Direct synthesis of N-terminal thiazolidine-containing peptide thioesters from peptide hydrazides

Article abstract of DOI:10.1039/c8cc03591a

We report a simple and promising synthetic method to oxidize peptide hydrazides containing N-terminal thiazolidine as a protected cysteine. This yields the corresponding thioester via a peptide azide without decomposition of the thiazolidine ring. The new

Full text of DOI:10.1039/c8cc03591a

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DOI: 10.1039/C8CC03591A
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Direct synthesis of N-terminal thiazolidine-containing peptide
thioesters from peptide hydrazides†
a
a
a
ab
abc
Kohei Sato,* Shoko Tanaka, Kazuki Yamamoto, Yosuke Tashiro, Tetsuo Narumi and
Received 00th January 20xx,
Accepted 00th January 20xx
abc
Nobuyuki Mase
DOI: 10.1039/x0xx00000x
www.rsc.org/
8
We report a simple and promising synthetic method to oxidize
Recently, peptide hydrazides, developed by Liu et al. as
peptide hydrazides containing N-terminal thiazolidine as a peptide thioester precursors, are beginning to replace
protected cysteine. This yields the corresponding thioester via a traditional thioesters. In this protocol, a peptide hydrazide is
peptide azide without decomposition of the thiazolidine ring. The
newly developed protocol was validated by the synthesis of the
bioactive peptide LacZα.
Chemical synthesis of proteins has attracted considerable
1
attention as an important tool in the field of structural biology,
2
3
drug discovery and chemical biology. Although the total- or
semi-synthesis of a protein remains challenging, recent
advances, including chemoselective ligations and use of related
protecting groups are bringing the chemical synthesis of
proteins into the mainstream in synthetic chemistry. In
particular, native chemical ligation (NCL), developed by Kent et
al. is a powerful technique for the chemoselective ligation
between a peptide thioester and an N-terminal cysteinyl
peptide, yielding the ligated product with high efficiency. A key
to the success of the chemical synthesis of a large protein is how
more than two peptide segments can be assembled in a
4,5
6
sequential and convergent manner. In sequential NCL strategy,
the N-terminal Cys residue of the middle segment must be
protected in order to prevent cyclization caused by
intramolecular reactions with the thioester moiety (Figure 1).
7
Among the protecting groups that have been developed, the
1
,3-thiazolidine-4-carboxo (Thz) group is a reliable choice
because of its facile convertibility to Cys and its commercial
availability, and it has been applied extensively to the synthesis
6,7c
of various proteins.
a.Department of Engineering, Graduate School of Integrated Science and
Technology, Shizuoka University, 3-5-1 Johoku, Hamamatsu, Shizuoka 432-8561,
Japan. Email: sato.kohei@shizuoka.ac.jp
Fig. 1 (A) Conversion of peptide hydrazide bearing protected cysteine to the
corresponding thioester. (B) Direct conversion of Thz-peptide hydrazide to the
corresponding thioester using TFA as a solvent and subsequent sequential NCLs. PG:
protecting groups; Tbeoc: 2-(tert-butyldisulfanyl)ethyloxycarbonyl; Dobz: p-
b.
Graduate School of Science and Technology, Shizuoka University, 3-5-1 Johoku,
Hamamatsu, Shizuoka 432-8561, Japan
Research Institute of Green Science and Technology, Shizuoka University, 3-5-1
c.
Johoku, Hamamatsu, Shizuoka 432-8561, Japan
dihydroxyborylbenzyloxycarbonyl;
trifluoroacetyl.
Tfacm:
trifluoroacetamidomethyl;
Tfa:
†
Electronic Supplementary Information (ESI) available: Experimental details of
syntheses including HPLC charts. See DOI: 10.1039/x0xx00000x
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J. Name., 2013, 00, 1-3 | 1
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prepared by conventional 9-fluorenylmethyloxycarbonyl chlorotrityl chloride resin using
Journal Name
a
previously reported
View Article Online
11
DOI: 10.1039/C8CC03591A
(
Fmoc) solid-phase peptide synthesis (SPPS) on hydrazine- protocol.
incorporated resin and can be converted into a thioester by triisopropylsilane (TIS)-H
treatment with NaNO under acid conditions, followed by hydrazide 1a. Aqueous NaNO
The peptidyl resin was treated with TFA-
2
O (95:2.5:2.5) to give the peptide
solution (approximately 5
2
2
thiolysis of the resulting peptidyl azide (Figure 1A). This equivalents relative to the hydrazide) was added to the
technique has seen widespread use and the number of ligations resulting mixture, affording the corresponding azide
carried out using hydrazides as an acyl donor represents 30% of intermediate 2a. After a 20 min reaction at -10 °C followed by
9
the total ligations reported in 2016. This method however has trituration with Et
2
O, the formed precipitate was dissolved into
buffer containing sodium 2-mercaptoethanesulfonate
thioesters due to the decomposition of the Thz moiety in the (MESNa).
not been used widely in the synthesis of N-terminal Thz-peptide
a
10a
hydrazide in the acidic activation step. To circumvent this
HPLC analysis indicated that the desired thioester 3a had
problem, alternative protections for Cys have been examined. been formed, while the hydrazide 1a and the nitrated thioester
The functionalized Cys derivatives that are called for by these 4a were also observed (Table 1, entry 1). Higher temperatures
10
methods must be prepared (Figure 1A). Thus, there is an during the azidation were not effective (entry 2). The nitration
increasing demand for the development of practical methods occurs in the phenol ring of Tyr residue, but was not observed
for the direct synthesis of Thz-containing peptide thioesters during the reaction of peptide hydrazides with NaNO
2
in
12
from peptide hydrazides (Figure 1B).
aqueous media. This side reaction was suppressed by the
Peptides with a Thz group are stable during cleavage addition of m-cresol which is commonly used as an additive in
reactions mediated by HF or TFA, and the moiety can be global deprotection of peptides (entry 3). Surprisingly, the
converted to the corresponding Cys residue under acidic addition of thioanisole, which is also used as an additive for
7c
aqueous conditions with MeONH
speculated that Thz-containing peptide hydrazides would be hydrazide (entry 4). Use of the both additives resulted in
compatible with the NaNO -mediated activation step using a complete conversion and no nitrated product was formed
2
. Based on these facts, we peptide deprotection, accelerated the conversion of the
13
2
TFA-based cocktail. Since TFA is usually used as a cleavage (entry 5). This condition was applied to the conversion of the
reagent in Fmoc SPPS, we also envisioned that the cleavage and peptide hydrazide with a Thz group at the N-terminus,
conversion of the hydrazide could be achieved in a one-pot successfully producing the N-terminal Thz-containing peptide
reaction.
In our initial attempts, the peptide hydrazide H-LYRAG
2
NHNH (1a) lacking a Thz group at N-terminus was used as a intermolecular ligations were not observed (entry 6). To the
thioester in 68% isolated yield. Side reactions, including opening
-
of the thiazolidine and subsequent intramolecular or
substrate (Table 1). The peptide hydrazide was elongated on 2- best of our knowledge, this is the first example of the direct
synthesis of an N-Thz peptide thioester from the peptide
Table 1 Conversion of peptide hydrazide to thioester during global deprotectiona
hydrazide without any additional protecting groups.
With the optimal conditions in hand, we evaluated the
effects of the C-terminal amino acids with the exception of Asp,
Asn and Gln, of which thioesters are applicable to NCL
17b
reactions
but the corresponding hydrazides cannot be
prepared due to intramolecular cyclization between the
8a
hydrazide and side-chain during SPPS. As summarized in Table
, in all cases, a peptide hydrazides was successfully converted
to the corresponding thioester with a modest to high purity
63–95%). Sterically hindered amino acids (Val and Ile) afforded
2
(
relatively low 70% and 63% yields, respectively, as a result of
Curtius rearrangement of the azide to an isocyanate, affording
global deprotection (v/v)
TFA TIS m-cresol thioanisole
HPLC area ratio [%]
entry
1
2
3
H
2
O
1
58
61
54
0
3
4
17
12
0
13
0
14
thiocarbamate derivatives (entries 3 and 4). This side reaction
also occurred at a higher level than 5% with Glu, Arg and Trp
(entries 11, 18 and 19). This probability of formation of a Curtius
rearrangement product is consistent with a previous report.14
The epimerization levels of C-terminal amino acids (Ala, Phe,
Ser, Cys, Tyr, and His) were assessed. Peptides containing Ser
and His were more sensitive to epimerization whereas the
95
95
90
90
80
80
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
0
0
5
0
5
5
0
0
0
5
10
10
20
16
39
77
87
93
b
4
c
5
6
0
de
0
0
a
All reactions were performed as follows: the peptidyl resin was treated under
global deprotection conditions at rt for 2 h. The reaction mixture was then epimerization was suppressed under more acidic thiolysis
incubated at -10 °C for 20 min after addition of 10% aqueous NaNO
. The peptide conditions (entries 8 vs 9 and 15 vs 16).
azide precipitated by the addition of cold Et O was subjected to thiolysis with
2
2
The synthetic applicability of our protocol was confirmed
MESNa (3% w/w) in 6 M Gn·HCl, 0.2 M sodium phosphate buffer (pH 7.3) at rt for
.5 h. The product was analysed by HPLC. The activating step with NaNO
performed at 0 °C. 69% isolated yield. N-Terminal Thz-containing peptide (H-
through the synthesis of the LacZ alpha subunit (LacZα) as
b
0
2
was
15
c
d
shown in Figure 2. Since LacZα has no Cys residue, use of an
) was used as a substrate. 68% isolated yield. HPLC charts: see NCL-desulfurization strategy, formally enabling the assembly of
e
Thz-LYRAG-NHNH
ESI.†
2
16
peptides at Xaa-Ala junctions, was envisioned. Peptide
2
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Table 2 Scope of the conversion of peptide hydrazides to peptide thioestersa
View Article Online
DOI: 10.1039/C8CC03591A
HPLC area ratio [%]
epimerization
(L:D) [%]c
entry
Xaa
product
b
3
5
1
2
3
4
5
6
7
8
Gly
Ala
Val
Ile
3a
3b
3c
3d
3e
3f
3g
3h
3h
3i
3j
3k
3l
3m
3n
3n
3o
3p
3q
87
90
70
63
89
90
89
67
90
72
76
79
95
82
72
82
93
78
81
n.d.
<1
8
13
3
-
>99:1
-
-
-
>99:1
-
88:12
>99:1
Fig. 2 (A) Primary sequence of synthesized LacZα. (B) Synthetic scheme for LacZα by
sequential NCLs-desulfurization.
Leu
Phe
Pro
Ser
Ser
Thr
Glu
Cys
Met
Tyr
His
His
Lys
Arg
Trp
2
n.d.
n.d.
n.d.
n.d.
7
n.d.
<1
2
n.d.
n.d.
n.d.
5
site.17 Kent et al. demonstrated that the use of an increased
concentration of MPAA and/or high pH can prevent such by-
d
9
1
7b
product formation, but although we tried to optimize ligation
conditions, pH or concentration of peptides and MPAA,
suppression of the side reaction could not be achieved.
1
1
1
0
1
2
-
-
>99:1
-
>99:1
84:16
e
1
3
After being purified by HPLC, the ligated peptide 10 was
subjected to the next NCLs. We initially envisioned a one-pot
1
1
4
5
reaction consisting of sequential ligations between peptides
and followed by desulfurization, yielding the desired
product 12. Peptides and 10 in 6 M Gn·HCl, 0.2 M sodium
phosphate, 40 mM methyl thioglycolate as a non-aryl thiol
7,
d
1
6
>99:1
1
0
6
1
1
1
7
8
9
-
-
-
7
10
18
additive, 20 mM TCEP·HCl (pH 7.2) were coupled smoothly.
a
All reactions were performed under similar conditions to that of Table 1 except The coupled material was converted to the corresponding Cys
for global deprotection conditions (TFA-TIS-H O-m-cresol-thioanisole
0:2.5:2.5:5:10). n.d. = not detected. Standard material as an epimerized
2
=
form with the aid of MeONH
was added to the resulting mixture to yield the full-length
polypeptide. Howev er, the thioester reacted preferentially
and no ligated product was obtained.
We examined the use of [Pd(allyl)Cl] as a reagent to convert
2
·HCl, and the peptide thioester 6
b
c
8
peptide was separately synthesized using the corresponding D-isomer, and
epimerization ratio was determined by HPLC analysis. Buffer solution containing
1
d
6
e
O-m-cresol-thioanisole = 75:2.5:2.5:5:15 with the residual MeONH
0% MESNa (pH 4) was used. TFA-TIS-H
2
2
was used as a global deprotection cocktail.
2
19
Thz to a free Cys, but this did not give the desired product.
These results led us revise our synthetic design, which is based
segments
prepared as peptide hydrazide for segments
for by the conventional Fmoc-SPPS (Figure 2B). After
construction of the peptide segments, the peptide hydrazides
were converted to the corresponding thioesters by the
established protocol. This procedure afforded the desired
peptide thioesters in 31% isolated yield for
for . Notably, no decomposition of N-terminal Thz group in
segments and during the activating step was observed.
The first NCL between bearing a Cys residue at the N-
terminus and with a Glu-thioester at the C-terminus in 6 M
6
–9
covering the entire sequence of LacZα were
on an NCL-deprotection between
of MeONH and subsequent one-pot NCL-desulfurization
between peptide and 11 using imidazole-aided NCL conditions
7 and 10 followed by removal
6–8
or peptide acid
2
9
6
2
0
(
Figure 2B).
The reaction sequence for the synthesis of LacZα is shown in
Figure 3A–C. Ligation between the Thz-containing thioester
peptide and Cys-peptide 10 followed by deprotection of the
6, 34% for 7 or 64%
7
8
Thz ring was conducted under the optimum conditions,
affording the three-component ligated product 11 (Figure 3B).
After purification by HPLC, the ligated peptide was subjected to
7
8
9
8
the next NCL reaction with thioester
6 under imidazole-aided
guanidine hydrochloride (Gn·HCl), 0.2 M sodium phosphate
buffer (pH 7.3) in the presence of 4-mercaptophenylacetic acid
NCL conditions, allowing us to conduct subsequent free-radical-
based desulfurization of three Cys residues in a one-pot
reaction which proceeded efficiently to afford LacZα (12) in 85%
isolated yield (Figures 3C and D).
(
MPAA) (100 mM) and tris(2-carboxyethyl)phosphine
hydrochloride (TCEP·HCl) (50 mM) at 37 °C was complete within
h but HPLC analysis of the products of the ligation followed by
conversion of Thz to Cys by the addition of MeONH ·HCl showed
two peaks with the expected molecular weight of the product
(Figure 3A, denoted by 10’). The formation of γ-linked side
7
Finally, β-galactosidase activity of the complex between the
synthesized LacZα and expressed LacZ omega subunit (LacZω)
2
in the conversion of o-nitrophenyl-β-
D-galactopyranoside
1
0
(ONPG) was evaluated (Figure 4A). Incubation of ONPG with the
products had been observed previously in an NCL at the Glu-Cys
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Fig. 4 (A) Enzymatic hydrolysis of ONPG by -D-galactosidase. (B) α-Complementation
assay using ONPG with synthesized LacZα and crude proteins of E. coli DH5α expressing
LacZω. Full length of LacZ expressed by E. coli W3110 was used as a positive control.
Chem., 2017, 25, 4926; (c) S. B. H. Kent, Chem. Soc. Rev.,
2
009, 38, 338.
6
7
L. Raibaut, N. Olivier and O. Melnyk, Chem. Soc. Rev., 2012
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(a) J. A. Camarero, G. J. Cotton, A. Adeva and T. W. Muir, J.
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4
Pept. Res., 1998, 51, 303; (b) D. Bang, N. Chopra and S. B. H.
Kent, J. Am. Chem. Soc., 2004, 126, 1377; (c) D. Bang and S.
B. H. Kent, Angew. Chem. Int. Ed., 2004, 43, 2534; (d) S.
Ueda, M. Fujita, H. Tamamura, N. Fujii and A. Otaka,
ChemBioChem, 2005, 6, 1983.
(a) G.-M. Fang, Y.-M. Li, F. Shen, Y.-C. Huang, J.-B. Li, Y. Lin,
H.-K. Cui and L. Liu, Angew. Chem. Int. Ed. 2011, 50, 7645; (b)
J.-S. Zheng, S. Tang, Y.-K. Qi, Z.-P. Wang and L. Liu, Nat.
Protoc., 2013, 8, 2483; (c) J.-S. Zheng, S. Tang, Y.-C. Huang
and L. Liu, Acc. Chem. Res., 2013, 46, 2475.
Fig. 3 HPLC analyses of reactions for 12: (A) ligation of 8 and 9 followed by deprotection
of Thz (t = 7 h and 11 h); (B) ligation of 7 and 10 followed by deprotection of Thz (t = 5
h and 3 h); (C) ligation of 6 and 11 followed by desulfurization (t = 11 h and 16 h). (D)
HPLC analysis and mass spectrum of purified 12. The calculated mass is 6990.83 Da.
Deconvolution of the mass spectrum yielded an observed mass of 6991.98 Da as a
proton adduct. Reaction and HPLC conditions: see ESI.† * MPAA.
8
synthetic material and bacterial homogenate of Escherichia coli
DH5α as a source of LacZω resulted in a colour change resulting
from the formation of o-nitrophenol through the enzymatic
reaction, whereas absence of LacZα or LacZω did not affect the
colour of the reaction solution (Figure 4B). This result clearly
indicated that the synthesized LacZα can exhibit α-
9
1
V. Agouridas, O. E. Mahdi, M. Cargoët and O. Melnyk, Bioorg.
Med. Chem., 2017, 25, 4938.
0 (a) G.-M. Fang, J.-X. Wang and L. Liu, Angew. Chem. Int. Ed.,
2
012, 51, 10347; (b) J. Li, Y. Li, Q. He, Y. Li, H. Li and L. Liu,
Org. Biomol. Chem., 2014, 12, 5435; (c) M. Pan, Y. He, M.
Wen, F. Wu, D. Sun, S. Li, L. Zhang, Y. Li and C. Tian, Chem.
Commun., 2014, 50, 5837; (d) S. Tang, Y.-Y. Si, Z.-P. Wang, K.-
R. Mei, X. Chen, J.-Y. Cheng, J.-S. Zheng and L. Liu, Angew.
Chem. Int. Ed., 2015, 54, 5713; (e) Y.-C. Huang, C.-C. Chen, S.
Gao, Y.-H. Wang, H. Xiao, F. Wang, C.-L. Tian and Y.-M. Li,
Chem. Eur. J., 2016, 22, 7623.
1
5
complementation.
In summary, we have developed the simple and practical
protocol for converting N-Thz-peptide hydrazides into the
corresponding thioesters. A key to our developed protocol is the
use of TFA as a reaction medium for NaNO -mediated activation 11 G. Stavropoulos, D. Gatos, V. Magafa and K. Barlos, Lett.
2
Pept. Sci., 1995, 2, 315.
in the presence of thioanisole and m-cresol, which can
accelerate the desired reaction and suppress the side reaction,
respectively. Application of the developed protocol to the
synthesis of more-complex proteins will be presented in due
course.
This research was supported in part by a Grant-in-Aid for
Young Scientists (B) (No. 16K21077) from the Japan Society for
the Promotion of Science (JSPS) and research grants from the
Futaba Electronics Memorial Foundation.
1
2 The nitration is likely to occur through the coupling of the
phenoxyl radical with nitrogen dioxide derived from
oxidation of nitric oxide. These species could be generated
by homolysis of aromatic O-nitroso derivatives. See a
reference: D. Koley, O. C. Colón and S. N. Savinov, Org. Lett.,
2
009, 11, 4172.
1
3 Although we have yet to elucidate the role of thioanisole in
the conversion, one potential explanation for the effect of
thianisole is that thianisole reacts with nitrosonium cation to
afford sulfonium that might work as a more effective oxidant
than nitrosonium itself. See a reference: G. I. Borodkin, V. A.
Podryvanov, M. M. Shakirov and V. G. Shubin, J. Chem. Soc.,
Perkin Trans. 2, 1995, 1029.
Conflicts of interest
There are no conflicts to declare.
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8 Aryl thiol hampers radical-mediated desulfurization. See
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9 M. Jbara, S. K. Maity, M. Seenaiah and A. Brik, J. Am. Chem.
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