10.1002/anie.201906486
Angewandte Chemie International Edition
COMMUNICATION
We also applied the threonine-forming monomer for the
synthesis of a domain of the muscle protein Titin. Human Titin
consists of about 320 individually folded protein domains, all of
which have slightly different amino acid sequences. We selected
one of these domains, TI I27, a β-sandwich protein consisting of
89 amino acid residues with no disulfide bridges present. [23-27]
To accommodate future work we mutated Cys47 and Cys63 to
Ser, and to avoid aspartimide formation we also mutated Asp51
to a Glu residue. Both the α-ketoacid segment 16 and the
hydroxylamine segment were accessible by standard SPPS
conditions. The attachment of the threonine monomer, resin
cleavage and Fmoc removal with N,N-diethylamine proceeded
well to give 17. These two segments were cleanly ligated in
NMP/H2O at room temperature for 6 hours to give the desired
product, which was isolated by preparative HPLC in 42%
isolated yield. The ligated product 18 was denatured in 6 M
Gdn/HCl (pH 4.4) and then dialyzed against PBS-buffer at 4 °C
to give the folded product (Scheme 6).
that this variant of the KAHA ligation will be particularly useful in
situations where the introduction of a homoserine residue is not
acceptable or ligations under milder conditions are required.
Experimental Section
Experimental, characterization details for all compounds and experiments
are given in the supporting information.
Acknowledgements
This work was supported by the Swiss National Science
Foundation (169451) and ETH Zürich. S. Shimura and R.
Hofmann are acknowledged for their help with protein folding
and enzymatic evaluation. We thank the MS, NMR and SMocc
services of the Laboratorium für Organische Chemie at ETH
Zürich for analyses and Haewon Song for helpful comments.
a) Sequence and marked fragments of Titin I27:
LIEVEKPLYG10 VEVFVGETAH20 FEIELSEPDV30 HGQWKLKGQP40 LTASPDSEII50
Keywords: ligation • hydroxylamines • amides • proteins •
peptides
EGGKKHILIL60 HNSQLGNleTGE70 VSFQAANAKS80 AANLKVKEL89
[1]
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b) Assembly of TI I27:
c) Ligation of 16 and 17:
CH3
Other amide-forming ligations include the traceless Staudinger and the
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S. S. Kulkarni, J. Sayers, B. Premdjee, R. J. Payne, Nature Reviews
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H3C
O
O
T = 6 h
H
N
TI I27 (43-89)
TI I27 (1-40)
18
NH2
H2N
N
H
N
H
OH
O
O
T = 1 h
16
16
17
CH3
T = 0 h
17
NMP/H2O (9:1),
5 mM, RT
42%
d) Purified TI I27 and
mass spectrum:
18
CH3
H3C
O
H
N
H
TI I27 (43-89)
N
TI I27 (1-40)
NH2
N
H
H2N
H
O
H3C
OH
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J. W. Bode, R. M. Fox, K. D. Baucom, Angew. Chem. Int. 2006, 45,
1248–1252.
18
[4]
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J. W. Bode, Acc. Chem. Res. 2017, 50, 2104-2115.
e) CD-spectrum of folded 18:
f) CD-spectrum of wild type TI I27:
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Scheme 6. a) Primary structure of Titin I27, b) Assembly of TI I27 by KAHA
ligation, c) Ligation traces for ligation of peptide segments 16 and 17, d) HPLC
trace and mass spectrum of synthetic TI I27. e) CD-spectrum of folded
synthetic TI I27 (18), f) CD-spectrum of wild type TI I27.
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In conclusion, we have developed a synthetic route to a
new hydroxylamine monomer that yields threonine upon KAHA
ligation. It affords a canonical amino acid residue at the ligation
site and, in contrast to 5-oxaproline, directly forms the amide
product. We applied it to the chemical synthesis of ubiquitin-
conjugating enzyme UbcH5a and Titin domain TI I27. We expect
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