D
Synlett
M. C. Pirrung, N. A. Bakas
Letter
7
tein. Further, peptides derived from the N-terminal 10–15
tial for oxidation of the homoserine at the ligation junction
to a native aspartic acid residue. Threonine (and serine)
peptide ligation technologies are also known, but typically
for unprotected peptides in dilute denaturing aqueous me-
dia. Combined with past work on serine peptide assembly
(SPA),1 hydroxylated amino acid assembly reactions are
now applicable to ca. 18% of the residue positions among
known protein sequences. This feature should facilitate
their use in assembly of a broad range of targets.
residues of LTNF retain much of its activity; LTNF-10 has
the sequence LKAMDPTPPL. Biotechnology has been used
to produce LTNF-derived peptides, but we viewed LTNF-10
as an excellent proving ground for chemical synthesis using
threonine peptide assembly. It involves a challenging junc-
tion with a C-terminal proline that is hindered and poorly
reactive in native chemical ligation but has good perfor-
mance in assembly.
8
9
11
LTNF-10 was prepared from two subpeptides, LKAMDP
and TPPL. Using Fmoc SPPS on 2-chlorotrityl resin, these
two fragments were synthesized and freed from the sup-
port, the former in side-chain- and N-terminus-protected
form (Scheme 6). The N-terminal fragment was converted
into HIP ester 11 using a carbodiimide coupling. While we
typically prefer to form such fluorinated esters using car-
boxylate O-alkylation with a fluorinated triflate owing
to the inability of that step to trigger racemization of the
C-terminus, racemization is not an issue here – proline
is widely recognized as a racemization-resistant residue.
The C-terminal fragment was converted into ester 12 by
O-alkylation with benzyl bromide and Fmoc removal.
Our long-term goal for hydroxylated amino acid assem-
bly is the preparative production of peptide therapeutics
via segment condensations. Segments can be prepared by
SPPS, which is more reliable with relatively short subse-
3
quences, or other technologies under development. The
manufacture of the 36 amino acid HIV drug enfuvirtide
(Fuzeon®) by Trimeris/Roche is the pinnacle achievement
of synthetic production of therapeutic peptides and uses
12
SPPS methods with following segment condensations. Es-
tablishing methods for threonine assembly makes its appli-
cation to LTNF-10 pertinent even though it could also be
made efficiently by conventional SPPS.
The utility of threonine peptide assembly was demon-
strated in the preparation of a useful target with a particu-
larly challenging Pro-Thr junction. As peptide assembly
technology matures, such demanding junctions should be-
come more synthetically tractable. As in our past work on
serine assembly, the sustainability of threonine and homo-
serine assembly is high owing to reagent-less coupling re-
actions at high segment concentrations.
t
Fmoc-Leu-Lys(Boc)-Ala-Met-Asp(O Bu)-Pro-OHIP +
11
DMF / cat. AcOH
ambient / 5 d
H2N-Thr-Pro-Pro-Leu-OBn
12
30%
t
Fmoc-Leu-Lys(Boc)-Ala-Met-Asp(O Bu)-Pro-Thr-Pro-Pro-Leu-OBn
13
H2 / Pd•C 4-methylpiperidine TFA / TES
LKAMDPTPPL 54%
(LTNF-10)
H2O
Funding Information
Scheme 6 Synthesis of LTNF-10 by threonine peptide assembly
This work was supported by a grant from the NSF (CHE 1362737). Dvsoin of
C
h
e
mstiry
C(
H
E
1
3
6
2
7
3
7)
The assembly of 11 and 12 was initially studied in THF.
Reactant solubility was poor at a 0.25 M concentration, but
reaction still proceeded, albeit giving only a 25% yield of 13
over 5 d at ambient temperature. This assembly did not re-
spond to microwave heating as others have, giving 13 in
only 10% yield after 5 h. The solvent was changed to DMF,
which we have earlier used when reactant solubility is an
issue. Here, full dissolution could be achieved at a 0.33 M
concentration, and reaction proceeded in a 30% yield over 5
d at ambient temperature. As in our past experience with
assemblies, the unreacted starting materials are readily re-
covered. A straightforward three-step process deprotects
the assembly product 13, giving LTNF-10 in 54% overall
yield, whose structure was verified by mass spectrometry
and purity was verified by HPLC.
Supporting Information
Supporting information for this article is available online at
https://doi.org/10.1055/s-0036-1589123.
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References and Notes
(1) Pirrung, M. C.; Schreihans, R. S. Eur. J. Org. Chem. 2016, 5633.
(
2) (a) Varfolomeev, S. D.; Uporov, I. V.; Fedorov, E. V. Biochemistry
(Mosc). 2002, 67, 1099. (b) Li, W.; Kinch, L. N.; Karplus, P. A.;
Grishin, N. V. Protein Sci. 2015, 24, 1075. (c) Agouridas, V.; El
Mahdi, O.; Cargoët, M.; Melnyk, O. Bioorg. Med. Chem. 2017, 25,
4938.
(3) Pirrung, M. C.; Zhang, F.; Ambadi, S.; Ibarra-Rivera, T. R. Eur. J.
Org. Chem. 2012, 4283.
This work reports peptide assembly with two addition-
al hydroxyl amino acids, threonine and homoserine, the lat-
ter enabling segment condensations to introduce aspartic
acid at the C-terminal side of assembly junctions. The gen-
eration of homoserine peptides in KAHA native chemical li-
gation has been reported by Bode.1 However, because that
process yields unprotected peptides, it offers limited poten-
(
4) (a) Pratt, R. C.; Lohmeijer, B. G. G.; Long, D. A.; Waymouth, R. M.;
Hedrick, J. L. J. Am. Chem. Soc. 2006, 128, 4556. (b) Kiesewetter,
M. K.; Scholten, M. D.; Kirn, N.; Weber, R. L.; Hedrick, J. L.;
Waymouth, R. M. J. Org. Chem. 2009, 74, 9490.
5) Ac-Ala-Thr-OMe (3)
(
0
Protocol A
Threonine methyl ester (1 equiv) was dissolved in THF (1 M)
©
Georg Thieme Verlag Stuttgart · New York — Synlett 2017, 28, A–E