Modulating oxytocin activity and plasma stability by disulfide bond engineering

Article abstract of DOI:10.1021/jm100989w

Disulfide bond engineering is an important approach to improve the metabolic half-life of cysteine-containing peptides. Eleven analogues of oxytocin were synthesized including disulfide bond replacements by thioether, selenylsulfide, diselenide, and ditelluride bridges, and their stabilities in human plasma and activity at the human oxytocin receptor were assessed. The cystathionine (K_i = 1.5 nM, and EC₅₀ = 32 nM), selenylsulfide (K_i = 0.29/0.72 nM, and EC₅₀ = 2.6/154 nM), diselenide (K_i = 11.8 nM, and EC₅₀ = 18 nM), and ditelluride analogues (K_i = 7.6 nM, and EC₅₀ = 27.3 nM) retained considerable affinity and functional potency as compared to oxytocin (K_i = 0.79 nM, and EC₅₀ = 15 nM), while shortening the disulfide bridge abolished binding and functional activity. The mimetics showed a 1.5-3-fold enhancement of plasma stability as compared to oxytocin (t _1/2 = 12 h). By contrast, the all-d-oxytocin and head to tail cyclic oxytocin analogues, while significantly more stable with half-lives greater than 48 h, had little or no detectable binding or functional activity.

Full text of DOI:10.1021/jm100989w

J. Med. Chem. 2010, 53, 8585–8596 8585
DOI: 10.1021/jm100989w
Modulating Oxytocin Activity and Plasma Stability by Disulfide Bond Engineering
Markus Muttenthaler, Asa Andersson, Aline D. de Araujo, Zoltan Dekan, Richard J. Lewis, and Paul F. Alewood*
Institute for Molecular Bioscience, The University of Queensland, 306 Carmody Road, 4072 St. Lucia, Brisbane, Queensland
Received August 2, 2010
Disulfide bond engineering is an important approach to improve the metabolic half-life of cysteine-
containing peptides. Eleven analogues of oxytocin were synthesized including disulfide bond replace-
ments by thioether, selenylsulfide, diselenide, and ditelluride bridges, and their stabilities in human plasma
and activity at the human oxytocin receptor were assessed. The cystathionine (Ki=1.5 nM, and EC50=
32 nM), selenylsulfide (Ki = 0.29/0.72 nM, and EC50 = 2.6/154 nM), diselenide (Ki=11.8 nM, and
EC50=18 nM), and ditelluride analogues (Ki=7.6 nM, and EC50=27.3 nM) retained considerable affinity
and functional potency as compared to oxytocin (Ki=0.79 nM, and EC50=15 nM), while shortening the
disulfide bridge abolished binding and functional activity. The mimetics showed a 1.5-3-fold enhance-
ment of plasma stability as compared to oxytocin (t1/2=12 h). By contrast, the all-D-oxytocin and head
to tail cyclic oxytocin analogues, while significantly more stable with half-lives greater than 48 h, had
little or no detectable binding or functional activity.
Introduction
Modification of the proteolytic recognition sites within the
peptide is the underlying principle of most strategies aimed at
improving proteolytic stability. However, steric distortions
employed to increase stability often affect the biological activity
and selectivity.7-10 Presently, no general approach has been
identified that works for all target peptides, and understand-
ing their structure-activity relationships (SAR)a is funda-
mental for identifying the appropriate strategy for each class
of peptide. While cyclization, truncation, and terminal cap-
ping are widely applied techniques in the pharmaceutical indus-
try for stabilizing short linear peptides as well as disulfide-
rich peptides, this approach is only applicable to target peptides
where the N and C termini are not part of the pharmacophore.
Replacement of selected residues by D-amino acids has been
another successful approach to enhance the half-lives of peptide
drugs,11 exemplified with octreotride, a somatostatin analo-
gue used in the treatment of gastrointestinal tumors,12 and
with desmopressin (1-desamino-8-D-Arg-vasopressin) for the
treatmentof diabetes insipidusand bedwetting.13-16 Disulfide
bond engineering represents another complementary approach
that protects peptides from reduction or scrambling by thiol-
containing molecules such as glutathione, serum albumin, or
oxido-reductases. Various disulfide bond replacements have
been investigated, including lactam,17,18 dicarba,19-21 thio-
ether,22-26 and diselenide bridges,27-35 but there exists no
direct comparison of the efficacy of these modifications within
the same peptide, making it difficult to draw conclusions on
their relative merits. In this work, we evaluate various con-
servative disulfide bond replacements on the single-disulfide
bond peptide oxytocin (OT) and assess the effects of these
disulfide bond mimetics on peptide stability and biological
activity.
Peptides have emerged as a commercially relevant class of
drugs that offer the advantage of greater specificity and potency
and lower toxicity profiles over traditional small molecule
pharmaceuticals.1 They offer promising treatment options for
numerous diseases, such as diabetes, HIV, hepatitis, cancer,
and others, with physicians and patients becoming more
accepting of peptide-based medicines.2 On the other hand,
peptides also have several well-known drawbacks that have
prevented them from becoming a mainstream source of drug
candidates, including short circulation half-life, poor proteo-
lytic stability, and low oral bioavailability.3,4 A series of
formulation and conjugation strategies have been developed
to address delivery disadvantages,3,4 while the use of unnatural
and D-amino acids, cyclization, terminal capping, truncation,
and disulfide bond engineering can improve stability to pro-
teolytic degradation.5 Peptides are metabolized by three major
mechanisms: Endopeptidases break peptide bonds of non-
terminal amino acids, exopeptidases cleave N- or C-terminal
amino acids, and thiol-protein-disulfide oxido-reductases
inactivate the peptides due to loss of secondary structure.6
*To whom correspondence should be addressed. Tel: þ61 7 3346
2982. Fax: þ61 7 3346 2101. E-mail: p.alewood@imb.uq.edu.au.
a Abbreviations: *, C-terminal amide; cAMP, cyclic adenosine mono-
phosphate; AVP, vasopressin, arginine-vasopressin, antidiuretic hormone;
Boc, tert-butyloxycarbonyl; CNS, central nervous system; DIEA, diiso-
propylethylamine; DTT, dithiothreitol; ESI-MS, electrospray ionization-
mass spectrometry; Fmoc, fluorenylmethyloxycarbonyl; GPCR, G protein-
coupled receptor; GSH, reduced glutathionine; GTP, guanosine tripho-
sphate; HATU, 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium
hexafluorophosphate; HBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyl
uronium hexafluorophosphate; HOAt, 1-hydroxy-7-azabenzotriazole;
HTRF, homogeneous time-resolved fluorescence; IP, inositol phosphate;
LC-MS, liquid chromatography-mass spectrometry; MBHA, 4-methyl-
benzhydrylamine; OT, oxytocin; OTR, oxytocin receptor; PyAOP, 7-aza-
benzotriazol-1-yloxy-tri(pyrrolidino)phosphonium hexafluorophosphate;
SAR, structure-activity relationship; Sec, U, selenocysteine; SPPS, solid-
phase peptide synthesis; Tec, tellurocysteine.
OT and vasopressin (AVP) are closely related, highly con-
served, multifunctional nonapeptides with N-terminal cyclic
six-residue ring structures stabilized by an intramolecular
disulfide bond and a flexible 3-residue C-terminal tail.36
r
2010 American Chemical Society
Published on Web 11/30/2010
pubs.acs.org/jmc

8586 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Table 1. Summary of Synthesized OT Analoguesa
Muttenthaler et al.
a U, selenocysteine.
OT and AVP mediate their biological function by acting on
four known receptors: the OT (uterine), the vasopressin V1a
(vasopressor), the V1b (pituitary), and the V2 (renal) receptors,
which are all members of the G protein-coupled receptor
(GPCR) family.36 Activation of the OT, V1a, and V1b recep-
tors leads to a measurable intracellular increase of inositol
phosphate one (IP-one), while V2R activation results in a
measurable intracellular increase of cyclic adenosine mono-
phosphate (cAMP).36 OT in the periphery is involved in
uterine smooth muscle contraction during parturition, ejacu-
lation, and milk ejection during lactation.36,37 In the central
nervous system (CNS), OT functions as a neurotransmitter
involved in complex social behaviors, maternal care, partner-
ship bonding, stress, and anxiety.36-38 Recently, the OT
receptor has been shown to play a role in breast cancer tumor
growth39,40 and autism,36,41 where metabolically more stable
OTanalogues wouldbeof advantage. Thus, OTwas chosen as
a starting point in this study with the additional advantage to
be the most studied peptide in the literature with extensive
structural, biological, and therapeutic data available for com-
parison. Importantly, the biological characterization of activ-
ity (cell-based assays, animal models, and clinical studies) is
well established, and synthetic access is readily achieved via
solid-phase peptide synthesis (SPPS). Its single disulfide bond
with its conformational flexibility is critical for secondary
structure and agonistic activity.21,42 NMR and crystallography
studies of unbound OT and desamino-OT indicate a flex-
ible structure, which crystallizes into two well-defined con-
formers.42 Recognition motifs such as the type II β-turn
between the backbone -NH of Tyr20 and the carbonyl Asn50
and the type III β-turn between the Cys60 carbonyl and the
Gly90 imino group are present when free but are not observed
when OT is bound to its carrier protein neurophysin,43 sug-
gesting that binding occurs stepwise via a receptor-induced fit.
Additionally, NMR studies show high conformational mobi-
lity around Tyr20 and of the carboxylic tripeptide tail, with the
disulfide bridge conformation tied to their orientation and
consequently their agonist activity.44,45 Even minor distortions
such as an increase of the 20-membered ring by one methylene
group,46 reducing the disulfide bond flexibility by substitution
of the cysteine at position 1 by penicillamine45 or replacement
of the disulfide bond by an amide18 or dicarba bond,21,47,48
lead to a significant decrease in biological activity together with
either a switch from an agonist to antagonist or loss of receptor
selectivity. Hence, OT can be considered as a highly sensitive
model for structural modification studies, where even small
changes in bond length or dihedral angles may result in bio-
logical activity changes, and thus provides an ideal platform
for the SAR study of disulfide bond mimetics.
This study investigated 11 OT analogues (Table 1) including
nine that contain thioether, selenylsulfide, diselenide, and dite-
lluride bonds to evaluate the impact of disulfide bond mimetics
on biological activity at the human OT receptor and on their
metabolic stability in human plasma. In addition, backbone
cyclized and all-D-amino acid OT analogues were synthe-
sized to place the disulfide engineering strategy in context
with other commonly used strategies to enhance the half-life
of peptides.
Results
The native neuropeptides OT and AVP (1 and 2) as well as
the all-D-analogues (12 and 13) were synthesized via tert-
butyloxycarbonyl (Boc) chemistry SPPS using the 2-(1H-
benzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluor-
ophosphate (HBTU)-mediated in situ neutralization protocol
for chain assembly.49 The seleno analogues (6-8) were synthe-
sized as described by Muttenthaler et al.,27,50 and the N to C
cyclic analogues (10 and 11) followed the procedure described
by Clark et al.9 Synthesis of the thioether analogues proved to
be more challenging, and a novel on-resin strategy was devel-
oped to produce the thioether analogues (3-5). The synthe-
sis of the ditelluride analogue (9) involved novel chemistry
and will be reported in detail elsewhere. Metabolic stability
was determined by human plasma degradation studies,
and the biological activity was investigated via radioligand
binding and IP-one functional studies on the human OT
receptor.
Synthesis of OT Thioether Analogues (3-5). Two thioether
OT analogues, cystathionine (5) and lanthionine (3 and 4)
OT, have been prepared as outlined in Scheme 1. The cyst-
athionine analogue (5) was synthesized following the proce-
dure described by Mayer et al.,51 where thioether cross-
linking was achieved by on-resin cyclization/alkylation of
an internal cysteine thiol with a N-terminal bromo-homo-
alanine residue.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8587
Scheme 1. Lanthionine Building Block Synthesis Followed by Assembly and Formation of Lanthionine OT (3 and 4)a
a The structure of cystathionine OT (5) is also shown.
In a different approach, the thioether linkage of lanthio-
nine OT was constructed prior to resin assembly. For this
purpose, the orthogonally protected lanthionine building
block 15 was synthesized and later incorporated into the
solid-phase synthesis. The lanthionine building block 15 was
generated following the methodology reported by Zhu and
Schmidt,52 where the thioether bond was formed by reaction
of protected β-bromo-L-alanine with cysteine derivatives
under phase-transfer conditions at pH 8.5 (Scheme 1). Their
proposed route was somewhat modified here to suit the
installation of the more appropriate orthogonal protecting
groups: fluorenylmethyloxycarbonyl (Fmoc), tBu, pNZ,
and allyl. Although this method has been reported to give
enantiopure lanthionine compounds,52 the thioalkylation
step in our case afforded a mixture of two diastereoisomers
in a ratio of 2:3 as indicated by NMR spectroscopy. Race-
mization at the R-carbon position of the pNZ-β-Br-OtBu
precursor is believed to have occurred as a result of an
elimination-Michael addition process. The isomers could
not be separated by flash chromatography; therefore, the
lanthionine building block was further employed as an
isomeric mixture.
The synthesis of lanthionine OT was carried out by standard
Fmoc/tBu-based SPPS, using HBTU/DIEA activation pro-
tocols (Scheme 1). The linear peptide was assembled manually
on a Rink amide 4-methylbenzhydrylamine (MBHA) resin. The
building block 15 was incorporated at the fourth residue
followed by linear extension of the peptide chain. Removal
of the allyl protecting group with Pd(0) and Fmoc group
with piperidine was followed by intramolecular amide bond
cyclization assisted by the 7-azabenzotriazol-1-yloxy-tri-
(pyrrolidino)phosphonium hexafluorophosphate (PyAOP)/
1-hydroxy-7-azabenzotriazole (HOAt)/DIEA coupling
cocktail.53 Attempts to remove the N-terminal pNZ protect-
ing group while on resin using the procedure reported by
Albericio54 were unsuccessful. Deprotection was achieved
as follows: The cyclic peptide was first cleaved from the resin
in the pNZ-protected form where two products having the
same expected molecular weight for the lanthionine OT
were obtained. The crude material was then submitted to
hydrogenolysis to afford two isomers (isomers 3 and 4) of
the fully unprotected lanthionine OT that could be separated
by RP-HPLC. As the two lanthionine OT isomers showed
considerable reduction in activity (Table 2), we were dis-
couraged to further investigate their stereochemical proper-
ties in detail.
Biological Activity. The peptides were tested for activity at
the human OT receptor (Table 2 and Figure 1). Binding data
were obtained via radioligand binding assays, which measured
the displacement of 3H-OT from the oxytocin receptor (OTR)
expressed in COS-1 cells. The ability of the analogues to signal
through Gq and generate IP-one in COS1-cells transfected
with the OTR was investigated using an HTRF (homogeneous
time-resolved fluorescence) assay.55 Hill slope analysis was
done using the sigmoidal curves (variable slope) fitted to
individual data points by nonlinear regression using the
software package Prism (GraphPad Software) showing that
the Hill slopes were not statistically significantly different
from -1.
Thioether Analogues (3-5). Both lanthionine [--S]-OT iso-
mers showed a 1000-fold drop in binding affinity (Ki =
856 nM, 1547 nM) at the OTR as compared to OT (Ki =
0.79 nM). The lanthionine isomers also failed to stimulate IP-
one signaling at concentrations up to 10 μM. Cystathionine OT,

8588 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Muttenthaler et al.
Table 2. Potencies of OT Analogues at the OTR Tested in Radioligand Binding Assay and Functional IP-One Assay and Metabolic Half-Lives in
Human Seruma
human OT receptor
functional EC50 (nM)
human serum
half-life (h)
no.
analogues
binding Ki (nM)
1
2
OT
vasopressin
0.79 ( 0.16
12 ( 2.8
15 ( 2.5
41 ( 3.6
>105
11.6 ( 1.9
1.7 ( 0.6
ND
3
[--S]-OT iso 1
[--S]-OT iso 2
[CH2-S]-OT
1547 ( 457
856 ( 201
1.5 ( 0.29
0.29 ( 0.02
0.72 ( 0.31
11.8 ( 4.1
7.6 ( 2.7
1404 ( 243
>105
4
>105
ND
5
32.0 ( 9.4
2.6 ( 1.0
154 ( 86
18.0 ( 12
27.3 ( 7.4
>105
36.9 ( 2.8
18.6 ( 3.0
ND
6
[Se-S]-OT
[S-Se]-OT
7
8
[Se-Se]-OT
[Te-Te]-OT
25.3 ( 5.9
24.2 ( 2.2
>48
9
10
11
12
13
N-C cyclic-OT
N-C cyclic-OT-AGAGAG
all-D-retroinverse-OT
all-D-OT
>105
>48
5.5 ( 1.3
>48
>105
>105
>105
>105
a ND, not determined; n = 3-6.
Figure 1. Binding and functional data of the disulfide bond mimetics of OT on the human OT receptor. Affinity data (A1 and A2) were
obtained via radioligand binding assay, which measured the displacement of 3H-OT by the analogues from the human OT receptor expressed in
COS-1 cells. The ability of the analogues to signal through Gq and generate IP-one in COS-1 cells transfected with the human OT receptor was
investigated using a homogeneous time-resolved fluorescence assay (B1 and B2). The exact Ki and EC50 values of each analogue are listed in
Table 2. Results are the means ( SEs of three separate experiments, each performed in triplicate.
[CH2-S]-OT, retained binding affinity (Ki = 1.5 nM) and the
ability to signal (EC50 = 32 nM) at the OTR.
Stability in Human Plasma. The stability of peptides 1, 2, 5,
6, and 8-13 in human plasma was assessed by LC-MS and
RP-HPLC analysis (Figure 2 and Table 2). AVP showed a
short half-life of 2 h as compared to OT with 12 h and was not
detectable by analytical RP-HPLC or LC-MS after 4 h. As
the positive controls, OT and AVP were incubated in phos-
phate buffer at pH 7.4 and 37 °C, and no degradation was
observed over a period of 48 h. The selenium (6 and 8), tellu-
rium (9), and thioether (5) analogues exhibited an 1.5-3-fold
increase in stability in relation to OT, although not as distinct
as the two bicyclic analogues (10 and 11) with half-lives greater
than 48 h. While all-D-OT was the most stable analogue (only
20% degradation after 48 h), the all-D-retroinverse OT surpris-
ingly exhibited a half-life of only 4 h and was less stable than
L-OT. No traces of linear reduced analogues were observed
throughout the study.
Selenium Analogues (6-8). [Se-S]-OT and [S-Se]-OT retained
both their binding affinity and [Se-S]-OT also its functional
efficacy, while a 10-fold decrease was observed with [S-Se]-OT
(EC50 = 154 nM) as compared to OT (EC50 = 15 nM). [Se-Se]-
OT showed a 10-fold decrease in binding affinity (Ki = 11.8 nM)
as compared to OT (Ki = 0.79 nM); however, its functional
efficacy was retained (EC50 = 18 nM).
Tellurium Analogue (9). The binding affinity of [Te-Te]-
OT decreased 10-fold (Ki = 7.6 nM) when compared to
OT, although functional activity was maintained (EC50
27.3 nM).
=
N- to C-Terminal Cyclic and D-OT Analogues (10-13). The
bicyclic and the all-D OT analogues all showed drastic de-
creases in binding affinities (>2000-fold) and also failed to
stimulate signaling at concentrations up to 10 μM on the
human OTR.
Reduction and Alkylation Study. Peptides 1, 5, and 8 were
exposed to a variety of reduction and alkylation conditions

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8589
Figure 2. Metabolic stability of OT analogues in human serum. OT vs AVP (A), OT vs D-analogues all-D-OT and all-D-retroinverse-OT (B),
OT vs selenium analogues [Se-S]-OT and [Se-Se]-OT (C), and OT vs cyclic analogues cyclic-OT and cyclic-OT-AGAGAG (D).
to compare the stability of the disulfide, diselenide, and thio-
ether bond in reducing environments (Figure 3). The thioether
bond (Figure 3, A3-D3) was stable to all tested reduction
and alkylation conditions, including 100-fold excess dithio-
threitol (DTT) and 1000-fold excess glutathione. The diselenide
bond (Figure 3, A2-D2) was completely reduced and alkyl-
ated by a 10-fold excess of DTT, followed by the addition of
excess of iodoacetamide at pH 7.4 and 37 °C. The reduced
species (selenol) could not be detected by analytical means of
MS, LC-MS, nor HPLC, indicating that reduction/reoxida-
tion occurs rapidly. The disulfide bond (Figure 3, A1-D1)
was readily reduced and alkylated by a 10-fold excess of DTT
or a 1000-fold excess of reduced glutathione (GSH) in the
presence of iodoacetamide. Reduction with 10-fold DTT or
1000-fold GSH of [Te-Te]-OT resulted in a complex mixture
of GSH adducts and products with tellurium deletions (data
not shown).
thioether, selenylsulfide, diselenide, and ditelluride substitutions
can be considered conservative disulfide bond replacements as
full binding and functional activity ((10-fold) was retained in
these analogues (Table 2 and Figures 1, 4, and 5). The affinity
and functional activity of OT at the human OT receptor
correlate well with literature values, displaying binding affi-
nity ∼10-fold higher than agonist potency.55-58 Surprisingly,
[Se-S]-OT enhanced both binding affinity and potency 3-
6-fold, while [S-Se]-OT had no affect on binding affinity but
produced a 10-fold reduction in agonist activity as compared
to OT (Table 2). Even the ditelluride analogue with the largest
˚
changes inbondlength (þ0.65A) retainedfull bindingaffinity
and functional efficiency. Reduction in ring size by deletion of
one sulfur in the bridge as in case of the lanthionine analogues
(3 and 4) resulted in a drastic loss of binding and functional
activity. The cystathionine on the other hand maintained full
binding and functional activity. N- to C-terminal cyclization
of OT (10) led to a complete loss of activity, which has also
been observed in earlier work.59 Incorporation of a six-residue
linker (AGAGAG, 11) to induce more flexibility also resulted
in an inactive analogue.
Discussion and Conclusion
Eleven OT analogues were synthesized and characterized
on the human OT receptor and in human serum to determine
the impact of disulfide bond engineering on biological activity
and stability (Table 1). Modifications to OT included replace-
ment of the disulfide bond by thioether, selenylsulfide, disel-
enide, and ditelluride bridges, as well as bicyclic and all-D-amino
acid analogues selected as controls for metabolic stability
assessment. Given that OT activity appears highly sensitive to
structural changes (Table 2), it permitted a close evaluation of
isosteric replacements of either one or both sulfur atoms in the
bridge with closely related elements such as carbon, selenium,
and tellurium (Table 3). Changes in the bond lengths of the
disulfidebridgereplacementsrangedfromapproximately0.77
The observed 10-100-fold shift between binding affinity
and agonist potency of OT and analogues has been observed
previously for other agonists of GPCRs including vasopressin
analogues acting at the V1b receptor, where small changes in
ligand structure resulted in a 30-fold shift between binding
affinity and potency to stimulate IP.60 The affinity/potency
difference observed in these studies could arise from cross-talk
between G-proteins, receptors, and ligand that influences the
conformation of the receptor in a well-described guanosine
triphosphate (GTP)-dependent manner.61-64 Hill slope anal-
ysis showed no significant evidence for two binding sites or
cooperative binding effects.
˚
(lanthionine analogues) to 2.68 A (ditelluride analogues), and
changes in torsion angles [from 180° (thioether bridge) to 89-
98° (disulfide, diselenide, and ditelluride bridges)] gave insight
into the influence of bond length and torsion angles on con-
formational flexibility and activity.
The biological analysis of the disulfide bond mimetics
on the human OT receptor confirmed the hypothesis that
Overall, it is interesting to observe that changes in bond
˚
˚
length of the disulfide bridge by -0.21 A up to þ0.65 A can be
achieved without any major losses in activity, even in such
sensitive systems as OT, where β-turns need to be preserved
for receptor recognition.44,65 The observed changes due
to torsion angles or individual positions (Se in position 1

8590 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Muttenthaler et al.
Figure 3. Reduction and alkylation study of OT (1), diselenide OT (8), and cystathionine OT (5). This study illustrates that the thioether bond
(A3-D3) is stable to all tested reduction and alkylation conditions. The diselenide bond (A2-D2) seems to be stable to reduction up to 1000-
fold GSH and 100-fold DTT according to MS/LCMS/HPLC analysis, but by addition of alkylating agent, the diselenide bond is reduced and
alkylated, indicating that reduction/reoxidation occurs rapidly and cannot be detected by MS/LCMS/HPLC. The disulfide bond is readily
reduced by 10-fold DTT or 100-fold GSH. Conditions A: Compounds 1, 5, and 8 in PBS buffer, pH 7.4, room temperature at time = 0 min.
Conditions B: 1000-fold glutathione at 37 °C, pH 7.4, after 12 h. Conditions C: 100-fold DTT at 37 °C, pH 7.4, 1 h. Conditions D: 10-fold DTT
at 37 °C, pH 7.4, 1 h, followed by addition of 100-fold iodoacetamide (IA) and incubated for 1 h at 37 °C, pH 7.4.
Table 3. Physicochemical Properties of Carbon, Sulfur, Selenium, and Tellurium27,34,80,81a
properties
˚
X = carbon
X = sulfur
X = selenium
X = tellurium
covalent radius (A)
0.77
1.54
1.54
1.70
NA
NA
1.02
1.82
2.03
1.80
180
98
1.17
1.35
2.40
2.68
2.06
180
89
β
γ
˚
bond length C -X (A)
1.95-1.99
2.33
1.90
γ
δ
˚
bond length X -X (A)
˚
van der Waals radii (A)
torsion angle χ3-Cβ-Cγ-Xδ-Cε- (°)
174
94
torsion angle χ3-Cβ-Xγ-Xδ-Cε- (°)
a NA, not available.
vs position 6) further confirm the enormous sensitivity of the
OT ligand-receptor system, making it an ideal system for
SAR studies.
mimetics. A comprehensive structural and functional study
of various R-conotoxins containing selenocysteine replace-
ments illustrated that such a modification had no significant
impact on torsion angles, activity, or receptor subtype selec-
Previous to this study, the two-disulfide bond containing
R-conotoxins represented the most-studied system for disulfide
bond mimetics, although the disulfide bond replacements had
been conducted on different R-conotoxins, thus making inter-
pretation and comparison of their impact difficult. Disulfide
bonds stabilize and orientate the secondary loop structure
(R-helix) in the R-conotoxins, which contain the residues impor-
tant for receptor interaction.66 Nevertheless, replacement of
one of the disulfide bonds in R-conotoxin SI by a lactam bridge
resulted in either complete or 60-70-fold loss of activity,
depending on which disulfide bond was modified.17 Substitu-
tion of a single disulfide bond in R-ImI with an unsaturated
dicarba bridge resulted in both cis and trans isomers, with one
retaining significantly reduced biological activity, while the
other was inactive.19 Thioether replacement of one disulfide
bond in R-GI also resulted in 260-800-fold loss of activity of
the two obtained isomers.22 Similarly to our study, it appears
that the most promising approach is the generation of selenotoxin
tivity of this class of peptides.27 X-ray analysis at 1.4 A res-
˚
olution of R-selenoconotoxin PnIAshowed thatthe diselenide
˚
˚
bond was 0.3 A longer than the disulfide bond (2.03 A) with
torsion angles of 93.9 and 83.1°, respectively. Additionally, it
was shown that the increased hydrophobicity and surface
exposure of the diselenide bond had a beneficial effect on the
activity in some of the analogues.
The second objective of this study was to compare disulfide
bond mimetics with other commonly employed metabolic sta-
bility improvement strategies such as backbone cyclization
and the use of D-amino acids. The driving concept behind disul-
fide replacement is that disulfide bond reduction and scram-
bling by thiol-oxido-reductases or similar free thiol-containing
molecules generally leads to biological inactivation.6 Thus,
implementation of disulfide bond mimetics with redox-stable
cross-links such as thioether or dicarba bridges should yield
metabolically more stable analogues.67-69 This was exemplified

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8591
Figure 5. Functional data of OT analogues at the human OTR
expressed in COS-1 cells. Functional data were measured as the
accumulation of IP-one in the cell after stimulation by OT analo-
gues. * indicates statistically significant changes based on OT (p <
0.05). Results are the means ( SEs of 3-6 experiments. Each
experiment was performed in triplicate.
Figure 4. Binding data of OT analogues at the human OTR expressed
in COS-1 cells. Binding affinities were measured as the displacement
of H3-OT radioligand by OT analogues. * indicates statistically sig-
nificant changes based onOT (p < 0.05). Resultsare the means ( SEs
of 3-6 experiments. Each experiment was performed in triplicate.
in carbetocin, clinically used to treat postpartum hemorrhage,
where a thioether bridge results in a longer acting agonist than
its disulfide bond counterpart.25,26 A similar strategy replaces
sulfur by selenium, as diselenides also exhibit a lower redox
potentialthandisulfideswitha difference of 111-166 mV.30,70
There exists only limited data on ditelluride compounds
regarding redox potentials, in particular within peptides; how-
ever, the ditelluride bridge is expected to have a lower redox
potential than the diselenide bond. While there are currently
no in vivo studies that assess a diselenide bond impact on half-
life or longer lasting effects, recent studies on R-conotoxins
showed that a single diselenide bond substitution in this two-
disulfide bond system efficiently suppressed drug inactivation
via scrambling. However, nosignificanthalf-life improvement
was observed in plasma degradation studies, mainly dueto the
fact that plasma degradation occurs largely by endo- and
exopeptidases rather than by oxido-reductases.27,31 More-
over, a question remains as to whether the increased stability
of the diselenide is due to its lower redox potential or to its
rapid reoxidation kinetics. To answer this question, we con-
ducted a comprehensive reduction and alkylation study to-
gether with stability studies in human serum.
Initially, the influence of the bridge redox potential on plasma
half-life was investigated and was compared to other commonly
used stabilization strategies such as backbone cyclization and
the use of D-amino acids. Well-established plasma stability assays
were employed, which are frequently used in peptide drug
optimizationprocesses:71,72 Metabolic stabilityimprovements
were observed that correlate with the redox potential of the
disulfide bond substitution, yielding half-lives of 12 h for OT,
19 h for [Se-S]-OT, 24 h for [Te-Te]-OT, 25 h for [Se-Se]-OT,
and 37 h for [CH2-S]-OT (Figure 2C and Table 2). This 1.5-
3-fold enhancement in half-life, however, is quite moderate as
compared to other strategies employed; for example, the bicy-
clic analogues (10 and 11) led to an increase in half-life (>48 h),
and all-D-OT (13) resulted in the greatest stability increase
of all tested compounds, showing only 20% degradation over
48 h. In general terms, the cyclization approach and the use of
unnatural or D-amino acids can be considered as the more effec-
tive strategies to improve the proteolytic half-lives of peptides
than the often more challenging disulfide bond modifications.
This is particularly the case given that plasma degradation by
endo- and exopeptidases occurs more readily than by oxido-
reductases or other reducing molecules.73 However, consider-
ing the complete loss of activity of the bicyclic analogues, head
to tail cyclization will only have a selective use in OT agonist
design but might be practical for the development of long-
acting antagonists where free N and C termini are not necessa-
rily important for function due to distinct SARs and topo-
graphical features regarding receptor binding.45,74-77 Selective
and strategic residue exchanges with D-amino acids in combi-
nationwithdisulfidebond engineering canbe considered asan
effective approach to improve the proteolytic stability of OT
analogues significantly. It has to be noted that the circulation
half-life of OT in vivo is only 1-5 min due to effective renal
clearance; however, proteolytic stability is important for other
biomedical applications including CNS-related disorders
(Autism, anxiety, andstress)36,41 orbreast cancer tumor detec-
tion,40 where the route of administration eludes renal clearance.
The plasma stability study also revealed that although OT
and AVP only differ in two amino acids (isoleucine to phenyl-
alanine change at position three and a leucine to arginine
change atposition eight);OTwitha half-life of12h was found
to be six times more stable than AVP with a half-life of 2 h
(Figure 2A). It seems that the L8R substitution renders AVP
susceptible to Pro-Arg-cleavage by trypsinlike serine proteases
such as thrombin, factor Xa, and plasmin, which are abundant
in blood and play an important role in blood coagulation.73
Replacement of arginine in position 8 by its D-amino acid
analogue to increase the metabolic stability was successfully
employed within the drug Desmopressin (1-desamino-8-D-
Arg-vasopressin), resulting in increased stability and longer
lasting effects.14-16 All-D-retroinverse-OT showed a surpris-
ingly short half-life of only 5.5 h as compared to the 12 h of
L-OT, suggesting that the secondary structure (two β-turns) has
significant contribution on the peptide’s overall stability. The
reduction and alkylation study on compounds 1, 5, and 8 was
conducted to evaluate the stability of the disulfide, thioether,
and diselenide bonds in a reducing environment. The thioether

8592 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Muttenthaler et al.
bridge was as expected stable to reduction by a 1000-fold
excess of GSH and DTT, while the disulfide bond was readily
reduced by a 100-fold excess of GSH or a 10-fold excess of
DTT. The reduction of the diselenide bond, however, could
only be measured indirectly by reduction in the presence of an
alkylating agent. It was not possible to observe the reduced
species directly by means of MS, LC-MS, or HPLC analysis
due to the high reactivity of the selenolate, which reoxidizes
rapidly to the diselenide bond. However, complete reduction
and alkylation were observed within 1 h in the presence of a
10-fold excess of DTT and excess of iodoacetamide, demon-
strating that the diselenide bond is readily reduced by a small
excess of reducing agent, but is reoxidized immediately if no
other molecules are present to trap the reduced species.
In conclusion, a set of conservative disulfide bond mimetics
of OT, including thioether, selenylsulfide, diselenide, and dite-
lluride bridges, were analyzed for binding and functional
activity on the human OT receptor and metabolic stability
in human plasma. Single sulfur substitution by a methylene or
selenium was well tolerated, along with the longer diselenide
and ditelluride bond analogues, which also retained their bio-
logical activity. These analogues represent enhanced mimetics
as compared to earlier studied lactam and dicarba analogues,
whereagonist activitywas lost. Incontrast, deletionof a sulfur
atom from the bridge led to complete loss of activity, under-
scoring theimportanceofring size for receptor activation. The
disulfide bond mimetics showed a 1.5-3-fold improvement of
metabolic plasma stability; yet, degradation in plasma occurs
largely by endo- and exopeptidases,73 and strategies such as
backbone cyclization and the use of D-amino acids were
shown to outperform disulfide bond engineering in plasma.
This study establishes a robust platform for future OT/AVP
agonist design and is fully compatible with other important
classes of disulfide-rich peptides, such as neurotransmitters,
growth factors, hormones, enzyme inhibitors, antimicrobial
peptides, and venom toxins to create analogues with enhanced
metabolic stability.
and FuGENE 6 Transfection Reagent were from Roche
Diagnostics (Castle Hill, NSW, Australia). [Tyrosyl-2,6-3H]-OT;
46.3 Ci/mmol, 2200 Ci/mmol, FlashBLUE GPCR scintillating
beads, TopSeal-A 96-well sealing film, and 384-well white optiplates
were from PerkinElmer Life Sciences (Knoxfield, VIC, Australia).
Costar 96-well white plates with clear bottoms were obtained from
Corning (Lindfield, NSW, Australia). The IP-one HTRF assay kit
ꢀ
was from CisBio International (30204 Bagnols-sur-Ceze, France).
Peptide Synthesis. Peptides 1, 2, 6-8, and 10-13 were
assembled manually via Boc-SPPS using the HBTU-mediated
in situ neutralization protocol49 following the procedure of
Muttenthaler et al.27 for the monocyclic (1, 2, 6-8, 12, and 13)
and the procedure of Clark et al.9 for the bicyclic OT analo-
gues (10 and 11). After HF cleavage, the crude peptides were
˚
purified by preparative RP-HPLC (Vydac C18 column, 300 A,
250 mm ꢀ 21.2 mm) using a linear gradient of 0-50% B (A,
H2O/0.05% TFA; B, 90% CH3CN/10% H2O/0.043% TFA)
in 50 min at 8 mL/min while monitoring UV absorbance at 226
nm. The selenium-containing peptides were already oxidized
after HF cleavage.27 Folding and cyclization of the disulfide
bond containing analogues 1, 2, and 10-13 were carried out in
0.1 M NH4HCO3 buffer, pH 8.4, at 25 °C and a peptide
concentration of 100 μM. Peptide oxidation, deprotection,
and purity were monitored by mass spectrometry (MS) on a
LCT-TOF mass spectrometer equipped with an electrospray
ionization source, by analytical RP-HPLC using a Vydac C18
˚
column (300 A, 5 μm, 250 mm ꢀ 4.6 mm) at 214 nm, and by
ESI-LCMS on a Phenomenex Jupiter liquid chromatography
˚
mass spectrometry (LC-MS) C18 column (90 A, 4 μm, 250 mm ꢀ
2 mm) on a SCIEX QSTAR Pulsar QqTOF mass spectro-
meter equipped with an atmospheric pressure ionization source,
running a linear gradient of 0-50% solvent B over 50 min
with a flow rate of 1 mL/min for RP-HPLC and 200 μL/min
for LC-MS analysis. The synthesized peptides and organic
compounds were of >98% purity, unless stated otherwise.
The cystathionine analogue (5) was synthesized following the
procedure described by Mayer et al.,51 where thioether cross-
linking was achieved by on-resin cyclization/alkylation of an
internal cysteine thiol with a N-terminal bromo-homoalanine
residue. The synthesis of the ditelluride analogue 9 will be
reported elsewhere.
Synthesis of Lanthionine OT Isomers (3 and 4). Nr-(9-
Fluorenylmethoxycarbonyl)-L-cysteine tert-Butyl Ester (Fmoc-
Cys-OtBu). To a solution of (Fmoc-Cys-OtBu)2 (0.91 g, 1.14
mmol) in DCM (20 mL), 1,4-DTT (263 mg, 1.71 mmol) and
triethylamine (0.239 mL, 1.71 mmol) were added. After it was
stirred at room temperature for 1 h, the reaction mixture was
concentrated and purified by flash chromatography (petro-
leum ether:EtOAc 4:1). The product was obtained as a color-
less oil in quantitative yield (0.90 g, 99%). Analytical data for
this compound are in accordance with literature.78
Experimental Section
Materials. NR-Boc- and NR-Fmoc-L-amino acids, Fmoc-Rink
amide MBHA resin, and reagents used during chain assembly
were peptide synthesis grade purchased from Novabiochem (Merck
Pty., Kilsyth, VIC, Australia) and Bachem (Bubendorf, Switzerland).
L-Selenocystine and Boc-L-Sec(MeBzl)-OH were synthesized as
described by Muttenthaler et al.27 MBHA resin was purchased
from the Peptide Institute (Osaka, Japan). HBTU and PyAOP
were purchased from Fluka (Buchs, Switzerland), 2-(1H-7-
azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluoro-
phosphate (HATU) was purchased from GenScript Corp.
(Piscataway, NJ), and diisopropylethylamine (DIEA), trifluoro-
acetic acid (TFA), dichloromethane (DCM), and N,N0-dimethyl-
formamide (DMF) were purchased from Auspep (Melbourne,
VIC, Australia). Anhydrous hydrogen fluoride (HF) was pur-
chased from BOC Gases (Sydney, NSW, Australia), and p-cresol
and p-thiocresol, human plasma, as well as all other organic
reagents and solvents, unless stated otherwise, were purchased
in their highest purity from Sigma-Aldrich (Sydney, NSW,
Australia). All solvents for SPPS were of peptide synthesis grade
and used without further purification. HPLC grade acetonitrile
(Lab Scan, Bangkok, Thailand) and water measuring 18.2 MΩ
(ELGA, Melbourne, VIC, Australia) were used for the prepara-
tion of all solvents for liquid chromatography.
Nr-para-Nitrobenzyloxycarbonyl-L-serine Allyl Ester (pNZ-
Ser-OAll). p-Nitrobenzyl chloride (6.15 g, 28.5 mmol) was
dissolved in dioxane (12 mL), and a solution of NaN3 (2.22 g,
34.2 mmol) in 9 mL of H2O was added. The resulting emulsion
was stirred for 2.5 h. Serine (3.0 g, 28.5 mmol) was dissolved in
35 mL of dioxane/2% Na2CO3 (1:1) and added dropwise. The
resulting emulsion was stirred for 2 days keeping the pH between
9 and 10 by addition of 10% Na2CO3 solution. Water was
added, and the suspension was washed with diethylether (3ꢀ).
The aqueous phase was acidified to pH 2 by adding 3 N HCl,
and the product was extracted with 3ꢀ EtOAc. The organic
layers were combined, dried over MgSO4, and concentrated.
The crude product (6.25 g of pNZ-Ser-OH, 77% yield) was used
without further purification. To a solution of pNZ-Ser-OH (3.0 g,
10.5 mmol) in DMF (20 mL), K2CO3 (1.85 g, 13.4 mmol) and
allyl bromide (1.7 mL, 15.8 mmol) were added. The mixture was
stirred overnight at room temperature. Water was added, and
the product was extracted 3ꢀ with EtOAc. The combined
organic layers were washed with water (2ꢀ), brine, dried over
OTR cDNA was obtained from Origene technologies inc
(Rockville, United States). Dulbecco’s modified Eagle’s med-
ium and Lipofectamine2000 were purchased from Invitrogen
(Mulgrave, VIC, Australia). Complete protease inhibitor cocktail

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8593
MgSO4, and concentrated. The desired product was recrystal-
lized from EtOAc/petroleum ether to give light yellow crystals
(2.3 g, 68% yield, > 98% purity by HPLC). MS (m/z): MS
(calcd): 325.1036 [M þ H]; MS (found): 325.1022 [M þ H]. mp
4.21 (1H, t, J = 7.0 Hz, CH Fmoc), 4.25-4.31 (3H, m, RCH þ
CH2 Fmoc), 4.57 (2H, d, J = 4.7 Hz, CH2), 5.17 (1H, d, J =
14 Hz, CHdCH2), 5.18 (2H, s, CH2), 5.28 (1H, d, J = 17 Hz,
CHdCH2), 5.83-5.89 (1H, m, CHdCH2), 7.30 (2H, t, J =
7.4 Hz, Ar Fmoc), 7.39 (2H, t, J = 7.4 Hz, Ar Fmoc), 7.58 (2H, d,
J = 8.4 Hz, Ar pNZ), 7.71 (2H, d, J = 7.3 Hz, Ar Fmoc), 7.74/
7.77 (1H, d, J = 8.5 Hz, NH), 7.87 (2H, d, J = 7.5 Hz, Ar Fmoc),
8.00/8.02 (1H, d, J = 7.5 Hz, NH), 8.21 (2H, d, J = 8.5 Hz, Ar
pNZ). 13C NMR (150 MHz, DMSO-d6) δ (ppm): 33.1/33.3,
33.4/33.6, 47.0, 54.3/54.4, 54.5/54.7, 64.9, 65.6, 66.2,118.2,
120.6, 124.0, 125.7, 127.5, 128.1, 128.5, 132.6, 141.1, 144.2,
145.3, 147.4, 156.2, 156.5, 170.7, 172.6.
1
81-83 °C. H NMR (600 MHz, CDCl3) δ (ppm): 2.34 (1H, s,
OH), 4.00 (2H, m, βCH2), 4.46 (1H, m, RCH), 4.67 (2H, d, J =
5.5 Hz, CH2), 5.21 (2H, m, CH2), 5.26 (1H, d, J = 10.4 Hz,
CHdCH2), 5.34 (1H, d, J = 17.2 Hz, CHdCH2), 5.86-5.92
(2H, m, CHdCH2 þ NH), 7.50 (2H, d, J = 8.3 Hz, Ar), 8.20
(2H, d, J = 8.5 Hz, Ar). 13C NMR (150 MHz, CDCl3) δ (ppm):
56.1, 63.1, 65.6, 66.4, 119.1, 123.8, 128.1, 131.2, 143.5, 147.6,
155.7, 170.0.
Nr-para-Nitrobenzyloxycarbonyl-β-bromo-L-alanine Allyl Es-
ter (pNZ-β-Br-Ala-OAll). pNZ-Ser-OAll (3.23 g, 9.96 mmol)
and CBr4 (5.29 g, 15.9 mmol) were dissolved in 50 mL of DCM,
and the solution was cooled down to 0 °C. PPh3 (5.22 g,
19.9 mmol) was added portion-wise, and the reaction mixture
was stirred at 0 °C for 20 min. Diethyl ether was added, and the
precipitate was removed by filtration. The resulting diethyl ether
mother solution was washed with saturated solution of NaH-
CO3 (2ꢀ) and brine, dried over MgSO4, and concentrated. Puri-
fication of the product was performed by flash chromatography
eluting with petroleum ether:EtOAc (6:1 to 2:1) to give 1.66 g
(43% yield, >98% purity by HPLC) of a white solid. MS (m/z):
MS (calcd): 387.0191/389.0171 [M þ H]; MS (found): 387.0171/
389.0128 [M þ H]. mp 70-72 °C. 1H NMR (600 MHz, CDCl3)
δ (ppm): 3.81 (2H, m, βCH2), 4.70 (2H, m, CH2), 4.81 (1H, m,
RCH), 5.23 (2H, m, CH2), 5.29 (1H, d, J = 10.4 Hz, CHdCH2),
5.36 (1H, d, J= 17.2 Hz, CHdCH2), 5.77 (1H, d, J= 7.6 Hz, NH),
5.91 (1H, m, CH=CH2), 7.52 (2H, d, J = 8.4 Hz, Ar), 8.21 (2H, d,
J = 8.0 Hz, Ar). 13C NMR (150 MHz, CDCl3) δ (ppm): 33.4, 54.4,
65.6, 66.9, 119.5, 123.8, 128.1, 131.0, 143.4, 147.7, 155.1, 168.4.
Fully Protected Lanthionine Building Block (14). Fmoc-Cys-
OtBu (730 mg, 1.83 mmol) and pNZ-β-Br-Ala-OAll (710 mg,
1.83 mmol) were dissolved in EtOAc (20 mL), and 20 mL of a
saturated solution of NaHCO3 and 2.49 g of tetrabutylammo-
nium hydrogensulfate (7.32 mmol) were added. The reaction
mixture was allowed to stir overnight at room temperature. EtOAc
was added, and the organic phase was washed with saturated
solution of NaHCO3 (3ꢀ) and brine, dried over MgSO4, and
concentrated. The product was purified by flash chromatogra-
phy (petroleum ether:EtOAc, gradient of 5:1 to 1:1) to afford
900 mg of a colorless oil (70% yield, >99% purity by HPLC).
MS (calcd): 706.2434 [M þ H]; MS (found): 706.2452 [M þ 1],
Assembly of OT Isomers(3 and 4). The OT thioether was assem-
bled by Fmoc/tBu-based manual SPPS on a Rink amide MBHA
resin (Novabiochem, loading 0.64 mmol/g) using HBTU/DIEA
to activate the standard residues as well as the building block 15,
and treatment with 50% piperidine/DMF (2 ꢀ 1 min) for Fmoc
deproctection. Couplings were carried out with 4 equiv of Fmoc-
amino acid, 4 equiv of HBTU, and 8 equiv of DIEA in DMF for a
minimum of 10 min and monitored by the Kaiser test.79 After
assembly of the Gly, Leu, and Pro residues, the lanthionine build-
ing block was coupled to the peptide chain by using 2 equiv of 15,
2 equiv of HBTU, and 4 equiv of DIEA. Following Fmoc deprotec-
tion, residues Asn, Gln, Ile, and Tyr were coupled as usual. Prior
to peptide cyclization, the allyl group of the lanthionine moiety
was removed by treatment with 3 equiv of Pd[PPh3]4 in 6 mL of
CHCl3:AcOH:N-methyl morpholine (37:2:1) for 2.5 h under
argon and subsequent washing of the resin with DMF (5ꢀ),
0.5% DIEA in DMF (2 ꢀ 1 min), 0.5% dithiocarbamate in
DMF (3 ꢀ 1 min), and finally DMF (5ꢀ). Removal of the
N-terminal Fmoc protecting group was followed by overnight cycli-
zation with 5 equiv of PyAOP, 5 equiv of HOAt, and 10 equiv of
DIEA in DMF. Cleavage of the thioether from resin was per-
formed under standardconditions (TFA:triisopropylsilane:water,
95:2.5:2.5, 2 h). Final deprotection of pNZ group was accom-
plished by hydrogenolysis in a Parr hydrogenation apparatus
where the crude peptide was dissolved in EtOH:MeOH (1:1) and
submitted to hydrogenation at 30 psi pressure in the presence of
Pd-C (10% g/g) for 2 h. After removal of the Pd catalyst by
filtration through Celite, the OT thioether was purified by RP-
˚
HPLC (Phenomenex C18 column, 300 A, 10 mm, 250 mm ꢀ 21.2
mm, 0-30% solvent B in 60 min at 8 mL/min flow). Two isomers
of the desired lanthionine OT (8 and 9, ratio 2:3) having the same
molecular weight {MS (calcd): 974.5, MS (found): 975.5 [M þ 1]}
were obtained in pure form. Their stereochemical properties were
not investigated. All steps of lanthionine assemblywere monitored
by MS and analytical RP-HPLC.
1
650.2005 [M-tBu]. H NMR (600 MHz, DMSO-d6) δ (ppm):
1.37 (9H, s, tBu), 2.75-2.86 (2H, m, βCH2), 2.90-3.00 (2H, m,
βCH2), 4.06-4.10 (1H, m, RCH), 4.21 (1H, t, J = 7.0 Hz, CH
Fmoc), 4.26-4.32 (3H, m, RCH þ CH2 Fmoc), 4.57 (2H, d, J =
1.0 Hz, CH2), 5.17 (1H, d, J = 12 Hz, CHdCH2), 5.19 (2H, s,
CH2), 5.29 (1H, d, J = 17 Hz, CHdCH2), 5.83-5.90 (1H, m,
CHdCH2), 7.30 (2H, t, J = 7.4 Hz, Ar Fmoc), 7.39 (2H, t, J =
7.4 Hz, Ar Fmoc), 7.59 (2H, d, J = 8.4 Hz, Ar pNZ), 7.69 (2H, d,
J = 7.3 Hz, Ar Fmoc), 7.77/7.79 (1H, d, J = 8.4 Hz, NH), 7.87
(2H, d, J = 7.5 Hz, Ar Fmoc), 8.01/8.02 (1H, d, J = 8.2 Hz,
NH), 8.21 (2H, d, J = 8.6 Hz, Ar pNZ). 13C NMR (150 MHz,
DMSO-d6) δ (ppm): 28.0, 33.1/33.3, 33.4/33.5, 47.1, 54.4/54.6,
54.9/55.1, 64.9, 65.6, 66.2, 81.6, 118.2, 120.6, 123.9, 125.7, 127.5,
128.1, 128.5, 132.6, 141.2, 144.2, 145.3, 147.4, 156.2, 156.4,
170.3, 170.7.
Lanthionine Building Block (15). Fully protected lanthionine
14 (730 mg, 1.06 mmol) was dissolved in 10 mL of DCM and
cooled to 0 °C. TFA (10 mL) was added, and the reaction mix-
ture was stirred at 0 °C for 30 min and then allowed to react at
room temperature for 1.5 h. TFA was removed by coevapora-
tion with toluene, and the product was dissolved in 50% ACN
and lyophilized. The product was obtained as a white amor-
phous solid (quantitative, >98% purity by HPLC). MS (m/z):
MS (calcd): 650.1808 [M þ H]; MS (found): 650.1822 [M þ H].
1H NMR (600 MHz, DMSO-d6) δ (ppm): 2.77-2.87 (2H, m,
βCH2), 2.96-3.00 (2H, m, βCH2), 4.12-4.17 (1H, m, RCH),
Stability Assay. Three hundred microliters of human plasma
(Sigma Aldrich) was incubated at 37 °C for 30 min. Fifty micro-
liters of peptide sample (0.3 mM) in 0.1 M phosphate buffer, pH
7.2, was added to the human plasma. The vortexed mixture was
incubated at 37 °C. Aliquots (30 μL) were taken at 1, 2, 3, 4, 12,
24, and 48 h, quenched with extraction buffer (70 μL) consisting
of 50% ACN/H2O, 0.1 M NaCl, and 1% TFA, chilled on ice for
5 min, centrifuged (14000 rpm, 10 min), and analyzed (2 ꢀ 20 μL
injection) by RP-HPLC and LC-MS. Data analysis (n = 3-6)
was performed using Prism Version 5 a nonlinear fit one-phase
decay model.
Reduction/Alkylation Study. Condition A: PBS buffer, pH
7.4, and room temperature. Condition B: 1000-fold GSH: 10 μL
of a 1.2 mM OT, 720 μM [Se-Se]-OT, and 800 μM [CH2-S]-OT
solution was added to 90 μL of a 136, 80, and 90 mM solution of
glutathione in PBS buffer, respectively, and incubated at 37 °C
and at pH 7.4 over a period of 24 h. Condition C: 100-fold DTT:
5 μL of a 1.2 mM OT, 720 μM [Se-Se]-OT, and 800 μM [CH2-S]-
OT solution was added to 75 μL of a 8, 4.8, and 5.3 mM solution
of DTT in PBS buffer, respectively, and incubated at 37 °C and
at pH 7.4 over a period of 12 h. Condition D: 10-fold DTT-
100-fold iodoacetamide: 5 μL of a 1.2 mM OT, 720 μM [Se-Se]-
OT, and 800 μM [CH2-S]-OT solution was added to 75 μL of a

8594 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Muttenthaler et al.
800, 480, and 530 μM solution of DTT in PBS buffer, respec-
tively, and incubated at 37 °C and at pH 7.4. After 1 h, 15 μL of a
40 mM iodoacetamide in methanol solution was added and
incubated at 37 °C, pH 7.4, for another 2 h. Samples were taken
every hour and analyzed by RP-HPLC and LC-MS, running a
linear gradient of 2%/min of solvent B from 0 to 40% solvent B.
Transfection and Membrane Preparation. COS-1 cells grown
in Dulbecco’s modified Eagle’s medium and 5% fetal bovine serum
in 150 mm plates were transiently transfected with plasmid DNA
(25 μg) encoding OTR using Lipofectamine2000 reagent (50 μL).
The cells were harvested 48 h post-transfection and homogenized
using an Ultra turrax homogenizer (22000/min) in OT buffer
(50 mM Tris-HCl, 5 mM MgCl2, pH 7.4) with complete protease
inhibitor cocktail. The homogenate was centrifuged at 484g
(2000 rpm) for 10 min, and the resulting supernatant was centri-
fuged at 23665g (14000 rpm) for 30 min. The pellet was resus-
pended in OT buffer without protease inhibitor containing 10%
glycerol and stored at -80 °C until assayed.
Radioligand Binding Assay. Receptor binding assays were
performed using FlashBLUE GPCR scintillation beads. Reac-
tions containing increasing concentrations of competing OT
analogue (10 pM to 10 μM), FlashBLUE GPCR beads (100 μg),
OT membrane preparation (5 μg protein), and radioligand 3H-
OT (2 nM) in assay buffer with 0.1% BSA were established in
96-well white polystyrene plates with clear flat bottoms. The
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Acknowledgment. We acknowledge Alun Jones for the help
with MS. This work was supported by the National Health and
Medical Research Council (NHMRC) of Australia (Program
Grant 351446), the Australian Research Council (DP0774245),
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