.
1
H NMR (CDCl ), d (ppm): 8.15 (br, 1H), 7.18–7.31 (m, 5H), 6.61 ether/ethylacetate. Slight yellow solid, yield: 95% (570 mg, 1.41
3
1
(
s, 1H), 5.89–5.89 (d, 1H), 4.99 (s, 1H), 4.43 (s, 1H), 3.78 (s, 3H), mmol). H NMR (CDCl
3
), d (ppm): 1.45 (s, 9H), 2.88 (s, 2H), 3.74
1
3
3
3
1
C
3
.11–3.16 (m, 2H), 1.40 (s, 9H); C NMR (CDCl ), d (ppm): 28.32, (s, 3H), 3.76 (s, 2H), 3.78 (s, 3H), 4.56–4.61 (t, 1H), 5.28–5.30 (d,
1
3
3
8.30, 53.00, 56.64, 80.64, 109.48, 127.19, 128.88, 129.32, 1H), 6.81–6.83 (d, 2H), 7.19–7.21 (d, 2H); C NMR (CDCl ),
30.72, 136.39, 155.50, 164.13, 170.38; ESI-MS: m/z calcd for d (ppm): 25.9, 27.5, 28.4, 52.6, 53.5, 55.4, 80.2, 114.1, 130.1,
+
77
18
H
24
N
2
O
5
Na [M + Na] : 371.1583, found: 371.3443.
3
130.7, 155.2, 158.7, 171.8; Se NMR (CDCl ), d (ppm): 215.3;
+
Compound 17. Pale yellow solid, yield: 30% (430 mg, 1.11 ESI-MS: m/z calcd for C H NO SeNa [M + Na] : 426.0796;
1
7
25
5
1
mmol). H NMR (CDCl ), d (ppm): 0.83–0.89 (m, 6H), 1.06–1.13 found: 426.0714.
3
(m, 1H), 1.35–1.37 (d, 3H), 1.39 (s, 9H), 1.49–1.54 (m, 1H), 1.82
Compound 9a. Eluent for column chromatography: 20% Pet.
(
5
1
4
br, 1H), 3.80 (s, 3H), 3.98–4.0 (d, 1H), 4.62–4.65 (dd, 1H), 5.34– ether/ethylacetate. Thick viscous oil, yield: 90% (621 mg, 1.35
1
.36 (d, 1H), 5.87 (s, 1H), 6.52 (s, 1H), 7.0–7.01 (d, 1H), 8.54 (s, mmol). H NMR (CDCl
H); C NMR (CDCl
3
3
), d (ppm): 1.41 (s, 9H), 2.85–2.86 (m,
), d (ppm): 11.2, 15.5, 17.7, 24.8, 28.3, 37.3, 2H), 3.69 (s, 3H), 3.71 (s, 2H), 3.73 (s, 3H), 4.79–4.84 (m, 1H),
9.6, 52.9, 59.1, 79.9, 109.7, 131.2, 156.0, 164.2, 171.1, 172.3; 5.43–5.46 (t, 1H), 6.77–6.79 (m, 2H), 7.14 (br, 1H), 7.16–7.17 (d,
1
3
+
13
ESI-MS: m/z calcd for C18
4
H
31
N
3
O
6
Na [M + Na] : 408.2111; found: 2H); C NMR (CDCl
55.2, 80.1, 114.0, 130.0, 130.5, 156.1, 169.6, 171.2; Se NMR
Compound 13. Compound 12 was synthesized by following (CDCl ), d (ppm): 216.4; ESI-MS: m/z calcd for C H N O SeNa
3
), d (ppm): 25.1, 27.4, 28.3, 44.2, 52.0, 52.6,
7
7
08.2048.
3
19 28 2 6
27
+
the reported procedure. To a solution of 12 (500 mg, [M + Na] : 483.1010; found: 483.2105.
1
K
.15 mmol, 1 equiv.) in DMF (15 mL) was added 5 equiv. of
Compound 9b. Eluent for column chromatography: 20% Pet.
ꢀ
1
2
CO
3
(794 mg, 5.74 mmol) at 0 C and stirred for 15 min, then ether/ethylacetate. Yield: 90% (639 mg, 1.34 mmol) H NMR
added 5 equiv. of 1,4 diiodobutane (152 mL, 5.75 mmol) and (CDCl
stirred for 6 h at 27 C. The completion of reaction was moni- 2H), 3.72 (d, 3H), 3.73 (s, 2H), 3.77 (s, 3H), 4.19–4.20 (br, 1H),
tored by TLC. Aer completion of the reaction, 1 N HCl was 4.80–4.84 (m, 1H), 5.09–5.11 (br, 1H), 6.79–6.82 (dd, 2H), 6.85–
3
), d (ppm): 1.33–1.36 (dd, 3H), 1.43 (d, 9H), 2.83–2.94 (m,
ꢀ
1
3
added to neutralize the reaction mixture and extracted with 7.0(br, 1H), 7.17–7.19 (d, 2H); C NMR (CDCl ), d (ppm): 17.96,
3
ethylacetate (3 ꢂ 25 mL), washed with brine (5 ꢂ 15 mL) to 18.04, 24.53, 26.73, 26.78, 27.91, 49.59, 49.65, 51.75, 52.02,
remove the DMF. The organic layer was dried over anhydrous 52.05, 54.71, 77.35, 79.23, 113.53, 129.61, 130.13, 130.24,
7
7
Na
2
SO
4
, ltered and concentrated under reduced pressure. The 155.04, 155.11, 158.08, 170.81, 170.93, 172.73; Se NMR
), d (ppm): 214.9, 215.7; ESI-MS: m/z calcd for C20
product was puried by column chromatography using 100–200 (CDCl
mesh silica and 5% dichloromethane and methanol as eluent to
3
30
H -
+
N
2
O
6
SeNa [M + Na] : 497.1167; found: 497.4734.
afford the product 13 as light yellow oil. Yield: 90% (415 mg,
Compound 9c. Eluent for column chromatography: 20% Pet.
1
1
1
2
1
.03 mmol) H NMR (CDCl
3
), d (ppm): 1.37 (s, 9H), 1.93–2.14 (m, ether/ethylacetate. Yield: 85% (658 mg, 1.27 mmol) H NMR
H), 2.39 (brs, 2H), 3.68 (s, 3H), 3.72 (s, 3H), 4.06 (s, 2H), 4.25 (s, (CDCl ), d (ppm): 0.2–0.93 (t, 3H), 1.42–1.43 (d, 9H), 1.45–1.47
H), 5.38 (s, 1H), 6.37 (s, 1H), 7.35 (s, 1H), 8.16 (s, 1H); C NMR (m, 1H), 1.65–1.70 (m, 1H), 2.86–2.91 (m, 2H), 3.72–3.74 (d, 3H),
3
1
3
(CDCl ), d (ppm): 28.8, 30.2, 33.8, 42.1, 52.9, 53.0, 53.4, 80.5, 3.77 (s, 2H), 3.79 (s, 3H), 4.08–4.15 (br, 1H), 4.81–4.85 (m, 1H),
3
1
03.8, 134.3, 156.1, 164.9, 170.7, 171.5, 173.3; ESI-MS: m/z calcd 4.90–4.94 (t, 1H), 6.80–6.86 (dd, 2H), 6.88–6.97 (br, 1H), 7.18–
+
13
for C17
H
27
N
3
O
8
Na [M + Na] : 424.1696; found: 424.4928.
7.20 (d, 2H); C NMR (CDCl
3
), d (ppm): 21.67, 21.75, 22.64,
2
5
1
2.73, 24.41, 24.49, 24.72, 26.90, 26.97, 28.06, 40.96, 40.99,
1.88, 52.15, 52.19, 52.77, 54.88, 77.35, 79.40, 113.68, 129.76,
30.25, 130.40, 155.39, 155.48, 158.25, 170.94, 171.02, 172.63;
General procedure for the synthesis of Sec derivatives
NaBH (4 equiv., 6.0 mmol) was added portion-wise to a solu-
tion of p-methoxy benzyldiselenide or o-NO –C –CH SeCN (2
77
Se NMR (CDCl ), d (ppm): 213.5, 214.0; ESI-MS: m/z calcd for
3
+
C
H
36
N
O
2
SeNa [M + Na] : 539.1636; found: 539.1557.
4
23
6
2
6
H
4
2
Compound 9d. Eluent for column chromatography: 20% Pet.
1
equiv., 3.0 mmol) in degassed MeOH (1 mL per 0.03 mmol) at ether/ethylacetate. Yield: 80% (619 mg, 1.20 mmol) H NMR
ꢀ
0
2
3
C under N atmosphere. An immediate evolution of hydrogen (CDCl ), d (ppm): 0.89–0.95 (m, 6H), 1.08–1.18 (m, 1H), 1.43–
gas was observed. Aer 5 min, this solution was added dropwise 1.44 (d, 9H), 1.46–1.50 (m, 1H), 1.84–1.97 (m, 1H), 2.87–2.95 (m,
to Dha containing aminoacid (1)/dipeptides (5a–e)/tripeptides 2H), 3.73 (s, 3H), 3.74 (s, 2H), 3.78 (s, 3H), 3.97–4.05 (m, 1H),
(
(
13, 17) (1 equiv., 1.50 mmol) in 0.5 M sodium phosphate 4.82–4.87 (m, 1H), 5.01 (br, 1H), 6.55–6.72 (m, 1H), 6.81–6.23
pH-8) (1 mL per 0.003 mmol) and stirred at 27 C for 4–6 h. The (dd, 2H), 7.18–7.20 (d, 2H); C NMR (CDCl ), d (ppm): 11.44,
3
ꢀ
13
reaction mixture was treated with EtOAc and washed subse- 11.51, 15.46, 15.66, 24.57, 24.74, 25.11, 27.25, 27.37, 28.31,
quently with 1 M aq. HCl, saturated NaHCO
, water and brine. 37.33, 37.39, 51.95, 51.99, 52.49, 52.53, 55.22, 59.16, 79.81,
The organic layers were combined and dried over anhydrous 114.00, 130.01, 130.44, 130.54, 155.64, 155.72, 158.59, 171.08,
3
7
7
2
4
3
Na SO , ltered, and concentrated under reduced pressure. The 171.17, 171.48, 171.53; Se NMR (CDCl ), d (ppm): 213.7, 214.0;
+
crude residue was puried by column chromatography (100– ESI-MS: m/z calcd for C23
00 mesh silica gel for compounds 8, 9a–9e, and 230–400 mesh found: 540.4047.
H
37
N
2
O
6
SeNa [M + Na + H] : 540.1715;
2
silica gel for compound 18, 19 & 22) to afford the protected
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Compound 9e. Eluent for column chromatography: 20% Pet. CSIR for a research fellowship. G. M. acknowledges the SERB for
1
ether/ethylacetate. Yield: 85% (696 mg, 1.26 mmol) H NMR the award of J. C. Bose National fellowship (SB/S2/JCB-067/2015).
(
CDCl
3
), d (ppm): 1.40 (s, 9H), 2.73–2.87 (m, 2H), 3.07–3.11 (m,
H), 3.68–3.72 (m, 5H), 3.78 (s, 3H), 4.39 (br, 1H), 4.77–4.83 (m,
H), 5.00 (br, 1H), 6.61–6.66 (m, 1H), 6.81–6.83 (d, 2H), 7.17–
2
1
7
2
5
1
1
Notes and references
1
3
.29 (m, 5H), 7.30 (d, 2H); C NMR (CDCl ), d (ppm): 24.77,
3
4.84, 27.02, 27.08, 28.07, 38.23, 51.86, 52.04, 52.27, 52.30,
4.97, 55.37, 77.36, 79.71, 113.78, 126.58, 128.31, 129.16,
29.82, 130.28, 130.40, 136.52, 136.62, 155.20, 155.30, 158.35,
1 (a) T. C. Stadtman, Science, 1974, 183, 915–922; (b) J. E. Cone,
R. M. Del R ´ı o, J. N. Davis and T. C. Stadtman, Proc. Natl. Acad.
Sci. U. S. A., 1976, 73, 2659–2663.
7
7
70.78, 170.96, 171.25; Se NMR (CDCl
3
), d (ppm): 217.9, 218.6;
2 (a) A. B ¨o ck, K. Forchhammer, J. Heider and C. Baron, Trends
Biochem. Sci., 1991, 16, 463–467; (b) A. Bock,
K. Forchhammer, J. Heider, W. Leinfelder, G. Sawers,
B. Veprek and F. Zinoni, Mol. Microbiol., 1991, 5, 515–520.
3 (a) F. Zinoni, A. Birkmann, T. C. Stadtmant and A. B ¨o ck, Proc.
Natl. Acad. Sci. U. S. A., 1986, 83, 4650–4654; (b) I. Chambers,
J. Frampton, P. Goldfarb, N. Affara, W. McBain and
P. R. Harrison, EMBO J., 1986, 5, 1221–1227; (c) B. J. Lee,
P. J. Worland, J. N. Davis, T. C. Stadtman and
D. L. Hateld, J. Biol. Chem., 1989, 264, 9724–9727.
4 (a) L. Floh ´e , E. A. G u¨ nzler and H. H. Schock, FEBS Lett., 1973,
32, 132–134; (b) J. T. Rotruck, A. L. Pope, H. E. Ganther,
A. B. Swanson, D. G. Hafeman and W. G. Hoekstra, Science,
1973, 179, 588–590; (c) H. Sies, Angew. Chem., Int. Ed.,
1986, 25, 1058–1071; (d) D. Behne, A. Kyriakopoulos,
H. Meinhold and J. K ¨o hrle, Biochem. Biophys. Res.
Commun., 1990, 173, 1143–1149; (e) T. Tamura and
T. C. Stadtman, Proc. Natl. Acad. Sci. U. S. A., 1996, 93,
1006–1011; (f) G. Mugesh, W.-W. du Mont and H. Sies,
Chem. Rev., 2001, 101, 2125–2180; (g) J. K ¨o hrle, Methods
Enzymol., 2002, 347, 125–167; (h) G. Roy, B. K. Sarma,
P. P. Phadnis and G. Mugesh, J. Chem. Sci., 2005, 117, 287–
303; (i) M. Conrad, M. Schneider, A. Seiler and
G. W. Bornkamm, Biol. Chem., 2007, 388, 1019–1025; (j)
K. P. Bhabak and G. Mugesh, Acc. Chem. Res., 2010, 43,
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10074–10076; (l) S. Mondal, K. Raja, U. Schweizer and
G. Mugesh, Angew. Chem., Int. Ed., 2016, 55, 7606–7630.
5 (a) A. P. Arnold, K. S. Tan and D. L. Rabenstein, Inorg. Chem.,
1986, 25, 2433–2437; (b) B. J. Byun and Y. K. Kang,
Biopolymers, 2011, 95, 345–353.
+
34 2 6
ESI-MS: m/z calcd for C26H N O SeNa [M + Na] : 573.1480;
found: 573.3649.
Compound 18. Eluent for column chromatography: 10%
dichloromethane/methanol. Yield: 80% (718 mg, 1.19 mmol) H
1
NMR (CD OD), d (ppm): 1.42 (s, 9H), 1.85–1.96 (m, 1H), 2.04–
3
2
3
4
1
2
7
1
.22 (t, 2H), 2.69–2.75 (m, 1H), 2.89–2.97 (m, 1H), 3.69 (s, 3H),
.71 (s, 2H), 3.75 (s, 3H), 3.79 (s, 2H), 3.95–3.96 (br, 2H), 4.15–
.18 (q, 1H), 6.81–6.83 (d, 2H), 7.21–7.24 (d, 2H), 8.28–8.31 (t,
1
3
3
H); C NMR (CD OD), d (ppm): 24.77, 24.84, 27.02, 27.08,
8.07, 38.23, 51.86, 52.04, 52.27, 52.30, 54.97, 55.37, 77.36,
9.71, 113.78, 126.58, 128.31, 129.16, 129.82, 130.28, 130.40,
36.52, 136.62, 155.20, 155.30, 158.35, 170.78, 170.96, 171.25;
7
7
Se NMR (CD OD), d (ppm): 217.9, 218.6; ESI-MS: m/z calcd for
3
+
C H N O SeNa [M + Na] : 626.1593; found: 626.9387.
2
5
37 3 9
Compound 19. Eluent for column chromatography: 10%
1
dichloromethane/methanol. Yield: 85% (743 mg, 1.26 mmol) H
NMR (CDCl ), d (ppm): 0.86–0.93 (m, 6H), 1.08–1.16 (m, 1H),
.24 (s, 1H), 1.36–1.39 (dd, 3H), 1.42–1.43 (d, 9H), 1.48 (br, 1H),
3
1
1
3
.73 (br, 1H), 1.86–1.90 (m, 1H), 2.83–2.94 (m, 2H), 3.72 (s, 3H),
.73–3.74 (d, 2H), 3.78–3.79 (d, 3H), 3.94–3.97 (t, 1H), 4.49–4.54
(m, 1H), 4.78–4.80 (m, 1H), 5.04–5.06 (d, 1H), 6.53–6.94 (m, 1H),
1
3
6.81–6.84 (dd, 2H), 7.18–7.20 (dd, 2H); C NMR (CDCl3),
d (ppm): 11.26, 15.48, 15.53, 18.48, 24.63, 24.79, 24.97, 27.12,
2
5
1
7.23, 28.27, 29.54, 37.40, 48.61, 52.13, 52.32, 52.39, 55.11,
9.01, 79.47, 113.89, 129.92, 130.49, 130.54, 155.82, 158.46,
7
7
71.07, 171.13, 171.73, 171.80, 172.09, 172.18; Se NMR
), d (ppm): 221.8, 222.8; ESI-MS: m/z calcd for C26
(CDCl
3
41
H -
+
N O SeNa [M + Na] : 610.2007; found: 610.1074.
3
7
Compound 23. Eluent for column chromatography: 20% Pet.
1
ether/ethylacetate. Yield: 35% (217 mg, 0.52 mmol) H NMR
CDCl ), d (ppm): 1.44 (s, 9H), 2.90–3.01 (m, 2H), 3.73 (s, 3H),
.06–4.15 (q, 2H), 4.59–4.61 (d, 1H), 5.32–5.34 (d, 1H), 7.36–7.41
(
3
6 (a) W. Leinfelder, E. Zehelein, M. A. MandrandBerthelot and
A. B ¨o ck, Nature, 1988, 331, 723–725; (b) A. A. Turanov,
X. M. Xu, B. A. Carlson, M. H. Yoo, V. N. Gladyshev and
D. L. Hateld, Adv. Nutr., 2011, 2, 122–128; (c)
E. G. Varlamova, M. V. Goltyaev, S. V. Novoselov,
V. I. Novoselov and E. E. Fesenko, Mol. Biol., 2013, 47, 558–
567.
4
1
3
(
(
1
m, 2H), 7.51–7.55 (m, 1H), 8.01–8.03 (dd, 1H); C NMR
CDCl ), d (ppm): 25.4, 27.1, 28.8, 53.1, 54.0, 80.7, 126.3, 128.6,
3
32.4, 133.8, 135.9, 148.3, 155.5, 171.9; Se NMR (CDCl ),
3
7
7
+
d (ppm): 233; ESI-MS: m/z calcd for C H N O SeNa [M + Na] :
1
6
22 2 6
441.0541; found: 441.3427.
7
(a) S. Palioura, R. L. Sherrer, T. A. Steitz, D. S ¨o ll and
M. Simonovic, Science, 2009, 325, 321–325; (b)
K. Forchhammer and A. Bock, J. Biol. Chem., 1991, 266,
Conflicts of interest
¨
6324–6328.
There are no conicts of interest to declare.
8
9
(a) P. M. Masters, Calcif. Tissue Int., 1985, 37, 236–241; (b)
Z. Wang, B. Lyons, R. J. W. Truscott and K. L. Schey, Aging
Cell, 2014, 13, 226–234.
(a) P. J. Knerr and W. A. van der Donk, Annu. Rev. Biochem.,
2012, 81, 479–505; (b) Y. Shi, X. Yang, N. Garg and
W. A. van der Donk, J. Am. Chem. Soc., 2011, 133, 2338–2341.
Acknowledgements
This study was supported by the Science and Engineering
Research Board (SERB), New Delhi, India. K. M. R. thanks the
42 | RSC Adv., 2019, 9, 34–43
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1
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