Construction of Challenging Proline-Proline Junctions via Diselenide-Selenoester Ligation Chemistry

Article abstract of DOI:10.1021/jacs.8b07877

Polyproline sequences are highly abundant in prokaryotic and eukaryotic proteins, where they serve as key components of secondary structure. To date, construction of the proline-proline motif has not been possible owing to steric congestion at the ligatio

Full text of DOI:10.1021/jacs.8b07877

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Article
Construction of Challenging Proline-Proline Junctions
via Diselenide-Selenoester Ligation Chemistry
Jessica Sayers, Phillip M. T. Karpati, Nicholas J. Mitchell, Anna M.
Goldys, Stephen M. Kwong, Neville Firth, Bun Chan, and Richard J Payne
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 21 Sep 2018
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Construction of Challenging Proline-Proline Junctions via
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Diselenide–Selenoester Ligation Chemistry
Jessica Sayers,a‡ Phillip M. T. Karpati, a‡ Nicholas J. Mitchell, a† Anna M. Goldys,a Stephen M. Kwong,b
Neville Firth,b Bun Chan,c Richard J. Payne a*
a School of Chemistry, The University of Sydney, Sydney, NSW 2006, Australia
b School of Life and Environmental Sciences, The University of Sydney, Sydney, NSW 2006, Australia
c Graduate School of Engineering, Nagasaki University, Bunkyo 1-14, Nagasaki 852-8521, Japan
ABSTRACT: Polyproline sequences are highly abundant in prokaryotic and eukaryotic proteins where they serve as key compo-
nents of secondary structure. To date, construction of the proline-proline motif has not been possible owing to steric congestion at
the ligation junction, together with an n→* electronic interaction that reduces the reactivity of acylated proline residues at the
C-terminus of peptides. Here, we harness the enhanced reactivity of prolyl selenoesters and a trans-γ-selenoproline moiety to access
the elusive proline-proline junction for the first time through a diselenide–selenoester ligation–deselenization manifold. The effi-
cient nature of this chemistry is highlighted in the high yielding one-pot assembly of two proline-rich polypeptide targets, submax-
illary gland androgen regulated protein 3B and lumbricin-1. This method provides access to the most challenging of ligation junc-
tions, thus enabling the construction of previously intractable peptide and protein targets of increasing structural complexity.
terminal -branched residue e.g., valine (Val), threonine
(Thr) or isoleucine (Ile).11 Furthermore, thioesters bearing
a C-terminal proline (Pro) residue have proven to be un-
reactive acyl donors in traditional NCL reactions, includ-
ing at Cys, selenocysteine (Sec) and thiol/selenol amino
acids.12-13 This lack of reactivity has been attributed to a
reduction in electrophilicity of the thioester due to a stabi-
lizing n → π* electron donation from the proximal amide
carbonyl to the carbonyl of the thioester moiety.14-15 This
stabilizing interaction is enabled by the rigid trans-
configured peptide bond induced by the cyclic Pro resi-
due.14-15 Attempts to overcome the challenge of perform-
ing ligation chemistry at Pro thioesters have focused on
enhancing the reactivity of the acyl donor component.
One approach has involved substituting the prolyl thioe-
ster with a more reactive selenoester functionality
(Scheme 1A).16 These selenoester acyl donors have been
reported to react with N-terminal Cys-containing pep-
tides, albeit in the presence of a selenol catalyst and a
large molar excess of the acyl donor fragment. More re-
cently, Dong et al. have designed a prolyl thioester
INTRODUCTION
Peptide ligation chemistry has revolutionized protein science
by providing a means to access large polypeptide and pro-
tein targets,1-3 including those that cannot be generated by
recombinant expression technologies. This includes the
ability to site-specifically incorporate non-proteinogenic
amino acids, post-translational modifications and/or iso-
topic or fluorescent labels.2 The most widely adopted
method for the convergent assembly of peptide fragments
to afford large polypeptides and proteins is undoubtedly
native chemical ligation (NCL). This technology, first
reported by Kent and co-workers,4 involves the chemose-
lective reaction of a peptide possessing a C-terminal thi-
oester functionality with a second fragment bearing an N-
terminal cysteine (Cys) residue. Whilst this technology
has enabled access to numerous protein targets to date,1-3
the conventional reaction is limited by the requirement of
a Cys residue at the ligation junction. This restriction has,
however, been recently addressed through the develop-
ment of NCL at thiol-derived amino acids,5 selenocyste-
ine6-8 as well as a number of synthetic selenol-derived
amino acids9 that can serve as cysteine surrogates in reac-
tions with peptide thioesters. Importantly, following the
ligation reaction, these thiol and selenol auxiliaries can be
removed through desulfurization or deselenization chem-
istry, respectively, to afford native polypeptides and pro-
teins.10 One further limitation of the NCL manifold is that
reactions typically proceed slowly at sterically encum-
bered thioesters, particularly those containing a C-
whereby the -position of the Pro ring is functionalized
with a thiol moiety (Scheme 1B).17 This modified Pro
thioester reacts via a bicyclic thiolactone intermediate
which leads to activation of the carbonyl through the gen-
eration of a highly strained cyclic thioester. While this is
a very elegant strategy, the -thiol auxiliary must be re-
moved via a post-ligation desulfurization protocol and
therefore lacks chemoselectivity in the presence of native
Cys residues found elsewhere in the sequence. Danishef-
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sky and co-workers have reported the use of both thiopro-
example of the construction of challenging and hitherto
inaccessible Pro-Pro junctions through ligation chemistry
(Scheme 1D), as well as the application of this method in
the synthesis of two large Pro-rich polypeptide targets.
line and selenoproline residues in NCL reactions with
peptide thioesters (Scheme 1C).13, 18 These ligation reac-
tions worked well at unhindered sites, but were inefficient
at more sterically encumbered C-terminal thioesters, e.g.,
Val, even with the enhanced nucleophilicity of the sele-
noproline moiety. Importantly, when peptides bearing N-
terminal thioproline or selenoproline residues were react-
ed with peptide thioesters bearing a C-terminal Pro no
reaction was observed. This inability to forge Pro-Pro was
attributed to the deactivation of the Pro thioester compo-
nent through the n → π* interaction from the adjacent
carbonyl (vide supra).
RESULTS AND DISCUSSION
Design and Synthesis of -Selenoproline Building Block.
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Before embarking on the development of the DSL meth-
odology at Pro, we first needed to prepare a suitably pro-
tected selenol-derived Pro residue that would be compati-
ble with solid-phase synthesis protocols and the ensuing
ligation chemistry. Danishefsky and co-workers have
previously reported the preparation of a -selenoproline
diselenide amino acid.13 We initially replicated this syn-
thetic route (see Supporting Information), but as reported
for other diselenide building blocks,12 we observed that
only one half of the diselenide amino acid was able to
couple to a resin-bound peptide chain (i.e. the other sele-
noproline unit within the diselenide dimer was unable to
couple to another peptide N-terminus). This is particularly
problematic in larger immobilized targets or when pep-
tides are more highly loaded on resin supports (> 0.5
mmol/g). In order to mitigate these issues, we sought to
synthesize a monomeric selenoproline building block
whereby the -selenol moiety was orthogonally protected
with a -methoxy benzyl group (PMB). The synthesis
began with the C-terminal protection of commercially
1,

p
available trans
-

-hydroxyproline
2
as a methyl ester
3
(Scheme 2A). Conversion to cis
performed under Mitsunobu conditions with inversion of
-

-iodoproline
4
was then
stereochemistry at the position. Direct treatment of io-
dide
4 with 4-methoxybenzyldiselenide 5 in the presence
Scheme 1. Ligation reactions between: A) A prolyl selenoes-
ter and cysteinyl peptide; B) Thiolated prolyl thioester and
cysteinyl peptide that proceeds via a thiolactone intermediate;
C) Thioesters and thio- or selenoproline peptides; D) Prolyl
selenoester and selenoproline peptides via diselenide–
selenoester ligation (DSL).
of NaBH4 then provided trans-γ-selenoproline methyl
ester . Finally, saponification of the methyl ester afford-
ed the target trans
yield over the 4 steps.
6
-

-selenoproline amino acid
1
in good
Synthesis of Peptides Bearing -Selenoproline.
We have recently reported the development of an addi-
tive-free peptide ligation reaction between peptides bear-
ing a C-terminal selenoester and peptides containing an
N-terminal selenocystine residue (the oxidized form of
Sec).12 Importantly, these represent the fastest ligation
reactions known, proceeding efficiently in aqueous buffer
without the addition of external catalysts or reductants to
afford selenopeptide products in minutes (including at
With monomeric selenoproline building block 1
in hand,
we next investigated the efficiency of incorporation into
the N-terminus of Rink amide resin immobilized pen-
tapeptide
pleased to observe complete coupling to
slight excess of (1.2 equiv.) under standard coupling
conditions [HOAt (1.2 equiv.), DIC (1.2 equiv.), in DMF]
to generate resin-bound selenopeptide . Acidolytic side-
7
loaded at 0.7 mmol/g (Scheme 2B). We were
7
using only a
1
8
chain deprotection with concomitant cleavage from the
resin followed by deprotection of the PMB group (using
selenoesters bearing C-terminal -branched amino acids).
Upon reaction completion, and without purification, the
selenocystine at the ligation junction can be cleanly con-
verted to alanine or serine through reductive19-20 or oxida-
20% DMSO, 5%
reverse-phase HPLC provided the model diselenide dimer
peptide in 50% yield (based on the resin loading of the
first amino acid).
iPr3SiH in TFA) and purification via
tive deselenization,21-22 respectively. This diselenide
–
9
selenoester ligation (DSL) manifold has been extended to
alternative selenol-derived amino acids including
selenoaspartate, -selenoleucine and -selenoglutamate

-


and has been successfully employed for the rapid and
high yielding synthesis of a number of protein and sele-
noprotein targets.23-24 Following the establishment of the
DSL technology, we were interested in probing whether
the method could be used for the construction of peptides
at more challenging junctions. Herein, we describe our
efforts to extend the application of DSL
–deselenization to
amino acid junctions containing Pro; we outline the first
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Table 1. Model Ligation–Deselenization reactions between
diselenide dimer 9 and selenoesters 10–14
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Entry
Ac-LYRANX-
SePh 10 14 (X=)
Ligation
Time
(min)
Yield for One-Pot
Ligation–
Deselenization
–
1
2
3
4
5
Ala (10
)
5
56% (15
52% (16
60% (17
51% (18
54% (19
)
)
)
)
)
Met (11
)
10
10
10
45
Tyr (12
)
Leu (13
)
Val (14
)
Ligation: 9 and 10–14 in ligation buffer (6 M Gdn•HCl, 0.1 M
Na2HPO4), rt, pH 6.2, [9] = 2.5 mM, [10–14] = 6.5 mM, 5–45
min. in situ deselenization: Hexane extraction followed by addi-
tion of an equivalent volume of TCEP (250 mM) and DTT (20
mM) in buffer (6 M Gdn·HCl, 0.1 M Na2HPO4, pH 4.5–5.5)
Scheme 2. A) Synthetic route to trans-γ-selenoproline 1; B)
Incorporation of trans-γ-selenoproline 1 building block into
model peptide 7 via Fmoc-SPPS.
It should be noted that a fine precipitate of diphenyl
diselenide (DPDS) is produced during the ligation reac-
tion which serves as a visual indication of reaction com-
pletion.12 Upon completion of the ligation reaction, the
DPDS precipitate was removed via hexane extraction and
the deselenization step was effected by treatment with
tris-(2-carboxyethyl)phosphine (TCEP, 250 mM) and
dithiothreitol (DTT, 20 mM) in 6 M Gdn•HCl, 0.1 M
One-Pot DSL–Deselenization at -Selenoproline.
With model peptide
9 in hand, the ability of the dise-
lenoproline motif to participate in additive-free peptide
ligation chemistry with a range of model selenoesters
(Ac-LYRANX-SePh) was investigated (Table 1). We first
assessed the reaction of
9 with peptide selenoesters 10–14
bearing C-terminal Ala (A), Met (M), Tyr (Y), Leu (L)
and Val (V) residues as a representative selection of the
proteinogenic amino acids (see Supporting Information
for details on peptide selenoester synthesis). Specifically,
phosphate buffer at pH 5.0. In situ deselenization of the -
selenoproline moiety proceeded to completion within
16 h to afford the desired native Pro-containing peptides.
Purification via reverse-phase HPLC provided 15
excellent yields (51–60%) over the two steps. Having
established that the -selenoproline moiety is competent
in additive-free DSL deselenization chemistry, we next
investigated whether the method could be expanded to
include C-terminal Pro selenoesters in an attempt to forge
previously intractable Pro-Pro junctions. Importantly,
polyproline sequences are highly abundant in peptides
and proteins across all taxa; more than 33% of E. coli
proteins25 and ca. 25% of human proteins (see Supple-
mentary Table) possess one or more polyproline motifs. A
method to construct such junctions would therefore be
highly valuable. As an initial assessment of the ligation
efficiency, Ac-LYRANP-SePh 20 (2.0 equiv.) was react-
–19 in
diselenide dimer
9
and peptide selenoesters 10
–14 (1.3
equiv. with respect to monomeric
9) were simply dis-

–
solved in 6 M Gdn•HCl, 0.1 M phosphate buffer at pH 6.2
without the addition of any other exogenous additives.
Equal volumes of the solutions were combined to a final
concentration of 2.5 mM diselenide dimer
selenoester 10 14. In all cases the ligation reached com-
pletion within 5 45 min (as judged by UPLC-MS analy-
9 and 6.5 mM
–
–
sis) to afford the ligation product as a mixture of the
symmetrical diselenide, and the product additionally ac-
ylated at the γ-selenol moiety via
a
trans-
selenoesterification reaction with excess selenoester start-
ing material (also observed with the parent reaction at
Sec,12 see Supporting Information). The mixture of liga-
tion products formed is ultimately inconsequential as all
products converge to the desired native peptide following
in situ deselenization.
ed with diselenide dimer peptide
spect to the monomer) in 6 M Gdn•HCl, 0.1 M phosphate
buffer at pH 6.2. Whilst the reaction proceeded more
9 (1.0 equiv. with re-
slowly than with selenoesters 10–14, we were delighted to
observe clean and complete conversion within 16 h
(Scheme 3A). Extraction of the DPDS precipitate and in
situ deselenization then provided the desired Pro-Pro con-
taining peptide 21 in 54% yield over two steps. To our
knowledge, this represents the first successful example of
a peptide ligation at the sterically encumbered and elec-
tronically disarmed Pro-Pro junction, thus providing a
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unique advantage of the DSL reaction manifold. Encour-
aged by the successful Pro-Pro ligation under the DSL
manifold, we next explored the scope of the chemistry at
lenging Pro-Pro junctions. We also envisaged that the
reductant would be capable of regenerating phenylseleno-
late from DPDS which would perform selenolysis of the
product selenoesters that are generated during the reac-
tion, thus enabling the reduction of the molar excess of
the selenoester component required (2.0 equiv. under ad-
ditive-free conditions, vide supra). We chose to use TCEP
as the reductant and DPDS as the radical trap, which can
be easily extracted prior to in situ deselenization. Liga-
another Pro-derived residue, namely
-hydroxyproline
(Hyp), a common post-translationally modified residue
found in proteins, e.g., collagens. Studies began with the
synthesis of model peptide selenoester 22 possessing a C-
terminal Hyp residue (Ac-LYRANP(OH)-SePh, see Sup-
porting Information for details). Pleasingly, the additive-
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tions between peptide diselenide dimer
9 and selenoesters
free DSL reaction between 22 and
9 also proceeded to
20 and 22 were repeated in the presence of TCEP (50
mM) and DPDS (20 mM) using 1 equiv. of the selenoes-
ter component (Scheme 3A). These ligations proceeded to
completion with only ca. 3 – 5% deselenization of the
completion within 16 h and, following in situ deseleniza-
tion and purification by HPLC, peptide product 23 was
isolated in 55% yield (Scheme 3A). In order to show that
DSL reactions at prolyl selenoesters were also competent
with other selenoamino acids, we next performed ligation
between peptide selenoester 20 and peptides bearing N-
starting diselenide dimer
9 over a period of 16 h, but in-
terestingly, did lead to substantial deselenization of the
ligation product. Nonetheless, upon extraction of the ex-
cess DPDS with hexane and treatment with TCEP and
DTT, a single deselenized product could be obtained.
Purification via reverse phase HPLC afforded the desired
peptide products 21 and 23 bearing Pro-Pro and Hyp-Pro
motifs in 51% and 64% yield, respectively. Importantly,
terminal selenocystine and -selenoaspartate residues as
the corresponding diselenide dimers (see Supporting In-
formation). These reactions proceeded cleanly within 16 h
and subsequent deselenization provided products contain-
ing Ala and Asp at the ligation junction in good yield.
these experiments demonstrate that DSL reactions at -
selenoproline can be performed using reductants in the
presence of a radical quenching agent (in this case
DPDS), however, there is no benefit to the rate of the li-
gation reaction. Finally, we envisaged that the use of ad-
ditives in forging Pro-Pro junctions via DSL chemistry
would be beneficial for substrates possessing unprotected
Cys residues. Specifically, the inclusion of TCEP and
DPDS would enable any unproductive thioesters that may
form to be selenolyzed with phenylselenolate. Towards
this end, reactions were performed with and without addi-
tives (TCEP, DPDS) on a model peptide containing an
unprotected internal Cys residue (see Supporting Infor-
mation). Gratifyingly, the reactions proceeded smoothly
and at similar rates under both additive- and additive-free
manifolds. Following chemoselective deselenization19 of

-Se-Pro, native peptides bearing an intact internal Cys
residue were generated in good yields (see Supporting
Information).
Effects of -Selenoproline Stereochemistry on Ligation Effi-
ciency.
Scheme 3. A) Ligation–deselenization between diselenide
dimer 9 bearing an N-terminal trans--selenoproline and se-
lenoesters 20 and 22 under additive-free and additive condi-
tions (1 equiv. 20 or 22 used in the additive reaction with 50
mM TCEP, 20 mM DPDS); B) Ligation between cis--
selenoproline diselenide dimer 25 and selenoesters 20 and 22
leading to unrearranged selenoester intermediates 26 and 27.
Having shown that the reaction was tolerant of a num-
ber of selenoester reactants, we were next interested in
investigating the effect of the stereochemistry at the
position of the selenoproline unit on the productivity of
the ligation. As such, we prepared cis -selenoproline
building block 24 using a similar route to that developed
for (Scheme 3B). Stereochemical inversion at the γ-
position was effected by treatment of a tosylated variant
of -hydroxyproline with diselenide under reducing
-
-

1
Pro-Pro Ligation in the Presence of Additives.
Together with the additive-free DSL
chemistry, we have previously shown that diselenide pep-
tides (with the exception of those bearing N-terminal
selenoaspartate or
-selenoglutamate residues22) can be
–deselenization

5
conditions (see Supporting Information for full synthetic

-
details). The cis-γ-selenoproline 24 was subsequently
coupled to resin-bound peptide 7 and subjected to
acidolytic cleavage, side chain deprotection and PMB
removal. The final HPLC-purified diselenide dimer
peptide 25 bearing cis--selenoproline was obtained in
66% yield (based on the loading of the first amino acid).
Interestingly, ligation of 25 to both Ac-LYRANP-SePh
20 and Ac-LYRANP(OH)-SePh 22, under both additive-
free and additive conditions, failed to afford the desired
ligation product over 16 h. Instead, formation of

successfully ligated with peptide selenoesters in the pres-
ence of additives, specifically the phosphine reductant
TCEP. Since TCEP is able to facilitate deselenization
reactions, a radical trapping agent such as ascorbic acid26
or DPDS must also be used to avoid this deleterious side
reaction (until the ligation has reached completion). We
were interested in exploring whether the inclusion of ad-
ditives would accelerate the ligation reaction at the chal-
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unrearranged selenoesters of 25 (i.e. branched
algorithm together with molecular mechanics. These
structures were then optimized with density functional
theory (DFT) using the PBE/6-31G(d) method; improved
single-point energies were subsequently obtained using
the higher-level M06-2X/6-311G(2df,p) method (see
Supporting Information). Using this method we found
selenoesters 26 and 27) were observed as the major
products (Scheme 3B). This was confirmed upon
treatment of 26 and 27 with aqueous hydrazine to effect
cleavage of the side-chain selenoester to reafford 25 and
form the acyl hydrazide of 20 and 22 (see Supporting
Information). Importantly, the results of this study sug-
that the low-energy structures for the trans-isomer (e.g.,
in Figure 1B) have the carbonyl carbon of the selenoester
moiety in close proximity to the Pro -NH and at a suita-
I
gest that the normally rapid SeN acyl shift is unable to
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proceed when the γ-seleno moiety is in the (
configuration i.e. cis-configured with respect to the
amine moiety.
S)-


-
ble geometry to facilitate the intramolecular acyl shift, a
pathway that is observed experimentally. Logically, one
can envisage that inversion of the stereochemistry at the
γ-selenoproline unit would place the Pro
-NH away
from this more optimal location. Indeed, despite sampling
hundreds of conformations (e.g., II) for the cis-isomer,
none place the carbonyl carbon of the selenoester in a
position that would lead to a favourable Se
the Pro -NH. It is interesting to note that our calcula-
tions suggest that the Se N acyl shift would be further
N shift with


favored for the trans-isomer from an acyl-selenonium
species if generated under the reaction conditions (see
Supporting Information for data).12 In summary, our DFT
results explain the experimental observation that the se-
lenoesters, 26 and 27, are formed but do not rearrange.
Synthesis of Proline-rich Polypeptides.
Having successfully developed a robust method for the
one-pot DSL–deselenization at -selenoproline, and more
importantly, demonstrated the first successful ligation at
Pro-Pro, we next turned our attention to the application of
this technology for the preparation of larger polypeptides.
For this purpose we first chose to target the synthesis of
the Pro-rich polypeptide SMR3B (submaxillary gland
androgen regulated protein 3B 28) using the additive-free
manifold.27 The 57-amino acid sequence of SMR3B is
Cys-and Ala-free and contains a total of 30 Pro residues
(53%), 7 of which form a centrally located polyproline
stretch. We envisaged a disconnection between Pro27 and
Pro28 within this motif that would facilitate a simple two-
fragment assembly via the Pro-Pro DSL–deselenization
method. Fragment 29 (SMR3B 1–27) bearing a C-
terminal proline selenoester and 30 (SMR3B 28–57) con-
Figure 1. Proposed selenoester intermediates for trans- (I, not
observed experimentally) and cis- (II, observed experimental-
ly) -selenoproline isomers; A) Chemdraw representations, B)
low-energy structures. Hydrogen atoms are omitted for clari-
ty.
taining an N-terminal trans--selenoproline diselenide
dimer were first synthesized via Fmoc-SPPS on 2-
chlorotrityl chloride resin (see Supporting Information).
The resulting fragments 29 (10 mM final conc.) and 30 (5
mM final conc. with respect to the monomer), were then
subjected to ligation in 6 M Gdn•HCl, 0.1 M phosphate
buffer at a final pH of 6.4. The reaction was monitored by
UPLC-MS and reached completion within 16 h. Extrac-
tion of the precipitated DPDS with hexane, followed by in
situ deselenization with TCEP and DTT successfully gen-
erated the native polypeptide. Purification via reverse-
phase HPLC then provided SMR3B 28 in 52% yield.
In addition to SMR3B, we also chose to prepare the
62-amino acid antimicrobial peptide lumbricin-1 via one-
Computational Studies.
In order to help rationalize the strict stereochemical re-
quirement for the SeN acyl shift of the selenoester in-
termediate, and therefore productive ligation at Pro-Pro,
we turned to computational studies. Specifically, using
model systems for the selenoester intermediate generated
with trans- and cis--selenoproline diastereoisomers (Fig-
ure 1A), we extensively examined the possible conforma-
tional space for both to determine the likelihood of the
acyl shift proceeding. Towards this end, we assessed a
large number of initial conformers using a Monte Carlo
pot DSL
–deselenization chemistry at-selenoproline.
Lumbricin-1 is a Pro-rich peptide (15% Pro content) iso-
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completion within 16 h. On
this occasion extraction of
the DPDS, followed by in
situ deselenization with
TCEP and DTT did not
proceed to completion,
attributed to the inability of
DTT to thiolyze the prod-
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uct selenoester generated in
the reaction. Therefore the
ligation was repeated and,
upon completion, the crude
reaction mixture was treat-
ed with 2 vol.% hydrazine
for 10 min which led to
complete hydrazinolysis of
the product selenoester,
generating diselenide liga-
tion product 34 exclusively.
Hexane extraction of resid-
ual DPDS, in situ deseleni-
zation with TCEP and DTT
and purification via re-
verse-phase HPLC then
Scheme 4. A) Primary structure of SMR3B; B) Synthesis of SMR3B (28) via a one-pot addi-
tive-free ligation-deselenization of fragments 29 and 30; C) analytical HPLC and ESI mass
spectrum (inset) of purified synthetic SMR3B; D) MALDI-TOF mass spectrum of purified
synthetic SMR3B.
afforded lumbricin-1 31 in
52% yield over two steps.
The synthetic lumbricin-1
lated from the earthworm Lumbricius rubellus 28
It has
.
was subsequently assessed for activity against
Staphylococcus aureus (SH1000 strain) and exhibited
activity consistent with that reported for the material
been shown to exhibit antimicrobial activity in vitro
against a broad spectrum of microorganisms including
Gram-positive (e.g., Staphylococcus aureus) and Gram-
negative bacteria (e.g., E. coli) as well as some fungi
(e.g., Candida albicans). Given the absence of Cys or
suitably positioned Ala residues in the sequence, together
with the abundance of Pro,
isolated from the earthworm (IC50 = 80 
M).28
CONCLUSIONS
lumbricin-1 was considered a
second ideal target to show-
case the DSL–deselenization
method at Pro-Pro.
We chose to disconnect lum-
bricin-1 (31) centrally be-
tween Pro33 and Pro34 for
assembly through additive-
free DSL–deselenization in
one-pot. This led to two pep-
tide targets for synthesis, the
33-amino acid peptide 32
bearing a C-terminal Pro se-
lenoester and peptide dimer
33 bearing an N-terminal
trans
-

-selenoproline.
Both
fragments were synthesized
on 2-chlorotrityl chloride res-
in via Fmoc-SPPS (see Sup-
porting Information for de-
tails). Fragments 32 (10 mM
final conc.) and 33 (5 mM
final conc. with respect to the
monomer) were then ligated
Scheme 5. A) Primary structure of lumbricin-1; B) Synthesis of lumbricin-1 (31) via a
one-pot additive-free ligation-deselenization of fragments 32 and 33; C) analytical HPLC
and ESI mass spectrum (inset) of purified synthetic lumbricin-1; D) MALDI-TOF mass
spectrum of purified synthetic lumbricin-1.
by dissolving in
6
M
Gdn•HCl, 0.1 M phosphate
buffer at a final pH of 6.2.
The reaction was monitored
by UPLC-MS and reached
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In summary, an efficient one-pot DSL
–deselenization
AUTHOR INFORMATION
strategy has been developed at -selenoproline that has

Corresponding Author
*richard.payne@sydney.edu.au
enabled access to Pro-Pro ligation sites for the first time.
The reaction was shown to be equally efficient with or
without the inclusion of reductive additives. However, the
Present Addresses
† School of Chemistry, University of Nottingham, University
Park, Nottingham NG7 2RD, UK
stereochemistry of the -selenol moiety was shown to be
crucial for peptide bond formation. The power of this
technology was demonstrated via a one-pot synthesis of
submaxillary gland androgen regulated protein 3B and the
antimicrobial peptide lumbricin-1 in excellent overall
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Author Contributions
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the manu-
script. ‡These authors contributed equally.
yields. The simplicity and efficiency of the ligation
–
deselenization chemistry developed here should see this
technology applied to the synthesis of numerous Pro-rich
peptide and protein targets in the future, particularly those
which can only be accessed via Pro-Pro/Hyp-Pro liga-
tions, e.g., antimicrobial peptides and collagens.
ACKNOWLEDGMENT
This work was supported by an ARC Discovery Project
(DP160101324), a Northcote Scholarship (J.S.) and a John Lam-
berton Research Scholarship (J.S.).
EXPERIMENTAL
One-pot Additive-free DSL–Deselenization at Proline-
Proline. Peptide prolyl selenoesters (2.0 equiv.) and N-terminal
selenoproline diselenide dimer peptides (1.0 equiv. with respect to
the monomer) were dissolved separately in ligation buffer (6 M
Gdn•HCl, 100 mM Na2HPO4, pH 7.2). The solutions were com-
bined to give an overall concentration of 10 mM with respect to
the peptide selenoester and 5 mM with respect to monomeric
selenoproline peptide, and the pH was adjusted to 6.2–6.5 with
aqueous 1 M NaOH. The ligation progress was monitored by
UPLC-MS until the selenoproline peptide was completely con-
sumed. The precipitated DPDS was extracted from the crude liga-
tion solution with hexane (x3). The solution was then thoroughly
degassed with Ar (g) sparging. To effect deselenization, an equal
volume of degassed buffer containing TCEP (0.25 M) and DTT
(20 mM) at pH 4.5–5.5 was added. Following the completion of
the reaction (16 h), the solution was purified via semi-preparative
reverse-phase HPLC (see Supporting Information for column and
gradient information). All peptides were isolated as white solids
following lyophilization.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website. Experimental, reaction, characteriza-
tion and computational data (PDF) and human proteins possessing
polyproline sequences (Supplementary Table).
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Cho, J. H., Park, C. B., Yoon, Y. G., Kim, S. C., Lumbricin
R = H or OH
O
SePh
O
+
Se
2
N
O
N
O
LIGATION
Se
2
R
N
NH
O
H
N
N
H
O
R
ONE-POT
DESELENIZATION
trans- -selenoproline
Se
O
N
O
N
NH
O
OH
N
R
Boc
O
MeO
Access to Pro-Pro Junctions
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