Thiol-assisted one-pot synthesis of peptide/protein C-terminal thioacids from peptide/protein hydrazides at neutral conditions

Article abstract of DOI:10.1039/c4ob01885k

An efficient thiol-assisted one-pot synthesis of peptide/protein C-terminal thioacids was achieved by using peptide/protein hydrazides precursors at neutral pH and room temperature (about 20 °C). The transformation from hydrazides to thioacids was shown t

Full text of DOI:10.1039/c4ob01885k

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Thiol-assisted one-pot synthesis of peptide/
protein C-terminal thioacids from peptide/protein
Cite this: DOI: 10.1039/c4ob01885k
hydrazides at neutral conditions†
Chenchen Chen,‡a,b Yichao Huang,‡c Ling Xu,b Yong Zheng,b Huajian Xu,a
Qingxiang Guo,b Changlin Tian,b Yiming Li*a,c and Jing Shi*b
An efficient thiol-assisted one-pot synthesis of peptide/protein C-terminal thioacids was achieved by
using peptide/protein hydrazides precursors at neutral pH and room temperature (about 20 °C). The
transformation from hydrazides to thioacids was shown to be efficient for different C-terminal amino
acids and was racemization-free. The in situ formed peptide-thioacids were further used for protein
chemical synthesis and site-specific labelling successfully.
Received 4th September 2014,
Accepted 30th September 2014
DOI: 10.1039/c4ob01885k
www.rsc.org/obc
resin is time-consuming. Moreover, the temperature (37 °C)
and the long time required for achieving considerable yields of
Introduction
Peptide C-terminal thioacids were considered as important product still needs to be optimized.
functional groups for protein chemoselective reactions.1 It has
Because peptide thioacids can be obtained from thioesters,
been widely used for protein site-specific modification and it is possible to be produced from hydrazides precursor in one-
labeling, also converted to the peptide-thioesters for protein pot approach. As easy prepared equivalents of thioesters,
chemical synthesis.2 Most early strategies reported for prepar- peptide/protein hydrazides have been verified for protein
ing peptide thioacids were mainly based on Boc solid phase chemical synthesis and modification.9 Herein, we report for
peptide synthesis (SPPS).3 Another progress that utilized intein first time an efficient and facile one-pot synthesis of both
thioesters to make protein thioacids need relatively high pH peptide and recombinant protein C-terminal thioacids by
and very long time (16 h).4 Recently, C. F. Liu et al. developed a using its hydrazides as precursors at neutral pH and room
simple and efficient method to produce peptide thioacids temperature (about 20 °C) (Scheme 1). The interesting point of
through hydrothiolysis of thioesters.5 This method has been this in situ transformation is the catalysis of disulfide inter-
successfully used for protein chemical synthesis and site- mediate, which was possibly generated from thiol groups.
specific labeling.6 However, despite the high yields of the Then, the peptide thioacids can be formed after the thioesteri-
thioacids formation from thioesters, relatively high pH (more fication of peptide hydrazides within 2 h. Another important
than 8) and temperature (42 °C) was necessary. In addition, advantage of this method is that peptide or protein hydrazides
the preparation of peptide thioesters through Fmoc based can be easily obtained through standard protocol as reported
SPPS for subsequent hydrothiolysis remain challenging till previously.10
now.7 To further acquire peptide thioacids by mild methods,
bis(2-sulfanylethyl) amido (SEA) peptides were developed to
react with triisopropylsilylthiol in water at neutral pH.8 Never-
Results and discussion
theless, the reagent, such as triisopropylsilylthiol, used for the
synthesis of SEA peptide is expensive and the preparing of SEA
We started with hydrazides based thioacids synthesis of the
model peptide Leu-Tyr-Arg-Ala-Gly-NHNH2 (LYRAG-NHNH2). It
was first converted to C terminal thioesters by treating with
MESNa (2-mercaptoethane sulfonic acid sodium salt) as thiol
using standard protocol (NaNO2, pH 3.0; MESNa 30 mM, pH
5.0–6.0), and subsequently in situ turned to thioacids through
hydrothiolysis of thioesters. It is surprising to find that thio-
acids can be easily achieved in high yield under very mild con-
dition (pH 7.0, 20 °C, 1 h), which was quite different to
previously reported methods.5 The yield of Leu-Tyr-Arg-Ala-
aSchool of Medical Engineering, School of Chemistry and Chemical Engineering,
Hefei University of Technology, Hefei, Anhui 230009, China.
E-mail: lym2007@mail.ustc.edu.cn, ymli@hfut.edu.cn
bDepartment of Chemistry, University of Science and Technology of China, China.
E-mail: shijing@ustc.edu.cn
cDepartment of Chemistry, Tsinghua University, Beijing 100084, China
†Electronic supplementary information (ESI) available: Experimental details.
See DOI: 10.1039/c4ob01885k
‡These authors contributed equally to this work.
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Scheme 1 One-pot synthesis of peptide C-terminal thioacids by using
hydrazides as precursors. (a): NaNO2, pH 3.0; MESNa 30 mM, pH
5.0–6.0; (b): TCEP, pH 7.0, 37 °C, HS-Si(iPr)3, tBuOH, MPAA 60 mM; (c):
Na2S 100 mM, pH 7.0, 20 °C.
Gly-SH, as determined by HPLC, was very high (48% isolated
yield). To determine whether this result was because of the
excess of thiol groups (MESNa) existed in thioesterification
reaction system, we first purified the peptide thioesters and
hydrothiolysis of thioesters in two independent procedures.
Similar as C. F. Liu et al. reported, Fig. 1b indicated that the
conversion yield was very low when Na2S (100 mM) only was
added for hydrothiolysis of thioesters at neutral condition (pH
7.0, 20 °C). As compared, when MESNa (30 mM) was added
with Na2S, it was very easy to obtain thioacids in quantitative
yield at similar condition. Decrease in the loading of MESNa
to 1 mM produced nearly the same HPLC conversion rate
(Fig. S2†). The results demonstrated that the added thiol
groups facilitated the hydrothiolysis of thioesters.
Fig. 1 (a) HPLC (λ = 214 nm) analysis of the conversion of peptide
thioacids from hydrazides precursors. ESI-MS mass of LYRAG-NHNH2 is
593.3 (calc. 592.6); LYRAG-MESNa is 703.3 (calc. 701.6); LYRAG-SH is
595.3 (calc. 594.6); (b) HPLC (λ = 214 nm) analysis of the conversion of
peptide thioacids from thioesters with (R) or without (L) added MESNa
(30 mM, pH 7.0, 20 °C).
It has been reported that thiol group, including GSH and
Mesna, can generate disulfide or persulfide (RSSH).11 Thus, it
is worthy to speculate whether the high efficiency of hydra-
zides based thioacids synthesis was possible because of the
formation of disulfide intermediate. As shown in Fig. 2, when
TCEP (tris(2-carboxyethyl)phosphine) was added, which could
reduce the disulfide, decreased the conversion from thiol to
thioacids (about 40%) in one hour. As compared, thiol can be
converted into thioacids almost completely without added
TCEP. Therefore, we hypothesized that the thiol group should
generate disulfide intermediate and thereby, increase the
efficiency of the reaction. Hence collectively, our conclusion
indicated that hydrazides based one-pot synthesis of thioacids
is a novel thiol assisted strategy with high efficiency.
Fig. 2 HPLC (λ = 214 nm) analysis of the conversion of peptide thio-
acids from thioesters with (L) or without (R) TCEP (50 mM). All the reac-
tions were performed at MESNa 30 mM, Na2S 100 mM, pH 7.0, 20 °C.
(4-mercaptophenylacetic Acid) and EDT (1,2-dithioethane). We
found that treating with MPAA as ester group for 1 h also leads
to near quantitative conversion to the thioacids, whereas rela-
tive long time (3 h) was needed for EDT. Moreover, different
pH conditions were compared for the synthesis of thioacids.
This one-pot hydrazides based thioacids synthesis was
further explored by using different thiols, including MPAA
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Table 1 Synthesis of thioacids under different pH and thiols
HPLC/Isolated
Entry
pH
Thiols
Time(h)
yielda [%]
1
2
3
4
5
6
7
7.0
7.0
7.0
7.0
6.0
8.0
9.0
MESNa
MPAA
EDT
1.0
1.0
3.0
1.0
5.0
0.75
0.75
94/48
93/44
96/41
21/13
90/34
95/51
92/44
No
MESNa
MESNa
MESNa
a 5 mM peptide hydrazides Leu-Tyr-Arg-Ala-Gly-NHNH2.
At pH 6.0, R.T., the conversion rate was decreased accordingly,
whereas prolonging the reaction for 5 h led to near complete
conversion of the thioesters to thioacids. Increasing the pH
(from pH 8.0 to 9.0) increased the reaction rate in similar way
as the previous results (Table 1, Fig. S1†). Therefore for practi-
cal reasons, the one-pot reactions were all performed at pH
7.0, R.T. in subsequent experiments.
To demonstrate the general utility of this method, a
number of peptide hydrazides with different C terminal resi-
dues were tested (Leu-Tyr-Arg-Ala-X-NHNH2). We chose six
different amino acids to test the conversion rate of thioacids.
All reactions were conducted under conditions of pH 7.0, R.T.
and 100 mM aqueous Na2S. Table 2 shows that C-terminal Ser,
Phe, Ala and Leu hydrazides can transform to thioacids within
3 h to nearly 100% HPLC yield and high isolated yield. The
large steric hindrance residues, such as Pro and Val, can also
achieve better conversion rates in a relatively longer time
(Fig. S3†).
Fig. 3 Synthesis of trifolitoxin using thioacids captured ligation. (a)
Sequence of trifolitoxin[Ala23Cys]. (b) Procedure for ligation. (c) HPLC (λ
= 214 nm) and ESI-MS for the final purified trifolitoxin were performed.
Observed mass is 4437.5 Da (calc. 4438.3 Da). The MS data was taken
across the whole UV peak.
Racemization of the C-terminal amino acid is another
important issue for the synthesis of the polypeptide-thioacids.
To test the possibility of racemization of the synthetic thio-
acids converted form hydrazides, we compared double-benzyl-
protected tripeptide Bn-Leu-Tyr(Bn)-Phe(L)-SH and Bn-Leu-Tyr-
(Bn)-Phe(D)-SH. Through RP-HPLC analysis with either L- or
D-Phe at the C-terminus, we confirmed that the formation of
thioacids was epimerization-free (Fig. S4†).
To verify the application of the above in situ formed thioa-
cids, we selected a 42 amino acid antimicrobial peptide trifoli-
toxin [Ala23Cys] for chemical synthesis (Fig. 3). The ligation
site was chosen to be Ala22-Cys23. We first synthesized
peptide hydrazide 1 (M1-A22-NHNH2) and Npys modified
peptide 2 (C23-A42). After one-pot thioesterification and hydro-
thiolysis, we obtained peptide thioacids 3 (M1-A22)-SH (43%
isolated yield). Then, by using previously reported thioacid
capture ligation,5 we found that the ligation proceeds provide
the desired product in a good HPLC yields. Finally, after de-
Acm protecting group, we obtained a full-length trifolitoxin
[Ala23Cys] with high purity and homogeneity.
Finally, the functionality of synthetic thioacids was further
expanded to recombinant protein labeling. We first obtained
LC3-NHNH2 (microtubule-associated protein 1A/1B-light chain 3),
an autophagosomal marker protein, from recombinant LC3-
intein-CBD.12 LC3-SH was easily obtained as aforementioned
in high yields, because the C terminal residue of this model
protein is Gly. Then, LC3 thioacids (0.34 mM) reacted with 10
folds dansyl azide molecules overnight at room temperature,
having pH 7.0. The final labeled product was purified by using
desalting column for two times. As shown in Fig. 4, ESI-MS
and gel electrophoresis experiments proved that the fluo-
rescent molecules were site-specific labeled with LC3 protein.
Another important protein Ub-NHNH2 was also proved to be
converted to C-terminal thioacids and labeled with dansyl
azide (Fig. S6†).
Table 2 Synthesis of thioacids with different C-terminal residues
HPLC/Isolated
Entry
X
Time (h)
yielda [%]
1
2
3
4
5
6
Ser
Phe
Ala
Leu
Val
Pro
1.5
1.5
2.5
3.0
7.0
>12
97/51
93/50
92/46
95/47
88/32
90/35
a 5 mM peptide hydrazides Leu-Tyr-Arg-Ala-X-NHNH2.
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Conclusions
To summarize, we devised a facile and efficient one-pot syn-
thesis of peptide thioacids approach at neutral conditions by
using peptide hydrazides precursors. It was demonstrated that
existing thiol group was essential for the in situ formation of
thioacids in high yields. This method was further proved to be
efficient for different C-terminal amino acids and racemization
free. Finally, the synthetic thioacids was successfully used for
the chemical synthesis of antimicrobial peptide trifolitoxin
and recombinant protein labeling.
Experimental section
Synthesis of hydrazine 2CTC resin
2-Chlorotrityl chloride resin (loading = 1.0 mmol g−1) (2 g) was
swelled in CH2Cl2–DMF (15/15 mL) at 0 °C. NH2NH2·H2O
(1 mL, 10 eq.) and DIEA (3.3 mL, 20 eq.) were then added. The
reaction was conducted from 0 °C to room temperature, over-
night. Methanol (2 mL) was added to quench the remaining
2-chlorotrityl chloride resin. After reaction, the resin was
washed with DMF, H2O, methanol, ethyl ether and kept under
high vacuum for 3 h. Then, the resin was stored at 4 °C.
Synthesis of peptide hydrazine
Hydrazine 2CTC and 2CTC resins were first swelled in DMF–
DCM (1/1) for 10 min before use. The coupling step was
carried out at 30 °C by standard Fmoc chemistry (4 eq. pro-
tected amino acid; 3.6 eq. HBTU or HCTU, 8 eq. DIEA). Each
coupling step required 45 min and the resin was washed with
DMF, DCM and DMF. If the reaction required two coupling,
the time was changed to 30 min × 2. For 2CTC resin, the first
amino acid was coupled with 4 eq. protected amino acid, 8 eq.
DIEA in DMF–DCM (1 : 1) in 2 h and the resin was capped with
methanol. Deprotection reagent was 20% piperidine/DMF
(5 min, 10 min). The resin was washed with DMF, DCM and
DMF. Cleavage reagent selected reagent B: TFA–phenol–water–
TIPS (88/5/5/2). Reaction time was about 2–3 hours. Peptide
purification: The crude peptides was dissolved in acetonitrile
(0.08% TFA)–water (0.1% TFA), analyzed by analytical HPLC
and purified by semi-preparative HPLC and lyophilized
immediately.
Synthesis of thioacids from H-Leu-Tyr-Arg-Ala-Gly-NHNH2
H-Leu-Tyr-Arg-Ala-Gly-NHNH2 (0.5 mg, 1 mM) was dissolved in
800 μL buffer (6 M Gn-Cl, 100 mM Na2HPO4, pH 3.0) and
cooled in an ice bath (−10 °C). 100 μL of aqueous 50 mM
NaNO2 solution was added and the reaction was incubated for
20 min. MESNa (5 mg) or MPAA (5 mg) or EDT (2.5 μL) was
added and the pH was adjusted to 5.0–6.0. Reaction was incu-
bated for 20 min and the pH was adjusted to about 1.0. 100 μL
of aqueous 1 M Na2S was added and then the pH was adjusted
to 6.0 or 7.0 or 8.0 or 9.0 at room temperature (20 °C). The
reaction was detected by analytical RP-HPLC.
Fig. 4 Fluorescence labeling of LC3 by thioacids based ligation.
(a) Generation of C terminal labeling of LC3 through thioacid-sulfona-
zides ligation; (b) HPLC (λ = 214 nm) analysis of the conversion of LC3
thioacids from hydrazides precursors; (c) SDS-PAGE of fluorescent
labeling of LC3-SH; (d) ESI-MS for purified LC3-SH is 14 028.1 Da
(calc. 14 029.2) and labeled LC3 is 14 245.4 Da (calc. 14 244.2).
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Comparison of the synthesis of thioacids from H-Leu-Tyr-Arg-
Ala-Gly-MESNa with or without thiol
standard Fmoc chemistry and cleaved for 2 h in the presence
of 4 eq. of 2,2′-dithio-bis-(5-nitropyridine) which was to add
the Npys group to Cys thiol. H-M1-A22-SH (2.4 mg, 1 mM) and
Npys-C23-A42-OH (the Cys 40 was modified by Acm) (3.3 mg,
1.5 mM) were dissolved in 1 mL 10% ACN–H2O and the pH
was adjusted to 5.0–6.0. Reaction was incubated for 30 min
and then AgOAc (8.3 mg) was added for overnight. 1 mL of
aqueous 1 M DTT was added to reduce the reaction. The reac-
tion was detected by analytical RP-HPLC.
H-Leu-Tyr-Arg-Ala-Gly-MESNa was converted from hydrazides
as standard protocol and further purified by HPLC. Then, it
(0.7 mg, 1 mM) was dissolved in 900 μL buffer (6 M Gn-Cl,
100 mM Na2HPO4). MESNa (5 mg, 1 or 30 mM) was added in
reaction B (not added in reaction A) and the pH was adjusted
to about 1.0. 100 μL of aqueous 1 M Na2S was added and then
adjusted the pH to 7.0 at 20 °C. The reaction was detected by
analytical RP-HPLC.
Fluorescence labeling of protein by thioacids-based ligation
Comparison of the synthesis of thioacids with different
residues
Dansyl chloride (270 mg, 1 mmol, 1 eq.) was dissolved in
acetone (6 mL). NaN3 (97.5 mg, 1.5 mmol, 1.5 eq.) in water
(2 mL) was then added. The reaction was kept at R.T. for 4 h.
The resulting solution was diluted with NH4Cl (15 mL) and
extracted by using EtOAc (30 mL). The organic phase was
washed with sat. NH4Cl, brine, dried over Na2SO4. Concen-
tration in vacuo afforded dansyl azide (270 mg, 0.97 mmol,
97%). LC3-NHNH2 (3 mg, 1 mM) was dissolved in 160 μL
buffer (6 M Gn-Cl, 100 mM Na2HPO4, pH 3.0) and cooled in an
ice bath (−10 °C). 25 μL of aqueous 50 mM NaNO2 solution
was added and the reaction was incubated for 20 min. MESNa
(5 mg) was added and the pH was adjusted to 5.0–6.0. Reaction
was incubated for 20 min and the pH was adjusted to about
1.0. 20 μL of aqueous 1 M Na2S was added and then the pH
was adjusted to 7.0. The reaction was detected by analytical
RP-HPLC and isolated by semi-preparative RP-HPLC.
H-Leu-Tyr-Arg-Ala-X-NHNH2 (1 mM) was dissolved in 800 μL
buffer (6 M Gn-Cl, 100 mM Na2HPO4, pH 3.0) and cooled in an
ice bath (−10 °C). 100 μL of aqueous 50 mM NaNO2 solution
was added and the reaction was incubated for 20 min. MESNa
(5 mg) was added and the pH was adjusted to 5.0–6.0. The
reaction was incubated for 20 min and the pH was adjusted to
about 1.0. 100 μL of aqueous 1 M Na2S was added and then
the pH was adjusted to 7.0. The reaction was detected by
analytical RP-HPLC. All HPLC gradients were conducted at
1%–91% (ACN)/30 min/1.2 mL/C18.
Racemization test
Peptide Leu-Tyr-Phe(L/D)-NHNH2 was obtained by standard
Fmoc-SPPS (0.25 mmol resin), after assembly of the peptide
chain, 10 mmol benzyl bromide and 20 mmol DIEA were
added. The reaction was conducted for overnight and cleaved
from resin by reagent B. After it was purified by HPLC and lyo-
philized, we obtained the Bn-Leu-Tyr(Bn)-Phe(L/D)-NHNH2·
Bn-Leu-Tyr(Bn)-Phe(L/D)-NHNH2 (0.6 mg, 1 mM) was dissolved
in 800 μL buffer (6 M Gn-Cl, 100 mM Na2HPO4, pH 3.0) and
cooled in an ice bath (−10 °C). 100 μL of aqueous 50 mM
NaNO2 solution was added and the reaction was incubated for
20 min. MESNa (5 mg) was added and the pH was adjusted to
5.0–6.0. Reaction was incubated for 20 min and the pH was
adjusted to about 1.0. 100 μL of aqueous 1 M Na2S was added
and then the pH was adjusted to 7.0. The reaction was
detected by analytical RP-HPLC. All HPLC gradients were con-
ducted at 1%–91% (ACN)/30 min/1.2 mL/C18.
LC3-SH (1 mg) was dissolved in 100 μL buffer (6 M Gn-Cl,
100 mM Na2HPO4, pH 7.0) and 100 μL DMSO. 4.2 μL of 0.6 M
Dansyl-N3 and 0.2 μL 2, 6-lutidine were added. The reaction
was detected by analytical RP-HPLC.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (21102083, 21372058 to Y. M. Li;
21272223 to Q. X. Guo).
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