Hydroxymethyl Salicylaldehyde Auxiliary for a Glycine-Dependent Amide-Forming Ligation

Article abstract of DOI:10.1021/acs.orglett.5b02350

A new amide-forming ligation that requires a glycine or a primary amine at the linkage site is described herein. The distinguishing feature of this ligation is its reliance on an O-hydroxymethyl salicylaldehyde ester at the C-terminus which allows, via an N,O-acetal intermediate, the formation of a native peptide bond.

Full text of DOI:10.1021/acs.orglett.5b02350

Letter
pubs.acs.org/OrgLett
Hydroxymethyl Salicylaldehyde Auxiliary for a Glycine-Dependent
Amide-Forming Ligation
Marianne Fouche,* Florence Masse,† and Hans-Jorg Roth*
́
̈
Global Discovery Chemistry, Novartis Institute for BioMedical Research, Basel CH-4002, Switzerland
S
* Supporting Information
ABSTRACT: A new amide-forming ligation that requires a
glycine or a primary amine at the linkage site is described
herein. The distinguishing feature of this ligation is its reliance
on an O-hydroxymethyl salicylaldehyde ester at the C-terminus
which allows, via an N,O-acetal intermediate, the formation of
a native peptide bond.
ith their unique biological properties and their ability to
Wtarget protein−protein interactions, peptides are receiv-
ing a great deal of interest within the pharmaceutical industry as
potential therapeutic agents.1,2 Naturally, this has led to a greater
focus on the development of new synthetic methodologies for
generating peptides and proteins of increasing size and complex-
ity. The most remarkable breakthrough in the field of peptide
synthesis was made by Merrifield in 1963, with the “solid-phase-
peptide-synthesis” (SPPS) concept.3 Today, SPPS still plays a
major role in the preparation of peptides, but this technique also
suffers from its extreme linearity, making the synthesis of long
peptides (40−50 amino acid residues) very difficult. Therefore, in
order to gain access to much larger peptides and proteins,
chemoselective ligation protocols were further developed
throughout the 1990s. The Native Chemical Ligation (NCL),
developed by Kent and co-workers, proved to be a very powerful
method for the synthesis of complex peptides and proteins,4,5 and
despitemanyotherrecentcontributions,6−10 NCLstillrepresents
the state-of-the-art technique in the field of peptide ligation.
However, a major drawback of NCL is its reliance on cysteine at
the ligation site. This is particularly problematic considering the
occurrence of cysteine in natural proteins is only 1.1%. Even
though desulfurization reactions could overcome this limita-
tion,11 thereisstillaneedfordiversificationofligationstrategiesin
order to meet the synthetic challenge for generating peptides and
proteins with increasing complexity.
More recently, Li and co-workers developed a new serine/
threonine dependent approach based on the functionalization of
peptides at their C-terminus with a salicylaldehyde auxiliary.12,13
This methodology, which is an extension of the work by Tam et
al.,14 caught our attention due to its selective linkage (serine and
threonine are abundant residues in proteins), leading to the
formation of a native peptide bond (Figure 1a).
Figure 1. C-Terminal auxiliary promoted ligations.
salicylaldehyde auxiliary mediated ligation,12,13 we decided to
develop a method that relies on the same concept. Thus, we
envisaged building a new auxiliary by transferring the CH2-OH
moiety from the N-terminal Ser/Thr side chain onto the C-
terminal salicylaldehyde. This new auxiliary should deliver a
ligation method independent from the presence of an N-terminal
Ser/Thr at the linkage site (Figure 1b). The ligation would follow
a similar mechanism with first, imine formation followed by
cyclization with the auxiliary hydroxyl group, and subsequent O-
to-N acyl shift via a 6-membered ring intermediate. Finally, the
N,O-acetal intermediate should be cleaved under acidic
We believed that the field of peptide research would benefit
from a new chemical ligation method which utilizes a different
linkage site from those that have already been reported in the
literature.4−10 Moreover, because several literature examples have
already demonstrated the robustness and generality of the
Received: June 10, 2015
© XXXX American Chemical Society
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DOI: 10.1021/acs.orglett.5b02350
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Scheme 1. Synthesis of Fmoc-Gly-Gly-OMe
Table 1. Scope and Limitations of the Ligation between
Monomers
conditions to afford the ligation product with a native peptide
bondatthelinkagesite. Herein, wedescribeoureffortstoward the
development of this auxiliary as a new tool for the synthesis of
peptides.
To validate our starting hypothesis, a ligation between two
glycine monomerswas investigated (Scheme1). Auxiliary115 was
coupled to Fmoc-Gly using HATU in DMF to give ester 2.
Oxidative cleavageof thePMB ether was carried outwith DDQ. It
is worth noting that without 2,6-di-tert-butylpyridine, partial
degradation of the product was observed.16 Our choice to install a
PMB was driven by the need to have a protecting group with
cleavage conditions compatible with those used with the more
common protecting groups found in peptide chemistry. NMR
analysisconfirmedtheformation ofthehemiacetal intermediate 4
after deprotection (Scheme 1a). Following the isolation of 4, a
ligation was attempted with Gly-OMe (5). A mixture of pyridine/
acetic acid (1:2) proved to be the best solvent system to promote
the formation of intermediate 6.17 The crude mixture was then
subjected to acidic treatment and the desired dipeptide 7 was
isolated in excellent yield over two steps (Scheme 1b).
In order to demonstrate the scope and limitations of this
method, sterically demanding amino acids were investigated
(Table 1). It is well established that bulky residues at the C-
terminal amino acids are a limiting factor for ligation reactions.18
Therefore, it was decided to test a range of α- and β-branched
amino esters, bearing the o-hydroxymethyl salicylaldehyde
auxiliary at their C-terminus, for ligation with Gly-OMe.19 Even
though β-branched amino acids, for instance, proline and valine,
significantly affected the rate of the ligation, all reactions (Table 1,
entries 1−6) showed high conversions, and after cleavage of the
auxiliary, the desired dipeptides were isolated in good yield and
excellent enantiomeric purity.20
Conditions: All reactions were performed at room temperature, 20
mM. Step 1: pyridine/acetic acid (1:2). Step 2: TFA (20 equiv) in
MeCN. The HCl salt form was used. Intermediate: conversion was
a
b
checked by LC−MS and was calculated on the basis of consumption of
c
AA-Aux(OH). Product: yield corresponds to the isolated product
d
after chromatography purification. The ee was obtained by chiral
HPLC by comparison between Fmoc-L-AA-Gly-OMe and Fmoc-D-
AA-Gly-OMe retention times.
Scheme 2. Chemoselectivity Studies between Gly and Phe/Ala
However, a limitation regarding N-terminal amino acids was
observed: when the ligation was carried out with Ala-OBz or Phe-
OBz (Table 1, entries 7 and 8), formation of the desired
intermediate was unsuccessful. Mild heating resulted mainly in
hydrolysis of the ester but also in its direct aminolysis. In order to
better understand this limitation and to check if the imine
intermediate was formed, the reaction mixture was treated with
the reducing agent NaCNBH3. This led to the formation of the
corresponding amine showing that the imine formation did occur
but that the following imine capture or migration failed due to the
presence of sterically demanding N-terminal amino acids.21 We
actually believe that the O-to-N acyl shift is responsible for
disrupting the ligation, as it proceeds through an energetically
unfavorable 6-membered ring intermediate. In this context, the
ligation was also tested with an N-terminal β-amino-acid and
proved to be successful (Table 1, entry 9), demonstrating again
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Table 2. Scope and Limitation of the Ligation Using
Unprotected Side-Chain Residues
Conditions: All reactions were performed at room temperature, 20
mM. Step 1: pyridine/acetic acid (1:2). Step 2: TFA (20 equiv) in
a
b
MeCN. The HCl salt form was used. Intermediate: conversion was
checked by LC−MS and was calculated on the basis of consumption of
c
peptide-Aux(OH). Product: yield corresponds to the isolated product
d
after chromatography purification. The de was obtained by chiral
e
HPLC. The ratio of regioisomers was determined by LC−MS.
f
Cleavage of the auxiliary was carried out in neat TFA and led to the
tBu-deprotected peptide.
that steric hindrance at the α-position is a key factor of this
method.
In order to showcase the difference in reactivity of glycine
versus alanine or phenylalanine at the N-terminus, a competition
experiment was carried out (Scheme 2). Although in the presence
of other potential ligation partners, only Gly-OMe readily reacted
with 4 to form the corresponding intermediate with 100%
conversion.22 With these experiments, the o-hydroxymethyl
salicylaldehyde auxiliary has demonstrated that it can offer a
new linkage site for the synthesis of a target peptide and also
provides a chemoselective method in a mixture of peptides.
Furthermore, even though a ligation reaction with N-terminal
serine was successful (Table 2, entry 1) the chemoselectivity of
this ligation toward Gly-OMe in the presence of Ser-OMe was
demonstrated inasimilar competitionexperiment whereby, upon
Figure 2. Epimerization determination between the ligation reaction of
Fmoc-L-Phe-Aux(OH)/Fmoc-D-Phe-Aux(OH) and Gly-Leu-Tyr-OH.
(A) Chiral HPLC profile of crude ligation products using Fmoc-L-Phe-
Aux(OH). (B) Chiral HPLC profile of crude ligation products using
Fmoc-D-Phe-Aux(OH). (C) Co-injection of crude ligation products.
completion, the formation of a majority of the glycine
intermediate 6 was observed (glycine intermediate/serine
intermediates 9:1).22
The synthesis of tri-, tetra-, penta-, and decapeptides from
unprotected or partially protected smaller units was then
Table 3. Scope and Limitation of the Ligation between Peptidic Fragments
a
b
c
d
entry
R1
R2 (equiv)
I (%) (time, h)
yield P (%) (de, %)
e
1
2
3
4
Boc-Phe-Phe-Gly
Gly-His-OH (2)
100 (18)
100 (48)
100 (60)
100 (60)
64
e
Boc-Phe-Phe-Gly
Gly-Ser-OH (2)
48 (100)
e
Boc-Phe-Phe-Gly
Boc-Phe-Thr(OtBu)-Phe
Gly-Ile-Ser-Thr-Pro-Val-Ile-Phe-OH (1.5)
Gly-Trp-OH (2)
66
f
46 (100)
a
Conditions: All reactions were performed at room temperature, 20 mM. Step 1: pyridine/acetic acid (1:2). Step 2: TFA (40 equiv) in MeCN. The
b
HCl salt form was used. Intermediate: conversion was checked by LC−MS and was calculated on the basis of consumption of peptide-Aux(OH).
c
d
e
Product: yield corresponds to the isolated product after chromatography purification. The de was obtained by chiral HPLC. Cleavage of the
f
auxiliary led to the Boc-deprotected peptide. Cleavage of the auxiliary was carried out in HCl in dioxane (4 M) and led to the fully deprotected
peptide.
C
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investigated (Tables 2 and 3). To our delight, peptide fragments
with unprotected serine, glutamine, arginine, histidine, and
tryptophan were tolerated. However, as expected, unprotected
lysine residue competed with the N-terminus of glycine and led to
the formation of two regioisomers (Table 2, entry 6). For reasons
that are unclear to us, the product corresponding to the ligation
with the N-terminal glycine proved to be the major one compared
to the product formed by the ligation with the amino group of the
lysine side-chain.
Tripeptides bearingthe C-terminal auxiliary werealsoprepared
and ligated successfully with various peptides. Boc- and tert-butyl-
protected peptides (Table 3) could be deprotected during the
cleavage of the auxiliary, giving rise to a straightforward synthesis
of fully deprotected peptides in good yields. It is noteworthy that
thesynthesis of adecapeptide (Table3, entry3) wasalso achieved
in good yield.
The reported peptides (Table 1, entries 1−5, and Table 2,
entries 1−5 and 7, and Table 3, entries 1−4) were isolated in
excellent diastereoisomeric purity.23 Epimerization was also
further investigated with the ligation between Fmoc-L-Phe-
Aux(OH) and Gly-Leu-Tyr-OH that was compared to the
ligation with Fmoc-D-Phe-Aux(OH) (Figure 2). During this
experiment, epimerization was not detected.24
In conclusion, a new amide-forming ligation has been reported.
This strategy, which requires a glycine or a primary amine at the
linkage site, enables the use of unprotected side-chain peptide
fragments (excepted lysine). At this stage, the auxiliary has to be
installed after resin cleavage but, as demonstrated for the
salicylaldehyde auxiliary, SPPS synthesis could be envisaged.25
In the near future, we aim to demonstrate the usefulness of this
method with the synthesis of longer and/or cyclic peptides.
REFERENCES
■
(1) (a) Sato, A.; Viswanathan, M.; Kent, R. B. Curr. Opin. Biotechnol.
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(10) For an aspartate-assisted ligation−desulfurization: Thompson, R.
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2013, 52, 9723.
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2014, 12, 3768. (b) Wong, C. T. T.; Lam, H. Y.; Song, T.; Chen, G.; Li, X.
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ASSOCIATED CONTENT
■
S
* Supporting Information
(14) (a) Liu, C.-F.; Tam, J. Proc. Natl. Acad. Sci. U. S. A. 1994, 91, 6584.
(b) Liu, C.-F.; Tam, J. P. J. Am. Chem. Soc. 1994, 116, 4149. (c) Tam, J. P.;
Miao, Z. J. Am. Chem. Soc. 1999, 121, 9013.
(15) The synthesis of auxiliary 1 is described in the Supporting
Information, section 2.1.
(16)Kim,H.M.;Kim,I. J.;Danishefsky, S.J. J.Am. Chem.Soc. 2001,123,
35.
(17)SeetheSupportingInformation, section2.4,forcharacterization of
the intermediate.
(18) Hackeng, T. M.; Griffin, J. F.; Dawson, P. E. Proc. Natl. Acad. Sci. U.
S. A. 1999, 96, 10068.
(19) All C-terminal 6-(hydroxymethyl)salicylaldehyde esters were
prepared according to Scheme 1. Chiral HPLC of Fmoc-Phe-
Aux(OPMB) proved that the auxiliary can be installed racemization
free; see the Supporting Information, section 2.
TheSupportingInformationisavailablefreeofchargeontheACS
Publications website at DOI: 10.1021/acs.orglett.5b02350.
Experimental details, spectral and analytical data, and 1H
NMR and 13C NMR spectra for new compounds (PDF)
AUTHOR INFORMATION
■
Corresponding Authors
*E-mail: marianne.fouche@novartis.com.
*E-mail: hans-joerg.roth@novartis.com.
Present Address
†Actelion Pharmaceuticals, Ltd., Allschwill CH-4123, Switzer-
land.
(20) The ee was obtained by chiral HPLC by comparison between
Fmoc-L-AA-GlyOMe and Fmoc-D-AA-GlyOMe retention times; see the
Supporting Information, section 2.4.
Notes
(21) The reactions were followed by LC−MS; see the Supporting
Information, section 3.
The authors declare no competing financial interest.
(22) The competition experiments were followed by LC−MS; see the
Supporting Information, section 4.
ACKNOWLEDGMENTS
■
(23) For all synthesized peptides (expect Table 2, entries 1 and 2, Table
3, entries 1 and 3), chiral HPLC was performed to determine the ee/de.
Otherwise (Table 2, entries 1 and 2, and Table 3, entries 1 and 3), NMR
analysis showed the presence of one diastereoisomer.
(24) See the Supporting Information, section 2.4.
We thank Eric Francotte, Thomas Wolf, and Christophe Bury
(GDC, NIBR, Basel) for purifications as well as Monique Kessler
and Dan Huynh (GDC, NIBR, Basel) for chiral analysis. We
acknowledge Regis Denay and Elodie Osmont (GDC, NIBR,
Basel) for their help with recording NMR and IR spectra and
Sylvie Chamoin and Darryl Jones (GDC, NIBR, Basel) for
proofreading.
(25) Wong, C. T. T.; Lam, H. Y.; Li, X. Org. Biomol. Chem. 2013, 11,
7616.
D
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