An Electrochemical Approach to Designer Peptide α-Amides Inspired by α-Amidating Monooxygenase Enzymes

Article abstract of DOI:10.1021/jacs.1c05718

Designer C-terminal peptide amides are accessed in an efficient and epimerization-free approach by pairing an electrochemical oxidative decarboxylation with a tandem hydrolysis/reduction pathway. Resembling Nature's dual enzymatic approach to bioactive primary α-amides, this method delivers secondary and tertiary amides bearing high-value functional motifs, including isotope labels and handles for bioconjugation. The protocol leverages the inherent reactivity of C-terminal carboxylates, is compatible with the vast majority of proteinogenic functional groups, and proceeds in the absence of epimerization, thus addressing major limitations associated with conventional coupling-based approaches. The utility of the method is exemplified through the synthesis of natural product acidiphilamide A via a key diastereoselective reduction, as well as bioactive peptides and associated analogues, including an anti-HIV lead peptide and blockbuster cancer therapeutic leuprolide.

Full text of DOI:10.1021/jacs.1c05718

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An Electrochemical Approach to Designer Peptide α‑Amides
Inspired by α‑Amidating Monooxygenase Enzymes
Yutong Lin and Lara R. Malins*
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ABSTRACT: Designer C-terminal peptide amides are accessed in
an efficient and epimerization-free approach by pairing an
electrochemical oxidative decarboxylation with a tandem hydrol-
ysis/reduction pathway. Resembling Nature’s dual enzymatic
approach to bioactive primary α-amides, this method delivers
secondary and tertiary amides bearing high-value functional motifs,
including isotope labels and handles for bioconjugation. The
protocol leverages the inherent reactivity of C-terminal carbox-
ylates, is compatible with the vast majority of proteinogenic
functional groups, and proceeds in the absence of epimerization,
thus addressing major limitations associated with conventional coupling-based approaches. The utility of the method is exemplified
through the synthesis of natural product acidiphilamide A via a key diastereoselective reduction, as well as bioactive peptides and
associated analogues, including an anti-HIV lead peptide and blockbuster cancer therapeutic leuprolide.
evaluation of the structural and functional consequences of this
modification. Direct late-stage amidation (Scheme 1B),
through activation of a peptide C-terminal carboxylic acid
followed by acyl substitution with an amine, requires
orthogonal protecting group strategies and is notoriously
plagued by epimerization at the C-terminal residue. Although
considerable resources have been dedicated to the develop-
ment and optimization of hundreds of new coupling reagents
to address the latter challenge,8 practitioners generally agree
that there is no “one-size fits all” approach to overcoming
epimerization in the activation process and to facilitating
difficult couplings. Specialized resin linkers for the solid-phase
peptide synthesis (SPPS) of C-terminally amidated peptides
have also been explored.2,9 Aldehyde resins (Scheme 1B), for
example, introduce the amide substituent at an early stage via
reductive amination.4c,10 Alternatively, latent resin linkages
may be activated toward aminolysis to afford amide products
(e.g., sulfonamide or “safety-catch” resins4a,11 can be alkylated
with excess iodoacetonitrile or TMS-diazomethane). Though
conceptually appealing, resin-based methods are often limited
to nonbulky, nucleophilic amines, which considerably hampers
access to tertiary amides. Furthermore, challenging acylation
steps in the initial resin loading may contribute to lower yields.
INTRODUCTION
■
Peptide α-amidation is a deceptively simple post-translational
modification that characterizes ∼50% of bioactive peptides and
has profound impacts on function.1 Relative to the
corresponding C-terminal carboxylic acid, the α-amide
functionality decreases peptide polarity and alters both
isoelectric point and hydrogen bonding patterns, dramatically
influencing binding and stability.2 Nature’s sophisticated α-
amidating machinery leverages the dual catalytic ability of the
enzyme peptidylglycine α-amidating monooxygenase (PAM)
which converts a propeptide 1 bearing a C-terminal glycine
(Gly) extension into the corresponding truncated primary
amide 2 via liberation of glyoxylic acid (Scheme 1A).
Mechanistically, this oxidative cleavage involves two enzymatic
processes, oxidation to the α-hydroxy Gly derivative 3 and
subsequent hydrolysis to the target peptide α-amide.3
Although Nature’s monooxygenase-mediated α-amidation
strategy exclusively affords primary amides, there is consid-
erable interest in the inherent tuneability of “designer” amides
bearing more complex substitution patterns, including for
applications in drug discovery.2,4 For example, a synthetic α-
monomethyl amidated variant of human parathyroid hormone
(hPTH) has shown superior binding capabilities relative to
both the corresponding α-acid and primary α-amide.5
Moreover, blockbuster cancer therapeutic leuprolide, a
structural analogue of gonadotropin releasing hormone
(GnRH), notably features a C-terminal N-ethylamide.6
Received: June 2, 2021
Published: July 21, 2021
Yet despite both the significance and simplicity of
substituted amides, there are remarkably few chemical
strategies enabling access to a broad diversity of α-amidated
peptides,7 particularly tertiary amides, which has limited critical
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Scheme 1. (a) Biosynthetic Approach to Primary α-Amides; (b) Contemporary Strategies for C-Terminal Amidation; (c)
Proposed Electrochemical Approach
a
PDB entry 5WJA was used to generate figure.
A robust approach to substituted α-amidated peptides would
significantly accelerate the construction of C-terminally
modified peptides for myriad applications.
pathway, previously noted by Seebach to afford simple primary
amides,12b is reminiscent of the two-step biosynthetic approach
mediated by the enzyme PAM. In contrast to the primary
amide products afforded by the enzymatic process, however,
extending this chemistry to diverse N-alkylated variants
presents an opportunity to access “designer” substituted
amides in an epimerization-free, bioresemblant approach.
We therefore envisaged generalizing this simple decarbox-
ylative strategy to designer C-terminal amides by pairing
electrochemical oxidation with a tandem hydrolysis (path a), or
alternatively, a facile reduction (path b) to deliver a suite of
secondary and tertiary amides (Scheme 1C). This post-
synthetic, divergent strategy would harness the inherent
reactivity of unactivated C-terminal acids, in which “propep-
tide” substrates bearing C-terminal amino acid or N-alkyl
amino acid extensions are converted to the corresponding
truncated amides. The conceptual novelty lies in the ability to
avoid entirely conventional activation chemistry (e.g., Scheme
1B), thus eliminating any risk of epimerization. At the outset of
this endeavor, we were encouraged by recent work on
decarboxylative peptide modifications,14 including photo-
catalytic approaches to decarboxylative reduction15 and
peptide N,O-acetals.16 Herein, we sought to exploit electro-
chemistry as a mild and tunable strategy17 for site-specific
oxidation. Indeed, a selection of recent methods has explored
electrochemistry in the context of peptides,18 although the
majority of these studies rely on programmed oxidation of
peptide electroauxiliaries or the reactivity of electrochemically
active small molecule reagents/catalysts. Moreover, our own
studies13 have focused exclusively on the electrochemical
oxidation of peptides with hydrophobic side-chains. As such,
the broad functional group compatibility of the proposed
anodic decarboxylation remains to be explored. We therefore
aimed to conveniently exploit the inherent reactivity of peptide
C-terminal acids, including in the presence of diverse side-
chain functionalities, to deliver high-value substituted amide
products.
Building on the pioneering work of Seebach and co-
workers,12 we recently reported an arylation strategy which
harnessed the reactivity of hydrophobic peptide N,O-acetals
generated through direct electrolytic decarboxylation of C-
terminal acids.13 In the course of these studies, we identified
hydrolysis of a sarcosine-derived N,O-acetal (Scheme 2A) as a
prevalent side-pathway, which delivered a truncated N-
methylamide variant of the natural product biseokeaniamide
B 4 as a single diastereomer. This oxidation−hydrolysis
Scheme 2. (a) N,O-Acetal Hydrolysis; (b) Analysis of
Electrochemical Oxidation−Hydrolysis via UPLC-MS
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Scheme 3. Scope of Electrochemical Oxidation−Hydrolysis
a
Triisopropylsilane (TIPS) was excluded (see Supporting Information).
Scheme 4. Scope of Electrochemical Oxidation−Reduction
a
b
c
Peptide was prepared from a C-terminal DL-amino acid. Hydrolysis conditions (TFA/TIPS/H2O) led to the reduction product. Additional
NH4I was added to the reduction (see Supporting Information).
examine the generalization of the oxidation-hydrolysis path-
way. Peptides were prepared using Fmoc-SPPS through initial
loading of Fmoc-N(Ra)-Gly-OH (Ra = hydrogen, methyl,
ethyl, benzyl, propargyl, allyl, cyclopropylmethyl) and
elongation using standard coupling protocols (see Scheme 3
RESULTS AND DISCUSSION
■
Hydrolysis. To begin our investigations, model tetrapep-
tides bearing various N-alkylated glycine residues at the C-
terminal position were designed as “propeptide” substrates to
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and the Supporting Information (SI) for details). Following
resin cleavage, peptides were dissolved in MeOH, treated with
Et3N, and oxidized at constant current in an undivided
electrochemical cell equipped with carbon electrodes to deliver
the intermediate N,O-acetals.12b,13 A rapid and facile TFA-
promoted hydrolysis under standard peptide cleavage con-
ditions (TFA/triisopropylsilane (TIPS)/H2O, 90:5:5, v:v:v)
then afforded the truncated N-alkylamides 5−11 (Scheme 3)
in appreciable yields over 2 steps (from the starting peptide
acid) or 10 steps (from the resin loading). UPLC-MS analysis
of the oxidation-hydrolysis of N-cyclopropylmethyl glycine
derivative SI-11 (Scheme 2B) highlights the efficiency of the
process, which afforded 5 in 76% yield over 2 steps. Peptides
containing C-terminal alkenyl (6), alkynyl (7), N-methyl (8),
N-ethyl (9), and N-benzyl (10) derivatives were readily
accessible. Intriguingly, in the case of N-benzyl derivative 10,
standard conditions led to competitive reduction of the N,O-
acetal affording the corresponding N-methyl-N-benzyl deriva-
tive; exclusion of TIPS afforded the desired N-benzyl product.
Simple primary amide 11 was likewise accessible beginning
with the quintessential PAM “propeptide” substrate (see
Scheme 1A) bearing a single glycine extension at the C-
terminus.
Although this bioresemblant oxidation−hydrolysis strategy
enables direct access to N-alkylamide variants, the method is
not without limitations. Drawbacks include the need to prepare
protected N-alkyl amino acids and difficulties encountered
during acylation of the secondary amine of the N-alkyl glycine
variants (c.f. the resin-based approaches outlined in Scheme
1B). In addition, SPPS yields of N-alkyl glycine-derived
peptides were generally moderate due to competitive
diketopiperazine formation, which occurred upon elongation
of the peptide.19
and, in some cases, complete loss of stereochemical integrity
at the C-terminal position (see SI, p 40).
“Propeptides” containing a variety of other native or
commercially available modified amino acids at their C-termini
were also viable (12−20, Scheme 4). Highlights include 12
(derived from readily available Fmoc-Ile-OH), which would be
difficult to synthesize via late-stage amidation given the cost of
the requisite chiral amine (e.g., (S)-(−)-2-methylbutylamine =
USD$156/g).21 Notably, the α-stereochemistry of the C-
terminal amino acid in the “propeptide” precursor is
inconsequential, as reduction abolishes the stereocenter.
Amino acids with undefined α-chirality could therefore be
readily incorporated (e.g., 13, 14, and 16 were derived from C-
terminal DL-amino acids). α,α-Disubstituted amino acids (e.g.,
17 and 18) were also successfully converted to secondary
amides albeit in slightly lower yields, likely attributed to
competitive hydrolysis of the corresponding substituted
iminium intermediates which are more sterically encumbered
and perhaps lead to slower rates of reduction. Surprisingly,
hydrolysis was largely suppressed in the oxidation−reduction
of a C-terminal tert-leucine peptide, which proceeded smoothly
to afford 15, even in the presence of water, with a standard
peptide cleavage cocktail (TFA/TIPS/H2O). Functional group
tolerance is exemplified by the incorporation of a biotin handle
from a commercially available biotin-lysine derivative. Initially,
standard reduction with TFA/TESH afforded 19 in 13% yield
over 10 steps based on the resin loading. We reasoned that
thioether oxidation was a likely deleterious side-pathway in the
electrochemical oxidation step. NH4I was therefore added to
the TFA/TESH reduction cocktail to effect concomitant
sulfoxide reduction,22 affording 19 in an optimized 48% yield.
Tertiary amides were also readily accessible (e.g., 20, derived
from Hyp).23 Oxidation−reduction of the N-alkylglycine
peptide precursors utilized in the hydrolysis approach (see
Scheme 3) provided an additional path to valuable tertiary
amides. For example, reduction of the N,O-acetal derived from
N-cyclopropylmethyl glycine peptide SI-11 (see Scheme 2)
afforded the N-cyclopropylmethyl-N-methyl amide 21 in good
yield (66% over 2 steps). N,N-Dimethyl (22), N-allyl-N-
methyl (23), and N-propargyl-N-methyl (24) peptides were
also accessible; each of these substrates would be challenging,
if not impossible, to prepare using standard solution- or solid-
phase methods due to difficulties accessing the requisite amine
precursor and challenges in forming the tertiary amide using
conventional activation-based coupling approaches.
Reduction. Given the above considerations, we therefore
aimed to exploit the availability, rich functional diversity, and
facile couplings of amino acids bearing side-chain rather than
N-alkyl substituents (e.g., Ra = H; Rb = alkyl, aryl; see Scheme
4). To this end, it was envisaged that electrochemical oxidation
to deliver α-substituted N,O-acetals could be followed by a
tandem reduction pathway,20 to afford substituted amide
products via retention of the side-chain functionality of the C-
terminal amino acid (e.g., path b, Scheme 1C). Notably, the
competitive, silane-mediated reduction observed in the
preparation of N-benzyl peptide 10 (Scheme 3) lent credence
to the viability of this pathway.
Accordingly, crude tetrapeptides with Gly, Ala, and phenyl-
glycine (Phg) residues at the C-terminus were directly
subjected to an electrolysis/reduction sequence after cleavage
from the resin. To our delight, amides 8 (N-Me), 9 (N-Et),
and 10 (N-Bn) were smoothly obtained upon treatment of the
crude N,O-acetal with BF3·Et2O/triethylsilane (TESH) or
TFA/TESHanhydrous reduction conditions which served to
minimize hydrolysis (see SI, pp 44−45 for optimization
tables). Importantly, unlike the N-alkylated peptide precursors
required for the hydrolysis approach, the “propeptides” utilized
in the oxidation−reduction were readily prepared by SPPS
without complication (e.g., diketopiperazine formation) and
able to be used without intermediary purification. Notably,
amidated products were also obtained as single diastereomers.
In contrast, attempts to synthesize N-Me amide 8 by simple
coupling of the corresponding tripeptide C-terminal acid with
N-methylamine using standard coupling reagents (EDC,
PyBOP, DIC/Oxyma) led to considerable epimerization,
In another valuable extension of the method, site-selective
deuteration was readily accomplished by adopting slightly
modified oxidation−reduction conditions, allowing for the use
of commercially available NaBD4 as a reductant. This process
provided 25 in good yield (24% over 10 steps) when the more
reactive AcOH-derived N,O-acetal13 was employed. The
MeOH-derived N,O-acetal in this case afforded the desired
product together with an inseparable byproduct. Importantly,
the optimized protocol for late-stage isotope incorporation
serves as a useful proof-of-principle study demonstrating facile
access to labeled peptides, including for structural or biological
assays.
Side-Chain Compatibility. Given that late-stage mod-
ification methods require broad compatibility with canonical
amino acid side-chain functionalities,24 a systematic evaluation
of the side-chain tolerance of the oxidation−reduction pathway
was next examined. To the best of our knowledge, an
exhaustive evaluation of the side-chain tolerance of anodic
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Scheme 5. Side-Chain Compatibility Studies
a
b
c
Yield over 10 steps from resin loading. MeOH/AcOH (10:1 v:v) was used for electrolysis. Yield following an additional TCEP reduction step.
d
e
f
NH4I was added to the reduction mixture (see SI). Is = 2,4,6-triisopropylbenzenesulfonyl. TMSBr/thioanisole/1,2-ethanedithiol cocktail was
added for Is-deprotection.
Scheme 6. Synthesis of Natural Product Acidiphilamide A
oxidation of C-terminal peptide acids has never been
undertaken.25 Protected and unprotected model tetrapeptides
bearing functionalized amino acids (e.g., Asp, Asn, Ser, Lys,
Arg, Cys, Met, Tyr, His, Trp) at the N-terminal position were
subjected to the standard oxidation−reduction conditions
(Scheme 5, side-chain protected, green; side-chain unpro-
tected, yellow). Notably, the acidic reduction conditions result
in concomitant cleavage of side-chain protecting groups to
deliver unprotected peptides, so both protected and
unprotected peptides are treated equivalently and deliver
identical peptide products. Pleasingly, the desired N-ethyl-
amide product (e.g., 26−35) was formed in all cases in the
presence of protected residues. The majority of residues were
well-tolerated (34−76%) while highly oxidizable aromatic side-
chains, as expected, gave lower yields (e.g., Trp(Boc) 35, 9%).
The nature of the protecting groups played a critical role in
modulating the oxidation potential of the side-chain function-
alities. For example, oxidation−reduction of 2,4,6-triisopropyl-
benzenesulfonyl (Is)-protected Trp afforded the Trp-protected
product in a remarkable 75% yield. We reasoned that the
highly electron-withdrawing nature of the sulfonyl protecting
group rendered the indole nucleus less prone to oxidation.
Unfortunately, the use of sulfonyl-protected Trp is complicated
by difficulties in removing the protecting group under standard
acidolytic cleavage conditions. A suitable one-pot oxidation−
reduction−deprotection protocol employing TMSBr26 for the
removal of the sulfone protecting group was devised but led to
a significant reduction in yield (17% over 2 steps). In this case,
there is a slight trade-off between the yield of product and the
operational simplicity of the method.
In the presence of unprotected Asp, the protocol was
selective for C-terminal decarboxylation, delivering 26 in 59%
over 2 steps. Unprotected amides and alcohols (Asn 27, Ser
28) were well-tolerated. Basic residues (Lys 29, Arg 30)
benefitted from the addition of acetic acid in the electrolysis
step,25a with protonation reducing unwanted oxidation.
Interestingly, unprotected Cys (e.g., 31) did not preclude the
desired transformation, likely owing to in situ oxidation to the
disulfide during electrolysis (see SI). Markedly improved yield
was obtained with the corresponding cystine substrate (31,
from thiol: 10%; from disulfide: 21%). Oxidation−reduction of
the Met thioether-containing peptide afforded 32 in 40% yield
over 10 steps. As with the biotin substrate (19; see Scheme 4)
the addition of NH4I to the standard reduction conditions
enabled concomitant reduction of side-chain sulfoxide formed
in the electrochemical step.22 Tyr, His, and Trp did not
tolerate electrochemical oxidation in the absence of protecting
groups.27 Nevertheless, as protected peptides are easily
accessible using common SPPS strategies and the acidic
reduction conditions enable concurrent cleavage of side-chain
blocking groups, we envisaged that the method would be
readily extendable to more complex peptide systems with
diverse side-chain functionalities.
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Scheme 7. Scope of Bioactive Peptides: (a) Potent Pentapeptide Binder of Cyclophilin A; (b) Synthesis of Leuprolide and
Structural Analogues
a
b
Yields based on resin loading. An additional deprotection step was carried out following treatment with NaBD4.
Applications. To further evaluate the method, the total
SI); successfully afforded the target N-ethylamide products
synthesis of acidiphilamide A (Scheme 6), a bioactive
secondary metabolite from the rare actinobacterial genus
Streptacidiphilus,28 was pursued with the intent to construct the
characteristic secondary amide using our electrochemical
oxidation−reduction pathway. Though conceivably accessible
via alternative approaches, we envisaged that acidiphilamide A
would serve to challenge our method due to the presence of an
unprotected C-terminal alcohol and the obligatory intermedi-
acy of a quaternary N,O-acetal. Pleasingly, following synthesis
of the requisite “propeptide” incorporating commercially
available DL-2-benzylserine at the C-terminus (36; see SI),
standard electrolysis and subsequent reduction with the more
reactive NaBH4 afforded the natural product 37 and epi-37 (dr
1:2.2) in synthetically useful yields (Scheme 6). In an effort to
improve the diastereoselectivity of the reduction step, we next
evaluated the use of a chiral reductant to direct the
stereochemical outcome of the reduction. Accordingly, (S)-
and (R)-CBS-oxazaborolidines were employed,29 leading to
selective formation of 37 and epi-37, respectively, and thus
laying the framework for the future exploration of stereo-
selective reductions.
The method was finally applied to the synthesis of bioactive
peptides. Pentapeptide 38 (Scheme 7A), bearing a C-terminal
benzylamide, was reported to be a potent binder of cyclophilin
A,4a which is implicated in HIV-1 replication. This therapeutic
lead was readily accessible via oxidation−reduction of a
phenylglycine (Phg) precursor in 36% yield over 14 steps
(see SI). The deuterated variant 39 was likewise obtained in
comparable yield (32% over 15 steps). In the context of drug
discovery, facile isotope-labeling may serve as a valuable tool
for understanding mechanisms of drug metabolism.30
over 20 steps, without the need for intermediary purification.
Importantly, 1H NMR analysis and UPLC-MS coinjection
(Scheme 7B) demonstrated that synthetic leuprolide 42 was
identical to a commercial sample of the drug. To further probe
the efficiency of the two-step electrochemical oxidation−
reduction, the purified [L-Leu6] precursor peptide was
subjected to the reaction conditions. This process delivered
41 in 25% isolated yield despite the presence of multiple
oxidizable side-chains, notably outperforming our model
tetrapeptide (e.g., the synthesis of 35 using Trp(Boc)
protection, which proceeded in 9% yield). We postulate that
the peptide microenvironment might influence side-chain
oxidation potentials. In the case of leuprolide, the Trp residue
is internally located on the peptide chain, whereas model
substrate 35 bears an N-terminal Trp. Future studies will aim
to deconvolute the link between peptide sequence and
propensity toward oxidation and, therefore, explore oppor-
tunities for sequence-specific electrochemical modifications.
CONCLUSIONS
■
In summary, we disclose herein an electrochemically enabled
peptide C-terminal amidation strategy. Broadly inspired by the
dual catalytic functionality of the α-amidating monooxygenase
enzyme PAM, an electrochemical oxidative decarboxylation
paired with a tandem hydrolysis or reduction facilitates
postsynthetic conversion of C-terminally extended “propep-
tides” into N-alkylamides. A broad array of C-terminal
alkylamides bearing valuable functional handles (e.g., alkene,
alkyne, biotin, isotope labels) is readily accessible. The method
is both highly functional group tolerant and, by design,
epimerization-free, thus overcoming an unsolved challenge in
the preparation of homogeneous, C-terminally modified
peptides. Several case studies have highlighted the preparation
of bioactive peptides and associated analogues, and we have
established proof-of-principle for the viability of a diaster-
eoselective reduction protocol. We therefore envisage that this
approach to designer C-terminal alkylamides will have broad
The nonapeptide cancer therapeutic leuprolide and several
structural variants (40−42) were also accessible. Leuprolide
contains a C-terminal N-ethylamide and a number of
challenging, oxidatively labile residues (His, Tyr, and Trp).
Strikingly, the oxidation−reduction of the requisite protected
“propeptides”, each employing standard Boc/tBu-protecting
group strategies (including the incorporation of Trp(Boc), see
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analogue that binds human cyclophilin A more efficiently than the
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application in the development and advancement of peptide-
based therapies.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.1c05718.
■
sı
Experimental details and analytical data for all new
compounds (PDF)
AUTHOR INFORMATION
Corresponding Author
■
Lara R. Malins − Research School of Chemistry and Australian
Research Council Centre of Excellence for Innovations in
Peptide and Protein Science, Australian National University,
Canberra, ACT 2601, Australia; orcid.org/0000-0002-
7691-6432; Email: lara.malins@anu.edu.au
Author
Yutong Lin − Research School of Chemistry, Australian
National University, Canberra, ACT 2601, Australia
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.1c05718
(7) For examples of enzymatic/biomimetic late-stage C-terminal
amidation, see: (a) Wu, B.; Wijma, H. J.; Song, L.; Rozeboom, H. J.;
Poloni, C.; Tian, Y.; Arif, M. I.; Nuijens, T.; Quaedflieg, P. J. L. M.;
Szymanski, W.; Feringa, B. L.; Janssen, D. B. Versatile peptide C-
terminal functionalization via a computationally engineered peptide
amidase. ACS Catal. 2016, 6 (8), 5405−5414. (b) Ranganathan, D.;
Saini, S. Transformation of C-terminal serine and threonine extended
precursors into C-terminal alpha-amidated peptides - a possible
chemical-model for the alpha-amidating action of pituitary enzymes. J.
Am. Chem. Soc. 1991, 113 (3), 1042−1044.
Funding
This work was supported by the Australian Research Council
(DE180100092; fellowship to L.R.M.).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We would like to thank Ms. Anitha Jeyasingham and Mr.
Joseph Boileau (ANU) for assistance with mass spectrometry,
Dr. Chris Blake (ANU) for support with NMR spectroscopy,
and Dr. Hideki Onagi and Mr. Daniel Bartkus (ANU) for
technical assistance.
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ABBREVIATIONS
■
UPLC, ultraperformance liquid chromatography; TFA, tri-
fluoroacetic acid; TESH, triethylsilane; EDC, N-(3-(dimethyl-
amino)propyl)-N′-ethylcarbodiimide hydrochloride; PyBOP,
(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluoro-
phosphate; DIC, N,N′-diisopropylcarbodiimide; Oxyma,
Oxyma Pure, ethyl (hydroxyimino)cyanoacetate; Hyp, trans-
4-hydroxy-L-proline; TCEP, tris(2-carboxyethyl)phosphine
hydrochloride
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