High-throughput synthesis of peptide α-thioesters: A safety catch linker approach enabling parallel hydrogen fluoride cleavage

Article abstract of DOI:10.1002/cmdc.201300524

Peptide α-thioesters are fundamental building blocks in peptide and protein science, providing powerful tools for peptide medicinal chemistry. The application of peptide α-thioesters in native chemical ligation reactions has enabled synthetic access to cysteine-rich peptides and proteins, cyclic peptides as well as labeled and chemically modified biomolecules. An efficient high-throughput synthesis of peptide α-thioester building blocks would be beneficial for many medicinal chemical applications that require peptides and proteins. Herein we present a novel synthetic route to cysteine-rich peptide α-thioesters using a safety catch linker that enables a parallel synthetic strategy for chemical protein synthesis. ACP(68-75), bradykinin and dynorphin(1-13) were synthesized via Boc chemistry in their thioester form on a safety catch amide linker (SCAL), employing polystyrene- or poly(ethylene glycol)-based resins, compartmentalized in tea bags. This compartmentalized resin/linker strategy facilitated a parallel hydrogen fluoride cleavage in which each peptide thioester was subsequently cyclized by native chemical ligation, demonstrating the utility of this approach. A naturally occurring bioactive cyclic peptide, the sunflower trypsin inhibitor SFTI-1, was synthesized to demonstrate the viability of this method to access important peptide biomolecules. A boost for NCL: A synthetic route to cysteine-rich peptide α-thioesters employing a safety catch linker is described. The linker strategy allows parallel Boc chemistry including parallel HF cleavage, and removes the bottleneck of peptide thioester accessibility.

Full text of DOI:10.1002/cmdc.201300524

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DOI: 10.1002/cmdc.201300524
High-Throughput Synthesis of Peptide a-Thioesters: A
Safety Catch Linker Approach Enabling Parallel Hydrogen
Fluoride Cleavage
Andreas Brust,* Christina I. Schroeder, and Paul F. Alewood[a]
Peptide a-thioesters are fundamental building blocks in pep-
tide and protein science, providing powerful tools for peptide
medicinal chemistry. The application of peptide a-thioesters in
native chemical ligation reactions has enabled synthetic access
to cysteine-rich peptides and proteins, cyclic peptides as well
as labeled and chemically modified biomolecules. An efficient
high-throughput synthesis of peptide a-thioester building
blocks would be beneficial for many medicinal chemical appli-
cations that require peptides and proteins. Herein we present
a novel synthetic route to cysteine-rich peptide a-thioesters
using a safety catch linker that enables a parallel synthetic
strategy for chemical protein synthesis. ACP(68–75), bradykinin
and dynorphin(1–13) were synthesized via Boc chemistry in
their thioester form on a safety catch amide linker (SCAL), em-
ploying polystyrene- or poly(ethylene glycol)-based resins,
compartmentalized in tea bags. This compartmentalized resin/
linker strategy facilitated a parallel hydrogen fluoride cleavage
in which each peptide thioester was subsequently cyclized by
native chemical ligation, demonstrating the utility of this ap-
proach. A naturally occurring bioactive cyclic peptide, the sun-
flower trypsin inhibitor SFTI-1, was synthesized to demonstrate
the viability of this method to access important peptide
biomolecules.
Introduction
Native chemical ligation (NCL) has become the fun-
damental workhorse within modern protein chemis-
try for the study of biomolecular structure and func-
tion at the atomic level.[1] This technology has facili-
tated the total synthesis of cysteine-rich, small- and
medium-sized proteins and peptides as well as cyclo-
peptides,[2] representing targets for peptide drug dis-
covery. NCL is therefore a fundamentally important
enabling technology for peptide-based medicinal
chemistry. Fully synthetic access to bioactive peptides
and proteins allows a variety of chemical probes and
molecule modifications to be introduced, leading to
Scheme 1. Native chemical ligation; a-thioesters form an amide bond with N-terminal-
cysteine-containing peptides.
a vastly improved information flow for the study of
bioactive peptides.
NCL (Scheme 1) relies on the availability of a funda-
mental building block: the peptide a-thioester. NCL is a combi-
nation of two reaction steps in which 1) a C-terminal peptide
a-thioester reacts with an N-terminal cysteine-containing pep-
tide, resulting in trans-thioesterification and formation of a pep-
tide thioester bond to the cysteine sulfur atom, and 2) a rapid
intramolecular S- to N-peptidyl shift, which yields the two pep-
tide fragments linked together by a native amide bond. NCL is
chemoselective and takes place in aqueous solutions at pH 7,
usually in the absence of protecting groups, and does not re-
quire a large excess of reactants for completion. Furthermore,
thiol catalysts can be used to enhance the reaction rate. Since
the initial discovery[3] of this methodology, many variations
have been introduced, and a wide variety of potential ligation
sites have been employed.[1b,4] Apart from NCL, C-terminal pep-
tide thioesters, selenoesters,[5] and to some extent thioacids[6]
are also very useful synthons, due to their high reactivity. Pep-
tide thioesters have been successfully used as versatile reactive
building blocks for dendrimers,[7] the synthesis of protein mi-
croarrays, and selective peptide C-terminal and N-terminal
modifications.[8] Peptide thioesters transform readily with nu-
cleophiles to generate esters, amides, hydrazides, hydroxamic
acids, or under reductive conditions to peptide alcohols.[9] This
versatility makes peptide thioesters important reactive inter-
mediates in peptide and protein bioscience and drug discov-
ery, e.g., for the introduction of labels[10] or for anchoring on
[a] Dr. A. Brust, Dr. C. I. Schroeder, Prof. P. F. Alewood
Institute of Molecular Bioscience
The University of Queensland, Brisbane, 4072 (Australia)
E-mail: a.brust@imb.uq.edu.au
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solid supports.[11] As the synthetic potential of peptide thioest-
ers has grown, there has been an ongoing search for efficient
synthetic methods, although a high-throughput approach has
yet to be established.
Classic Fmoc chemistry protocols[12] are unsuitable when in
concert with thioester linkers due to premature cleavage by
the highly nucleophilic piperidine. However, in the past 10
years, several developments have emerged to enable Fmoc
chemistry to generate peptide thioesters.[13] These include the
use of less nucleophilic cleavage cocktails,[14] ap-
proaches using thiol-labile safety catch linkers,[15]
SCAL followed by attachment of S-trityl-3-mercaptopropionic
acid as the thiol linker (Scheme 2). The trityl group was subse-
quently removed with TFA/TIPS/water (90:5:5 v/v/v) resulting
in resin 2 containing a free thiol moiety. This thiol was then
used for the formation of the peptide thioester resin linkage of
the synthesized peptide.
To evaluate the feasibility of the approach, test peptides
were assembled (sequences: VQAAIDYING, a; RPPGFSPFR, b;
and YGGFLRRIRPKLK, c; see Table 1), which were selected
Table 1. Yield and purity of peptide a-thioesters 6a–g synthesized on mercaptopro-
pionic acid SCAL resin 2 after parallel HF cleavage. Intramolecular NCL resulted in the
cyclization products obtained (7d–f, 8d–f and 9).
backbone linkers, and post-synthetic thioesterifica-
tion,[16] activation of protected peptides in solution,[17]
the application of O- to S-[18] and N- to S-acyl trans-
fer[19] and also on-resin linker transformation into acti-
vated esters reactive toward thiolysis.[20]
Mr [Da]
Sequence[a]
Entry
Purity [%][b]
Yield [%][c]
calcd
found
VQAAIDYING*
RPPGFSPFR*
YGGFLRRIRPKLK*
CCVQAAIDYING*
6a
6b
6c
6d
6e
6 f
6g
7d
7e
7 f
8d
8e
8 f
9
80
81
91
72
55
65
67
37
35
30
39
36
39
35
15.6/83[b]
14/78[b]
26/88[b]
2.6/75[b]
2.5/69[b]
3.0/74[b]
17/55[b]
0.3/10[c]
0.5/12[c]
0.4/14[c]
0.5/15[c]
0.7/1[c]
1207.3
1204.4
1748.1
1413.5
1410.6
1954.3
1677.8
1249.4
1246.5
1790.3
1251.4
1248.5
1792.3
1513.8
1207.6
1204.8
1748.4
1411.8[d]
1408.8[d]
1953.2[d]
1678.0
1249.8
1246.8
1790.2
1251.8
1248.8
1792.2
1514.2
Despite these efforts, the most effective approach
for the synthesis of peptide a-thioesters remains the
in situ neutralization chain assembly protocol for tert-
butoxycarbonyl solid-phase peptide synthesis (Boc-
SPPS)[12,21] using thioester linkers.[9,22] Whereas this
well-established methodology allows access to high-
quality material following a straightforward reaction
pathway, the throughput is limited due to the neces-
sity of a final hydrogen fluoride (HF) cleavage usually
performed in a sequential, time-limiting fashion. This
limitation has so far only been addressed by Hought-
en and co-workers, employing Boc chemistry in com-
bination with an aqueous HF volatizable support,
suitable for the production of peptide thioester libra-
ries.[23]
CCRPPGFSPFR*
CCYGGFLRRIRPKLK*
CTKSIPPICFPDGR*
cyclo[CCVQAAIDYING]
cyclo[CCRPPGFSPFR]
cyclo[CCYGGFLRRIRPKLK]
cyclo[CCVQAAIDYING]
cyclo[CCRPPGFSPFR]
cyclo[CCYGGFLRRIRPKLK]
cyclo[CTKSIPPICFPDGR]
0.6/21[c]
1.8/12[c]
[a] *Thioester=S-CH2CH2-CONH-Gly-NH2. [b] Purity of crude products is determined by
the peak area of HPLC at 214 nm. [c] Yields are based on the weight of the crude
product and are relative to the substitution of the resin; yield after purification
(>95%) is relative to the weight of crude product used for cyclization. [d] Vicinal-
disulfide-containing peptides were obtained after cleavage; underlined ’C’ represents
disulfide-bound cysteine residues.
Herein we present a more straightforward high-
throughput approach for the synthesis of peptide a-
thioesters that includes the inherent advantages of in
situ neutralization Boc-SPPS[21c] on thioester linkers[9]
combined with a safety catch amide linker (SCAL)[24] that is
stable to HF cleavage. The SCAL[24] is highly stable in both TFA
and HF, making it particularly useful for Boc chemistry applica-
tions. We have used this linker in previous work for the high-
throughput production of conopeptide amide libraries employ-
ing Fmoc chemistry[25] as well as for the synthesis of bioiso-
steric diseleno analogues of a-conopeptides employing Boc
chemistry.[26] Most recently, we used the SCAL to develop
a high-throughput synthetic approach toward peptide turn
mimetics.[27]
based on their difficulty of chain assembly as well as the di-
verse range of residues within their sequences, allowing poten-
tial amino acid side reactions to be observed. Peptide synthesis
was performed on resin 2, by employing in situ neutralization
Boc-SPPS chemistry[12,21] with HBTU as activating reagent,
throughout which led to the fully side-chain-protected peptid-
yl resins 3a–c.
A fragment of the acyl carrier protein, ACP(65–74) has been
known as a difficult peptide (VQAAIDYING, a) due to strong
chain aggregation and has been used in multiple studies as
a synthetically challenging target.[28] During the chain assembly
of ACP(65–74) (a) on resin 2, no difficult couplings were en-
countered. The coupling yields were monitored using the
quantitative ninhydrin test[29] and were close to quantitative
within 2 min coupling times. Further test peptides synthesized
on resin 2 were the nonapeptide bradykinin (RPPGFSPFR, b)
and dynorphin(1–13) (YGGFLRRIRPKLK, c), both of which exhib-
ited very good coupling yields throughout the sequence. The
amino acids used were side chain protected with protecting
groups commonly used in Boc-SPPS: Asn(Xan), Arg(Tos), Asp-
(OcHxl), Gln(Xan), Lys(ClZ), Tyr(BrZ), Thr(Bzl), and Ser(Bzl). As we
wished to cyclize peptides a–c, cysteine residues were intro-
Results and Discussion
Peptide thioester synthesis on a SCAL
As the synthesis of peptide a-thioesters remains limited by
low-throughput chemistry, we decided to remove this bottle-
neck by employing the SCAL. To achieve this we selected
a standard aminomethylated polystyrene (PS) resin and loaded
it with an Fmoc-SCAL using standard HBTU/DIEA activation.
Fmoc deprotection with piperidine/DMF (50%) gave SCAL-AM-
PS-resin 1. Boc-glycine was then attached as a spacer on the
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Scheme 2. General procedure for parallel synthesis of peptide a-thioesters (peptide sequences 3a–g to 6a–g, see Table 1): Thiol linker attachment via an
HF-stabile safety catch amide linker (SCAL, 1) allows for Boc-SPPS (2!3) and HF cleavage of multiple peptides, compartmentalized in tea bags, at one time
(3!4). Fully unprotected peptides 4 still attached to the solid support are released from the linker via NH4I/DMS/TFA treatment (4!6 via 5) due to reduction
of the linker sulfoxide group (4!5), rendering the linker TFA-labile. Peptides containing a vicinal cysteine pair form a vicinal disulfide bond during SCAL
cleavage; Pg=protecting group.
duced to allow intramolecular NCL. This was achieved by N-ter-
minal extension of the test peptides ACP(68–75) (a), bradykinin
(b) as well as dynorphin(1–13) (c) by coupling of two cysteine
residues (Cys(4-MeBzl)) to the N terminus. Again, good cou-
pling yields were observed during chain assembly employing
in situ neutralization Boc-SPPS, resulting in fully protected pep-
tidyl resins 3d–f (Sequence: CCVQAAIDYING, d; CCRPPGFSPFR,
e; and CCYGGFLRRIRPKLK, f).
obtained are not contaminated by other peptides cleaved
under the same conditions.
The assembled peptide-thioester resins 3a–f obtained after
N-terminal Boc-group removal and thorough washing with
DMF and dichloromethane were dried in vacuum and weigh-
ed. The dried peptide resins were then compartmentalized em-
ploying tea bags.[30] These were made from polypropylene
mesh sheets (Cole–Parmer, 105 mm pore size), as they can be
heat welded to size as required. The optimum HF cleavage
conditions were elaborated by performing multiple cleavage
experiments on small (10 mg) peptidyl-resin samples (d–f)
using p-cresol and p-thiocresol in 2–10% overall quantity and
variable ratios. These experiments have shown that p-cresol[31]
is the most suitable scavenger, avoiding premature cleavage
from the resin. Inclusion of p-thiocresol led to a partial reduc-
tion of the SCAL, resulting in some release of peptide thioester
(6a–f) during HF cleavage. The optimum cleavage conditions
were found to be 5% vol p-cresol in HF with a cleavage time
Parallel HF side chain cleavage of peptide thioester resins
To achieve simultaneous parallel HF cleavage of the peptide
thioester resins 3a–f, it was necessary to ensure a suitable
method of compartmentalization prior to selecting cleavage
conditions, which would only remove side chain protecting
groups, while avoiding premature peptide cleavage from the
solid support. It was also essential to ensure that the products
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of 1 h at 08C. For the final large-scale cleavage, all tea bags
containing peptide (3a–f) resin were transferred into a single
HF cleavage reactor. The cleavage was undertaken with 2.5 mL
of p-cresol,[31] in 50 mL HF at 08C for 1 h. After evaporation of
the excess HF under vacuum, the tea bags were washed with
cold diethyl ether (2ꢁ) and dichloromethane (2ꢁ) to remove
residual p-cresol and protecting group quencher adducts. The
organic wash fractions were collected, concentrated, and inves-
tigated by LC–ESMS. No peptide thioester (6a–f) were detect-
ed, indicating that the SCAL as well as the thioester linkage is
sufficiently stable under the selected reaction conditions.
ing vicinal cysteine residues.[25,27] This was also observed for
peptides 6d–f (Figure 1). In all cases, the formation of a vicinal
disulfide bond was observed, and the peptide thioester prod-
ucts 6d–f were formed preferentially in the disulfide form, as
was confirmed by mass spectrometry (Table 1). This oxidation
is attributed to the formation of an iodosulfonium cation inter-
mediate during NH4I sulfoxide reduction.[25,33] Further disulfide
formation promoters are likely trace amounts of iodine formed
during the cleavage step.[25,33] Iodine in the presence of acid is
an excellent reagent for disulfide bond formation. Furthermore,
DMSO is also formed during the NH4I reduction from the DMS
scavenger used,[25,33] and DMSO in neat TFA is also an excellent
reagent for disulfide formation.[25]
After removal of excess solvent under vacuum, the tea bags
containing the dried unprotected peptide resins (4a–f) were
transferred into separate glass reactors. SCAL activation by re-
duction of the sulfoxide group was performed as previously
described using NH4I/DMS in TFA.[25] The resulting peptidyl
resins (5a–f) are labile in TFA, and the peptides were released
from the resin into solution over a period of 18 h. The TFA so-
lution was transferred into cold diethyl ether resulting in pre-
cipitation of the desired peptide thioesters 6a–f. Repeated
centrifugation and washing steps with cold diethyl ether re-
sulted in crude peptide, which was readily dissolved in acetoni-
trile/water/0.1% TFA (50%) freeze-dried, weighed, and ana-
lyzed by ESI LC–MS (Table 1). No cross-contamination of the
crude peptides with other peptide thioesters was observed,
demonstrating that the methodology is well suited for parallel
peptide thioester library production. All peptides synthesized
were obtained in good yield and purity. The HPLC traces of the
obtained crude peptide thioester 6a–f are shown in Figure 1,
Intramolecular NCL of peptide thioesters 6d–f
To confirm that peptide thioesters 6d–f were useful as build-
ing blocks for NCL chemistry, cyclization experiments were per-
formed. To evaluate if the impurities resulting from the SCAL
cleavage cocktail may influence the ability of the obtained
thioester to be used in ligation reactions, we performed cycli-
zation using crude peptide thioesters 6d–f. Peptide thioesters
6d–f were dissolved in NH4HCO3 buffer (pH 7.4), and the prog-
ress of the cyclization was monitored by LC–MS analysis. A
single reaction product was formed with a molecular mass of
162ꢀ0.5 amu less than the starting thioester, indicating intra-
molecular cyclization with loss of a S-(CH2)2-CO-Gly-NH2 moiety.
In all experiments, cyclization was complete within 3 h. No re-
sidual starting thioester was observed, suggesting that the vici-
nal disulfide bond must undergo partial reduction to conse-
quently enable intramolecular ligation. In addition, the release
of the thiol linker autocatalyzes disulfide–thiol exchange, thus
enabling intramolecular “thia zip”[34] NCL, driving the equilibri-
um toward the cyclized products (Figure 2). The final products
obtained are cyclic peptides containing a backbone amide link-
age and a disulfide bond formed between the vicinal cysteine
residues (7d–f). Alternatively, peptides 6d–f were also cyclized
in the presence of an additional thiol catalyst, thought to help
vicinal disulfide–thiol exchange as well as trans-thioesterifica-
tion, which may be required to achieve more efficient cycliza-
tion. Under these conditions, (MESNA, 5 equiv NH4HCO3 solu-
tion, pH 7.4) cyclization was accelerated, and cyclized peptide
was obtained with the difference that the disulfide bond was
not formed due to the excess thiol used. The cyclic reduced
peptides 8d–f were obtained as main products under these
conditions. Overall, a loss of 160ꢀ0.5 amu was observed, con-
sistent with the loss of the thioester moiety (S-(CH2)2-CO-Gly-
NH2) and the gain of two protons due to disulfide bond reduc-
tion. For all cyclic peptides (7d–f and 8d–f), hydrolysis experi-
ments with 0.1m NaOH solution were performed and moni-
tored by LC–MS. In none of the investigated cyclic products
was hydrolysis (Dm= +18) observed. This lead us to the con-
clusion that all products were backbone cyclized through an
amide bond with the intramolecular thioester intermediates
absent.
Figure 1. HPLC traces of obtained crude peptide a-thioesters 6a–f.
and results are summarized in Table 1. The SCAL cleavage
yields, obtained for peptide thioesters 6a–f, were strongly de-
pendent on cleavage time. Crude peptide workup after 2, 6,
and 18 h showed that the highest yields are obtained after
overnight cleavage. This long cleavage time did not influence
the quality of the obtained peptide thioester. No side products
were formed due to extended NH4I/DMS/TFA treatment which
is consistent with previous observations that peptide thioesters
are stable under the selected SCAL cleavage conditions.[32]
Cysteine oxidation during NH4I/TFA treatment has been de-
scribed previously.[25,33] Vileseca et al.[33] obtained disulfide
folded peptides as main products, whereas we observed oxida-
tion during SCAL cleavage, in particular with peptides contain-
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Synthesis of SFTI-1 (9)
The sunflower trypsin inhibitor
SFTI-1 (9) is a 14-residue cyclic
peptide cross-braced with one
disulfide bond and a network of
hydrogen bonds, giving it
a well-defined structure.[35] SFTI-
1, originally isolated from sun-
flower seeds, is a very potent in-
hibitor of trypsin and other pro-
teases. The inhibition of trypsin-
like enzymes, in particular serine
proteases, is of great interest in
medicinal science, due to impli-
cations in cancer propagation.[36]
These findings have initiated the
search for novel serine protease
inhibitors as potential cancer
therapeutics. This has prompted
the need for rapid access to
combinatorial libraries of SFTI-
1
analogues. Multiple ap-
proaches have been used to syn-
thesize such libraries.[35,37] The
novel technology presented in
this work enables improved
access to libraries of cysteine-
rich cyclic peptides and demon-
strates the potential of the ap-
proach for combinatorial-chemis-
try-based drug discovery.
The synthesis of SFTI-1 (9) was
performed on the aminomethyl-
PEG based resin (1-ChemMatrix)
resulting in superior product
quality. This resin has been used
in our hands very successfully
for difficult sequences[28d] as well
as for the synthesis of selenocys-
Figure 2. A) Cyclization of peptide a-thio-
esters 6d–f via intramolecular native liga-
tion: free thiol from thiol linker or added
as MESNA catalyzes vicinal disulfide thiol
exchange, thereby facilitating cyclization.
Cyclized and vicinal disulfide-containing
peptides 7d–f are formed in aqueous
buffer at pH 7.4, versus formation of cy-
clized and reduced cysteine-containing
peptides 8d–f with the thiol MESNA in
excess. B) HPLC–MS monitoring of cycliza-
tion of peptide a-thioester 6d ([M+H]+
1411.8), yielding simultaneous cyclized and
oxidized peptide 7d ([M+H]+ 1249.8) if re-
acted in a 0.1m NH4HCO3 buffer at pH 7.4.
In contrast, in the presence of excess
MESNA (5 equiv), cyclic reduced peptide
8d ([M+H]+ 1251.8) is formed.
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teine-containing a-conopeptides.[26] The ChemMatrix resin was
pretreated in DMF overnight, and a sequence of three glycine
residues was attached to improve the handling of the resin.
After attachment of the SCAL followed by a further glycine res-
idue, the mercaptopropionic acid linker was attached. The syn-
thesis of SFTI-1-thioester 3g was performed using in situ neu-
tralization Boc-SPPS[12] employing HBTU as the coupling re-
agent with good yields throughout. The sequence was modi-
fied to allow for NCL with an N-terminal cysteine and an argi-
nine residue adjacent to the thioester (sequence:
CTKSIPPICFPDGR). After completion of chain assembly, the
resin was transferred into a tea bag, and HF cleavage was per-
formed (3g!4g). The SFTI-1 resin 4g was then treated with
NH4I/DMS/TFA for 4 h to cleave the SCAL and generate the
SFTI-1 thioester 6g in a moderate yield of 55%. In contrast to
the PS-based resin used for the synthesis of 3a–f, extended
cleavage times did not improve the yield. The cyclization of
crude SFTI-1-thioester 6g was performed in NH4HCO3 solution
(pH 7.6) and did not require a thiol catalyst (Figure 3), giving
the fully oxidized SFTI-1 9, which co-eluted with an authentic
Figure 4. Comparison of the secondary a-shift NMR data of synthesized
SFTI-1 (9) with wild-type SFTI-1.
Conclusions
A general method for the synthesis of peptide a-thioester li-
braries has been established by employing a safety catch
amide linker (SCAL)[24] and compartmentalizing the peptide
resin during HF cleavage. The peptide thioesters synthesized
containing additional cysteine
residues were subsequently
readily cyclized employing intra-
molecular NCL. The synthetic
technology was then applied to
the synthesis of the biologically
important sunflower trypsin in-
hibitor 1 (SFTI-1, 9), a lead mole-
cule for the development of
serine protease inhibitors with
potential for cancer therapy. In
summary, we have successfully
developed
a
technology to
remove the bottleneck of pep-
tide thioester synthesis, making
access to cyclic peptide libraries
highly feasible.
Experimental Section
Materials and methods
All solvents and reagents were ob-
tained commercially and were
used without further purification.
Na-Boc-l-amino acids were pur-
chased from NovaBiochem (Merck
Pty., Kilsyth, Vic., Australia). The fol-
lowing side chain protected Boc-
Figure 3. Cyclization of crude peptide a-thioester 6g ([M+H]+ 1678.2) in 0.1m NH4HCO3 buffer at pH 7.6 yielding
simultaneous cyclized and oxidized peptide 9 (SFTI-1, [M+H]+ 1514.2).
sample.[35] To further confirm the identity of the product 9,
NMR was used to compare the secondary Ha chemical shifts
with wild-type SFTI-1 and demonstrated the structural identity
of both molecules (Figure 4). Furthermore, the activity of prod-
uct 9 as a protease inhibitor was confirmed by using a colori-
metric assay[38] with the substrate l-BAPNA, in which the prod-
uct 9 produced the same protease inhibition as did wild-type
SFTI-1.[38b]
amino acids were used: Arg(Tos), Asn(Xan), Asp(Chxl), Cys(4-MeBzl),
Gln(Xan), Tyr(2-BrZ), Thr(Bzl), Trp(For), Ser(Bzl), Lys(2-ClZ). N,N-Dime-
thylformamide (DMF), diisopropylethylamine (DIEA), and trifluoro-
acetic acid (TFA) were all peptide synthesis grade supplied by
Auspep P/L (Melbourne, Australia). 2-(1H-Benzotriazol-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate (HBTU), ammonium
iodide, triisopropylsilane (TIPS), dimethyl sulfide (DMS), HPLC-grade
acetonitrile, diethyl ether, methanol, bovine trypsin, N-a-benzoyl-
l-arginine 4-nitroanilide hydrochloride (BAPNA) and scavengers p-
cresol and p-thiocresol were supplied by Sigma–Aldrich (Australia).
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Anhydrous HF gas was purchased from BOC Gases (Sydney, NSW,
Australia). Aminomethyl polystyrene hydrochloride resin (loading=
0.17 mmolgꢁ1, 100–200 mesh, 1% DVB) was purchased from Nova-
Biochem (Merck Pty., Kilsyth, Vic., Australia). Aminomethyl Chem-
Matrix hydrochloride resin (loading=0.74 mmolgꢁ1, 35–100 mesh)
was purchased from Matrix Innovation (Montreal, QC, Canada).
Fmoc-SCAL was purchased from CSPS Pharmaceutical (San Diego,
CA, USA). Polypropylene mesh sheets for the assembly of tea bags
were purchased from Cole–Parmer (Vernon Hills, IL, USA).
the side chain protecting groups, but does not cleave the peptide
from the SCAL, was carried out in apparatus supplied by the Pep-
tide Institute (Osaka, Japan) using 50 mL HF covering all tea bags
and scavenger (p-cresol, 5% vol) for 1 h at 08C. After removal of
excess HF, the peptide resin tea bags were washed with diethyl
ether and CH2Cl2 and dried under vacuum.
SCAL activation/cleavage
The washed and dried peptidyl-CO-S-(CH2)2-CO-Gly-SCAL-AM-PS
resin or peptidyl-CO-S-(CH2)2-CO-Gly-SCAL-Gly3-AM-ChemMatrix
resins (4a–g), still in the tea bags, were separated into individual
cleavage vessels. To achieve safety catch linker activation 50 mg of
NH4I, 100 mL DMS and 2.5 mL neat TFA were added. The cleavage
was performed, while shaking, for 18 h (PS-based resin) or 4 h
(ChemMatrix resin) at RT. After SCAL activation/cleavage the pep-
tide solution was filtered, and cold diethyl ether (30 mL) was
added to the cleavage mixtures resulting in precipitation of the re-
spective peptide thioester (6a–g). The precipitate was collected by
centrifugation and subsequently washed with further cold diethyl
ether (2ꢁ20 mL). The final product was dissolved in 50% aqueous
acetonitrile/0.1% TFA (30 mL) and lyophilized to yield a white
solid. The crude peptide thioesters (6a–g) were examined by re-
versed-phase HPLC for purity, and the correct molecular weight
confirmed by electrospray ionization mass spectrometry (ESIMS).
For obtained crude yields and LC–MS data, see Table 1.
HS-CH2CH2-CO-Gly-SCAL-AM-PS resin (2-PS)
Aminomethyl polystyrene hydrochloride resin (5 g, 0.17 mmolgꢁ1
)
was conditioned overnight in DMF. After washing with DMF, the
resin was neutralized (1 min) with a 10% solution of DIEA in DMF
(10 mL, 2ꢁ5 min). Fmoc-SCAL (1.74 g, 2.7 mmol) was dissolved in
5.4 mL of a solution of HBTU in DMF (0.5m, 2.7 mmol). After activa-
tion with 470 mL (2.7 mmol) DIEA, the linker mixture was added to
the resin and coupled overnight. After Fmoc removal with piperi-
dine (50% in DMF, 2ꢁ2 min), Boc-glycine (2.7 mmol) was coupled
using HBTU/DIEA activation. After Boc deprotection with neat TFA,
the 3-trityl-mercaptopropionic acid linker (2.7 mmol) was attached
by HBTU (2.7 mmol)/DIEA (3.0 mmol) activation, followed by the re-
moval of trityl protection by treatment with a solution of 5% TIPS
and 5% water in TFA (10ꢁ1 min). The HS-(CH2)2-CO-Gly-SCAL-AM-
PS resin (2-PS) with a substitution value (SV) of 0.1 mmolgꢁ1 was
used for the synthesis of peptide thioester 6a–f. The obtained SV
was determined by using the quantitative ninhydrin test.[29]
Cyclisation of peptide thioester 6d–f
HS-CH2CH2-CO-Gly-SCAL-(Gly)3AM-ChemMatrix resin (2-Chem-
Matrix)
The crude peptide thioesters 6d–f were cyclized employing intra-
molecular native chemical ligation under two different conditions:
Aminomethyl-ChemMatrix hydrochloride resin (1 g, 0.74 mmolgꢁ1
)
A: cyclization in 0.1m NH4HCO3 (pH 7.6) was performed at a pep-
tide concentration of 1 mgmLꢁ1. The progress of the cyclization
was monitored by LC–MS. The cyclization resulting in a loss of an
S-(CH2)2-CO-Gly-NH2 moiety (Dm=ꢁ162 amu) was completed
within 3 h, yielding a single main product (7d–f), the backbone
amide cyclized with a disulfide bond formed as well.
was conditioned overnight in DMF. By employing in situ neutraliza-
tion Boc chemistry,[12] a triple glycine sequence (3ꢁ2.7 mmol) was
attached consecutively to achieve a less sticky resin that allowed
for better handling during ninhydrin testing.[29] Identical conditions
were employed as for the assembly of resin 2-PS to attach the
SCAL (2.7 mmol) as well as the thiol linker (2.7 mmol, see above).
The HS-(CH2)2-CO-Gly-SCAL-Gly3AM-ChemMatrix resin (2-Chem-
Matrix, SV=0.47 mmolgꢁ1) was used for the synthesis of peptide
thioester 6g.
B: cyclization in 0.1m NH4HCO3 in the presence of MESNA (5 equiv)
with a peptide concentration of 1 mgmLꢁ1. The progress of the
cyclization was monitored by LC–MS. (Dm=ꢁ160 amu). The cycli-
zation was completed within 2 h, yielding a single main product
identified as backbone cyclized peptide with reduced cysteine
(8d–f). For obtained crude yields and MS data, see Table 1.
Peptide synthesis
The chain assembly of the peptides (0.1 mmol resin used) was per-
formed on
a manual shaker system using Boc-amino acid
Cyclization of peptide thioester (6 g) to SFTI-1 (9)
(0.5 mmol)/HBTU (0.5 mmol)/DIEA (0.6 mmol) activation and in situ
neutralization protocols[12] to couple the Boc-protected amino acid
to the resins (2) prepared above. The Boc protecting group was re-
moved using 100% TFA (2ꢁ), and DMF was used as both the cou-
pling solvent and for flow washes throughout the cycle. The prog-
ress of the assembly was monitored by quantitative ninhydrin
assay.[29]
Crude peptide thioester (6g, 17 mg) was dissolved in 20 mL 0.1m
NH4HCO3 solution (pH 7.6) and stirred at room temperature. Moni-
toring by LC–MS indicated a straightforward cyclization and oxida-
tion to SFTI-1 (9) within 3 h (Dm=ꢁ164 amu). For obtained yields
and MS data, see Table 1.
HPLC analysis and purification
Parallel HF side chain deprotection
Analytical HPLC was performed using a Shimadzu HPLC system
LC10A with a dual-wavelength UV detector set at 214 and 254 nm.
A reversed-phase C18 column (Hypersil Gold C18, 3 mm, 100 mmꢁ
2.1 mm) was used at a flow rate of 0.3 mLminꢁ1. Gradient elution
was performed (408C) with the following buffer systems: A, 0.05%
TFA in water and B, 0.043% TFA in 90% acetonitrile in water, from
After completion of assembly of peptides 3a–g, subsequent re-
moval of the Na-Boc group, using TFA (2ꢁ1 min) and flow-washes
with DMF, CH2Cl2, and drying under nitrogen, the peptide resin
was transferred into individual tea bags. All tea bags were trans-
ferred into one HF reactor tube. The HF cleavage, which removes
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0% B to 80% B in 20 min. Absorbance was monitored at 214 and
254 nm, and crude purities are given by peak areas at 214 nm.
125 mL l-BAPNA. The plate was incubated in the dark for 10 min,
the reaction stopped by addition of 25 mL 30% acetic acid, and the
plate was read at 410 nm using a fluorescence plate reader.
Peptides were purified by preparative HPLC on a Shimadzu HPLC
system associated with a reversed-phase C18 column (Vydac C18,
25 cmꢁ25 mm) at a flow rate of 15 mLminꢁ1 with a 0.5% per min
increase in buffer B concentration from 10–60% B. The purity of
the final products was evaluated by analytical HPLC (Hypersil C18,
130 ꢂ, 5 mm, 250 mmꢁ4.6 mm, 1 mLminꢁ1 flow rate, gradient 10%
B to 60% B in 50 min).
Acknowledgements
The authors acknowledge financial support by the National
Health and Medical Research Council (NHMRC) of Australia (Pro-
gram Grant No. 351446).
ESIMS
Keywords: Boc chemistry
· high-throughput synthesis ·
Electrospray ionization mass spectra were collected inline during
analytical HPLC runs on an Applied Biosystems, quadrupole spec-
trometer (API-150) operating in the positive ion mode with a de-
clustering potential (DP) of 20 V, a focusing potential (FP) of 220 V,
and a turbospray heater temperature of 3508C. Masses between
300 and 1800 amu were detected (Step 0.2 amu, Dwell 0.3 ms).
peptides · solid-phase synthesis · thioesters
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1
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Bioassays
N-a-Benzoyl-l-arginine 4-nitroanilide hydrochloride,[38] (BAPNA) is
a colorless chromogenic trypsin substrate that turns yellow when
hydrolyzed, and light absorption can be determined at 410 nm.
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activity, and the readout is the percent inhibition of trypsin activity
displayed by the inhibitor, in this case SFTI, compared with a con-
trol, such as buffer. The inhibitory activity of SFTI-1 was assayed in
a 96-well plate against bovine trypsin using the colorimetric trypsin
substrate l-BAPNA.[38] Briefly, SFTI-1 (9) was diluted from a stock of
0.1 mm in 0.05m Tris·HCl (pH 8.2) with 0.02m CaCl2 (assay buffer).
Trypsin was dissolved in 1 mm HCl to
a concentration of
4.5 mgmLꢁ1. Immediately prior to use, the trypsin was diluted 1:10
in the assay buffer. l-BAPNA (0.435 mgmLꢁ1) was dissolved in
assay buffer with 1% DMSO. Peptide (15 mL, at various concentra-
tions), and trypsin (5 mL) were added to the plate followed by
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Received: December 13, 2013
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Published online on March 3, 2014
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ChemMedChem 2014, 9, 1038 – 1046 1046