Peptide 2-formylthiophenol esters do not proceed through a Ser/Thr ligation pathway, but participate in a peptide aminolysis to enable peptide condensation and cyclization

Article abstract of DOI:10.1039/c5ob00825e

Peptide thiol salicylaldehyde (SAL) esters unexpectedly do not follow a Ser/Thr ligation pathway to react with peptides containing N-terminal Ser/Thr, but proceed towards a peptide aminolysis in DMSO. The reaction takes place even at a low substrate concentration (1 mM). The method has been successfully used to synthesize several natural cyclic peptides, with a high ratio of monocyclic to dimeric products.

Full text of DOI:10.1039/c5ob00825e

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Peptide 2-formylthiophenol esters do not proceed
through a Ser/Thr ligation pathway, but participate
in a peptide aminolysis to enable peptide
condensation and cyclization†
Cite this: Org. Biomol. Chem., 2015,
13, 6922
Received 24th April 2015,
Accepted 8th May 2015
DOI: 10.1039/c5ob00825e
Chun Ling Tung,‡a,b Clarence T. T. Wong‡a,b and Xuechen Li*a,b
www.rsc.org/obc
Peptide thiol salicylaldehyde (SAL) esters unexpectedly do not
follow a Ser/Thr ligation pathway to react with peptides containing
N-terminal Ser/Thr, but proceed towards a peptide aminolysis
in DMSO. The reaction takes place even at a low substrate con-
centration (1 mM). The method has been successfully used to
synthesize several natural cyclic peptides, with a high ratio of
monocyclic to dimeric products.
Chemoselective peptide ligation enables two side chain un-
protected peptide segments to be joined together at the termini
with the generation of the natural peptidic linkage at the lig-
ation site, which has dramatically advanced the protein chemi-
cal synthesis.1 Alternatively, some partially chemoselective
aminolysis-based methods, which require the internal lysine
side chain to be protected, have also been used in the conver-
gent synthesis of peptides and proteins.2 These aminolysis-
mediated peptide condensation methods are often limited to
condense the peptides at the C-terminal glycine and proline
sites, due to the proneness to epimerization with other amino
acids. Recently, our laboratory has developed a chemoselective
Ser/Thr ligation (STL),3 in which one side chain unprotected
peptide segment with a C-terminal salicylaldehyde (SAL) ester
reacts with another side chain unprotected peptide segment
with N-terminal serine or threonine to form an N,O-benzyli-
dene acetal intermediate, followed by acidolysis to reveal the
natural Xaa-Ser/Thr linkage (Fig. 1). This method has been
successfully applied in protein synthesis, protein semi-syn-
thesis and cyclic peptide synthesis.3–5
Fig. 1 Peptide (thiol) salicylaldehyde ester-mediated reactions.
under the Ser/Thr ligation conditions and is the subject of this
communication. From the point of physical organic chemistry,
the thioester is a better leaving group than the O-ester counter-
part, thus we expect that a 1,5 S to N acyl transfer would be
more feasible than a 1,5 O to N acyl transfer (Fig. 1). To
this end, we first tested this idea by using Fmoc-Ala thiol-
SAL ester to react with a serine derivative. These two com-
ponents were dissolved in pyridine acetate buffer (AcOH : Pyr
1 : 1, mol : mol), and indeed the expected N,O-benzylidene
acetal product was obtained, which upon acidolysis gave rise
to Fmoc-Ala-Ser-Phe-OMe tripeptide, as analyzed by LC-MS
(Fig. 2a). However, when a heptapeptide (Ac-SPKMVQG thiol-
SAL ester) was reacted with a hexapeptide (NH2-SFAVGA-CO2H)
with N-terminal Ser under the same conditions as above, we
were surprised to see that no ligated product was observed and
A logical inquiry along the line of STL chemistry is to use
peptide thiol-salicylaldehyde (2-formylthiophenol) esters
aDepartment of Chemistry, State Key Laboratory of Synthetic Chemistry,
The University of Hong Kong, China. E-mail: xuechenl@hku.hk
bChong Yuet Ming Chemistry Building, Pokfulam Road, Hong Kong, China
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c5ob00825e
‡Authors share equal contributions.
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Fig. 3 RP-HPLC profile of epimerization determination between the
ligation reaction of Ac-FVGFSDTYGA thiol-SAL ester (10)/Ac-
FVGFSDTYGa thiol-SAL ester (11) with NH2-AVYAAPYLAGG-CO2H
(12). A. Co-injection of ligated products. B. Ligation products from using
C-terminal L-Ala thiol-SAL ester and C. Ligation products from using
C-terminal D-Ala thiol-SAL ester.
Fig. 2 Thiol-SAL ester mediated reactions.
Next, we evaluated the epimerization issue under these con-
ditions. Peptide 10 (Ac-FVGFSDTYGA thiol-SAL ester) and
peptide 11 (Ac-FVGFSDTYGa thiol-SAL ester) were synthesized
using the epimerization-free peptide hydrazine displacement
approach.8 These two peptide thiol SAL esters were then
reacted with peptide 12 (H2N-AVYAAPYLAGG-CO2H), respect-
ively. The ligation could go to completion within few hours.
However, we observed the significant epimerized product,
which was proved by comparing the retention time of C-term-
inal L- and D-alanine peptides (Fig. 3). Thus, like other amino-
lysis-mediated peptide condensation,2a,c this method will be
limited to condense the peptide segment at the site with the
C-terminal glycine or proline residue. In addition, this method
will not tolerate the unprotected lysine and cysteine residues
in the peptide sequence.
To further probe the optimal conditions, Ac-AFQIG thiol-
SAL ester (13) was allowed to react with NH2-GLVYA-CO2H (14)
under different conditions at a substrate concentration of
1 mM. Under neutral or basic aqueous solution, the hydrolysis
product predominated (entries 2 and 3, Table 1).9 Organic sol-
vents (DMSO, DMF and 1-methyl-2-pyrrolidone) were more
suitable. An increase of the amount of DIEA to 10 equiv.
caused side reactions. In addition to DIEA, other bases includ-
ing DBU and DEA were found to be unsuitable. Therefore, the
the starting material remained intact as analyzed by LC-MS
(Fig. 2b). We have varied the peptide sequences for the ligation
study, none of which gave the ligated product. The underlying
reason is unclear to us yet.
In the 1970s, Kemp had reported that aminolysis of
8-acetoxy-1-naphthaldehyde or 2-acetoxybenzaldehyde with
primary amines generated amides. Under the conditions, an
initial hemiaminal formation took place, followed by intra-
molecular 1,5 O to N acyl transfer to generate the amide.6
Following this strategy, Ito and coworkers utilized 2-formyl-
4-nitrothiophenol esters to facilitate the amide formation
by hemiaminal-mediated acyl transfer.7 These results were
very promising, however, all these studies were limited to
simple substrates (Scheme 1). These precedent examples
prompted us to explore the utility of peptide thiol SAL esters
for peptide condensation via hemiaminal formation-mediated
aminolysis.
Initially, when peptide 7 (Ac-VGDTYA thiol-SAL ester) and
peptide
8 (NH2-VEGFA-CO2H) were dissolved in DMSO
(10 mM) with 2 equiv. of DIEA, a peptide condensation was
indeed observed to give 9 within 6 h (Fig. 2c).6 As a com-
parison, Agrigento et al. have used peptides with C-terminal
p-chloro or p-nitro thiophenyl ester for the peptide aminolysis
(DIEA, DMSO), in which the reaction needed 20–96 h at the
substrate concentration of 60 mM.2f Thus, the hemiaminal for-
mation-mediated reaction helped expedite the aminolysis.
Table 1 Optimization of the reaction conditions
Entry
Solvent
Base
Conversion
1
2
3
4
5
6
7
8
PBS (pH 6)
PBS (pH 7.4)
PBS (pH 8.5)
DMSO
DMSO
DMSO
DMSO–NMM
DMSO
DMSO
DMSO
DMF
—
—
—
<5%
<5%
<5%
69%
67%
91%
90%
90%
<5%
<5%
82%
80%
38%
77%
22%
10 eq. DIEA
10 eq. Imid.
3 eq. DIEA
3 eq. DIEA
1 eq. DIEA
3 eq. DBU
3 eq. DEA
1 eq. DIEA
3 eq. DIEA
10 eq. DIEA
3 eq. DIEA
3 eq. DIEA
9
10
11
12
13
14
15
DMF
DMF
Scheme 1 Previous examples using a hemiaminal-mediated acyl trans-
fer strategy.
1-Methyl-2-pyrrolidone
50% DMSO/H2O
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optimal condition is to use 1–3 equiv. of DIEA in DMSO.
Under the same condition, a peptide thiol SAL ester (15)
reacted much faster than its peptide thiophenyl ester counter-
part (17) (Fig. 4).
As the C-terminal residue was limited to the glycine or
proline residue, we next explored the scope and limitations on
the N-terminal residues. The synthesis of peptides containing
C-terminal Gly/Pro thiol SAL esters was straightforward using
the direct coupling conditions. We selected to use the peptide
thiol-SAL ester of Ac-ITGEFNAX (X = Gly or Pro) (18a–b) as the
N-terminal peptide and the peptide NH2-ZGVFA-CO2H (Z =
amino acid) (19a–j) as the C-terminal peptide. The two peptide
segments were dissolved at 1 mM concentration in DMSO with
3 equiv. DIEA. The reaction progress was analyzed by LC-MS at
4 h and 8 h. As seen in Table 2, the reactions with the peptide
C-terminal Gly thiol-SAL ester could be completed or nearly
completed within 4 h, while the peptide C-terminal Pro thiol-
SAL ester required 8 h. We also observed a small amount of
the imine product for the peptide C-terminal proline thiol-SAL
ester, which was consistent with Kemp’s observation that
certain substrates stopped at the imine stage without inducing
acyl transfer.
Fig. 4 Comparison of the aminolysis rate between a peptide thiol SAL
ester (15) and peptide thiophenyl ester (17). Reaction A was performed
for 5 h at 10 mM while B for 20 h at 10 mM.
Table 2 Examples of thiol SAL-mediated aminolysis of C-terminal Gly
and Pro thiol-SAL ester with different N-terminal amino acids
To demonstrate the effectiveness of this method, we next
applied it to the synthesis of the natural cyclic peptides. Many
natural cyclic peptides exhibit broad biological activities,
including anti-cancer, anti-bacteria, and anti-virus; thus cyclic
peptides serve as a useful scaffold for the development of new
8 h therapeutic agents.10 As such, the development of different
Conversion
Entry
X
Z
Product
4 h
methods for the synthesis of natural and unnatural cyclic pep-
1a
2a
3a
4a
5a
6b
7b
8b
9b
10b
Gly
Gly
Gly
Gly
Gly
Pro
Pro
Pro
Pro
Pro
Asp
Glu
Pro
Ser
Gly
Asp
Glu
Pro
Ala
Gly
12a
12b
12c
12d
12e
12f
12g
12h
12i
77
80
75
82
85
65
60
61
79
90
78
tides is attracting much attention.11
89
We selected several natural cyclic peptides with rings made
of 7 to 11 amino acid residues, containing Gly or Pro in the
sequence. These peptides include phakellistatin-13,12 cyclo-
squamosin D,13 Dichotomin G,14 antamanide15 and Stelladelin
D.16 The cyclization was performed at Pro–Phe, Gly–Gly, Pro–
Leu, Pro–Leu, Pro–Ala and Gly–Gly sites, respectively, via the
thiol-SAL ester-mediated peptide cyclization. The cyclizations
were performed at 1 mM in DMSO, which proceeded smoothly
with completion in 4–8 h. The monocyclic products were
obtained as major or sole products (Table 3, Fig. 5). Unfortu-
nately, highly constrained tetrapeptides could not be cyclized
using this method, in which only the dimerized cyclic pro-
ducts were observed.
77
82
85
83
90
71
85
98
12j
a Reaction conditions: peptide thiol-SAL ester (18a–b) (1 equiv.),
C-terminal peptides (19a–j) (1.2–1.3 equiv.), DIEA (3.0 equiv.), DMSO
at a final conc. of 1 mM, r.t.; conversion was calculated by the ratio of
the product area over the product and the hydrolyzed peptide.
b Reaction conditions: peptide thiol-SAL ester (18a–b) (1.5 equiv.),
C-terminal peptides (19a–j) (1 equiv.), DIEA (3.0 equiv.), DMSO at a
final conc. of 1 mM, r.t.; conversion was calculated by the ratio of the
product area over the product and the remaining C-terminal peptide.
Table 3 Synthesis of natural cyclic peptides via peptide thiol-SAL ester-mediated cyclization
Entry
Name
Sequence
Ring size
M : D
Yielda
1
2
3
4
5
Phakellistatin-13
Cyclosquamosin D
Dichotomin G
Antamanide
Cyclo-[FGPTLWP]
7
8
9
10
11
8 : 1
99 : 1
9 : 1
99 : 1
99 : 1
41%
49%
50%
48%
45%
Cyclo-[GVVSYYPG]
Cyclo-[LPSTFPPIP]
Cyclo-[AFFPPFFVPP]
Cyclo-[VPSPYFPAAIG]
Stelladelin D
a Isolated by HPLC. M: monocyclic product. D: dimerized product.
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Notes and references
1 (a) V. R. Pattabiraman and J. W. Bode, Nature, 2011, 480,
471–479; (b) C. P. R. Hackenberger and D. Schwarzer,
Angew. Chem., Int. Ed., 2008, 47, 10030–10047; (c) L. Raibaut,
N. Ollivier and O. Melnyk, Chem. Soc. Rev., 2012, 41, 7001–
7015; (d) C. T. T. Wong, C. L. Tung and X. Li, Mol. BioSyst.,
2013, 9, 826–833; (e) P. E. Dawson, T. W. Muir, I. Clark-Lewis
and S. B. H. Kent, Science, 1994, 266, 776–779.
2 (a) S. Aimoto, Curr. Org. Chem., 2001, 5, 45–87;
(b) R. J. Payne, S. Ficht, W. A. Greenberg and C. H. Wong,
Angew. Chem., Int. Ed., 2008, 47, 4411–4415; (c) G. Chen,
Q. Wan, Z. Tan, C. Kan, Z. Hua, K. Ranganathan and
S. J. Danishefsky, Angew. Chem., Int. Ed., 2007, 46, 7383–
7387; (d) D. Crich and I. Sharma, Angew. Chem., Int. Ed.,
2009, 48, 2355–2358; (e) B. L. Wilkinson, C. K. Chun and
R. J. Payne, Biopolymers, 2011, 96, 137–146; (f) P. Agrigento,
F. Albericio, S. Chamoin, I. Dacquigneis, H. Koc and
M. Eberle, Org. Lett., 2014, 16, 3922–3925; (g) S. Popovic,
H. Bieräugel, R. J. Detz, A. M. Kluwer, J. A. A. Koole,
D. E. Streefkerk, H. Hiemstra and J. H. van Maarseveen,
Chem. – Eur. J., 2013, 19, 16934–16937; (h) H. Hojo,
Y. Murasawa, H. Katayama, T. Ohira, Y. Nakahara and
Y. Nakahara, Org. Biomol. Chem., 2008, 6, 1808–1813.
3 (a) X. Li, H. Y. Lam, Y. Zhang and C. K. Chan, Org. Lett.,
2010, 12, 1724–1727; (b) Y. Zhang, C. Xu, H. Y. Lam,
C. L. Lee and X. Li, Proc. Natl. Acad. Sci. U. S. A., 2013, 110,
6657–6662; (c) C. T. Wong, T. Li, H. Y. Lam, Y. Zhang and
X. Li, Front. Chem. Biol., 2014, 2, 28.
4 (a) C. L. Lee and X. Li, Curr. Opin. Chem. Biol., 2014, 22,
108–114; (b) H. Y. Lam, Y. Zhang, H. Liu, J. Xu, C. T. Wong,
C. Xu and X. Li, J. Am. Chem. Soc., 2013, 135, 6272–6279;
(c) C. T. T. Wong, H. Y. Lam, T. Song, G. Chen and X. Li,
Angew. Chem., Int. Ed., 2013, 52, 10212–10215; (d) C. Xu,
H. Y. Lam, Y. Zhang and X. Li, Chem. Commun., 2013, 49,
6200–6202; (e) Y. Zhang, T. Li and X. Li, Org. Biomol. Chem.,
2013, 11, 5584–5587.
Fig. 5 RP-HPLC profiles of the crude reaction mixture of the thiol SAL-
mediated cyclization. * = partial removal of Trp (Boc) protecting group.
Conclusions
5 (a) P. M. Levine, T. W. Craven, R. Bonneau and
K. Kirshenbaum, Org. Lett., 2014, 16, 512–515; (b) P. M. Levine,
T. W. Craven, R. Bonneau and K. Kirshenbaum, Chem.
Commun., 2014, 50, 6909–6912; (c) P. M. Levine, T. W. Craven,
R. Bonneau and K. Kirshenbaum, Org. Biomol. Chem., 2013,
11, 4142–4146.
6 D. S. Kemp and F. Vellaccio Jr., J. Org. Chem., 1975, 40,
3003–3004.
7 A. Ishiwata, T. Ichiyanagi, M. Takatani and Y. Ito, Tetra-
hedron Lett., 2003, 44, 3187–3190. In this study, the substrate
was limited to the Fmoc-Val-SAr.
In conclusion, the peptide thiol-SAL ester surprisingly could
not participate in Ser/Thr ligation, but could undergo a direct
aminolysis, likely through a hemiaminal-mediated acyl trans-
fer.17 This thiol-SAL ester mediated aminolysis method can
serve as an alternative approach for the convergent synthesis
of peptides and cyclic peptides. We have demonstrated the
effectiveness of this method in the synthesis of several natural
cyclic peptides. The application of this method towards
protein chemical synthesis is ongoing.
8 G. M. Fang, Y. M. Li, F. Shan, Y. C. Huang, J. B. Li, Y. Lin,
H. K. Cui and L. Liu, Angew. Chem., Int. Ed., 2011, 50, 7645–
7649.
Acknowledgements
This work was supported by the National Basic Research
Program of China (973 program, 2013CB836900), the General
9 Different from the result of ref. 7, in which aqueous buffers
could be used.
Research Fund of the Research Grants Council of Hong Kong 10 (a) A. T. Bockus, C. M. McEwen and R. S. Lokey, Curr. Top.
(HKU702813P), AoE/M-12/06 and the Peacock Program-Project
Development Fund (KQC201109050074A).
Med. Chem., 2013, 13, 821–836; (b) S. H. Joo, Biomol. Ther.,
2012, 20, 19–26.
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11 (a) C. J. White and A. K. Yudin, Nat. Chem., 2011, 3, 509– 14 H. Morita, A. Shishido, T. Kayashita, K. Takeya and
524; (b) J. P. Tam and C. T. Wong, J. Biol. Chem., 2012, 287,
27020–27025.
12 W. L. Li, Y. H. Yi, H. M. Wu, Q. Z. Xu, H. F. Tang,
H. C. Itokawa, J. Nat. Prod., 1997, 60, 404–407.
15 I. L. Karle, T. Wieland, D. Schermer and H. C. J. Ottenheym,
Proc. Natl. Acad. Sci. U. S. A., 1979, 76, 1532–1536.
D. Z. Zhou, H. W. Lin and Z. H. Wang, J. Nat. Prod., 2003, 16 Y. R. Zhou, J. Zhou, X. K. Wang, X. L. Huang, H. M. Wu
66, 146–148. and X. Cheng, Chin. Chem. Lett., 1996, 7, 237–240.
13 A. Dellai, I. Maricic, V. Kumar, S. Arutyunyan, A. Bouraoui 17 We do not have direct evidence for the formation of a
and A. Nefzi, Bioorg. Med. Chem. Lett., 2010, 20, 6020–6023. hemiaminal.
6926 | Org. Biomol. Chem., 2015, 13, 6922–6926
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