On-Resin Preparation of Allenamidyl Peptides: A Versatile Chemoselective Conjugation and Intramolecular Cyclisation Tool

Article abstract of DOI:10.1002/anie.202004656

The ability to modify peptides and proteins chemoselectively is of continued interest in medicinal chemistry, with peptide conjugation, lipidation, stapling, and disulfide engineering at the forefront of modern peptide chemistry. Herein we report a robust method for the on-resin preparation of allenamide-modified peptides, an unexplored functionality for peptides that provides a versatile chemical tool for chemoselective inter- or intramolecular bridging reactions with thiols. The bridging reaction is biocompatible, occurring spontaneously at pH 7.4 in catalyst-free aqueous media. By this “click” approach, a model peptide was successfully modified with a diverse range of alkyl and aryl thiols. Furthermore, this technique was demonstrated as a valuable tool to induce spontaneous intramolecular cyclisation by preparation of an oxytocin analogue, in which the native disulfide bridge was replaced with a vinyl sulfide moiety formed by thia-Michael addition of a cysteine thiol to the allenamide handle.

Full text of DOI:10.1002/anie.202004656

A Journal of the Gesellschaft Deutscher Chemiker
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Accepted Article
Title: On-resin Preparation of Allenamidyl Peptides: a Versatile
Chemoselective Conjugation and Intramolecular Cyclisation Tool
Authors: Margaret Anne Brimble, Alan J. Cameron, and Paul W. R.
Harris
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202004656
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10.1002/anie.202004656
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RESEARCH ARTICLE
On-resin Preparation of Allenamidyl Peptides: a Versatile
Chemoselective Conjugation and Intramolecular Cyclisation Tool
Alan J. Cameron*, Paul W. R. Harris and Margaret A. Brimble*
[a]
A. J. Cameron, P. W. R. Harris, M. A. Brimble
School of Chemical Sciences and School of Biological Sciences
The University of Auckland
23 Symonds St, Auckland,1142 (New Zealand)
E-mail: m.brimble@auckland.ac.nz
E-mail: alan.cameron@auckland.ac.nz
[b]
A. J. Cameron, P. W. R. Harris, M. A. Brimble
Maurice Wilkins Centre for Molecular Biodiscovery,
The University of Auckland, Auckland,1142 (NewZealand)
Supporting information for this article is given via a link at the end of the document
Abstract: The ability to modify peptides and proteins
chemoselectively is of continued interest in medicinal chemistry, with
peptide conjugation, lipidation, stapling and disulfide engineering at
the forefront of modern peptide chemistry. Herein we report a robust
method for the on-resin preparation of allenamide-modified peptides,
an unexplored functionality for peptides that provides a versatile
chemical tool for chemoselective inter- or intramolecular bridging
reactions with thiols. The bridging reaction is biocompatible, occurring
spontaneously at pH 7.4 in catalyst free aqueous media. Employing
this “click” approach, a model peptide was successfully modified with
a diverse range of alkyl and aryl thiols with excellent conversions.
Furthermore, this technique was demonstrated as a valuable tool to
induce spontaneous intramolecular cyclisation by preparation of an
oxytocin analogue where the native disulfide bridge was replaced by
a vinyl sulfide moiety that in turn was formed via thia-Michael addition
of a cysteine thiol to the allenamide handle.
notoriously difficult to couple or result in poor yields[3e, 4a, 4d, 5]. Our
search for a versatile chemical tool to access modified peptides
without the requirement for catalysis or exogenous linkers,
identified the allenamide functional group as an attractive
candidate given that its reported Michael addition with thiols
displayed biocompatibility, excellent kinetics, irreversibility and
met the requirements for a “click” reaction[6].
The allenamide functionality has recently gained interest in
the field of peptide chemistry after Loh and co-workers[6a] reported
it as a novel handle for chemoselective modification of thiols in
peptides or proteins (Scheme 1). Subsequently, Roush and co-
workers[7] expanded upon this, demonstrating the allenamide
moiety to be an excellent tool for the modification of
selenocysteine (Sec), a Cys isostere that has gained popularity in
both peptide chemistry and antibody engineering[2a, 8]. Using an
allenamide handle, Derda and co-workers were able to prepare a
photo-sensitive peptide macrocyclisation linker by employing a
bis-allenamide functionality[6b]. An allenamide has also been
utilised as an electrophilic warhead in the design of small
molecule irreversible kinase inhibitors[9] and allenamides were
found to react with thiols 7 to 28-fold faster than equivalent
acrylamides[9a]. The thiol-allenamide reaction kinetics are also
Introduction
With the rise of peptide-therapeutics[1], the ability to perform
chemoselective modification and conjugation of peptides is of
considerable importance in medicinal chemistry and drug
development. While a large variety of chemical tools have been
developed to meet these needs, there is on-going interest in the
development of novel chemical tools and “click” reactions with
broad applications[2]. Many of the current approaches for peptide
conjugation, such as disulfide bridge replacement and peptide
stapling rely on the use of exogenous reagents or catalysts that
greater than those of chloro- or iodoacetamides [6b]
.
Scheme 1. Modification of a model peptide (YDSQCFHRW) with C-substituted
terminal allenamides by Loh et al[6a]
2f, 3]
are often toxic or require additional purification steps.[2d,
.
Furthermore, many existing techniques lead to poorer solubility,
undesired mixtures of isomers or linkages that are reversible in
vivo due to the presence of glutathione (GSH) or serum albumin
[3e, 4]. Additionally, unlike catalytic reactions such as the Cu(I)-
catalysed azide–alkyne cycloaddition (CuAAC) or ring-closing
metathesis (RCM), halogenated bridging reagents and
maleimides (both targeting cysteine thiols) can also react with
other nucleophilic residues such as lysine and methionine,
potentially leading to complex product mixtures[3e, 4d]. Elegant on-
resin approaches have been developed, avoiding issues of
removing exogenous reagents or peptide complexation with metal
catalysts, however, they often require numerous on-resin
manipulations, complex building block synthesis and
incorporation of hindered α,α-dialkyl amino acids that are
Inspired by these reports, we postulated that incorporation
of an allenamide functionality to a peptide would provide a
versatile tool for the facile preparation of modified peptides,
requiring only simple one-step purifications. We therefore initially
sought to develop methodology to introduce the allenamide
functionality to a peptide on-resin. Establishing this methodology
posed a significant challenge as although the allenamide group
has been shown to react chemoselectively with thiols as an
exogenous linker, its incorporation as a reactive functional group
within
a peptide sequence hitherto remained unexplored.
Furthermore, on-resin peptide synthesis requires high conversion
rates with minimal by-products to avoid inseparable product
mixtures and acidic resin-cleavage poses a further challenge from
exposure to the several highly reactive species formed. Herein we
1
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10.1002/anie.202004656
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amino acid, PG = TFA labile protecting group, Dap = 2,3-diaminopropionic acid,
Dde = 1-(4,4-dimethyl-2,6-dioxacyclohexylidene)ethyl.
report the development of a synthetic strategy in which an
allenamide functionality can be successfully introduced to a
peptide on-resin, thus creating a synthetic toolkit for a diverse
range of peptide modifications using inter- or intra-molecular thia-
Michael additions. Adopting this robust new synthetic
methodology, we further demonstrate its use as a versatile “click”
chemistry platform to introduce peptide modifications with alkyl or
aryl thiols and to effect intramolecular cyclisation by substituting
the disulfide bridge of oxytocin with a vinyl sulfide linkage.
Initial Attempted Synthesis of Allenamidyl Peptides
Our initial efforts focused on introducing an allenamide moiety to
the free amino sidechain of the resin-bound oxytocin model
peptide 2*, which was obtained by SPPS in 92% purity as
determined by resin mini-cleavage (Scheme 3). Parent peptidyl
resin 2* was then acylated with 3-butynoic acid (4 equiv.) by
adapting the solution phase procedure from Loh and co-
workers[6a] using the Mukaiyama reagent (6 equiv.) for 2 h with
dropwise addition of Et3N (6 equiv.). Upon mini-cleavage of the
resulting peptidyl resin 3*, RP-HPLC showed the desired peptide,
albeit in poor conversion, along with an inseparable complex
mixture of by-products (Scheme 3c). Coupling as the symmetric
anhydride (with DIC) afforded little improvement and trialling
numerous resin cleavage conditions (data not shown) failed to
improve the outcome. It was clear that efforts to optimise the
coupling step were therefore necessary.
Results and Discussion
Synthetic strategy
Initially we sought to prepare the modified 2,3-diaminopropionic
acid (Dap) residue 1 (Scheme 2a) as an allenamide containing
building block and investigate methodology for its incorporation
during Fmoc solid-phase peptide synthesis (Fmoc-SPPS). In
recent literature, preparation of terminal allenamides by coupling
of 3-butynoic acid has generally been effected with 2-chloro-1-
methylpyridinium iodide (Mukaiyama reagent)[6a, 7, 9a]. Couplings
in SPPS are instead typically performed with uronium reagents
derived from hydroxybenzotriazoles such as 1-hydroxy-7-
azabenzotriazole (HOAt). Unfortunately in our hands, significant
by-product formation was observed during preliminary attempts to
couple 3-butynoic acid using hydroxybenzotriazoles or other N-
hydroxyl species, even under base free conditions (Figure S12-
13). We reasoned these side reactions would preclude using
standard SPPS methods for peptide elongation after installing the
allenamide, leading us to abandon this strategy (Scheme 2a). A
new strategy was envisioned whereby late-stage introduction of
the allenamide to the resin-bound peptide is effected after
complete sequence assembly, enabled by orthogonal Dap β-
amino deprotection and subsequent coupling of 3-butynoic acid
(Scheme 2b). The reactive allenamide group is thus only exposed
to the reaction conditions of its own coupling step and the final
peptide cleavage procedure before the desired thia-Michael
addition.
Scheme 3. a) Synthetic procedure for initial attempt of coupling 3-butynoic acid
on-resin with Mukaiyama reagent and Et3N by adaption of the solution phase
procedure from Loh et al[6a]; b) RP-HPLC chromatogram (214 nm) of product
mixture upon resin mini- cleavage of 2*; c) RP-HPLC chromatogram (214 nm)
of product mixture upon resin mini-cleavage of 3*. Resin mini-cleavage was
effected with TFA/TIPS/H2O (95:2.5:2.5 v/v) for 2 h, 2 and 3 = the corresponding
desired peptide (C-terminal amide) upon concomitant deprotection and
liberation from the respective resins 2* and 3*. Dap = 2,3-diaminopropionic acid.
Scheme 2. General strategies to access allenamide modified peptides on-resin
via Fmoc-SPPS. a) Initial strategy employing building block 1. Adducts
observed during coupling of 3-butynoic acid with hydroxybenzotriazoles-derived
reagents were expected to preclude further peptide elongation by Fmoc-SPPS
(red dashed arrow) due to repeated exposure to these species (Figure S12-13);
b) revised strategy for late-stage allenamide modification of a peptide using an
orthogonal deprotection followed by coupling of 3-butynoic acid on-resin. AA =
2
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Table 1: Attempted reaction conditions and conversion rates for coupling of 3-butynoic acid with H2N-Phe-OtBu.HCl.
Reaction conditionsa
Entry
% areab
Coupling reagent
solvent
Base
H2N-Phe-OtBu
Prod (4)
1c
2
Mukaiyama reagent
Mukaiyama reagent
Mukaiyama reagent
Mukaiyama reagent
Mukaiyama reagent
Mukaiyama reagent
Mukaiyama reagent
Mukaiyama reagent
Mukaiyama reagent
TFFH
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
DMF
CH2Cl2
NMP
CH2Cl2
CH2Cl2
DMF
Et3N
Et3N
Et3N
-
80.5%
46%
10%
50%
59%
73%
14%
24%
63%
59%
51%
51%
43%
6%
44%
49%
36.3%
54%
6%
50%
53%
92%
17%
21%
16%
16%
22%
49%
74%
29%
6%
-
7%
-
65%
32%
23%
-
3d
4
NMM
5
6
iPr2EtN
sym-collidine
NMM
7
8e
9
-
sym-collidine
iPr2EtN
sym-collidine
iPr2EtN
sym-collidine
NMM
Et3N
NMM
sym-collidine
sym-collidine
NMM
10
12
13
14
15
16
17
18
19
20
21
22
23f
TFFH
TFFH
TFFH
TFFH
DIC
DIC
DIC
EDC.HCl
DMTMM
EEDQ
EEDQ
EEDQ
DMF
DMF
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
DMF
DMF
CH2Cl2
CH2Cl2
-
40%
5%
3%
4%
NMM
NMM
sym-collidine
a 3-butynoic acid (2 equiv.) dissolved in 400 μL of solvent with the specified base (2 equiv.) and transferred to coupling reagent (1.9 equiv.), after approx. 30 seconds
the solution was transferred to a 200 μL solution of H2N-Phe-OtBu.HCl (1 equiv.) which was pre-incubated with an additional 1 equiv. of base used to neutralise
the HCl salt. The reaction was shaken at r.t. for 18 h. The reaction progression was monitored by RP-HPLC (214 nm) at 2 h and 18 h, with reaction completion often
reached within 2 h. Reaction performed at 0.0292 mmol scale, relative to H2N-Phe-OtBu.HCl.
b % area determined at 214 nm and rounded to the nearest integer. Peaks noted if greater than 2% area. Peaks derived from coupling reagent by-products (unrelated
to starting materials) were ignored and identified from a Boc-Ala control reaction.
c Base and H2N-Phe-OtBu.HCl were prepared as a mixture in 400 μL of CH2Cl2 and then added dropwise to a mixture of 3-butynoic acid and the coupling reagent
(in CH2Cl2 200 μL), to replicate procedure of Loh et al[6a]
d Excess Et3N (8 equiv.) was added.
e 1 equiv. sym-collidine used to neutralise HCl salt of H2N-Phe-OtBu.HCl. No additional base was added.
f Reaction progress equivalent at 2 h and 18 h. Entry bolded to highlight as the optimised protocol, isolated yield = 83%.
Solution Phase Coupling Optimisation
reproducibility. Interestingly, reaction success varied significantly
with the choice of base and solvent, with the best results afforded
in CH2Cl2. Activation of 3-butynoic acid with Mukaiyama reagent
in CH2Cl2, employing Et3N (pKa: 10.8), or the weaker base N-
methylmorpholine (pKa: 7.4) both provided moderate conversions
to the desired allenamide 4 (46% and 50% respectively, Table 1,
entry 2 and 4). Substitution of the base for iPr2EtN (pKa: 10.8)
gave a modest improvement with 59% conversion (Table 1, entry
5). Interestingly, substituting the base for sym-collidine (pKa: 7.4)
provided a marked improvement with 73% conversion (Table 1,
entry 6), suggesting the influence of the base was independent of
the pKa. Furthermore, significant by-product formation was
observed for all three of the trialkylamine-bases, ranging 25-37%,
whilst less than 5% by-product formation was observed using
sym-collidine. Pleasingly, after substantial screening efforts, N-
To facilitate monitoring of the key allenamide coupling reaction we
designed a solution phase model to optimise the procedure
(Table 1), selecting phenylalanine tert-butyl ester (H2N-Phe-
OtBu.HCl) for the coupling partner as its functionalisation could
easily be monitored by RP-HPLC and it presented reasonable
steric hindrance. A variety of coupling reagents, bases and
solvents were trialled to effect coupling at room temperature (r.t.),
with conversions monitored by RP-HPLC (Table 1). We began
our optimisation studies with the Mukaiyama reagent, first under
conditions closely analogous to the procedure of Loh et al[6a], with
a mixture of Et3N and H2N-Phe-OtBu.HCl added dropwise to the
activated 3-butynoic acid. Despite dropwise addition of Et3N
outperforming the use of immediate or excess addition (Table 1,
entry 1-3), this procedure is tedious for on-resin chemistry and
performed poorly (vide supra, Scheme 3). Accordingly, our
further efforts aimed to develop a procedure without the
requirement for dropwise reagent additions, offering greater
ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline
(EEDQ)
was
identified as a superior reagent. In CH2Cl2 with sym-collidine,
EEDQ rapidly effected excellent conversion (92%) within 2 h
3
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(Table 1, entry 23). Employing this optimised procedure (Table 1,
entry 23), allenamide 4 was obtained in 83% isolated yield.
1
However, the H and 13C NMR spectra revealed the product to
exist as an approximately 8:2 mixture of allenic and
homopropargylic isomers respectively (Figure S3-4).
Synthesis of a Linear Allenamide-Modified Peptide
Encouraged by the optimised coupling procedure, we next set out
to introduce the allenamide functionality to the N-terminus of a
resin-bound model peptide. The well-known reporter peptide
LYRAG was modified to include a side chain amine (for a better
representative peptide sequence), giving the sequence LYRAK,
which was prepared by standard Fmoc-SPPS on 2-chlorotrityl
chloride (2CTC) functionalised polystyrene (PS) resin (Scheme
4).
Figure 1: RP-HPLC chromatogram (214 nm) of: a) crude peptide 7, b) purified
peptide 7, c) incubation of 7 with Boc-Cys-OMe (10 min) to afford product 7a
as specified in Table 2 entry 1, d) incubation of 7 with PhSH (10 min) to afford
product 7d as specified in Table 2 entry 4.
mixture of MeCN and phosphate buffer (10 mM) at r.t. revealed
no change in the RP-HPLC profile or observed mass of the
peptide, suggesting 7 was stable and that the allenamide
functionality was unreactive towards other side chains of the
peptide (e.g. amines or hydroxyls). Additionally, peptide 7 was
incubated with a ten-fold stoichiometric excess of Boc-Lys-OMe
for 18 h, again revealing no reaction with the free amine (Table 2,
entry 10). In contrast, peptide 7 was remarkably reactive towards
the thiol group of Boc-Cys-OMe, with 88% conversion to the
desired thiol adduct 7a effected within 10 min (Figure 1c, Table
2, entry 1). We next subjected peptide 7 to incubation with a
variety of structurally diverse thiols, both alkyl and aryl (Table 2).
Upon incubation of 7 in a 1:1 mixture of MeCN and pH 7.4
phosphate buffer (10 mM), conversions to the desired thiol
adducts 7a-e ranging from 86-89% were observed within 10-30
min (Table 2, entry 1-5). Of these, reactions towards 7a-d
reached completion within 10 min, exemplified by thiophenol
(PhSH) (Figure 1d). Interestingly, modification with 3-
mercaptopropionic acid (3-MPA) was found to be significantly
slower than other carbonyl containing alkyl thiols tested, whereby
a 2 h incubation afforded only 68% conversion (Table 1, entry 7).
However, upon raising the temperature to 45 oC, a conversion of
87% was achieved within 20 min (Table 2, entry 8). Applying the
elevated temperature to the reaction with sodium 2-
mercaptoethanesulfonate (MESNa) yielded an 81% conversion
within 5 min, comparable to the previous 86% conversion
observed at r.t. (30 min.). Dodecanethiol, a long chain alkyl thiol,
was also found to react particularly slowly at pH 7.4, with < 10%
conversion after 18 h, however, at pH 8.0, 85% conversion to 7g
was effected at r.t. within 2 h (Table 2, entry 9). Peptides 7a, 7b
Scheme 4: On-resin preparation of N-terminally modified allenamidyl peptide 7.
The allenamide modification is highlighted blue.
Upon complete assembly of resin-bound LYRAK peptide 5*, 3-
butynoic acid was coupled to the N-terminus of 5* with the
optimised protocol adjusted for on-resin application by increasing
the molar equivalents. 3-Butynoic acid (10 equiv.) was activated
with EEDQ (9.5 equiv.) and sym-collidine (9 equiv.) and coupled
for 18 h. Pleasingly, cleavage of peptidyl resin 6*, effected with a
cocktail of TFA/H2O/TIPS (95:2.5:2.5) for 2 h, provided the
desired allenamidyl peptide 7 in 73% purity (Figure 1a), which
could be readily purified by RP-HPLC to afford the pure peptide
(Figure 1b) in 33.2% isolated yield. NMR spectra of peptide 7
confirmed the presence of the allenic isomer (~80%, Figure S5-
6). Presence of the homopropygylic isomer (~20%) did not
impede subsequent reaction with thiols (vide infra).
Modification of Allenamidyl Peptide 7 with Thiols
With allenamidyl peptide 7 in hand, we set out to investigate its
chemoselective modification with thiols. While thia-Michael
addition to allenamides has previously been achieved employing
pH 8.0 or 8.5[6a, 6b], we sought to investigate reactivity under more
mild and physiologically relevant conditions. Incubation of
allenamidyl peptide 7 in pH 7.4 phosphate buffer (10 mM) or a 1:1
and 7d were then prepared from crude peptide
7 as
representative examples. In accordance with literature, 1H NMR
4
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Table 2: Modification of allenamidyl peptide 7 with thiolsa
.
Reaction
time
Observed m/z
Entry
Thiol (10 equiv.)
Prod
% conversionb
Temp
tR (min)c
[M+H]+ d
1
2
3
4
5
6
7
8
9
Boc-Cys-OMe
L-Cys
methylthioglycolate
PhSH
7a
7b
7c
7d
7e
7e
7f
88%
89%
87 %
89%
86%
81%
68%
87%
85%e
r.t.
r.t.
r.t.
10 min
10 min
10 min
10 min
30 min
5 min
2 h
22.1
14.0
18.8
22.6
16.0
16.0
17.3
17.3
33.0
951.4
837.4
822.4
826.3
858.3
858.3
822.4
822.4
918.6
r.t.
MESNa
MESNa
3-MPA
3-MPA
r.t.
45 oc
r.t.
7f
7g
45 oc
r.t.
20 min
2 h
Dodecanethiol
Amine (10 equiv.)
10
Boc-Lys-OMe
7
0%
r.t.
18 h
-
-
a Reaction performed in 1:1 MeCN / 10 mM phosphate buffer (pH 7.4). PhSH = thiophenol, MESNa = sodium 2-sulfanylethanesulfonate, 3-MPA = 3-
mercaptopropionic acid. For L-Cys (HCl salt) and 3-MPA, the thiol stock solution pH was brought to approx. 7.0 prior to adding thiol to reaction mixture.
b Conversion % from 7 to 7a-g estimated from RP-HPLC peak area (214 nm), peak identity confirmed by mass spectrometry. All crude reaction chromatograms
and mass spectra of products available in SI.
c Retention time of observed major peak.
d Mass observed by low resolution ESI-MS for the major peak collected from analytical RP-HPLC, which in all cases represented the desired thiol adduct. For
entry 10, incubation with free amine Boc-Lys-OMe resulted in starting material only, starting material m/z observed = 716.4.
e Reaction performed in 1:1 MeCN / 10 mM phosphate buffer (pH 8.0).
of Cys conjugated peptides 7a and 7b revealed the expected vinyl
sulfide linkage characterised by two vinylic proton signals (Figure
S7-8). Conjugation of peptide 7 with the aromatic thiol (PhSH) at
r.t. yielded 7d as a mixture of isomers (1H NMR), approx. 75% of
the expected vinyl sulfide kinetic product and two minor products
that were presumably cis- and trans-isomers of the
thermodynamic product proposed by Loh and co-workers[6a]. Upon
repeating the reaction at 4 oC, selectivity for the vinyl sulfide
isomer was 95%, (Figure S9) without sacrificing conversion (90
% conversion in 10 min). After HPLC purification, products 7a, 7b
and 7d were obtained in yields of 21.7 - 23.3 % with respect to
the initial resin-loading.
group can be orthogonally removed on-resin to reveal the free β-
amino group, allowing incorporation of the allenamide handle at
this position.
Starting with TentaGel S NH2 resin, functionalised with Rink
amide linker, the resin bound oxytocin analogue 8* was
assembled following standard Fmoc-SPPS protocols (Scheme
5a), incorporating the terminal Cys1 residue with Nα-Boc
protection and Dap(Dde) in position 6. Upon treatment of resin 8*
with a solution of hydrazine in DMF (2% v/v) the β-amino group of
Dap was liberated and the allenamide handle installed by a 2 h
coupling using our optimised reaction conditions to afford peptidyl
resin 3*. Cleavage of resin 3* yielded the crude allenamidyl
peptide 3 in 70% purity. Crucially, we were able to omit thiol-
based scavengers from the cleavage cocktail (to preserve the
allenamide) without significant formation of Cys-tert-butyl
adducts.
After purification by RP-HPLC at ca. pH 3, 5 min incubation
of pure allenamidyl peptide 3 at r.t. in a 1:1 mixture of MeCN and
pH 7.4 phosphate buffer (10 mM) resulted in a small but
quantitative shift in the RP-HPLC retention time (Scheme 5b)
presumed to be cyclic peptide 9. As both linear peptide 3 and the
desired cyclic peptide 9 share the same mass ([M+H]+ = 1058.50),
cyclisation was confirmed by a modified Ellman’s free thiol test[11]
(Figure S1) performed under acidic conditions to prevent the
competing thia-Michael cyclisation from occurring. Although the
yellow colour typical of the Ellman’s test is not observed under
acidic conditions, adduct formation was instead monitored by RP-
HPLC (Figure S1a-d). Upon a 5 min treatment of the pure linear
peptide 3 (Figure S1a) with the activated disulfide, 5,5′-
dithiobis[2-nitrobenzoic acid] (DTNB or Ellman’s reagent) at pH 3
approx., analysis by RP-HPLC revealed disappearance of the
starting material peak (tR = 12. 9 min) and appearance of two new
Disulfide Replacement via Intramolecular Thia-Michael
Cyclisation
Satisfied with the ability to modify model peptide 7 with a diverse
range of thiols in excellent conversions (> 85%) we turned our
attention towards the intramolecular reactivity of an allenamidyl
peptide. Oxytocin is a well-studied disulfide bridged peptide with
a particularly poor half-life in vivo, at least in-part due to reduction
of the disulfide bond which is generally thought to precede its
5b,
proteolytic degradation[3b,
10]. Taking oxytocin as a model
substrate, we envisioned replacing the disulfide bond with a vinyl
sulfide bridge by first introducing an allenamide handle to the
peptide on-resin and then effecting an intramolecular thia-Michael
cyclisation with a Cys residue by use of aqueous buffer. While
either of Cys1 or Cys6 in native oxytocin could be replaced with an
allenamide functionalised residue, we sought to further test our
methodology by incorporating the allenamide handle at the more
hindered, mid-sequence Cys6 position. A 2,3-diaminopropionic
acid (Dap) residue was selected as a substitution for Cys6,
prepared in the form of Fmoc-Dap(Dde)-OH, wherein the Dde
5
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peaks (tR = 14.8 and 16.4 min, Figure S1c). ESI-MS identified the
first of these peaks (tR = 14.8 min, observed m/z = 1255.7) as the
peptide-TNB adduct 10, and the second peak (tR =16.4 min,) was
identified as free TNB liberated during reaction with the peptide,
thus confirming the identity of 3 as the expected linear peptide.
Upon similar treatment of the putative cyclic peptide 9 (tR = 12.7
min, Figure S1b), no reaction with DTNB was observed (Figure
S1d), thus confirming its identity as the desired cyclic oxytocin
analogue 9.
Scheme 5: Synthesis of vinyl sulfide bridged oxytocin mimetic 9. a) On-resin preparation of linear allenamide-modified oxytocin precursor 3. The allenamide
modification of the Dap6 residue is highlighted in blue and analytical RP-HPLC of the crude product shown as inset. b) Cyclisation of allenamidyl peptide 3 to yield
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oxytocin mimetic 9 with partial RP-HPLC chromatograms (214 nm) shown below for the pure starting material 3 and crude product 9. Dap = 2,3-diaminopropionic
acid, Dde = 1-(4,4-dimethyl-2,6-dioxacyclohexylidene)ethyl.
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Incubating crude peptide 3 under the same cyclisation conditions
for 5 min yielded cyclic peptide 9 as the major product (Figure
S2b), confirmed by modified acidic DTNB test of a small sample
(Figure S2c). The purified cyclic oxytocin analogue 9 could thus
be afforded in an overall 8.5% yield (based on the initial resin
loading) after a single one-step purification by RP-HPLC.
Conclusion
While the allenamide functionality has been reported to exhibit
rapid, chemoselective and irreversible reactivity with thiols, it has
thus far remained unexplored as a reactive functionality for
incorporation into peptides. As a result of a thorough optimisation
study, an efficient method for the on-resin synthesis of allenamidyl
peptides was developed. We further demonstrate this allenamide
platform as a powerful “click” chemistry strategy, affording
excellent conversions for the modification/lipidation of a model
peptide with a diverse range of alkyl and aryl thiols, and
additionally enabling the thia-Michael cyclisation of an oxytocin
analogue with quantitative conversion. Unlike many existing
peptide modification strategies, thia-Michael addition to an
allenamide occurs spontaneously without catalysis at near neutral
pH and can be implemented for both intramolecular bridging
reactions and heterologous conjugations. The allenamide is inert
to amine nucleophiles during the thia-Michael addition procedure
and the addition reaction is irreversible, thereby addressing
common drawbacks of haloacetamides and maleimides
respectively. The allenamide handle can be installed onto the
requisite peptide on-resin in a simple one step procedure with low-
cost off the shelf reagents, eliminating the need for building block
synthesis. Furthermore, installation of the allenamide handle onto
the free amino group of a resin bound peptide provides flexibility
to adjust the length of the bridging unit by using lysine isoteres
(e.g. ornithine) or incorporating additional amino-alkyl chains (e.g.
6-aminohexanoic acid). We envision allenamidyl peptides will find
future application in peptide lipidation or adjuvant conjugation,
disulfide bond replacement, peptide stapling, conjugation of
peptide-based payloads or reporters to proteins or antibodies and
as an electrophilic warhead in the development of irreversible
inhibitors of protein-protein interactions (PPIs).
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Acknowledgements
A. J. Cameron thanks Lottery Health Research for a postdoctoral
Fellowship.
Keywords: Allenamide • Oxytocin • Click chemistry • Michael
addition • Allenes
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7
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Entry for the Table of Contents
We report methodology for the on-resin preparation of allenamide-modified peptides, which can subsequently undergo
chemoselective thia-Michael addition with thiols. We further demonstrate this approach as a versatile tool to modify peptides with
thiols and to prepare cyclic peptides with excellent conversions.
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