Visible-Light-Induced Specific Desulfurization of Cysteinyl Peptide and Glycopeptide in Aqueous Solution

Article abstract of DOI:10.1021/acs.orglett.6b00292

Visible-light-induced specific desulfurization of cysteinyl peptides has been explored. The photocatalytic desulfurization catalyzed by Ru(bpy)₃²⁺ can proceed efficiently at room temperature in aqueous solution or in binary mixtures of aqueous/organic solvent and be compatible with the presence of residues of amino acids, carbohydrates, and various sulfur-containing functional groups. This approach was successfully applied to synthesize linear and cyclic peptides through the ligation-desulfurization protocol.

Full text of DOI:10.1021/acs.orglett.6b00292

Letter
pubs.acs.org/OrgLett
Visible-Light-Induced Specific Desulfurization of Cysteinyl Peptide
and Glycopeptide in Aqueous Solution
Xiao-Fei Gao, Jing-Jing Du, Zheng Liu, and Jun Guo*
Key Laboratory of Pesticide & Chemical Biology of Ministry of Education, CCNU-uOttawa Joint Research Centre,
College of Chemistry, Central China Normal University, 152 Luoyu Road, Wuhan, Hubei 430079, P. R. China
*
S Supporting Information
ABSTRACT: Visible-light-induced specific desulfurization of cysteinyl peptides has been explored. The photocatalytic
2
+
desulfurization catalyzed by Ru(bpy)3 can proceed efficiently at room temperature in aqueous solution or in binary mixtures of
aqueous/organic solvent and be compatible with the presence of residues of amino acids, carbohydrates, and various sulfur-
containing functional groups. This approach was successfully applied to synthesize linear and cyclic peptides through the
ligation−desulfurization protocol.
1
0
ver the past several decades, methods of chemical synthesis
of peptides and proteins have been greatly developed. The
aqueous phase. Raney nickel or palladium under H has been
2
4a,11
O
utilized to desulfurize peptides,
but these desulfurization
most remarkable breakthrough was made by Merrifield in 1963
for applying solid phase peptide synthesis (SPPS). Stepwise
conditions are often haunted by their tolerance of special
1
4d,12
functional groups or low recovery of product.
In 2007, Wan
SPPS is limited to peptides bearing less than ca. 50 amino acids,
because longer peptide synthesis is often plagued by side
products and coupling efficiency. Thus, a segment condensation
strategy has been employed to facilitate longer peptides and
proteins. The most widely used method for condensation of
peptide segments is a native chemical ligation (NCL) protocol,
and Danishefsky developed an important protocol of heat-
10
induced radical desulfurization for peptide synthesis, which
involved tris(2-carboxyethyl)phosphine (TECP, Figure 1) and
2
developed by Kent and co-workers in the 1990s. The general
mechanistic process of NCLtransthioesterification and S to N
acyl transferis outlined in Scheme 1. Even though the NCL
Figure 1. Structures of photocatalysts and phosphines.
Scheme 1. Native Chemical Ligation and Desulfurization of
Unprotected Peptide Segments
radical initiator. In this protocol, an elevated temperature
(
37−65 °C) is required to activate the initiator and trigger the
4d,10
reaction,
and a large excess of initiator (VA-044 or V-50) and
TCEP is usually introduced to complete the desulfurization
6
,10,13
process of peptides.
Visible-light-photoredox catalysis has recently become an
important strategy for organic synthesis. A milestone was
14
3
achieved in this field due to the seminal research by MacMillan,
protocol has led to many proteins being chemically synthesized,
15 16 17
Yoon, Stephenson, and other groups. This photoredox
catalysis, which is based on the ability of transition metal com-
plexes or organic dyes to engage in the single-electron transfer
(SET) process with substrates upon photoexcitation, has been
the low abundance of cysteine in peptides and proteins has
limited the ligation site of NCL. To overcome the limitation of
cysteine dependence, synthesis and application of cysteine
4
5
surrogates or selenium analogs have emerged, which expanded
1
4−17
widely used in organic synthesis.
The thiyl radical, which
the peptide ligation at more amino acid sites through a ligation−
18
6
plays important roles in various fields, can be generated by
desulfurization/deselenization protocol.
Hoffmann and co-workers have reported desulfurization
reactions between mercaptans and trialkylphosphites under
visible-light-photoredox catalysis from thiol-containing sub-
strates to perform additive or oxidative reactions.
1
9
7
reflux or ultraviolet irradiation conditions. It was then suggested
8
that the reaction is a radical process. However, these reported
Received: January 28, 2016
7
−9
conditions
failed to effectively desulfurize peptides in an
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.6b00292
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
Based on these results, we tried to explore a specific
desulfurization method using visible-light-photoredox catalysis
for peptide chemistry. Therefore, we examined the reaction of
slightly improved as the amount of TPPTS increased (entry 5).
Trace product (<3%) was detected when TPPTS was replaced
by TCEP in this photoreaction (entry 6). As for different
solutions applied, not only water and phosphate buffer (entries 4
and 7) but also mixtures of PB/organic solvent (entries 8−9) are
suitable for this photoredox reaction, and all provide the desired
product in good yields. This photoredox catalysis is also com-
patible with tert-butyl mercaptan (TBM) and guanidine
hydrochloride (Gn·HCl) (entries 10−11), and a longer reaction
time is required for a high concentration of Gn·HCl. For different
photocatalysts (see Supporting Information, Table S1, entries
cysteine with water-soluble phosphine and 5 mol % Ru(bpy) Cl2
3
(
1) in D O, under visible-light irradiation, and monitored the
2
1
reaction process by H NMR. Initially, the most common water-
soluble phosphine (TCEP) was utilized, and only a trace amount
of desired product (<3%) was detected. Surprisingly, when
another water-soluble phosphine -3,3′,3″-phosphinidynetris-
(
benzenesulfonic acid) trisodium salt (TPPTS, Figure 1) was
used, the cysteine was almost quantitatively converted into
alanine (Figure 2 and Figure S1).
1−3), Ru(bpy)
Cl (1) was found to be the most efficient
catalyst; Ru(bpz) (PF ) (2) also resulted in the desired product,
3
2
3
6 2
but in a relatively lower yield; fac-Ir(PPy) (3) only led to the
3
desired product in a very low yield, probably due to its poor
solubility in aqueous solution. The scavenger reagents were also
evaluated (see Supporting Information, Table S1, entries 4−6),
and TBM was found to be a good scavenger reagent. The
reaction completed within 5 h when the concentration of
substrate is 1 mM (see Supporting Information, Table S1, entries
7
−9). Therefore, for this photoredox desulfurization reaction the
optimized conditions consist of 1 mM peptide substrate, 50 mM
of TPPTS, 5 mol % of 1, and additives at room temperature.
With the optimized conditions in hand, we next investigated
the longer peptide substrates, beginning with peptide 2a. In order
to reduce the reaction time, 1 mM of peptide substrate was used.
Under household bulb irradiation for 5 h, the cysteinyl peptide 2a
completely disappeared, and the expected alanyl peptide 2b was
obtained with an isolated yield of 89%. The process was
monitored by HPLC and ESI-MS as shown in Figure 3. Notably,
Figure 2. Model study of the visible-light-induced desulfurization of
cysteine by H NMR. Conditions: cysteine (20 mM) with TPPTS
1
(
50 mM), Ru(bpy) Cl (5 mol %), in D O at rt after (A) 0 h, (B) 8 h,
3 2 2
and (C) 16 h under the irradiation of a 36 W household bulb.
Encouraged by this result, we continued to optimize the reac-
tion conditions using glutathione (GSH) as a model substrate
(Table 1). In the absence of phosphine TPPTS, photocatalyst
a
Table 1. Screening and Control Experiments
b
entry phosphine source (mM)
solvent
sulfur additive yield (%)
1
2
3
4
5
6
7
8
9
1
1
−
H O
−
−
−
−
−
−
−
−
N.D.
N.D.
N.D.
85
2
c
50
50
50
100
100
50
50
50
50
50
H O
2
d
H O
2
H O
2
H O
2
88
e
H O
2
<3
PB
81
Figure 3. Model study of the visible-light-induced desulfurization of 2a
by HPLC and ESI-MS. Conditions: 2a (1 mM), TPPTS (50 mM),
Ru(bpy) Cl (5 mol %), and TBM (80 mM), in phosphate buffer
PB/CH CN
87
3
PB/MeOH
−
TBM
84
3
2
0
1
PB
PB
83
(200 mM, pH = 7.4) at rt after (A) 0 h, (B) 2.5 h, and (C) 5 h under the
f
TBM
79
irradiation of a 36 W household bulb.
a
Reaction conditions: 1a (20.0 mM), TPPTS, photocatalyst 1
b
(
5 mol %), phosphine, sulfur additive (80.0 mM), 16 h. Yield was
1
c d
for this photoredox desulfurization procedure, 50 mM of TPPTS
was used as a phosphine reagent, while for the conventional
determined by H NMR. In the absence of light. In the absence of 1.
e
f
The TPPTS was replaced by TCEP. Gn·HCl (200 mM). PB =
4
d,10
phosphate buffer (pH = 7.4, 200 mM), TBM = tert-butyl mercaptan,
desulfurization procedure with radical initiator,
a higher
N.D. = not detected.
concentration of TCEP (250−500 mM) was always used. If
TPPTS was replaced by TCEP in this photoredox desulfurization
reaction, only a high concentration of TCEP (500 mM) can give
the product 2b effectively. The different reactivity between
TPPTS and TCEP in the photoredox reactions is probably
caused by their different electronic effect. Compared with the
Ru(bpy) Cl , or visible light, no product was observed (entries
3
2
1
−3). These findings suggest that all these conditions are
essential in this system. The yield (85%) of product was good
when 50 mM TPPTS was employed (entry 4). The yield was
B
DOI: 10.1021/acs.orglett.6b00292
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
a
Scheme 2. Visible-Light-Induced Transformation of Cysteinyl Peptide to Alanyl Peptide
a
Desulfurization conditions: Cysteinyl peptide (1 mM), TPPTS (50 mM), Ru(bpy) Cl (5 mol %), TBM (80 mM), PB or PB mixed with organic
3
2
b
c
d
e
solvent, 36 W household bulb irradiation, 5 h; yield of isolated product. In PB. In PB, Gn·HCl (200 mM). In CH CN/PB (1:1, v/v). In MeOH/PB
3
(
1:1, v/v). PB = phosphate buffer, 200 mM, pH = 7.4.
heat-induced radical desulfurization, the present visible-light-
induced radical desulfurization has merits such as proceeding at
room temperature, with low concentrations of initiator and
phosphine (for more details, see Table S2).
Scheme 3. Synthesis of Linear and Cyclic Peptides through
Ligation/Desulfurization Protocol
Next, the visible-light-induced desulfurization procedure was
applied to a range of peptides with different functional residues,
and all the corresponding desulfurization peptides were obtained
in good yields (Scheme 2). Remarkably, this protocol removed
cysteine’s thiol group in the presence of other sulfur-containing
functional groups, such as thioether (Met in 7b, Cys(Acm) in 8b,
and Thz in 9b) and thioester (in 10b). The protected and
unprotected carbohydrate was intact during the phtotoredox
catalysis (in 11b and 12b). Furthermore, we prepared and
evaluated peptide 13a bearing a range of suspicious sensitive
functional groups (Met, Acm, Thz, thioester, and carbohydrate);
the desired desulfurization peptide 13b was obtained with a
good yield of 76%. Moreover, this desulfurization protocol can
proceed in both aqueous buffer and a mixture of aqueous buffer
and organic solvent (CH CN or MeOH).
3
We then combined this photodesulfurization protocol with
native chemical ligation (NCL) for peptide synthesis (Scheme 3).
The linear peptide 14a and the cyclic peptide 15a were
successfully prepared by native chemical ligation and were desul-
furized to the desired peptide 14b and Heterophyllin J (15b), a
20
natural compound isolated from plant Dianthus chinensis, in
good yield via photoredox desulfurization reaction.
7
−9,19c
Scheme 4. Proposed Mechanism of [Ru]2+ Catalyzed
Desulfurization
Based on a previous study,
a plausible mechanism for
this visible-light induced desulfurization process is outlined in
2
+
Scheme 4. Initially, photoactive catalyst [Ru] accepts a photon
2
+
from visible light to populate the excited state [Ru] * via metal-
to-ligand charge transfer (MLCT), and this activated [Ru] *
2+
intermediate will be efficiently reduced by the thiol group to
+
+
generate thiol radical cation and [Ru] . The [Ru] can be
2
+
21,22
oxidized back to the [Ru] by the oxidation species,
which
rejuvenated the photoactive catalyst, and the thiol radical cation
deprotonates to generate a thiyl radical. Concurrent with the
photoredox pathway, in another catalytic cycle the addition of the
thiyl radical to the phosphine generates a phosphoranyl radical.
Subsequently, β scission of the phosphoranyl radical provides an
23
alkyl radical, which abstracts a hydrogen atom from a mercaptan
to generate the desulfurization product and thiyl radical.
C
DOI: 10.1021/acs.orglett.6b00292
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
In conclusion, we have developed a visible-light-initiated
desulfurization method using a photocatalyst. This mild and
specific desulfurization reaction is compatible with various
related functional groups, able to incorporate into ligation of
polypeptides and glycopeptides synthesis, and will be further
applied in complex peptide and protein chemistry.
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ASSOCIATED CONTENT
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S
Supporting Information
(
2
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.orglett.6b00292.
́
2
Detailed experimental procedures, HPLC traces, ESI-MS
and characterization data (PDF)
(
9
(
2
AUTHOR INFORMATION
■
(
Corresponding Author
Kunishige, H. Tetrahedron 1997, 53, 12883−12894.
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The authors declare no competing financial interest.
(
ACKNOWLEDGMENTS
■
(
We are grateful to the National Natural Science Foundation of
China (Nos. 21272084, 21402058) and Central China Normal
University for support of this research.
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DOI: 10.1021/acs.orglett.6b00292
Org. Lett. XXXX, XXX, XXX−XXX