10.1002/anie.201810712
Angewandte Chemie International Edition
COMMUNICATION
Peptide ligations using aryloxycarbonyl-o-methylaminoanilides:
chemical synthesis of palmitoylated-Sonic Hedgehog.
Judith Palà-Pujadas, Fernando Albericio, Juan B. Blanco-Canosa*,+
Abstract: A simple procedure for C-terminal activation of peptides in
solution and its application in native chemical ligation and protein
synthesis is described. This method involves a mild thioesterification
based on the conversion of an aryloxy-o-methylaminoanilide to
thioester under aqueous conditions and in situ ligation with an N-
terminal cysteine peptide. The versatility is shown in pH-controlled
sequential ligations. To illustrate the usefulness of this methodology,
we synthesized the palmitoylated N-terminal domain of human Sonic
Hedgehog, a morphogen protein that binds the transmembrane
receptor Patched and activates the Hedgehog signaling pathway,
involved in embryonic development and in the proliferation of
multiple tumors. This approach extends the chemical toolset of
chemical protein synthesis based on o-aminoanilide and o-
methylaminoanilide peptides.
aminoanilides,[11] have been disclosed. These masked-thioesters
can be activated in solution enabling the transformation of
inactive precursors into thioesters through transthioesterification
of S or N-acyl peptides.[3] The use of thioester precursors allows
a peptide featuring a free N-terminal Cys and a C-terminal
masked-thioester to ligate with a second peptide-thioester faster
than through the intramolecular cyclization or oligomerization.
Conceptually, it resembles the idea of kinetically controlled
ligation (KCL), which was initially reported on the basis of the
different reactivities of thioalkyl and thioarylesters.[12] Overall,
these approaches depict the interest in the development of novel
and mild methods for the selective generation of thioesters.
3,4-diaminobenzoyl derivatives, either the first generation
3,4-diaminobenzoic (Dbz)[6] or the second 3-amino-4-
(methylamino)benzoic (MeDbz)[7] that is more selective and
avoids overacylation, have been developed as thioester
precursors for NCL. After peptide elongation, the Dbz and
MeDbz linkers are acylated on solid phase with p-nitrophenyl
chloroformate, followed by base-induced intramolecular
cyclization rendering Nbz and MeNbz peptides. Following
cleavage from the solid support, the peptide can undergo
thiolysis to generate the desired peptide thioester for NCL
(Scheme 1, route A).[6,7] Alternatively, unprotected peptide o-
aminoanilides (1) can be directly oxidized at low pH via sodium
nitrite to N-acylbenzotriazoles, which are strong acylating
species and render thioesters in the presence of thiols (Scheme
1, route B).[11] On the contrary, the MeDbz is not able to yield
The chemical synthesis of proteins requires the use of
chemoselective reactions for assembling the distinct peptide
segments.[1] Mainly, native chemical ligation (NCL)[2] ligates
unprotected C-terminal thioester with N-terminal cysteine
peptides, thereby streamlining access to proteins containing
proteogenic or non-proteogenic amino acids and post-
translational modifications (PTMs) in high homogeneity and
purity. Significantly, the irruption in the last years of new
synthetic avenues based on sequential ligations from the N
towards the C direction has facilitated chemical protein synthesis
(CPS) in convergent strategies where in the final step are
assembled two fragments synthesized using both N-to-C and C-
to-N approaches.[3]
benzotriazoles.
Herein,
we
show
that
C-terminal
aryloxycarbonyl-o-methylaminoanilide peptides (2) can also be
transformed into the corresponding MeNbz derivatives in
aqueous solution under mild conditions (pH 7.4-7.6, temperature
around 25-37 °C), which following the thiol capture provide
thioester-peptides for NCL and KCL (Scheme 1, route C).
Importantly, the reactivity is controlled by variation of pH and/or
temperature and the activation/NCL is compatible with the
presence of arylthiols and phosphines, enabling one-pot
sequential reactions.
The preparation of peptide-thioesters using conventional
Fmoc solid-phase peptide synthesis (Fmoc-SPPS) is an area of
active research in CPS. Apart from the use of HOBt-esters,[4] N-
methylsulfonylamides[5] or N-acylbenzimidazolinones (Nbz),[6,7]
other methods based on masked precursors, namely peptide-
hydrazides,[8] SEA[9] and SEAlide[10] peptides and peptide o-
[*]
J. Palà-Pujadas, Prof. Dr. F. Albericio, Dr. J. B. Blanco-Canosa[+]
Institute for Research in Biomedicine (IRB Barcelona)
The Barcelona Institute of Science and Technology (BIST)
Baldiri Reixac 10, 08028 Barcelona (Spain)
Initial studies with p-nitrophenyloxycarbonyl MeDbz (pNO2-
Phoc-MeDbz) peptides, obtained through acidolytic cleavage of
the corresponding peptide-bound Rink resins, indicated that the
pNO2-Phoc-MeDbz cyclized to MeNbz with concomitant trapping
of the peptide by 4-mercaptophenol (MPOH), forming a peptide-
thioester (Figure S1). Unfortunately, during this process the
pNO2-Phoc-MeDbz was partially hydrolyzed (up to 25%) to
MeDbz, leading to unproductive peptides for NCL. We surmised
that this hydrolysis was enhanced by the high acidic character of
the pNO2-phenolate and that, in principle, a poorer leaving group
would eliminate this side-reaction.[13] After a short screening, it
was found that p-cyanophenyloxycarbonyl MeDbz (pCN-Phoc-
MeDbz) led to an efficient conversion with no hydrolysis (Figure
S2).
Prof. Dr. F. Albericio
CIBER-BBN, Networking Centre on Bioengineering, Biomaterials
and Nanomedicine
Baldiri Reixac 10, 08028 Barcelona (Spain)
Department of Organic Chemistry
University of Barcelona, 08028 Barcelona (Spain)
School of Chemistry and Physics, University of KwaZulu-Natal
University Road, Westville, Durban 4001 (South Africa)
Present address: Spanish National Research Council (CSIC),
Institute of Advanced Chemistry of Catalonia (IQAC)
Department of Biological Chemistry
[+]
Jordi Girona 18-26, 08034 Barcelona (Spain)
Supporting information for this article is available on the WWW
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