Peptide Ligation at High Dilution via Reductive Diselenide-Selenoester Ligation

Article abstract of DOI:10.1021/jacs.9b12558

Peptide ligation chemistry has revolutionized protein science by providing access to homogeneously modified peptides and proteins. However, lipidated polypeptides and integral membrane proteins- A n important class of biomolecules-remain enormously challenging to access synthetically owing to poor aqueous solubility of one or more of the fragments under typical ligation conditions. Herein we describe the advent of a reductive diselenide-selenoester ligation (rDSL) method that enables efficient ligation of peptide fragments down to low nanomolar concentrations, without resorting to solubility tags or hybridizing templates. The power of rDSL is highlighted in the efficient synthesis of the FDA-approved therapeutic lipopeptide tesamorelin and palmitylated variants of the transmembrane lipoprotein phospholemman (FXYD1). Lipidation of FXYD1 was shown to critically modulate inhibitory activity against the Na⁺/K⁺ pump.

Full text of DOI:10.1021/jacs.9b12558

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Article
Peptide Ligation at High Dilution via
Reductive Diselenide-Selenoester Ligation
Timothy S. Chisholm, Sameer S. Kulkarni, Khondker R. Hossain,
Flemming Cornelius, Ronald J. Clarke, and Richard J Payne
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b12558 • Publication Date (Web): 16 Dec 2019
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Peptide Ligation at High Dilution via Reductive Diselenide-Selenoes-
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ter Ligation
Timothy S. Chisholm,1‡ Sameer S. Kulkarni,1‡ Khondker R. Hossain,1 Flemming Cornelius,2 Ronald
J. Clarke,1,3 and Richard J. Payne1*
1 School of Chemistry, The University of Sydney, Sydney, NSW 2006, Australia
2 Department of Biomedicine, University of Aarhus, DK-8000 C, Denmark
3 The University of Sydney Nano Institute, Sydney, NSW 2006, Australia
Supporting Information Placeholder
range of other reactions that rely on the reaction of pep-
tide thioesters with peptides bearing thiol-derived
amino acids or thiol auxiliaries on their N-termini.2a, 2d, 4
Other mechanistically distinct ligation manifolds that
have been successfully employed in chemical protein
synthesis include the Staudinger ligation,5 α-ketoacid-
hydroxylamine/potassium acyltrifluoroborate liga-
tions,6 serine/threonine ligation,7 and the diselenide-se-
lenoester ligation (DSL).8 Together these methods have
enabled the synthesis of several hundred proteins of var-
ying complexity,9 including mirror image proteins
through the fusion of peptide segments containing all D-
amino acids.10 One key application of ligation chemistry
has been in the generation of homogeneously modified
proteins which have contributed significantly to the un-
derstanding of how certain post-translational modifica-
tions (PTMs) influence polypeptide structure and func-
tion.2, 5c, 11
ABSTRACT: Peptide ligation chemistry has revolution-
ized protein science by providing access to homogene-
ously modified peptides and proteins. However, lipi-
dated polypeptides and integral membrane proteins – an
important class of biomolecules – remain enormously
challenging to access synthetically owing to poor aque-
ous solubility of one or more of the fragments under typ-
ical ligation conditions. Herein we describe the advent of
a
reductive diselenide-selenoester ligation (rDSL)
method that enables efficient ligation of peptide frag-
ments down to low nanomolar concentrations, without
resorting to solubility tags or hybridizing templates. The
power of rDSL is highlighted in the efficient synthesis of
the FDA-approved therapeutic lipopeptide tesamorelin
and palmitylated variants of the transmembrane lipo-
protein phospholemman (FXYD1). Lipidation of FXYD1
was shown to critically modulate inhibitory activity
against the Na+/K+ pump.
Despite the enormous advances in chemical protein syn-
thesis, lipidated and integral membrane proteins are a
subset of the modified proteome which remain recalci-
trant to synthesis under standard ligation conditions.
This challenge is owing, in major part, to the poor aque-
ous solubility of the reacting fragments in solution and
their propensity to aggregate and form colloidal struc-
tures.12 The introduction of organic solvents or deter-
gents to ligation buffers can be used to assist in fragment
solubilization, but these can negatively impact the rate of
ligation and subsequent purification steps.13 An alterna-
tive approach to inhibit the formation of colloidal struc-
tures and/or improve solubility of peptides is to intro-
duce a range of synthetic modifications such as the deri-
vatization of backbone amide nitrogens to eliminate hy-
drogen bonding,14 the introduction of O-acyl isopeptide
bonds,15 or the incorporation of removable solubilizing
tags.16 There have also been several reports of the use of
DNA or peptide nucleic acid tags17 to template pseudo-
INTRODUCTION
Peptides and proteins underpin virtually all biological
processes in living systems.1 A number of robust meth-
ods have been developed to access these biomolecules,
either recombinantly or synthetically, that have helped
fuel the generation of ‘biologics’ and contributed to our
understanding of protein structure and function. The
majority of approved biologics are produced using re-
combinant expression methods; however, a unique ben-
efit of chemical synthesis is the ability to introduce mod-
ifications (both natural and non-natural) via the
chemoselective ligation of synthetic modified peptides to
afford larger polypeptides and proteins. Over the past 25
years a number of peptide ligation technologies have
been developed for protein synthesis.2 The most widely
adopted method has been native chemical ligation
(NCL), first reported in 1994.2b, 3 The efficiency and util-
ity of this reaction has inspired the development of a
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intramolecular ligation reactions at lower concentra-
buffering ability at lower pH. It was hypothesized that re-
tions. Whilst these tactics have assisted in the successful
synthesis of a small number of challenging targets to
date, they each require additional synthetic manipula-
tions for the installation and removal of the tags or tem-
plates. Furthermore, in the case of solubility tags, the op-
timal location for tag installation on the peptide back-
bone to improve solubility and prevent aggregation can
be difficult to predict.
actions would need to be performed at more acidic pH to
limit competing hydrolysis of the acyl donors over the
extended reaction periods necessary for ligations at high
dilution. As expected, NCL reactions at the highest con-
centrations of 1 proceeded cleanly to afford ligation
product 5 (3 h at 5 mM and 24 h at 500 μM) but failed to
reach completion (65% conversion after 24 h) at 50 μM
due to competing thioester hydrolysis (Scheme 1B; see
also Scheme S1, Figures S1 and S2 in the ESI). NCL reac-
tions between peptide 1 and phenyl selenoester 3 pro-
ceeded more rapidly (even at pH 5.1) reaching comple-
tion within 1 h at 5 mM of 1, 8 h at 500 μM of 1, and 88%
conversion after 24 h at 50 μM. It is worth noting that at
50 μM complete conversion was not possible due to a
small amount of selenoester hydrolysis over the long (24
h) reaction time (Scheme 1C; see also Scheme S2, Figures
S2 and S3 in the ESI).
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In the work described herein we explore the potential of
the DSL methodology to facilitate efficient ligation at
high dilution in aqueous media without the intervention
of co-solvents, templates, or solubility tags. In its origi-
nal form the DSL reaction possesses rapid kinetics at low
millimolar concentrations of reacting peptide fragments
(reactions complete in minutes) but has poor kinetics at
micromolar concentrations.8a We have now addressed
this limitation through the development of a reductive
diselenide-selenoester ligation (rDSL) methodology
which enables efficient ligation down to concentrations
as low as 50 nM. In addition, we demonstrate that this
method can be coupled with a highly efficient photode-
selenization process to rapidly afford native polypep-
tides. The power of the one-pot rDSL-photodeseleniza-
tion manifold is exemplified through the efficient synthe-
sis of the FDA-approved lipidated therapeutic tesamo-
relin and two stable palmitylated variants of the integral
membrane protein phospholemman (FXYD1).18 These li-
pid modifications of FXYD1 led to inhibition of the
ATPase activity of the Na+/K+ pump.
Scheme 1. (A) NCL between Cysteinyl Peptide 1 and
Peptide Trifluoroethyl Thioester 2 or Peptide Phe‐
nyl Selenoester 3. (B) Kinetics of NCL between Cyste‐
inyl Peptide 1 and Peptide Thioester 2 at a Variety of
Concentrations. (C) Kinetics of NCL Between Peptide
1 and Peptide Selenoester 3 at a Variety of Concen‐
trations.
LYRANV
2 eq. Ac-
2
-XR
3:
(A)
( : XR = SCH2CF3, XR = SePh)
6 M Gdn•HCl, 0.2 M BIS-TRIS,
50 mM TCEP, 37 °C
2
pH 6.2 (for reactions with
)
3
pH 5.1 (for reactions with
)
CSPGYS
LYRANV CSPGYS
5
H-
-NH2
Ac-
-NH2
1
RESULTS AND DISCUSSION
(C)
(B)
100
100
Concentration Limit of NCL and DSL Chemistry
50
0
50
0
5 mM
From the outset we performed a series of control ex-
periments to gauge the concentration dependence and
limits of NCL and DSL. Briefly, the NCL reaction was in-
terrogated using cysteinyl peptide H-CSPGYS-NH2 1,
which was ligated with the sterically hindered acyl do-
nor Ac-LYRANV-XR derivatized as a preformed trifluoro-
ethyl thioester 2 (XR = SCH2CF3) or phenyl selenoester 3
(XR = SePh) (Scheme 1). DSL reactions were performed
only with selenoester 3 and (H-USPGYS-NH2)2 dimer 4
(Scheme 2) as thioesters do not react with diselenides
under these conditions.8a In the first instance reactions
were performed at concentrations of 5 mM, 500 μM and
50 μM of 1 or 4 (based on monomeric selenopeptide) us-
ing two equivalents of acyl donors 2 and/or 3. Reactions
were performed in aqueous ligation buffer (6 M GdnꞏHCl,
0.2 M 2,2-bis(hydroxymethyl)-2,2′,2″-nitrilotriethanol
(BIS-TRIS)) at a final pH of 6.2 (for reactions with 2) and
5.1 (for reactions with 3) at 37 °C. In the case of the NCL
reactions, 50 mM tris(2-carboxyethyl)phosphine (TCEP)
was used as a reductant, whereas DSL reactions were
performed “additive-free”.8a, 8c It should be noted that
BIS-TRIS (pKa 6.5) was employed in place of tradition-
ally used phosphate buffer (pKa 7.2) due to its improved
5 mM
500 M
50 M
500 M
50 M
0
500
1000
1500
2000
0
500
1000
1500
2000
Reaction time / min
Reaction time / min
Scheme 2. (A) Additive‐Free DSL between Peptide
Diselenide Dimer 4 and Peptide Selenoester 3. (B)
Kinetics of Additive‐Free DSL between Peptide
Diselenide Dimer 4 and Peptide Selenoester 3 at pH
5.1 at a Variety of Concentrations.
(A)
LYRANV
3
4
2 eq. Ac-
-SePh (relative to monomeric )
6 M Gdn•HCl, 0.2 M BIS-TRIS,
37 °C, pH 5.1
2
2
USPGYS
4
LYRANV USPGYS
6
H-
-NH2
Ac-
-NH2
(B)
100
5 mM
500 M
50 M
50
0
0
200
400
Reaction time / min
2
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SePh
Se
We next turned our attention to the concentration
dependence of the additive-free DSL reaction (Scheme
2A). DSL reactions were performed at pH 5.1 at 5 mM,
500 μM and 50 μM of 4 (concentrations based on the
monomeric selenopeptide of 4) with two equivalents of
peptide selenoester 3. Reactions were carried out in the
absence of TCEP that would otherwise lead to deleteri-
ous deselenization of Sec.8a, 19 Additive-free DSL reac-
tions with sterically hindered selenoesters (i.e. selenoes-
ters bearing C-terminal valine, threonine, leucine and
isoleucine) proceed via a brief ‘initiation period’ where
no reaction product is observed for several minutes, fol-
lowed by ligation that proceeds to completion in 5-60
min at mM concentrations.8a With 5 mM of monomeric 4,
an initiation phase of 29 min was followed by complete
conversion to the ligation product within an additional
11 minutes. At 500 µM there was a longer initiation
phase for the reaction (2-4 h) and only 71% conversion
was observed after 25 h. However, at 50 µM no ligation
product was observed i.e. the reaction did not initiate
(Scheme 2B; see also Scheme S3 and Figures S4 and S5
in the ESI). Given the slow kinetics of NCL, and the failure
of additive-free DSL reactions to initiate at low concen-
trations, these reaction manifolds have limited utility for
peptide ligation at concentrations below 500 M.
O
H2N
H2N
O
O
SePh
O
Se
Se
O
H
IV
N
H2N
PhSe
NCL-like
mechanism
I
O
Se
H2N
II
Se
III
O
To probe this hypothesis we prepared a solution of
pre-formed phenylselenoate (I) for addition to DSL reac-
tions by prior reduction of diphenyldiselenide (DPDS) in
degassed ligation buffer using 0.9 equiv. of TCEP
(Scheme 4A). 1H NMR spectroscopy was used to quantify
the amount of phenylselenoate in solution (see Figure
S40), and 31P NMR spectroscopy was used to ensure that
the TCEP was fully oxidized to the corresponding phos-
phine oxide and was therefore not the species responsi-
ble for initiation of the reactions (see Figures S6 and S7
in the ESI). The preformed phenylselenoate solution
(0.025 equiv. with respect to monomeric 4) was added
directly to a DSL reaction mixture containing peptide
diselenide dimer 4 and peptide selenoester 3 in ligation
buffer. The reaction was initially performed at 5 mM con-
centration (with respect to monomeric 4) to assess the
effect of pre-formed phenylselenoate on the reaction
rate compared to the additive-free DSL manifold (see
Figure S8 in the ESI). Compared to additive-free DSL
(Scheme 2), the phenylselenoate-spiked reaction pro-
ceeded to completion on a much faster timescale (16 min
vs 40 min) with a diminished initiation period. Im-
portantly, the addition of DPDS to a DSL reaction without
prior reduction did not lead to any rate enhancement
over the additive-free method (see Scheme S5 and Figure
S10 in the ESI). Having observed an improved DSL reac-
tion rate with the addition of pre-formed phenylseleno-
ate, we next performed the ligation at 50 M (with re-
spect to monomeric 4) – a concentration at which addi-
tive-free DSL reactions did not initiate (Scheme 2B). Be-
cause of the lower concentration, here we chose to per-
form the reactions using a range of phenylselenoate
equivalents (0.025 equiv. to 4 equiv. based on mono-
meric 4). Pleasingly, these ligations were all productive
at 50 M with rates that were dependent on the
phenylselenoate concentration introduced. Specifically,
when 0.025 equiv. of phenylselenoate was employed the
reaction could now initiate, reaching 24% completion in
7 h (Scheme 4B). When 0.25 equiv. of phenylselenoate
was introduced to the reaction, the ligation proceeded to
79% completion after 1 h, whereas reactions were >95%
complete after 1 h with 4 equiv. of phenylselenoate (see
Scheme 4B, Figure S9 in the ESI). Taken together these
data support the proposed mechanism of the initiation of
additive-free DSL reactions and, importantly, demon-
strate that DSL can be performed at lower concentra-
tions if pre-reduced phenylselenoate is introduced into
the system.
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Mechanistic Considerations
Given the superior kinetics of the DSL reaction com-
pared to NCL at mM concentrations (1-2 orders of mag-
nitude improvement in rate), we were motivated to try
and rationalize why these reactions do not initiate at
lower concentrations. Mechanistically, the ‘initiation
step’ of DSL is proposed to proceed through a bimolecu-
lar reaction with the peptide diselenide dimer (Scheme
3).8a One proposed pathway invokes the liberation of a
small amount of phenylselenoate (I) from the peptide se-
lenoester that can partake in redox exchange with a pep-
tide diselenide dimer (II) to afford an equivalent of re-
duced selenopeptide (III); this equivalent of III can sub-
sequently react with a peptide selenoester IV under an
NCL-like mechanism (trans-selenoesterification fol-
lowed by an Se to N acyl shift), which generates a native
amide bond and propagates the DSL reaction.8a Based on
these mechanistic insights it is clear that the initial gen-
eration of very small amounts of I would have a low
probability of engaging in the bimolecular selenide ex-
change with II in DSL reactions performed at low con-
centrations. We therefore rationalized that if phenylse-
lenoate I was present at higher concentrations it would
potentially enable ligation at lower concentrations than
is possible under additive-free DSL conditions.
Scheme 3. Proposed Mechanism of the DSL Reaction.
NB: phenylselenoate I could be liberated through hydrol-
ysis, aminolysis, or acyl substitution of the peptide se-
lenoester (see Scheme S4 in the ESI).
Scheme 4. (A) Generation of Phenylselenoate. (B)
DSL Between Diselenide 4 and Selenoester 3 in the
Presence of Phenylselenoate. NB: For all these experi-
ments ligation buffer was thoroughly sparged with argon
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for 5-10 min prior to use. Figures in the box represent
1H NMR spectroscopy (see Figure S43) which was suffi-
conversions after 7 h (measured by analytical UPLC-MS,
see ESI); reactions would contain PhSeH in addition to
PhSe- (pKa of PhSeH = 5.9).
cient for inhibiting deleterious deselenization that would
otherwise be facilitated by the remaining TCEP. Gratify-
ingly, rDSL reactions also proceeded smoothly under
more dilute conditions, e.g. 16 min for 500 μM of mono-
meric 4. At 50 μM the rDSL proceeded to completion in
64 min (slightly faster than the reaction with 1.0 equiv.
of pre-reduced DPDS, see Scheme 5B). We next interro-
gated the concentration limit of the rDSL reaction by per-
forming ligations at 5 μM, 500 nM and 50 nM of the mon-
omer of 4 with 2 equivalents of 3 (10 μM, 1 μM, 100 nM)
in the presence of TCEP (30 mM) and DPDS (20 mM).
Due to the low concentration of peptide in solution, ali-
quots of the reaction mixture were quenched with hy-
drazine hydrate and concentrated through a plug of C18
silica prior to HPLC analysis. Gratifyingly, efficient liga-
tion was observed at all concentrations investigated
(Scheme 5D-E; Figures S13 and S14 in the ESI). Ligation
with 5 μM of monomeric 4 reached completion in 24 h,
and was complete in 48 h at 500 nM. Remarkably, even
reactions at 50 nM of monomeric 4 proceeded cleanly
and to completion within 96 h. To our knowledge this
represents the lowest concentration that a non-tem-
plated ligation reaction has been successfully performed
at to date.
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Development of the Reductive Diselenide‐Selenoes‐
ter Ligation (rDSL)
We next sought to improve the operational simplicity of
performing ligation reactions, whilst additionally explor-
ing the scope of reactions at even lower concentrations.
We proposed that this might be achieved by quantitative
generation of the monomeric selenopeptide (III in
Scheme 3) from the diselenide dimer starting material
(II). Under these conditions the reactions would likely
proceed through a simple bimolecular rate limiting
trans-selenoesterification step – akin to NCL.20 Here the
reaction should benefit from the vastly improved kinet-
ics engendered by the reactive selenoester acyl donor
and the enhanced nucleophilicity of the selenoate of se-
lenocysteine. In order to interrogate this reaction mani-
fold we sought to employ a strong reductant such as
TCEP (employed in the earlier ‘pre-reduction’ experi-
ments) to reduce the diselenide. However, unlike
phenylselenoate which is stable to TCEP treatment (due
to a strong Se-aryl C bond) the use of phosphine-based
reductants is known to lead to deselenization of the Sec
residue (vide supra).21 We therefore investigated the use
of TCEP combined with diphenyldiselenide (DPDS) as a
radical scavenger to suppress the deleterious deseleni-
zation pathway, and dubbed the resulting reaction con-
ditions “reductive DSL (rDSL)” (see Scheme S6 and Fig-
ures S11 and S12 in the ESI). In the first model rDSL re-
action, (H-USPGYS-NH2)2 4 (5 mM, 500 μM, 50 μM with
respect to the monomeric selenopeptide) and Ac-
LYRANV-SePh 3 (2 equiv.; 10 mM, 1 mM, 100 μM) were
Scheme 5. (A) rDSL between Peptide Diselenide Di‐
mer 4 and Selenoester 3. (B) Kinetics of rDSL be‐
tween Peptide Diselenide Dimer 4 and Selenoester 3
at Concentrations of 5 mM, 500 µM and 50 µM of
Monomeric 4. (C) HPLC‐FLR Chromatogram (Gradi‐
ent: 0 to 40% B over 30 Min, λex = 280 nm, λem = 347
nm) of the Hydrazine Quenched rDSL Reaction Con‐
taining 25 µM of 4 after 64 min. (D) Kinetics of rDSL
between Peptide Diselenide Dimer 4 and Selenoes‐
ter 3 at Concentrations of 5 µM, 500 nM and 50 nM of
Monomeric 4. (E) HPLC‐FLR Chromatogram (Gradi‐
ent: 0 to 40% B over 30 Min, λex = 280 nm, λem = 347
nm) of the Quenched rDSL Reaction Containing 25
nM of 4 after 96 h. 3* Peak Corresponds to the Acyl
Hydrazide Derivative of 3. 3# Peak Corresponds to
Hydrolyzed 3.
(A)
LYRANV
2 eq. Ac-
3
-SePh ( )
6 M Gdn•HCl, 0.2 M BIS-TRIS,
20 mM DPDS, 30 mM TCEP
pH 5.1, 37 °C
2
2
°
incubated at 37 C in ligation buffer comprising 6 M
USPGYS
4
H-
-NH2
LYRANV USPGYS
6
Ac-
-NH2
Gdn·HCl, 0.2 M BIS-TRIS, 30 mM TCEP and 20 mM DPDS
at pH 5.1. In this case the buffer was sonicated for 30 min
prior to use to ensure optimal dissolution of phenylse-
lenoate. Under these conditions rapid ligation was ob-
served (reaction complete in 4 min with 5 mM of mono-
meric 4 cf. 40 min for additive-free DSL at pH 5.1, vide
supra) with no observable initiation period nor deseleni-
zation (Scheme 5A-C; Figures S11 and S12 in the ESI). It
should be noted an excess of TCEP was employed in the
rDSL reactions to ensure reductive conditions were
maintained over long reaction times (without the need
for thorough degassing). The reaction conditions led to
23 mM of phenylselenoate in solution as determined by
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(C)
(B)
100
50
0
6
100
3*
50
0
5 mM
3#
500 M
50 M
0
10
20
30
0
20
40
60
Reaction time / min
Retention time / min
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(E)
(D)
Having demonstrated the efficiency of photodese-
lenization on a model peptide, we next moved to inte-
grate the method into a simple two-step one-pot rDSL-
photodeselenization protocol that would ultimately ena-
ble native peptides and proteins to be generated at low
concentrations (Scheme 7A). Towards this end, peptide
diselenide 4 at concentrations of 500 μM and 50 μM (rel-
ative to the monomeric selenopeptide) was reacted with
two equivalents of peptide selenoester 3 under the rDSL
conditions (6 M Gdn·HCl, 0.2 M BIS-TRIS, 30 mM TCEP,
15
100
80
60
40
20
0
6
10
5
3#
5 M
500 nM
50 nM
0
0
10
20
30
0
50
100
Reaction time / h
Retention time / min
Development of One‐Pot rDSL‐Photodeselenization
°
Platform
20 mM DPDS) at pH 5.1 and 37 C. Upon completion of
the ligation reactions to afford 6 (as determined by
UPLC-MS; 16 min for 500 M, 64 min for 50 M) DPDS
was extracted with Et2O. The reaction mixture was next
diluted with an equal volume of TCEP (400 mM) and GSH
(80 mM) in ligation buffer (pH 7.5) then exposed to UV
photoirradiation (λ = 254 nm, 35 W) for 2.0 min to 3.5
min. Reactions were concentrated then purified via RP-
HPLC to afford product 8 in comparable yields for all
concentrations (51-53%, Scheme 7B and 7C; Scheme
S11, Figure S19 in the ESI).
In addition to the benefits that phenylselenoate pro-
vides to the rate of DSL reactions at lower concentra-
tions, this additive can be simply extracted from the re-
action mixture with organic solvent, thus enabling in situ
deselenization following completion of a ligation reac-
tion. With this in mind, we next moved to further expand
the scope of the rDSL reaction through the development
of a one-pot rDSL-deselenization protocol. Towards this
end we interrogated the use of photochemistry for dese-
lenization, a strategy that we recently reported for the
rapid and clean desulfurization of peptides (see Scheme
6).22 It should be noted that preliminary photoirradia-
tion experiments on Sec performed by Metanis and co-
workers have also demonstrated conversion to Ala to-
gether with oxidative byproducts.19 To optimize the pro-
posed method, we first explored photodeselenization on
a model system (see Scheme S7-S10 and Figure S15-S18
in the ESI for optimization experiments). Optimized con-
ditions employed (H-USPGYS-NH2)2 4 (2.5 mM with re-
Scheme 7. (A) One‐Pot rDSL‐Photodeselenization.
(B) UPLC Trace (Gradient: 0 to 30% B over 5 Min, λ =
280 nm) for the Crude One‐Pot Protocol Using 500
µM of monomeric 4 and 1 mM of 3. (C) HPLC Chroma‐
togram with Fluorescence Detection (Gradient: 0 to
35% B over 30 Min, λex = 280 nm, λem = 347 nm) for
the Crude One‐Pot Protocol using 50 µM of mono‐
meric 4 and 100 µM of 3. 3^ Peak Corresponds to the
Glutathione Thioester of 3. 3# Peak Corresponds to
spect to monomeric 4), TCEP (200 mM) and reduced L-
Hydrolyzed 3.
glutathione (GSH, 40 mM) dissolved in ligation buffer
(pH 7.0) which was then exposed to UV photoirradiation
(254 nm, 35 W). Gratifyingly, under these conditions and
without degassing of the ligation buffer, complete photo-
deselenization to 7 was observed in just 30 s (Scheme 6,
see ESI for details). It is important to note that when the
same substrate (4) is subjected to deselenization under
standard conditions (TCEP and DTT) the reaction re-
quires 8 h to reach completion;8a, 8c this photodeseleniza-
tion method therefore provides a three orders of magni-
tude improvement in reaction rate.
(A)
6 M Gdn•HCl, 0.2 M BIS-TRIS
30 mM TCEP, 20 mM DPDS
pH 5.1, 37 °C
i: 16 min; ii: 64 min
2
2
USPGYS
4
H-
-NH2
LYRANVUSPGYS
Ac-
-NH2
6
not isolated
LYRANV
3
Ac-
-SePh
1)
2)
Et2O extraction
6 M Gdn•HCl, 0.2 M BIS-
i: 500 M monomeric 4, 1 mM 3
ii: 50 M monomeric , 100 M
TRIS, 200 mM TCEP,
40 mM GSH, pH 6.8,
 = 254 nm, 35 W
4
3
i: 2.0 min; ii: 3.5 min
LYRANVASPGYS
Ac-
-NH2
i: 51% yield
ii: 53% yield
8
(B)
(C)
0.4
0.3
0.2
0.1
0.0
600
Scheme 6. (A) Photodeselenization of 4. (B) Crude
UPLC Trace (Gradient: 0 to 15% B over 5 min, λ = 280
nm) of the Photodeselenization of 4 after 30 s with
Inset ESI‐MS Data.
8
8
400
200
0
3#
3^
3^
2
3
4
5
6
0
10
20
30
Retention time / min
Retention time / min
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(A)
Synthesis of Tesamorelin Lipopeptide via One‐Pot
rDSL‐Photodeselenization
2
Se
O
H
O
H
N
19
5
6
7
8
N
18
YADAIFTNSYRKVLGQL
H2
N
RKLLQDIMSRQQGESNQERGARARL
NH2
SePh
O
Having demonstrated the efficiency of the rDSL-
photodeselenization chemistry at a range of concentra-
tions, we next chose to implement the methodology for
the synthesis of larger targets where one or more of the
reacting peptide fragments possessed poor solubility in
aqueous buffer. We began with tesamorelin 9, a 44-resi-
due analogue of Growth Hormone Releasing Hormone
used for the treatment of HIV-associated lipodystro-
phy.23 Tesamorelin is modified with an N-terminal trans
2-hexenoyl moiety which protects the polypeptide from
proteolytic degradation and improves its biological ac-
tivity.24 The lipopeptide was disconnected near the mid-
dle of the sequence between Ser18 and Ala19 and there-
fore required the synthesis of two fragments, peptide 10
(tesamorelin 1-18) bearing a C-terminal phenyl se-
lenoester and peptide 11 (tesamorelin 19-44) bearing
an N-terminal selenocystine (the oxidized form of Sec) in
place of Ala19 in the native sequence. Both were synthe-
sized via modified Fmoc-SPPS conditions (see ESI for de-
tails). Having accessed the fragments, we first tested the
solubility limits of lipopeptide selenoester 10 in ligation
buffer (see Figure S31 in the ESI). Based on this investi-
gation 10 was dissolved at a concentration of 300 μM in
aqueous ligation buffer (pH 5.3) containing TCEP (30
mM) and DPDS (20 mM) and was ligated with diselenide
dimer 11 (at a final concentration of 125 μM with respect
to the dimer) (Scheme 8A). Pleasingly, the rDSL reaction
was complete within 20 min (Scheme 8B), at which point
residual DPDS was extracted with Et2O. An equal volume
of TCEP (400 mM) and GSH (80 mM) in ligation buffer
(pH 7.5) was then added. Upon irradiation ( = 254 nm,
35 W, 2 min) and RP-HPLC purification, tesamorelin 9
was isolated in excellent yield on a multi-milligram scale
(75%, Scheme 8C-E; Scheme S12 and Figures S28-S31 in
the ESI).
O
1
10
44
11
OH
300 M
1.2 equiv.
125 M
0.5 equiv.
6 M Gdn•HCl, 0.2 M BIS-TRIS
30 mM TCEP, 20 mM DPDS
pH 5.3, 37 °C, 20 min
9
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Se
H
O
O
H
N
N
18
YADAIFTNSYRKVLGQL
N
RKLLQDIMSRQQGESNQERGARARL
NH2
19
O
H
1
O
44
OH
12
[not isolated]
1)
2)
Et2O extraction
6 M Gdn•HCl, 0.2 M BIS-TRIS
200 mM TCEP, 40 mM GSH
pH 6.9,  = 254 nm, 35 W, 2 min
H
O
O
H
N
19
N
18
YADAIFTNSYRKVLGQL
N
RKLLQDIMSRQQGESNQERGARARL
NH2
O
H
1
O
44
OH
9
Synthesis of Lipidated Variants of the Membrane
Protein FXYD1 via One‐Pot rDSL‐Photodeseleniza‐
tion
We next turned our attention to a second target, the
membrane protein phospholemman (FXYD1), which
regulates the Na+/K+ pump and is natively S-pal-
mitoylated at two Cys residues. Previous work has high-
lighted the importance of palmitoylation for activity of
FXYD1 on the Na+/K+ pump,18 but samples of homogene-
ously lipidated variants of the protein have not been ac-
cessed to date. We envisioned synthesizing stable ana-
logues of FXYD1 bearing thioether-linked “palmityla-
tion” at Cys42 (incorporated during Fmoc-SPPS) and se-
lenoether-linked palmitylation at Sec40 that could be in-
corporated through late stage alkylation following a
rDSL reaction to assemble the full protein sequence.
Based on this synthetic strategy, peptide 13 (FXYD1 1-
39) bearing a C-terminal phenyl selenoester and S-pal-
mitylated peptide 14 (FXYD1 40-72) bearing an N-termi-
nal selenocystine were synthesized via Fmoc-SPPS.
These fragments were then dissolved in aqueous ligation
buffer (6 M Gdn·HCl, 0.2 M BIS-TRIS, 30 mM TCEP, 20
mM DPDS, pH 5.1). Given the hydrophobicity of the frag-
ments, the reductive DSL needed to be performed with
60 M of the diselenide dimer 14 and 240 M of the se-
lenoester 13 at 60 C (Scheme 9A). After 4 h complete
conversion of the diselenide fragment was observed,
Scheme 8. (A) Synthesis of Tesamorelin 9 via One‐
Pot rDSL‐Photodeselenization. HPLC Chromatogram
with Fluorescence Detection (Gradient: 0 to 80% B
over 30 min, λex = 280 nm, λem = 347 nm) for (B) rDSL
of 10 and 11 to 12, (C) One‐Pot Photodeselenization
of 12 to 9, and (D) Purified 9 upon RP‐HPLC Purifica‐
tion. (E) MALDI‐TOF Mass Spectrum of 9.
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generating the ligation product exclusively as the asym-
tivity was assessed by adding ATP and quantifying inor-
ganic phosphate production using an antimony-phos-
phomolybdate assay27 directly after addition of the
FXYD1 variant to the membrane preparation (t = 0 h)
and again 24 h after incubation (t = 24 h, Figure 1). Addi-
tion of rFXYD1 (that does not possess any lipid modifica-
tions) led to only a slight drop in ATPase activity when
incubated for 24 h (16% decrease in activity). In striking
contrast, incubation with mono-palmitylated FXYD1
(17) resulted in complete abolishment of Na+/K+-ATPase
activity. Interestingly, while the di-palmitylated FXYD1
(16) also displayed potent inhibition (75% inhibition of
Na+/K+-ATPase activity after 24 h) it was less effective
than the mono-palmitylated variant 17. While the reason
for the reduced inhibitory activity of 16 is unclear, it is
feasible that Cys42 provides the most critical binding in-
teractions with the 1 subunit of the pump and that lipi-
dation of the second site (Cys40) leads to reduced pump
inhibition by disrupting interactions with the 1 subu-
nit. Potential reasons for this could be an altered confor-
mation of FXYD1 when both palmityl chains are present,
or perhaps the doubly lipidated protein interacts more
favorably with the lipid membrane rather than the
pump. Future work in our laboratory will seek to solve
structures of complexes between Na+/K+-ATPase and
metric diselenide 15. After extraction of DPDS with Et2O,
the ligation mixture was treated with DTT (150 equiv.)
and the pH of the resulting solution was adjusted to 7.0.
Addition of 1-bromohexadecane in an equal volume of
DMF led to the formation of the palmitylated seleno-
ether, thus furnishing doubly palmitylated FXYD1 16 in
35% yield following HPLC purification (Scheme 9, Figure
S34). The divergence of this synthetic approach also en-
abled us to access a variant of FXYD1 with only a single
palmityl modification at Cys42 (Scheme 9, Figure S35).
Specifically, the rDSL between 13 and 14 could be per-
formed as above and after Et2O extraction of the DPDS,
the reaction mixture was subjected to in situ photodese-
lenization ( = 254 nm, 35 W, 1 h) using an equal volume
of a solution of TCEP (800 mM) and GSH (80 mM) in li-
gation buffer (pH 7.5). This provided mono-palmitylated
FXYD1 analogue 17 (a Cys40Ala mutant) in 32% yield
over the two synthetic steps following purification by
RP-HPLC. The synthetic palmitylated FXYD1 analogues
were characterized by UPLC, ESI-MS and MALDI-TOF MS
(Scheme 9B-D, see Figures S32-S37 in the ESI). CD spec-
tra of 16 and 17 were also obtained which were con-
sistent with that previously reported for recombinant
(unmodified) FXYD1.25
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 9. (A) One‐Pot rDSL‐Alkylation and rDSL‐Photodeselenization for the Synthesis of FXYD1 Analogues 16
and 17. UPLC Chromatogram (Gradient: 20 to 100% B over 5 min, λ = 214 nm) for (B) Purified 17 upon RP‐
HPLC Purification, (C) ESI‐MS of Purified 17, (D) CD Spectrum of 17.
With the mono- and di-palmitylated FXYD1 proteins
in hand, we next assessed their effect on the activity of
Na+/K+-ATPase by using a membrane preparation from
pig kidney outer medulla, purified as described by
Klodos et al.26 Accordingly, synthetic FXYD1 lipoproteins
16 and 17, and recombinant FXYD1 (rFXYD1),25 were in-
cubated with the Na+/K+-ATPase for 24 h to allow the
synthetic FXYD1 lipoproteins to displace the native
FXYD1 protein bound to the 1 subunit. The ATPase ac-
synthetic lipidated FXYD1 variants to rationalize the in-
hibitory activity observed in this study, which will aid in
understanding how post-translational palmitoylation of
FXYD1 regulates the Na+/K+ pump.
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7 min at rt the solution was acidified with 5 vol.% TFA in
H2O and DPDS extracted with hexane before UPLC anal-
ysis (see ESI for further details).
100
80
60
40
20
0
Diselenide‐Selenoester Ligation (DSL). Ligation
buffer (6 M Gdn·HCl, 0.2 M BIS-TRIS, pH 5.2 or pH 6.3)
was prepared. This solution was used to separately dis-
solve aliquots of model peptide diselenide dimer 4 and
model peptide phenyl selenoester 3 to the required con-
centration (for 4: 10 mM, 1 mM, 100 µM with respect to
the monomer; for 3: 20 mM, 2 mM, 200 µM) and the pH
adjusted to 5.1 or 6.2. Equal volumes of a solution of
diselenide 4 (1.0 equiv. with respect to the monomer)
and phenyl selenoester 3 (2.0 equiv.) were combined
and incubated at 37 °C.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
t = 0 h
t = 24 h
Figure 1. Effect of synthetic FXYD1 lipoproteins 16 and
17 on the ATPase activity of a Na+/K+ pump membrane
preparation from pig kidney outer medulla. NB: t = 0 data
is normalized to the ATPase activity measured in the
control experiment.
For ligations containing a starting concentration of 5 mM
of monomeric 4, 10 µL aliquots were quenched with 10 µL
of 10 vol.% N2H5·OH in H2O at specific time points. After 7
min at rt the solution was acidified with 25 µL of 5 vol.%
TFA in H2O and DPDS extracted with hexane (3 x 1 mL). The
aliquot was then treated with 4.5 µL of 0.5 M TCEP in H2O
immediately prior to UPLC analysis (λ = 280 nm, 0 to 30%
B over 5 min, 0.1 vol.% TFA).
CONCLUSIONS
For ligations containing a starting concentration of 500
µM and 50 µM of monomeric 4, 100 µL aliquots were
quenched with 10 µL of 40 vol.% N2H5·OH in H2O at specific
time points. After 7 min at rt the solution was acidified with
12 µL of 40 vol.% TFA in H2O and DPDS extracted with hex-
ane (3 x 1 mL). The aliquot was then treated with 12 µL of
0.5 M TCEP in H2O immediately prior to UPLC analysis (for
500 µM of monomeric 4: λ = 280 nm, 0 to 30% B over 5 min,
0.1 vol.% TFA) or analytical HPLC with fluorescence detec-
tion (for 50 µM of monomeric 4: λex = 280 nm, λem = 347 nm,
0 to 40% B over 30 min, 0.1 vol.% TFA).
In summary, we have described the development of
the first method that enables peptide ligation at nanomo-
lar concentrations in purely aqueous media, without the
need for solubility tags or a template. The use of rDSL fa-
cilitates efficient ligation down to 50 nM of selenopep-
tides. Subsequent one-pot photodeselenization allows
for polypeptide targets to be accessed in rapid time
scales in good yield. The power of this new technology
was exemplified through the high-yielding synthesis of
the FDA-approved therapeutic tesamorelin and stable
analogues of the regulatory protein FXYD1. The im-
portance of lipidation of FXYD1 for inhibition of the
Na+/K+ pump was also demonstrated. Given the effi-
ciency of the rDSL reaction at extremely low concentra-
tions, we envisage that this technology will find applica-
tion in the synthesis of a large range of hydrophobic and
lipidated targets in the future. Robust access to these bi-
omolecules provided by rDSL should in turn contribute
to a better understanding of how lipidation and other hy-
drophobic modifications modulate protein structure and
function.
Reductive Diselenide‐Selenoester Ligation (rDSL).
A suspension of 30 mM TCEP and 20 mM DPDS in liga-
tion buffer (6 M Gdn·HCl, 0.2 M BIS-TRIS, pH 5.2) was
prepared and sonicated for 30 min. The resulting solu-
tion was used to separately dissolve aliquots of model
peptide diselenide 4 and model peptide phenyl selenoes-
ter 3 to the required concentration (for 4: 10 mM, 1 mM,
100 µM, 10 µM, 1 µM, 10 nM with respect to the mono-
mer; for 3: 20 mM, 2 mM, 200 µM, 20 µM, 2 µM, 200 nM)
and the pH adjusted to 5.1. Equal volumes of a solution
of diselenide 4 (1.0 equiv. with respect to the monomer)
and phenyl selenoester 3 (2.0 equiv.) were combined
and incubated at 37 °C.
EXPERIMENTAL SECTION
Native Chemical Ligation (NCL). A solution of 50
mM TCEP in ligation buffer (6 M Gdn·HCl, 0.2 M BIS-
TRIS; peptide thioester 2: pH 6.4, peptide selenoester 3:
pH 5.2) was prepared. This solution was used to sepa-
rately dissolve aliquots of model cysteinyl peptide 1 and
model peptide acyl donor 2 or 3, to the required concen-
tration (for 1: 10 mM, 1 mM, 100 µM; for 2 and 3: 20 mM,
2 mM, 200 µM) and the pH adjusted (2: pH 6.2; 3: pH 5.1).
Equal volumes of a solution of cysteinyl peptide 1 (1.0
equiv.) and acyl donor 2 or 3 (2.0 equiv.) were combined
and incubated at 37 °C. Aliquots were quenched at spe-
cific time points with 10 vol.% N2H5·OH in H2O, and after
For ligations containing a starting concentration of 5 mM
of monomeric 4, 10 µL aliquots were quenched with 10
µL of 10 vol.% N2H5·OH in H2O at specific time points. Af-
ter 7 min at rt the solution was acidified with 25 µL of 5
vol.% TFA in H2O and DPDS extracted with hexane (3 x 1
mL) before UPLC analysis (λ = 280 nm, 0 to 30% B over
5 min, 0.1 vol.% TFA).
For ligations containing a starting concentration of 500
µM and 50 µM of monomeric 4, 100 µL aliquots were
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quenched with 10 µL of 40 vol.% N2H5·OH in H2O at spe-
diselenide dimer) in ligation buffer (6 M Gdn·HCl, 0.2 M
BIS-TRIS, 2.4 mL) containing 30 mM TCEP and 20 mM
DPDS was prepared at pH 5.3 and incubated at 37 °C. Af-
ter complete ligation in 20 min as confirmed by UPLC-
MS, DPDS and the reaction extracted with Et2O (5 x 0.5
mL) and an equal volume of a solution of 400 mM TCEP
and 80 mM GSH in ligation buffer (6 M Gdn·HCl, 0.2 M
BIS-TRIS, pH 7.4, 2.4 mL) was added. The resultant solu-
tion of pH 6.9 was photoirradiated inside a Rayonet RPR-
100 photoreactor (λ = 254 nm, 35 W) for 2 min. Follow-
ing purification by reverse-phase HPLC (Waters Sunfire
C-18 OBD, 5 µm, 19 mm x 150 mm, 0 to 10% B over 5 min
then 10 to 40% B over 60 min at 7 mL/min) and lyophiliza-
tion, tesamorelin 9 was afforded as a white solid (2.73
mg, 75% yield over 2 steps).
cific time points. After 7 min at rt the solution was acidi-
fied with 12 µL of 40 vol.% TFA in H2O, and DPDS ex-
tracted with hexane (3 x 1 mL) before UPLC analysis (for
500 µM of monomeric 4: λ = 280 nm, 0 to 30% B over 5
min, 0.1 vol.% TFA) or analytical HPLC with fluorescence
detection (for 50 µM of monomeric 4: λex = 280 nm, λem
=
347 nm, 0 to 40% B over 30 min, 0.1 vol.% TFA).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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40
41
42
43
44
45
46
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For ligations containing a starting concentration of 5 µM,
500 nM, or 50 nM of monomeric 4, aliquots (5 µM: 400
µL; 500 nM: 4 mL; 50 nM: 40 mL) were quenched with
40 vol.% N2H5·OH in H2O at specific time points (5 µM:
40 µL; 500 nM: 400 µL; 50 nM: 4 mL) for 7 min at rt. The
solution was acidified with 40 vol.% TFA in H2O (5 µM:
50 µL; 500 nM: 500 µL; 50 nM: 5 mL) and DPDS extracted
with Et2O (5 µM: 3 x 1 mL; 500 nM: 3 x 10 mL; 50 nM: 3
x 40 mL). The aqueous layer was loaded onto a plug of
C18 silica (50 mg) and excess salts removed via washing
with H2O with 0.1 vol.% TFA (10 mL). Any retained ma-
terial was then eluted using 1:1 v/v H2O/acetonitrile
with 0.1 vol.% TFA (6 mL), then acetonitrile with 0.1
vol.% TFA (6 mL). The combined eluents were lyophi-
lized and the resulting white solid analyzed using analyt-
ical HPLC with fluorescence detection (λex = 280 nm, λem
= 347 nm, 0 to 40% B over 30 min, 0.1 vol.% TFA).
One‐Pot Synthesis of Di‐Palmitylated FXYD1 16.
A solution of FXYD1 (1-39) phenyl selenoester 13 (0.35
µmol, 1.6 mg, 240 µM) and FXYD1 (40-72) diselenide di-
mer 14 (0.09 µmol, 0.75 mg, 60 µM with respect to the
dimer) in ligation buffer (6 M Gdn·HCl, 0.2 M BIS-TRIS;
1.45 mL) containing 30 mM TCEP and 20 mM DPDS was
prepared at pH 5.1 and incubated at 60 °C. After 4 h the
reaction had reached completion as confirmed by UPLC-
MS. The reaction was subsequently extracted with Et2O
(7 x 0.5 mL) and a solution of 270 mM DTT in ligation
buffer (6 M Gdn·HCl, 0.2 M BIS-TRIS, pH 5.5, 0.1 mL) was
added. The pH of the resultant solution was adjusted to
6.9-7.0, treated with a solution of 1-bromohexadecane
(27 µL, 500 equiv. in 1.5 mL DMF) and incubated at 37 °C
for 2 h. The reaction mixture was then passed through a
C4 silica plug for desalting; the lipoprotein was eluted
with 95:5 v/v acetonitrile:water (containing 0.1% TFA)
and lyophilized. Following purification by reverse-phase
HPLC (Symmetry 300 C4 OBD, 5 µm, 10 mm x 250 mm, 0
to 50% B over 5 min then 50 to 100% B over 40 min at 4
mL/min), the di-palmitylated analogue of FXYD1 16 was
isolated as a white solid (0.55 mg, 35% yield over 2
steps).
One‐Pot rDSL‐Photodeselenization. A suspension
of 30 mM TCEP and 20 mM DPDS in ligation buffer (6 M
Gdn·HCl, 0.2 M BIS-TRIS, pH 5.2) was prepared and soni-
cated for 30 min. The resulting solution was used to sep-
arately dissolve aliquots of model peptide diselenide 4
and model peptide phenyl selenoester 3 to the required
concentration (for 4: 10 mM, 1 mM, 100 µM with respect
to the monomer; for 3: 20 mM, 2 mM, 200 µM) and the
pH adjusted to 5.1. Equal volumes of a solution of
diselenide 4 (1.0 equiv. with respect to the monomer)
and phenyl selenoester 3 (2.0 equiv.) were combined
and incubated at 37 °C.
Upon complete ligation the reaction mixture was ex-
tracted with Et2O (x3) and residual Et2O removed under
a stream of nitrogen. The ligation solution was then com-
bined with an equal volume of 400 mM TCEP and 80 mM
GSH in ligation buffer (6 M Gdn·HCl, 0.2 M BIS-TRIS, pH
7.5) and irradiated in a Rayonet RPR-100 photoreactor
at λ = 254 nm (35 W, rt).
One‐Pot Synthesis of Mono‐Palmitylated FXYD1
(C40A) 17. A solution of FXYD1 (1-39) phenyl selenoes-
ter 13 (0.58 µmol, 2.6 mg, 240 µM) and FXYD1 (40-72)
diselenide dimer 14 (0.15 µmol, 1.25 mg, 60 µM with re-
spect to the dimer) in ligation buffer (6 M Gdn·HCl, 0.2 M
BIS-TRIS; 2.4 mL) containing 30 mM TCEP and 20 mM
DPDS was prepared at pH 5.1 and incubated at 60 °C. Af-
ter 4 h the reaction had reached completion as confirmed
by UPLC-MS. The reaction was subsequently extracted
with Et2O (7 x 0.5 mL) and an equal volume of a solution
of 800 mM TCEP and 80 mM GSH in ligation buffer (6 M
Gdn·HCl, 0.2 M BIS-TRIS, pH 7.4, 2.4 mL) was added. The
resultant solution of pH 6.9 was photoirradiated inside a
Rayonet RPR-100 photoreactor (λ = 254 nm, 35 W) for 1
h. The reaction mixture was then passed through a C4
silica plug for desalting. The lipoprotein was eluted with
75:25 v/v acetonitrile:water (containing 0.1% TFA) and
lyophilized. Upon reverse-phase HPLC purification
(Symmetry 300 C4 OBD, 5 µm, 10 mm x 250 mm, 0 to
25% B over 5 min then 25 to 100% B over 60 min at 4
The reaction solution was loaded onto a plug of C18
silica (50 mg) and excess salts removed via washing with
H2O with 0.1 vol.% TFA (10 mL). Any retained material
was then eluted using 1:1 v/v H2O/acetonitrile with 0.1
vol.% TFA (6 mL), then acetonitrile with 0.1 vol.% TFA
(6 mL). The combined eluents were lyophilized and the
resulting product analyzed using analytical HPLC with
fluorescence detection and UPLC.
One‐Pot Synthesis of Tesamorelin 9. A solution of
tesamorelin (1-18) phenyl selenoester 10 (0.72 µmol,
1.93 mg, 300 µM) and tesamorelin (19-44) diselenide di-
mer 11 (0.30 µmol, 2.33 mg, 125 µM with respect to the
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like to acknowledge scholarship support from a John Lamber-
mL/min), mono-palmitylated FXYD1 (C40A) 17 was iso-
lated as a white solid (0.8 mg, 32% yield over 2 steps).
ton Research Scholarship and the Research Training Program
scheme (T.S.C.). We would like to acknowledge Dr. Nick Pro-
schogo and Dr. Cody Szczepina from the University of Sydney
for technical support with mass spectrometry and HPLC, re-
spectively. We would also like to thank Charlotte Franck from
the University of Sydney for assistance with CD experiments
and Professor Gemma Figtree and Dr Owen Tang (the Kolling
Institute) for providing recombinant FXYD1.
Na+/K+‐ATPase activity assay. Recombinant FXYD
(rFXYD), mono-palmitylated FXYD1 (17) or di-pal-
mitylated FXYD1 (16) were initially added to 10 µL of a
pig kidney Na+/K+-ATPase membrane preparation26 (0.6
mg/mL) at a molar ratio of Na+/K+-ATPase:FXYD1 of
1:50. The mixture was then diluted into 1 mL of buffer
solution composed of 130 mM NaCl, 20 mM KCl, 30 mM
imidazole, 3 mM MgCl2, 1mM EDTA, pH 7.2, and incu-
bated for 0 or 24 hours at 4 °C. After incubation, the
ATPase activity was initiated by addition of 3 mM ATP at
37 °C for 15 minutes. Control experiments were per-
formed in the presence of 50 µM ouabain, a specific
Na+/K+-ATPase inhibitor, which showed at least 99% in-
hibition of activity, indicating that the entire measured
activity was due to the Na+/K+-ATPase. After 15 minutes
of reaction time, 100 µL aliquots were immediately
added to 900 µL of coloring solution (2.5 M H2SO4, 0.3 M
ascorbic acid, 4 mM potassium antimony (III) oxide tar-
trate and 24 mM ammonium heptamolybdate) contained
in glass disposable test tubes and incubated for another
15 minutes for color development.27 For each test sample
run in triplicate, two aliquots were taken to measure the
change in activity in comparison to that measured for
Na+/K+-ATPase alone. A calibration curve was deter-
mined by preparing standard solutions of inorganic
phosphate (Pi) from 1 or 0.1 mM aqueous stock solu-
tions. The absorbance was recorded at a wavelength of
850 nm in a 1 cm pathlength cuvette using a UV-2450
UV-visible spectrophotometer (Shimadzu, Kyoto, Japan).
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ABBREVIATIONS
ATP, adenosine triphosphate; BIS-TRIS, 2,2-bis(hy-
droxymethyl)-2,2′,2″-nitrilotriethanol; CD, circular di-
chroism; DMF, N,N-dimethylformamide; DPDS, diphenyl
diselenide; DSL, diselenide-selenoester ligation; EDTA,
ethylenediaminetetraacetic acid; FLR, fluorescence;
FXYD1, phospholemman; GSH, reduced L-glutathione;
HPLC, High-performance liquid chromatography;
MALDI-TOF, matrix-assisted laser desorption/ioniza-
tion-time of flight; NCL, native chemical ligation; OBD,
optimum bed density; Pi, inorganic phosphate; rDSL, re-
ductive diselenide-selenoester ligation; TCEP, tris(2-car-
boxyethyl)phosphine; TFA, trifluoroacetic acid; UV, ul-
traviolet.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: x. Experimental, all crude re-
action and ligation rate data, Figures S1-S43 and Schemes S1-
S14. (PDF).
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AUTHOR INFORMATION
Corresponding Author
*richard.payne@sydney.edu.au
Author Contributions
‡These authors contributed equally.
ORCID:
Diemer, V.; Cargoët, M.; Monbaliu, J.-C. M.; Melnyk, O. Native
chemical ligation and extended methods: mechanisms,
catalysis, scope, and limitations. Chem. Rev. 2019, 119 (12),
7328-7443.
Timothy S. Chisholm: 0000‐0002‐8693‐3797
Sameer S. Kulkarni: 0000‐0001‐5896‐9568
Ronald J. Clarke: 0000‐0002‐0950‐8017
Richard J. Payne: 0000‐0002‐3618‐9226
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Synthesis of proteins by native chemical ligation. Science 1994,
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Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
This work was supported by the Australian Research Council
through a Discovery Project (DP190101526). We would also
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