Stereoselective and Divergent Construction of β-Thiolated/Selenolated Amino Acids via Photoredox-Catalyzed Asymmetric Giese Reaction

Article abstract of DOI:10.1021/jacs.0c04994

Sulfur and selenium occupy a distinguished position in biology owing to their redox activities, high nucleophilicity, and acyl transfer capabilities. Thiolated/selenolated amino acids, including cysteine, selenocysteine, and their derivatives, play critic

Full text of DOI:10.1021/jacs.0c04994

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Stereoselective and divergent construction of ꞵ-thiolated/selenolated
amino acids via photoredox-catalyzed asymmetric Giese reaction
Hongli Yin1, Mengjie Zheng1, Huan Chen2, Siyao Wang1, Qingqing Zhou1, Qiang Zhang2 and Ping
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Wang1*
1Shanghai Key Laboratory for Molecular Engineering of Chiral Drugs, School of Chemistry and Chemical Engineering,
Frontiers Science Center for Transformative Molecules, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai
200240, China.
2Department of Chemistry, University at Albany, State University of New York, 1400 Washington Avenue, Albany, NY
12222, USA.
ABSTRACT: Sulfur and Selenium occupy a distinguished position in biology owing to their redox activities, high nucleophilicity
and acyl transfer capabilities. Thiolated/selenolated amino acids, including cysteine, selenocysteine and their derivatives, play critical
roles in regulating the conformation and function of proteins and serve as an important motif for peptide design and bioconjugation.
Unfortunately, a general and concise method to attain enantiopure ꞵ-thiolated/selenolated amino acids remains an unsolved problem.
Herein, we present a photoredox-catalyzed asymmetric method for the preparation of enantiopure ꞵ-thiolated/selenolated amino acids
using a simple chiral auxiliary, which controls the diastereoselectivity of the key alkylation step and acts as an orthogonal protecting
group in the subsequent peptide synthesis. Our protocol can be used to prepare a wide range of ꞵ-thiolated/selenolated amino acids
on a gram scale, which would otherwise be difficult to obtain using conventional methods. The impact of our chemistry was further
highlighted and validated through the preparation of a series of peptidyl thiol/selenol analogs, including cytochrome c oxidase subunit
protein 7C and oxytocin.
INTRODUCTION
acid scaffolds despite the significant research efforts toward the
preparation of ꞵ-thiolated/selenolated amino acids reported by
multiple research groups.12 Among the methods towards the
synthesis of ꞵ-thiolated/selenolated amino acids, using thiol/se-
lenol reagents and an electrophilic carbon to introduce S/Se is
the most representative approach with enantiopure ꞵ-hydroxyl
amino acids or Garner’s aldehyde utilized to stereoselectively
construct the C-S/Se bond (Fig. 1B). Unfortunately, modifica-
tion of β-hydroxyl amino acids approach is extremely impracti-
cal which requires expensive and rare commercially available
β-hydroxyl amino acids as the precursors (e.g. $200/gram for β-
hydroxy leucine), and it often leads to a diastereomeric mixture
of β-thiol products. The latter tactic could achieve moderate di-
astereoselectivity, yet, multistep manipulations are often re-
quired before incorporating β-thiol/selenol residues into solid
phase peptide synthesis.13 Therefore, a more general and con-
cise protocol to high-value enantioselective ꞵ-thio/selenolated
amino acids from readily accessible building blocks remains
elusive.
Cysteine (Cys) and its analog, selenocysteine (Sec) play irre-
placeable roles in protein folding and stability,1 enzymatic ac-
tivity,2 redox regulation.3 The intricate design of free thiol and
selenol is nature’s way of realizing these critical functions in
biology. The significance of cysteine and selenocysteine is ar-
guably more conspicuously presented in the domain of selective
chemical protein modification,4 the construction of native pep-
tide bonds (native chemical ligation, NCL),5 late-stage muta-
genesis (to Ala and Ser),6 disulfide bond engineering7 and de-
sign of peptidyl ligands.8 As a case in point, the combination of
the NCL-dechalcogenation strategy,9 one of the most efficient
approaches used to tether two peptidyl segments, completely
relies on the specific chemoselectivities exhibited by thio-
lated/selenolated proteinogenic amino acids (Fig. 1A). After li-
gation and selective dechalcogenation, peptides bearing the cor-
responding native residues are produced. Moreover, disulfide
bridges are crucial for the stability and activity of many im-
portant therapeutic peptides and proteins. Recent studies have
suggested the use of thiolated amino acids as disulfide precur-
sors can also enhance the stability and activity of peptides.10 In
addition, Cys is a fundamental element for functional peptide
design, thus fine-tuning the properties of synthetic peptide lig-
ands could be achieved with thiolated amino acids prepara-
tion.11 Nonetheless, the resultant ꞵ-thiolated/selenolated amino
acids, in which the ꞵ-carbon carries the thiol/selenol, are valua-
ble precursors to bioactive peptides and proteins.
Herein, we present a general and practical venue to access
enantiopure β-thiolated/selenolated amino acids via asymmetric
Giese reaction14 (Fig. 1C). Our strategy starts from enantiopure
selenozoline/thiazoline bearing a chiral pivalaldehyde acetal,
which is easily prepared from L-Cys/Sec on a large scale. Visi-
ble-light photocatalysis generated alkyl radicals (including pri-
mary, secondary, and tertiary radicals) furnishes thiazoline/se-
lenozoline to produce β-substituted thiol/ selenolated amino
acid framework. The chiral pivalaldehyde acetal of thia-
zoline/selenozoline controls the diastereoectivity of the radical
addition reaction and acts as a “smart” protecting group, which
is orthogonal to solid phase peptide synthesis conditions and
eliminates the need for protecting group manipulations. This
In contrast to these powerful applications derived from thio-
lated/selenolated amino acids, a major challenge for their wide-
spread utilities is the limited accessibility of these pre-requisite
enantiopure thiolated/selenolated amino acids. Currently, the
majority of methods are indirect and confined to certain amino
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strategy allows the concise stereoselective synthesis of a diverse
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handling labile Se during reactions prevents securing Se-amino
acids from the large quantity. We began our exploration with
the preparation of β-selenolated amino acids as a more practical
synthetic targets. The starting material, pivalaldehyde N,Se-ac-
etal selenozoline 1, was quickly prepared over three steps from
the diselenide derivative of L-Sec methyl ester on a 20-gram
scale. The N-formyl group was used to protect the nitrogen atom
as well as to improve the electrophilicity of the double bond.20
Under irradiation of blue LED light (10 W), Ru(bpy)3(PF6)2
(PCA, 1 mol%) catalyzed the coupling of 1 with iso-propyl N-
hydroxyphthalimide ester 2a (1.2 equiv.) provided the desired
product, trans-3a (β-selenolated Leu), as a single diastereoiso-
mer in the presence of Hantzsch ester (HE, 1.5 equiv.) and
DIPEA (2.0 equiv.). It is worth noting that 3a was obtained on
a gram scale as a mixture of rotamers in near quantitative yield
(97%) as confirmed by variable temperature 1H NMR and sol-
vent switching (see supplementary information page 10 for de-
tails) (Fig. 2B). Screening of the solvent system suggested that
dichloromethane (DCM) afforded the highest yield compared
to MeCN and DMF; other photoredox catalysts such as Eosin
Y (PCB) and Ir[dF(CF3)ppy]2(dtbbpy)- PF6 (PCC) could also
mediate same reaction with eroded yields. In all the case, only
a single diastereoisomer trans-3a was obtained (>99:1 d.r.).
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range of β-seleno/thiolated amino acids from a common precur-
sor at will with high stereopurity and high yield. Products could
be rapidly acquired on a multigram scale that are not easily ac-
cessible using conventional methods. Furthermore, the resulting
thiazolino protected amino acids can be directly used in peptide
synthesis after simple operation without complex protecting
group manipulation.
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An ideal method is that an inexpensive or readily accessible
starting material can be converted into a diverse family of mem-
bers with higher values via a single-step reaction. Subsequently,
a broad scope of carboxylic acids was evaluated, including pri-
mary, secondary and tertiary carboxylic acids with various
functionalities. RAEs derived from secondary and tertiary car-
boxylates were initially examined (Fig. 3A, Protocol A). Reac-
tion of selenozolidine 1 with a series of cyclic carboxylate N-
hydroxyphthalimide esters (cyclobutyl 2b, cyclohexyl 2c and
adamantyl 2d) and fluorinated cyclobutyl carboxylate ester 2e
yielded the desired products (3b-3e) in excellent yields and high
diastereoselectivity on a gram scale. Notably, product 3d bear-
ing an adamantyl substituent was obtained with >90% yield,
which suggested that generating sterically bulky quaternary
centers could be accomplished. The absolute configuration of
3d was confirmed via X-ray crystallography (Fig. 3B).
Figure 1. Development of a general method used for the prep-
aration of enantiopure β-thio/selenolated amino acids. A. NCL-
dechalcogenation. B. Representative methods for constructing
thio/selenolated amino acids. C. The photoredox-catalyzed
asymmetric Giese reaction as a general solution to this synthetic
problem.
RESULTS AND DISCUTION
In contrast to the thiol/selenol nucleophiles used to generate
the C-S/Se bond described in the existing methods, the stereose-
lective insertion of an alkylated group onto the ꞵ-carbon of
Cys/Sec is superior. It has been reported that using the concept
of “self-regeneration of chirality centres”,15 chiral S, N-acetyl
thiazoline served as a good starting point for the preparation of
β-thiolated amino acids via Michael-type alkylation.16 However,
this transformation remains limited in synthesis due to its poor
yield, low functional group tolerance and diversity, attributed to
the severe β-elimination of the thiol group when alkyl metal re-
agents are employed in the reaction. The challenges notwith-
standing, we envisioned that a radical 1,4-conjugate addition
under mild photoredox conditions may lead to superior results.
It is well established that convenient radical generation occurs
from carboxylic acids,17 redox-active esters (RAEs)18 and alkyl
halides19 in the presence of photoredox catalyst via single-elec-
tron transfer (SET). Mechanistically, production of alkyl radical
via SET oxidation (carboxylic acids) or reduction (RAEs and
alkyl halides) can lead to stereoselective radical addition from
the reverse face of the bulky t-butyl group in A, which generates
radical intermediate B. B is reduced via H-atom abstraction
(HAT) or SET followed by a diastereoselective enolate proto-
nation step to produce trans-product C, providing two new ste-
reocenters in one step (Fig. 2A). Subsequent deprotection may
furnish a broad family of β-thio/selenolated amino acids (D),
which are suitable for direct peptide coupling.
Coupling with selenozolidine 1 and primary alkyl carboxylic
acids could not be accessed effectively by PCA and PCC catal-
ysis. After extensive investigations, the optimized conditions
were obtained using a 40 W household compact fluorescent
light (CFL) bulb to irradiate 1 in the presence of 2.0 equiv. of
RAEs (2f-2i) and DIPEA (2.5 equiv.) in DCM using PCB (Eo-
sin Y), which gave the desired coupling products (3f–3i) in
good yield at room temperature (Fig. 3A, Protocol B).21
Moreover, carboxylic acids bearing a stabilizing α-hetero
atom (O, N and S) were evaluated. Compounds 2j-2l were
added to 1 in the presence of PCC directly, yielding products
3j-3l in excellent yields (Fig. 3A, Protocol C). In all cases, we
were pleased to find that excellent diastereoselectivity was ob-
tained under our reaction conditions.
We next sought to convert products 3 into target amino acids
4, which might be used directly as SPPS-compatible amino ac-
ids. Desired products (4a-4g, 4k and 4l) can be generated from
their parent compounds after saponification of methyl ester and
acidic removal of the N-formyl group over two steps
(Fig. 3A. condition a). Substrates 4a and 4l represent β-seleno
The importance of Se in biology renders selenolated amino-
acids in the high demand, conversely, the sheer difficulty of
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Figure 2. Design plan and optimization of the reaction conditions used in the preparation of β-selenolated Leu. A. Asymmetric Giese
reaction via photoredox catalysis. B. Standard conditions: PC (1 mol%), 2a (1.2 equiv.), HE (1.5 equiv.), DIPEA (2.0 equiv.), DCM,
blue LED (10 W), 25 °C. a18 h. b48 h. cIsolated yield. dThe diastereoselectivity was determined by 1H NMR and GC analysis of the
crude reaction mixture. PC, photoredox catalyst. HE, Hantzsch ester. DIPEA, N, N-diisopropylethylamine. DCM, dichloromethane.
DMF, N, N-dimethylformamide.
Figure 3. Asymmetric alkylation of selenozolidine under photoredox conditions and their derivatization. A. Scope of the β-seleno-
lated amino acids. Deprotection conditions: a. 1) LiOH (10.0 equiv.), H2O/MeOH (1:3 v/v), 25 °C, 6 h; 2) 6 N HCl in dioxane, 50 °C,
8 h. b. 1) LiOH (10.0 equiv.), H2O/MeOH (1:3 v/v), 25 °C, 6 h; 2) 6 N HCl in dioxane, 50 °C, 8 h; 3) (Boc)2O (1.0 equiv.), THF/H2O
(10:1 v/v), DIPEA (2.0 equiv.), 25 °C, 1 h. c. 1) LiOH (10.0 equiv.), H2O/MeOH (1:3 v/v), 25 °C, 6 h; 2) 6 N HCl in dioxane, 50 °C,
8 h; 3) N, N'-bis-Boc-1-guanylpyrazole (1.0 equiv.), DIPEA (2.0 equiv.), MeOH, 25 °C, 3 h. B. The absolute configuration of 3d was
i
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confirmed by X-ray crystallography. The diastereoselectivity was determined by GC analysis of the crude reaction mixture. brsm,
based on recovered starting material. iiiA mixture of diastereomers was found.
Leu and Met, respectively. After acidic treatment, epimeriza-
tion of the stereogenic center in the auxiliary was observed, and
the α, β-chiral center of the amino acids remained untouched.
In the case of 3h and 3j, a further Boc protection step was re-
quired to form β-selenolated Lys 4h and 4j (Fig. 3A. condition
b). Guanidinylation was performed to yield β-selenolated Arg
4i from ornithine 3i, (Fig. 3A. condition c). For most cases, only
a single purification was needed after these deprotection condi-
tions, minimizing time-consuming isolation process.
We further expanded the reaction scope to a number of β-
thiolated amino acids, which are listed in Figure 4. In line with
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Figure 4. Asymmetric alkylation of thiozolidine under photoredox conditions and derivatization. A. Scope of β-thiolated amino acids.
Deprotection conditions: a. 6 N HCl, 90 ℃, 8 h. b. 1) 0.5 N HCl in MeOH, 25 ℃, 12 h; 2) LiOH (10 equiv.), H2O/MeOH (1:3 v/v),
25 ℃, 6 h. c. 1) 6 N HCl, 90 ℃, 8 h; 2) (Boc)2O (1.0 equiv.), DIPEA (2.0 equiv.), THF/H2O (10:1 v/v), 25 ℃, 1 h. d. 1) 6 N HCl,
90 ℃, 8 h; 2) N,N'-bis-Boc-guanylpyrazole (1.0 equiv.), DIPEA (2.0 equiv.), MeOH, 25 ℃, 3 h. e. 1) 33% HBr in AcOH, 80 ℃, 1
h; 2) LiOH (10.0 equiv.), H2O/MeOH (1:3 v/v), 25 ℃, 6 h. f. 1) 6 N HCl, 90 ℃, 30 min; 2) p-TsOH (1.0 equiv.), isobutylene in DCM
(50.0 equiv.), 40 ℃, 12 h; 3) LiOH (10.0 equiv.), H2O/MeOH (1:3 v/v), 25 ℃, 6 h. g. 1) PhNHNH2 (2.0 equiv.), TFA/DCM (1:3 v/v),
25 ℃, 30 min, 97%; 2) 0.5 N HCl in MeOH, rt, 12 h; 3) LiOH (10.0 equiv.), H2O/MeOH (1:3 v/v), 25 ℃, 6 h. h. 1) p-MeO-PhNHNH2
(2.0 equiv.), TFA/DCM (1:3 v/v), 97%; 2) 0.5 N HCl in MeOH, 25 ℃, 12 h; 3) LiOH (10.0 equiv.), H2O/MeOH (1:3 v/v), 25 ℃, 6
h. B. Synthesis of D-β-thiolated amino acids from D-Cysteine. C. The absolute configuration of 9 was confirmed by X-ray crystal-
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lography. iThe diastereoselectivity was determined by GC analysis of the crude reaction mixture. brsm, based on recovered starting
material. iiiA mixture of diastereomers was found.
the β-selenolated amino acids preparation, all the RAEs (2a-2d,
2f-2i, 2m, 2n) and carboxylic acids (2j-2l, 2o) smoothly conju-
gated with thiozolidine 5, which was readily obtained from L-
cysteine methyl ester hydrochloride ($0.20/g) and pivalalde-
hyde ($0.25/ml) over three steps on a 100-gram scale (see sup-
plementary information page 4 for details). Products (6a-6d, 6f-
6o) were acquired in high yields using Protocol A, B and C (Fig.
4A). Notably, reaction of 5 with 2-iodoacetamide 2p under pho-
toredox dehalogenation conditions (Fig. 4A, Protocol A’)
yielded 6p in 63% yield. Synthetically valuable handles such as
the sulfide of methionine (6l), pyridine (6m), acetal (6n), ketone
(6o) and amide (6p) were well tolerated, and the incorporation
of an adamantyl nucleus (6d) offered a ready protocol to modify
peptide distribution and lipophilicity/hydrophobicity. The
structure and stereochemistry of 6d were confirmed by X-ray
crystallography. Pyridine-containing amino acids have signifi-
cant effects on the biological properties of peptides and are of-
ten found in therapeutic peptides, therefore substrate 6m was
used to demonstrate that the pyridine motif can be smoothly in-
troduced. Compound 6a, exist as a mixture of rotamers as con-
firmed by variable temperature 1H NMR and solvent switching.
Compared with delicate selenolated analogs, β-thiolated
amino acids are more robust in nature. Thus, methods used for
the deprotection of these compounds are more straightforward.
The deprotection conditions used to yield β-thiolated amino ac-
ids are summarized in Fig 4A. Hydrolysis using 6 N HCl led to
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Figure 5. Exploiting the utility of β-thio/selenolated amino acids. A, DSL-deselenization using β-selenolated amino acids. B, NCL-
desulfurization using β-thiolated amino acids. C, One-pot synthesis of cytochrome c oxidase subunit 7C 22. D, Crude HPLC trace of
final deselenization to give 22 and ESI mass spectra of 22. Conditions for auxiliary removal: a. 6 M GND•HCl, 0.2 M Na2HPO4, 0.2
M MeONH2, pH 4.0. b. 6 M GND•HCl, 0.2 M Na2HPO4, MPAA (1.5 equiv.), pH 6.8. Ligation: 13 (1.2 equiv.), 6 M GND•HCl, 0.2
M Na2HPO4, TCEP (2.0 equiv.), pH 6.2, 25 ºC, 16 h. Conditions for deselenization: c. 6 M GND•HCl, 0.2 M Na2HPO4, DTT (9.0
equiv.), TCEP (50 equiv.), pH 5.2, 25 º C, 16 h. d. 6 M GND•HCl, 0.2 M Na2HPO4, DTT (250 equiv.), TCEP (50 equiv.), pH 5.2, 25
ºC, 16 h. Conditions for desulfurization: e. 6 M GND•HCl, 0.2 M Na2HPO4, 200 mM TCEP, 10 mM VA-044, 50 mM GSH, 37 ºC,
16 h. f. 6 M GND•HCl, 0.2 M Na2HPO4, 500 mM TCEP, 30 mM VA-044, 150 mM GSH, 37 ºC, 16 h. iYield according to the original
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loading of the resin. Yield of ligation and deselenization. iiiYield of ligation and desulfurization. GND, guanidine. MPAA, 4-mer-
captophenylacetic acid. TCEP, tris-(carboxyethyl)phosphine. VA-044, 2,2'-azobis[2-(2-imidazolin-2-yl)propane]-dihydrochloride.
DTT, dithiothreitol. GSH, glutathione. Fmoc SPPS, Fmoc solid-phase peptide synthesis.
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pot diselenide-selenoester ligation (DSL)-deselenization23 be-
tween peptide selenoester 13 and selenolated peptides 12a, 12e,
12i and 12l were conducted. Peptides 14a, 14e and 14i were
obtained according to ligation and deselenization protocol in
good yields (Fig. 5A, condition c). Low yield of 14l was ob-
served during deselenization. The deselenization proceeded
well when excess equivalent DTT and TCEP were used (Fig.
5A, condition d). We further examined NCL-desulfurization us-
ing β-thiolated amino acids. Peptides 15c, 15d, 15n and 15o
were prepared via Fmoc-SPPS and TFA deprotection. Next,
auxiliary removal was achieved successfully using excess
amount of MeONH2, providing thiolated peptides (16c, 16d,
16n and 16o) in good yields (Fig. 5B, condition a, entry 1-4).
Ligation of peptide 16 with peptide 13 under NCL conditions,
followed by one-pot desulfurization, yielded peptides 17c, 17d
and 17o smoothly (Fig. 5B, condition e). Though peptide 17n
was obtained in 20% yield after desulfurization, the reaction
could be improved upon treatment of a large excess of TCEP
and VA-044 (Fig. 5B, condition f). Therefore, this method not
only significantly expanded the utility of NCL-
dechalcogenation to generate native peptides, but also provided
a simple strategy to access peptides containing unnatural amino
acids.6a, 9f, 24 For example, the synthesis of fluorinated peptide
14e represents a general strategy to incorporate fluorinated
amino acids into peptides, which opens up a new avenue to ac-
cess fluorinated peptides.25
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substrates (6a to 6d, 6f, 6g, 6o and 6l) directly in a single step
(Fig. 4A, condition a), including β-thiolated Leu (6a) and Met
(6l). Deformylation of 6k and 6m was carried out using 0.5 N
HCl followed by saponification to give 7k and 7m in high yields
(Fig. 4A, condition b). Similarly, after acidic removal of N-
formyl group, followed by hydrolysis of the methyl ester and
Boc group gave the free amine from 6h and 6j, which were re-
protected with Boc to give β-thiol Lys 7h and 7j in high yields,
respectively (Fig. 4A, condition c). After acidolysis and guani-
dinylation, β-thiol Arg 7i was produced in three steps starting
from β-thiolated Orn 6i (Fig. 4A, condition d). β-thiol Trp ana-
logs (7n and 7n’) were constructed via Fischer indole synthesis
from γ-aldehyde dimethyl acetal 6n (Fig. 4A, condition g and
h). β-thiol Gln 7p and Glu 7p’ were obtained in good yields
starting from 6p, respectively. Selective deformylation in the
presence of the side chain amide moiety in 6p was achieved us-
ing 33% HBr in acetic acid and a subsequent saphonication step
provided β-thiol Gln 7p in 70% yield (Fig. 4A, condition e).
Maintenance of the methylester during acidic removal of N-
formyl group together with hydrolysis of side-chain amide moi-
ety of 6p was possible using 6 N HCl at 90 ℃ for 30 min, leav-
ing the methyl ester remained untouched. Functional group ma-
nipulations eventually produced β-thiol Glu 7p’ in good yield
(Fig. 4A, condition f). Gram quantities of the β-thiolated amino
acids could be easily prepared in the majority of these cases.
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Mirror-image proteins have important therapeutic potential.22
Thus, we further explored the preparation of D-β-thiolated
amino acids. Starting from 8 (the enantiomer of 5), adamantyl
radical was introduced in the same manner. Compound 9 (the
enantiomer of 6d) was obtained and subjected to acidolysis to
produce 10 in high yield (Fig. 4B). The absolute configuration
of 9 was confirmed using X-ray crystallography (Fig. 4C).
To further exhibit the practicality of this method, cytochrome
c oxidase subunit protein 7C26 (22) was prepared by one-pot
synthesis using selenolated Lys building block 4h as an exam-
ple. As shown in Fig 5C, MPAA-thioester 18 and peptide 19
were combined under NCL condition to yield peptide 20 via in
situ production of MPAA disulfide from 18. Without seperation,
the crude reaction mixture was directly combined with se-
lenoester 21, followed by an in situ chemoselective deseleniza-
tion reaction using DTT and TCEP to give protein 22 in 38%
isolated yield over four steps. Overall, we have sucessfully syn-
thesized cytochrome c oxidase subunit 7C 22 via sequential
one-pot NCL-activation of auxiliary and DSL-deselenization
chemistry without any purification or solvent removal. The fac-
ile synthesis of 22 demonstrated the application of construction
of ꞵ-selenolated amino acids and the MPAA-mediated depro-
tection of selenozolidine could expand and enhance the seleno-
lated amino acids-mediated ligations.
With these β-thio/selenolated amino acids in hand, we next
explored their utility in peptide synthesis. Selenolated amino
acids 4a, 4e, 4i and 4l were introduced onto a solid supported
polypeptide N-terminus. After subsequent acidic cleavage from
the resin and removal of the side-chain protecting groups using
TFA, peptides 11a, 11e, 11i, 11l were prepared, respectively
(Fig. 5A). In all cases, no double coupling products were ob-
served due to the steric bulkiness of the neighboring t-Bu group.
Next, deprotection of the auxiliary in the presence of excess
MeONH2, diselenide dimer peptides 12a, 12e and 12l were ob-
tained in good yield after HPLC purification (Fig. 5A, condition
a, entry 1, 2 and 4). Unfortunately, the deprotection of the
pivalaldehyde acetal of β-selenolated Arg peptide (11i) failed in
the presence of MeONH2 due to the decomposition of peptide.
As such, we opted for the employment of the milder conditions.
Interestingly, we observed that this auxiliary was stable under
NCL conditions in the presence of MPAA and TCEP, yet, com-
plete acetal removal of N-terminal selenolated amino acids was
achieved in the presence of 1.5 equiv. of MPAA in 6 M
GND•HCl/0.2 M Na2HPO4 at pH 7.0, generating the MPAA-
adduct peptide 12i in good yield (Fig. 5A, condition b, entry 3).
Mechanistic studies suggested that the selenozolidine ring was
opened via the in situ produced disulfide of MPAA (see supple-
mentary information page 84 for details). To the best of our
knowledge, the deprotection of selenozolidine described here is
one of the mildest conditions reported to date. Meanwhile, one
Figure 6. Thermal stability of synthetic oxytocin analogs. A.
Synthetic oxytocin analogs. B. The thermal stability of oxytocin,
23a, 23d and 23h in water at 50 ℃. OT, oxytocin.
Disulfides have been extensively used to stabilize peptides
conformations and confine structure rigidity. Current methods
for disulfide bond engineering are limited to the replacement of
the disulfide bond with thioether, selenoether, diselenide and
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hydrocarbon bridges.27 In order to further understand the impact
of introducing cysteine analogs on protein stability, a series of
oxytocin (a hormone stimulating parturition and lactation drug)
analogs (23a, 23d, and 23h) were prepared via SPPS and sub-
sequent oxidative folding, which contain various N-terminal ꞵ-
thiolated amino acid residues (Fig. 6A). The thermal stability of
native oxytocin and its analogs were evaluated at 50 ℃ by
LCMS analysis.28 Native oxytocin degraded with a half-life of
3.9 d, whereas synthetic analogs demonstrate significant stabil-
ity (23a, 18.8 d; 23d, 68.6 d; 23h, 23.1 d) (Fig. 6B). The intro-
duction of ꞵ-thiolated amino acid residues is simple and
straightforward, more importantly, our approach enables the
construction of peptides bearing novel disulfide bridge with
high stability and structural diversity.
Shanghai Committee of Science and Technology (17JC1405300).
QZ thanks National Science Foundation (CHE 1710174) and Na-
tional Institute of Health (1R35 GM138336-01) for support.
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CONCLUSION
In summary, we have developed a practical and general strat-
egy for diastereoselective preparation of ꞵ-thio/seleno-lated
amino acids via photoredox-catalyzed asymmetric Giese reac-
tion. In contrast to existing methods using nucleophilic and
electrophilic thiol/selenol reagents to construct C-S/Se bond,
this method unprecendently combines the concept of “self-re-
generation of chirality centres” with modern photoredox catal-
ysis. Nevertheless, the described convenient approach allowed
the scalable and diverse synthesis of proteinogenic amino acid
analogs including β-thiolated amino acids (Leu, Glu, Gln, Lys,
Arg, Trp, Met), β-selenolated amino acids (Leu, Lys, Arg, Met),
and a broad scope of unnatural β-thio/selenolated amino acids
via a single step asymmetric alkylation on a multigram scale.
We believe that this methodology will not only significantly ex-
pand the utility of ligation-dechalcogenation strategy, but also
allow design and implementation of amino acids at will and
construction of disulfide-engineered peptides and proteins in
academic and pharmaceutical settings.
ASSOCIATED CONTENT
Supporting Information.
The Supporting Information is available free of charge on the
ACS Publications website.
Detailed experimental procedures and spectral data
AUTHOR INFORMATION
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Corresponding Author
*wangp1@sjtu.edu.cn
ORCID
Ping Wang: 0000-002-8640-1483
Notes
There are no competing financial interests in this work
ACKNOWLEDGMENT
Financial support for this work from National Natural Science
Foundation of China (91753102, 21672146 and 21907064) and
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