R. Beard et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
5
investigated through an accelerated stability test in which dOTmeta
was compared to OT, as a representative analog of the bridged pep-
tides. Peptides were dissolved in water to a concentration of 0.5
mg mLÀ1 and heated to 50 °C in a heat block for 10 days. After this
time aliquots were removed and analysed by RP-LCMS. The
amount of remaining peptide was calculated as a percentage of
the relevant peak remaining before heat exposure. In this study,
degradation was prevented by conjugation of dibromo-xylene
(Fig. 5).
equipped with XBridge C18 reverse-phase columns with dimen-
sions 4.6 mm  100 mm for analytical and 19 mm  100 mm for
preparative runs. Solvents were degassed with helium and supple-
mented with 0.1% formic acid prior to use.
4.3. Solid phase peptide synthesis
Peptides were synthesised using automated solid-phase peptide
synthesis with Rink amide Tentagel resin on a ResPep SL apparatus
(Intavis) using the supplied Multipep software. Synthesis was car-
3. Conclusions
ried out in peptide synthesis grade DMF. Resin (20
swelled in DMF for 30 min before synthesis. Subsequent steps were
conducted automatically. N- -amino Fmoc groups were depro-
tected using 20% (v/v) piperidine in DMF (400
L, 2 Â 5 min). The
Fmoc-protected amino acid (100 mol, 5.0 eq.; 200 L of 0.5 M
stock solution in NMP) for coupling was pre-activated with HBTU
(95 mol, 4.75 eq.; 190 L of 0.5 M stock solution in NMP) and
NMM (200 mol, 10 eq.; 50 L of 4 M stock solution in NMP). Cou-
lmol/well) was
We report rapid and effective synthesis of xylene-bridged ana-
logs of dOT and determined the impact on biological activity and
stability following modification. This flexible approach facilitated
access to a variety of isomeric xylene bridges, which led to diverse
modulation of receptor selectivity, potency and affinity, presenting
an alternative pharmacological profile and therapeutic potential to
OT. A key finding from this work was that ring size and geometry
influenced OTR binding, with meta-bridged analogs demonstrating
the most favorable binding and pharmacology profile towards OTR.
dOTmeta demonstrated an improved selectivity profile for both
binding and activation towards the OTR and showed minimal V1a
antagonist activity, a more selective pharmacological than the
widely used drug carbetocin.22,31 It was surprising that dOTortho
displayed preferential binding affinity and selectivity towards
a
l
l
l
l
l
l
l
pling was allowed to take place over 30–55 min and amino acids
were ‘double-coupled’ (i.e. the coupling step was repeated). To pre-
vent deletion sequences, any unreacted N-terminal amines were
acetylated with ‘capping mixture’ (400 lL of 5% (v/v) Ac2O in
DMF) for 10 min. The resin was washed with DMF (3 Â 1 mL)
between the deprotection, coupling and acylation steps. The typi-
cal cycle was repeated until the final (N-terminal) amino acid cou-
pling, when N-a-Fmoc deprotection at the final residue undertaken
V1a, which can function as both a OTR agonist and V1a antagonist,
under usual conditions. Following synthesis, peptides were washed
several times (3 Â 1 mL DMF, 3 Â 1 mL DCM, 3 Â 1 mL MeOH, 3 Â
1 mL Et2O) and dried overnight in a desiccator. For the deprotec-
tion and cleavage of all peptides, a mixture of 1.5 mL TFA:H2O:
DTT:TIS (94:2.5:2.5:1) was added to resin bound peptide for 3 h.
Crude peptides were precipitated using ice cold TBME, centrifuged
at 4000 rpm for 15 min at 4 °C, and the supernatant discarded. The
remaining peptides were washed with a fresh aliquot of TBME and
the process repeated. Precipitate was dried in a desiccator over sil-
ica gel to yield off white solids which were dissolved in a H2O:
MeOH mixture for purification by RP LC-MS. Following purifica-
tion, fractions containing pure peptide were combined and concen-
trated in the Genevac. Subsequently, pure peptides were re-
dissolved in water and freeze-dried overnight.
which could be further enhanced for dOT(L8R)ortho, incorporating
a basic residue at position 8, a mutation important for AVP receptor
engagement. Notably, dOT(L8R)ortho displayed improved binding
selectivity towards V1a over AVP, with substantial antagonist activ-
ity only towards this receptor subtype.
OT binds and activates OTR, V1a and V1b receptor subtypes,
which has contributed to limited or debated efficacy in clinical
practice.32,33 The molecules reported here have a defined binding
and pharmacology profile, making them potentially useful tools
for probing the pharmacological potential of OTR. Furthermore,
xylene-bridging proved useful in enhancing thermal stability,
demonstrating minimal degradation compared to native OT at ele-
vated temperatures. Consequently, the xylene bridged analogs rep-
resent an interesting modification to OT with increased stability,
worthy of further investigation.
4.4. Purification and characterization
4. Experimental
LC-MS Analytical gradient 1: 5–98% MeOH in H2O over 10 min,
98% MeOH was held for 2 min, MeOH was reduced from 98% to
5% over 1 min, and held at 5% until 18 min. LC-MS Analytical gradi-
ent 2: 5–98% MeCN in H2O over 10 min, 98% MeCN was held for 2
min, MeCN was reduced from 98% to 5% over 1 min, and held at 5%
until 18 min. LC-MS semi-preparative gradient: 5–25% MeOH in H2O
over 1 min, then increased to 75% MeOH over 10 min. MeOH was
further increased to 98% over 1 min, held at 98% MeOH for 1 min,
then reduced to 5% MeOH over 1 min where it was held for 4
min (18 min total).
4.1. Materials
General laboratory chemicals, were obtained from Sigma-
Aldrich Chemical Co. and used without further purification. Rink
amide Tentagel resin (0.71 mmol/g) was obtained from Rapp Poly-
mere while N,N-dimethylformamide (DMF), N-Methyl-2-pyrroli-
done (NMP), piperidine and trifluoroacetic acid (TFA) were
obtained from Merck Millipore. Fmoc (Fluorenylmethoxycar-
bonyl)-l-amino acids, HBTU, N,N-Diisopropylethylamine (DIPEA)
were purchased from AGTC bioproducts. Gases were from BOC.
Solutions and buffers were prepared with Ultra-pure water
obtained from a Millipore Elix Q-guard purification system.
4.5. Peptide modification
4.2. Equipment
4.5.1. S-S disulfide bridging
Cyclization between internal cysteine residues was mediated in
ammonium bicarbonate buffer (0.1 M, pH 8) at a final peptide con-
centration of 0.1 mg/mL. The solution was stirred for up to 3 days
in the presence of oxygen at room temperature, after which time
the solution was concentrated then lyophilized to yield pure
peptide.
Peptides were purified and analysed on a Waters LC-MS system
consisting of i) Waters 2767 autosampler for samples injection and
collection; ii) Waters 515 HPLC pump to deliver the mobile phase
to the source; iii) Waters 3100 mass spectrometer with ESI; and, iv)
Waters 2998 Photodiode Array (detection at 200–600 nm)),
Beard, Rhiannon
