Investigation of the ring-closing metathesis of peptides in water

Article abstract of DOI:10.1039/c2ob26938d

A systematic study of the ring-closing metathesis (RCM) of unprotected oxytocin and crotalphine peptide analogues in water is reported. The replacement of cysteine with S-allyl cysteine enables RCM to proceed readily in water containing excess MgCl₂ with 30% t-BuOH as a co-solvent. The presence of the sulfur atom is vital for efficient aqueous RCM to occur, with non-sulfur containing analogues undergoing RCM in low yields.

Full text of DOI:10.1039/c2ob26938d

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Investigation of the ring-closing metathesis of peptides
in water†
Cite this: Org. Biomol. Chem., 2013, 11,
630
Stephen A. Cochrane,‡ Zedu Huang‡ and John C. Vederas*
A systematic study of the ring-closing metathesis (RCM) of unprotected oxytocin and crotalphine peptide
analogues in water is reported. The replacement of cysteine with S-allyl cysteine enables RCM to proceed
readily in water containing excess MgCl2 with 30% t-BuOH as a co-solvent. The presence of the sulfur
atom is vital for efficient aqueous RCM to occur, with non-sulfur containing analogues undergoing RCM
in low yields.
Received 2nd October 2012,
Accepted 21st November 2012
DOI: 10.1039/c2ob26938d
www.rsc.org/obc
examples have been shown to retain their biological activity.7
Solution-phase RCM of peptides has been mostly limited to
Introduction
Disulfide bonds are common in naturally occurring peptides hydrophobic peptides in organic solvents.8 The RCM of resin
and often play an important role in their correct folding, stabi- bound peptides is an alternative to the solution phase
lity and biological activity.1,2 Two examples are the mamma- process,7,9 but aggregation of the peptide chain with the resin
lian hormone oxytocin (1), which controls mammary and or peptide complexation to the catalyst can hinder the reac-
uterine smooth muscle contraction,3 and the orally active tion.10 The RCM of unprotected peptides in an aqueous
analgesic crotalphine (2), isolated from the venom of the environment is an attractive strategy. Peptides generally have
South American rattlesnake Crotalus durissus terrificus (Fig. 1).4 high solubility in water, and performing these reactions in
As disulfide bonds are susceptible to reduction to free thiols, aqueous media could allow cyclisations to be performed that
which can result in scrambling of bridges and loss of activity, are not possible on-resin.
A major drawback with this
there is considerable interest in replacement of these moieties approach is the insolubility of commercially available ruthe-
in peptides with more stable linkages.5 Ring-closing meta- nium-based metathesis catalysts and the competing catalyst
thesis (RCM) in peptides6,7 allows for the replacement of disul- decomposition pathways in water.11 It is possible to chemically
fide bridges with a dicarba bridge. The resulting stapled modify ruthenium catalysts to solubilize them in water,
peptides are more stable than the natural analogues and many however this requires a series of chemical steps to make appro-
priate ligands.12
Lipshutz and co-workers recently reported the use of
aqueous micelles as media to achieve cross-metathesis (CM)
and RCM on small organic molecules.13 Several common
detergents including sodium dodecyl sulfate (SDS) and Triton
X-100 were used at critical micelle concentrations to perform
CM reactions using 2% Grubbs 2nd generation catalyst in
moderate yields.13a Davis and co-workers have also reported a
novel system for aqueous CM.14 A mutant of the serine pro-
tease subtilisin Bacillus lentus containing S-allyl cysteine (Sac)
at position 156 successfully underwent CM in high yields with
a number of substrates using 200 equivalents Hoveyda–Grubbs
2nd generation catalyst (HGII) and 10 000 equivalents MgCl2
Fig. 1 Structures of oxytocin (1) and crotalphine (2).
in water with 30% t-BuOH as a co-solvent. The authors found
that Mg2+ is required for success of this reaction, presumably
to disrupt non-productive chelation between the catalyst and
Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada
protein. They proposed that the sulfur atom in the allyl sulfide
T6G 2G2. E-mail: john.vederas@ualberta.ca
coordinates to the ruthenium centre of the catalyst and brings
the reactive alkene moieties of the protein and substrate into
close proximity, thereby enhancing the rate of reaction and
†Electronic supplementary information (ESI) available: 1H- and 13C-NMR
spectra, MALDI spectra and HPLC traces. See DOI: 10.1039/c2ob26938d
‡Stephen A. Cochrane and Zedu Huang contributed equally to this project.
630 | Org. Biomol. Chem., 2013, 11, 630–639
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Fig. 2 Proposed RCM of peptide in a micellar system.
competing with non-productive chelation to the protein. This
method was also used by Schultz and co-workers to perform
RCM on a large protein bearing adjacent genetically encoded
O-crotylserine residues.15
Fig. 3 Linear oxytocin analogues 3a–e and crotalphine analogues 4a–e.
protection of commercially available S-allylcysteine using a
standard Fmoc-protection procedure.18
We now report our studies on the RCM of oxytocin 3a–e
and crotalphine 4a–e analogues in water, using micelles or
t-BuOH as an additive to solubilise the catalyst. The micellar
systems can potentially protect the insoluble catalyst from
decomposition by water by sequestration inside the micelle
(Fig. 2). The alkene chains could then enter the lipophilic
environment for RCM. Given that oxytocin (1) is positively
charged and crotalphine (2) is negatively charged at neutral
pH, polar head groups on the micelle could affect the RCM of
these peptides. Three commercially available detergents were
therefore selected for study at their critical micelle concen-
trations; (1) anionic SDS, (2) neutral Triton X-100 and (3) cat-
ionic cetyl trimethylammonium bromide (CTAB). In addition,
30% t-BuOH was used in a non-micellar solvent system. The
Hoveyda–Grubbs 2nd generation catalyst was chosen for these
reactions as it displays superior air and moisture stability com-
pared to other commercially available catalysts. All peptides
were synthesised using standard Fmoc solid-phase peptide
synthesis (SPPS). Several variants were selected to test the
influence of chain length and of heteroatoms in the alkene-
bearing side chain (Fig. 3). The replacement of cysteines in
oxytocin and crotalphine with allylglycine (Agl) affords ana-
logues that have sulfur replaced directly with CH upon cycliza-
tion.7a,b S-Allylcysteine (Sac) and O-allylserine (Oas) containing
peptides allow catalyst coordination effects to be probed.14,15
Pentenylglycine (Pgl) incorporation can probe the effect of
chain length.
The RCM of oxytocin and crotalphine analogues were first
performed on-resin for comparison with the latter aqueous
reactions (Scheme 1). The resin bound Fmoc-protected pep-
tides were refluxed in CH2Cl2 with 20 mol% HGII for 24 hours,
followed by the addition of DMSO (50 equiv. relative to the
catalyst loading) and stirred for a further 12 hours at room
temperature.7b Deprotection of the Fmoc-carbamate, acidic
cleavage from resin and RP-HPLC purification were then per-
formed. The RCM of resin bound Agl oxytocin analogue 3a
proceeded in 50% yield. The 1 : 1 mixture of cis- and trans-
isomers were separable by RP-HPLC. The resin bound Pgl, Oas
and Sac oxytocin analogues 3b–d underwent complete conver-
sion to the desired cyclic products as an inseparable mixture
of cis–trans isomers. Attempts to cyclize the crotalphine
analogues on-resin were unsuccessful, presumably due to
complexation between the catalyst and amide backbone and/or
hydrophobic protecting groups of the peptides.10
Our efforts were then directed towards the RCM of fully
unprotected oxytocin and crotalphine analogues in water.
RCM reactions failed to proceed for any analogues when CTAB
or Triton X-100 were used as additives. MALDI analysis showed
some product formation in the 8.2 mM SDS system for the Pgl,
Oas and Sac analogues, however product was not successfully
isolated from this system. We therefore directed our attention
to the use of 30% t-BuOH as a co-solvent.
We first focused on the RCM of the Agl oxytocin analogue
3a and Agl crotalphine analogue 4a as the resulting olefin
bridge is an isostere of a disulfide-bond. Initial attempts
to perform RCM on these analogues using stoichiometric
amounts of HGII at 37 °C were unsuccessful. Performing the
reaction at higher temperatures (up to 80 °C) had no effect
on the reaction. The loading of HGII was then increased to
50 equiv. but MALDI analysis after 24 hours revealed only the
formation of ruthenium-peptide adducts. Davis and co-
workers observed a similar problem in their cross-metathesis
reactions on proteins, which was circumvented by adding a
Results and discussion
The synthesis of Fmoc-protected alkene containing amino
acids was required for the SPPS of the oxytocin and crotal-
phine analogues. Asymmetric synthesis of Fmoc-Pgl-OH was
accomplished using the chiral Ni(II) Schiff-base method
reported by Belokon et al.,16 Fmoc-Oas-OH was synthesised
from commercially available Boc-Ser-OH using a previously
reported literature protocol.17 Fmoc-Sac-OH was obtained by
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Scheme 1 Method for the on-resin RCM of peptide analogues.
large excess of MgCl2 (10 000 equiv.) to disrupt non-productive the yield. The major products were ruthenium-peptide adducts
chelation.14 Even with the addition of 10 000 equiv. MgCl2 no and addition of excess MgCl2 had little effect on disrupting
reaction occurred in our case.
the non-productive chelation. For the Pgl oxytocin analogue
To determine if the failure of Agl analogues to cyclize in 3b, trace amounts of a side-product 14 Da higher in mass than
aqueous media is due to side chain length, the Pgl oxytocin the RCM product were detected. Comparison of the LC MS/MS
analogue 3b and Pgl crotalphine analogue 4b were examined. spectra of the isolated +14 Da side-product with a synthetic
Cyclic products were detected by MALDI when the reactions standard (3k) showed that a piperidine ring had been formed
were performed in the t-BuOH system using 50 equiv. HGII at the N-terminus, possibly by a hydroamination reaction
however the isolated yields were typically <1%. Lower loadings between the N-terminal amine and a ruthenium carbene on
of HGII gave no product and higher loadings did not increase residue 1 (Fig. 4).
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ruthenium-peptide adducts but failed to cyclize, whereas both
the Pgl and Oas analogues underwent RCM, albeit in low
yields, in the t-BuOH system.
Conclusions
The RCM of oxytocin analogues containing allylglycine, pente-
nylglycine, O-allylserine or S-allylcysteine can be achieved in
good to excellent yields on-resin. None of the crotalphine
analogues could be cyclized by this method. S-Allylcysteine
analogues of oxytocin and crotalphine also undergo efficient
RCM in water containing excess MgCl2 and 30% t-BuOH as a
co-solvent. These solution-phase RCM reactions took place
on peptides containing unprotected hydroxyl, carboxyl and
primary amide moieties. The addition of MgCl2 proved vital to
disrupt non-productive chelation between the resulting ruthe-
nium-carbene and these coordinating heteroatoms. The sulfur
atom within S-allylcysteine drastically improved the efficiency
of RCM compared to pentenylglycine and O-allylserine ana-
logues. This methodology provides an alternative to the on-
resin cyclization of S-allylcysteine, which is particularly attrac-
tive in instances where on-resin cyclization is not possible.
Fig. 4 1-Pip-6-Pgl-oxytocin (3k).
The role of a heteroatom within the alkene side-chain was
studied by examining the Oas and Sac analogues. The Oas
oxytocin analogue 3c and Oas crotalphine analogue 4c dis-
played similar reactivity to the Pgl analogues. A +14 Da side
product was also detected in the RCM of Oas oxytocin ana-
logue 3c, presumably analogous to Pgl oxytocin side-product
3k. The peptide analogues containing S-allylcysteine under-
went remarkably faster and cleaner reactions than all the other
analogues (Scheme 2). The Sac crotalphine analogue 4d under-
went RCM with 50 mol% HGII in 63% isolated yield. It is
important to note that this reaction failed on-resin. Initial
attempts to cyclize Sac oxytocin analogue 3d failed because
it decomposed rapidly upon exposure to HGII. When the
N-terminus was protected as the Fmoc-carbamate, Sac oxytocin
underwent complete conversion (78% isolated yield) to the
desired cyclic product in 3 hours using 50 mol% HGII. The
enhanced reactivity of S-allyl containing peptides is in full
agreement with the results of Davis and co-workers. The ability
to perform these cyclisations using sub-stoichiometric
amounts of HGII allows the cost-effective synthesis of
valuable biologically relevant peptide analogues in milligram
quantities.
The enhanced reactivity of the sulfur containing analogues
led us to synthesize linear peptides 3e and 4e. Fmoc-SAgl-OH
(7) was synthesised from commercially available Fmoc-L-Agl-
OH in 3 steps (Scheme 3). Protection as a methyl ester was
necessary for effective purification in the subsequent step.
Cross-metathesis between 5 and allyl methyl sulfide was per-
formed, followed by an ester hydrolysis using Pascal’s con-
ditions,19 yielding Fmoc-SAgl-OH (7). The unnatural amino
acid 7 is analogous to S-allyl cysteine with the positions of the
sulfur and alkene switched. Performing RCM on these ana-
logues would yield the same cyclic products as the RCM of 3a
and 4a respectively. We hoped the sacrificial allyl sulfide
within 7 would facilitate aqueous RCM through coordination
between the ruthenium in the catalyst and sulfur on the side
chain of 7. Attempts to perform the RCM of resin bound SAgl
oxytocin analogue 3e and SAgl crotalphine analogue 4e failed
with only the linear starting materials recovered. The aqueous
reactions were also unsuccessful. Analysis of the MALDI
spectra after 24 hours revealed only ruthenium-peptide
adducts, suggesting that the chain length of the alkene side
chain is also an important factor in aqueous RCM. This
is highlighted by comparison of the reactivity of the Agl, Pgl
and Oas analogues. Agl analogues 3a and 4a also formed
Experimental section
General information
NMR spectra were recorded on a Varian Inova 600, Inova 500,
1
Inova 400, or Unity 500 spectrometer. For H NMR spectra, δ
values were referenced to CDCl3 (7.26 ppm), D2O (4.79 ppm),
(CD3)2SO (2.50 ppm), and for 13C NMR spectra, δ values were
referenced to CDCl3 (77.0 ppm), (CD3)2SO (39.5 ppm) as the
solvents. Infrared spectra (IR) were recorded on a Nicolet
Magna 750 FT-IR or Nic-Plan FT-IR Microscope spectrometers.
Cast refers to the evaporation of a solution on a NaCl plate.
Mass spectra (MS) were recorded on an Agilent Technologies
6220 oaTOF (HR-ES) or on a Perspective Biosystems VoyagerTM
Elite MALDI-TOF (LR-MALDI), or on Bruker 9.4T Apex-Qe
FTICR (HR-MALDI), or on Waters (Micromass) Q-TOF Premier
(LC-MS/MS). 4-Hydroxy-α-cyanocinnamic acid (HCCA) was
used as matrix. Optical rotations were measured on a Perkin
Elmer 241 polarimeter with a microcell (10 cm, 1 mL) at
ambient temperature. All commercially available reagents and
protected amino acids were purchased and used without
further purification. All the solvents used for reactions were
distilled over appropriate drying reagents prior to use. Com-
mercially available ACS grade solvents (>99.0% purity) were
used for column chromatography without any further purifi-
cation. All reactions and fractions from column chromato-
graphy were monitored by thin layer chromatography (TLC)
using glass plates with a UV fluorescent indicator (normal
SiO2, Merck 60 F254). Following method was used for visualiza-
tion: UV absorption by fluorescence quenching, staining with
phosphomolybdic acid in ethanol (10 g/100 mL) or staining
with Ninhydrin (Ninhydrin:acetic acid:n-butanol/0.6 g:6
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Scheme 2 Aqueous RCM of Fmoc-1,6-Sac-oxytocin (Fmoc-3d) and 7,14-Sac-crotalphine (4d) in the t-BuOH system.
HPLC pump, GX-271 liquid handler, 156 UV/vis detector and a
10 mL sample loop, or a Gilson Analytical HPLC system
equipped with a 322 HPLC pump, FC 203B fraction collector,
171 diode array detector and a 100 to 1000 μL sample loop, or
a Beckman System Gold chromatograph equipped with a
model 166 variable wavelength UV detector and a Rheodyne
7225i injector fitted with a 100 to 1000 μL sample loop.
The columns used were Vydac C18 (5 μm, 4.6 × 250 mm), Vydac
C8 (5 μm, 4.6 × 250 mm), Vydac C18 (5 μm, 10 × 250 mm),
Phenomenex C18 (5 μm, 21.2 × 250 mm) and Zorbax C8 (21.2 ×
250 mm). All HPLC solvents were filtered with a Millipore
filtration system under vacuum before use.
Scheme 3 Synthesis of Fmoc-SAgl-OH (7).
mL:200 mL) spray. Flash chromatography was performed
using Merck type 60, 230–400 mesh silica gel. High Perfor-
mance Liquid Chromatography (HPLC) was performed on a
Gilson Preparative HPLC system equipped with a model 322
(S)-Methyl-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-6-
(methylthio)hex-4-enoate (6). Fmoc-L-Agl-OMe (5) (1.00 g,
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2.85 mmol) was added to a flame-dried flask under a blanket subunits were coupled using O-benzotriazole-N,N,N′,N′-tetra-
of argon and dissolved in dry CH2Cl2 (12 mL, 0.25 M). Allyl methyl-uronium-hexafluoro-phosphate (HBTU) as the activat-
methyl sulfide (0.62 mL, 5.70 mmol) was added, followed by ing agent and heated at 70 °C (50 °C for cysteines) for a 5 min
the addition of Hoveyda–Grubbs 2nd generation catalyst coupling time. Fmoc residues were deprotected using a 20%
(182 mg, 10 mol%) in parts to avoid excessive effervescence. solution of piperidine in DMF and the dibenzofulvene adduct
The resulting green solution was refluxed under argon for absorption monitored at 301 nm using a UV monitor.
24 hours, concentrated in vacuo and purified by column
General procedure for cleavage and purification of peptides
chromatography (silica gel, 7 : 1 hexanes–EtOAc) to yield
methyl ester 6 as a yellow oil and mixture of E/Z isomers To simultaneously cleave the peptide from resin and remove
(750 mg, 64%). [α]D +21.97° (c 0.83, CH2Cl2); IR (KBr disc) side chain protecting groups a solution of 95 : 2.5 : 2.5 trifluoro-
3335, 3065, 2951, 1722, 1520 cm−1; 1H NMR (CDCl3, 500 MHz): acetic acid (TFA) : triisopropylsilane (TIPS) : H2O was added to
δ 7.77 (d, 2H, J = 7.5, Fmoc-H), 7.6 (m, 2H, Fmoc-H), 7.40 (app. the resin-bound peptide for 2 hours. The resin beads were
t, 2H, J = 7.5, Fmoc-H), 7.32 (app. t, 2H, J = 7.5, Fmoc-H), removed via filtration through glass wool and the filtrate was
5.55–5.36 (m, 2H, RCHvCHR′), 5.32 (d, 1H, J = 8.0 Hz, NH), concentrated in vacuo. The crude peptide was obtained by pre-
4.48–4.44 (m, 1H, Fmoc-CHCH2), 4.40 (d, 2H, J = 7.1 Hz, Fmoc- cipitation with cold Et2O. The crude peptide was then dis-
CHCH), 4.23 (t, J = 7.0 Hz, 1H, Hα), 3.76–3.74 (m, 3H, OCH3), solved in 10 mL water and purified by HPLC. The following
3.11–3.02 (m, 2H, Hε), 2.64–2.49 (m, 2H, Hβ), 2.04–2.00 (m, 3H, HPLC methods were used for purification. Oxytocin analogues
SCH3); 13C NMR (CDCl3, 125 MHz): δ 172.2, 155.7, 143.9, prep-scale (Method A): Zorbax C8, flow rate 10 mL min−1
,
143.8, 141.3, 130.9, 127.7, 127.1, 126.3, 125.1, 120.0, 67.1, 53.5, detected at 220 nm. Gradient: starting from 10% MeCN (0.1%
52.5, 47.2, 35.7, 35.3, 30.0, 14.4; HRMS (ES) Calcd for TFA) and 90% water (0.1% TFA) for 5 min, ramping up to 55%
C23H25NO4SNa [M + Na]+ 434.1385, found 434.1385.
MeCN over 20 min, staying at 55% MeCN for 10 min, ramping
(S)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-6-(methyl- up to 95% MeCN over 4 min, staying at 95% MeCN for 1 min,
thio)hex-4-enoic acid (Fmoc-X-OH) (7). Methyl ester 6 (680 mg, ramping down to 10% MeCN over 2 min and staying at 10%
1.65 mmol) was dissolved in CH2Cl2 (2 mL), i-PrOH (22.5 mL) MeCN for 3 min. Crotalphine analogues prep-scale (Method
and water (10.5 mL). CaCl2·2H2O (4.11 mg, 28.0 mmol) was B): Phenomenex C18 column, flow rate 10 mL min−1, detected
then added and the reaction mixture stirred until all solids at 220 nm. Gradient: starting from 5% MeCN (0.1% TFA) and
had dissolved. 0.5 M NaOH (6.8 mL) was added and the result- 95% water (0.1% TFA) for 5 min, ramping up to 40% MeCN
ing suspension stirred for 75 min at room temperature. The over 25 min, then ramping up to 95% MeCN over 5 min,
reaction mixture was concentrated to remove i-PrOH, acidified staying at 95% MeCN for 5 min, ramping down to 5% MeCN
to pH 1 with 6 M HCl (∼25 drops) and washed with EtOAc (2 × over 1 min, then staying at 5% MeCN for 5 min. Oxytocin
40 mL). The EtOAc extracts were combined, dried over anhy- analogues analytical-scale (Method C): Analytical C18 column,
drous Na2SO4, filtered and concentrated in vacuo to yield the flow rate 1.0 mL min−1, detected at 220 nm. Gradient: starting
Fmoc amino acid 7 as a white solid (530 mg, 81%). [α]D from 10% MeCN (0.1% TFA) and 90% water (0.1% TFA) for
+13.31° (c 0.40, CH2Cl2); IR (KBr disc) 3313, 3065, 2916, 1721, 5 min, ramping up to 55% MeCN over 20 min, staying at 55%
1
1523 cm−1; H NMR (CDCl3, 400 MHz): δ 7.77 (d, 2H, J = 7.5, MeCN for 10 min, ramping up to 95% MeCN over 4 min,
Fmoc-H), 7.6 (m, 2H, Fmoc-H), 7.40 (app. t, 2H, J = 7.3, Fmoc- ramping down to 10% MeCN over 2 min and staying at 10%
H), 7.32 (app. t, 2H, J = 7.4, Fmoc-H), 5.62–5.40 (m, 2H, MeCN for 1 min. Crotalphine analogues prep-scale (Method
RCHvCHR′), 5.30 (d, 1H, J = 8.0 Hz, NH), 4.57–4.40 (m, 3H, D): Vydac C18 column, flow rate 1.0 mL min−1, detected at
Fmoc-CHCH2, Fmoc-CHCH2), 4.23 (t, J = 6.6 Hz, 1H, Hα), 220 nm. Gradient: starting from 5% MeCN (0.1% TFA) and
3.13–3.04 (m, 2H, Hε), 2.62–2.53 (m, 2H, Hβ), 2.04–1.98 (m, 3H, 95% water (0.1% TFA) for 5 min, ramping up to 40% MeCN
SCH3); 13C NMR (CDCl3, 125 MHz): δ 175.8, 155.9, 143.8, over 25 min, then ramping up to 95% MeCN over 5 min,
143.7, 141.3, 131.2, 127.8, 127.1, 126.0, 125.1, 120.0, 67.2, 53.3, staying at 95% MeCN for 5 min, ramping down to 5% MeCN
47.1, 35.7, 35.0, 14.4; HRMS (ES) Calcd for C22H23NO4SNa [M + over 1 min, then staying at 5% MeCN for 5 min. Semi-Prep
Na]+ 420.1240, found 420.1233.
method (Method E): Vydac C18 (5 μm, 10 × 250 mm) 5.0 mL
min−1, detected at 220 nm. Gradient: starting from 10% MeCN
(0.1% TFA) and 90% water (0.1% TFA) for 5 min, ramping up
Peptide syntheses
All peptides were synthesized on a CEM Liberty 1 Microwave to 40% MeCN over 20 min, ramping up to 95% MeCN
Peptide Synthesizer. Solid phase synthesis was carried out on a over 4 min, staying at 95% MeCN for 1 min, ramping down
0.1 mmol scale using Fmoc chemistry on Rink Amide to 10% MeCN over 2 min and staying at 10% MeCN for 3 min.
NovaGel™ resin (0.63 mmol g−1 loading) for oxytocin ana- The peptide was collected, concentrated in vacuo to remove
logues, 2-chlorotrityl resin (0.80 mmol g−1 loading) for 7,14- acetonitrile and lyophilized to give the final product.
Sac-crotalphine and 7,14-SAgl-crotalphine, and Wang resin
Procedure for loading amino acids onto 2-chlorotrityl resin
(0.65 mmol g−1 loading) for the remaining crotalphine ana-
logues. Commercially available asymmetrically protected In
a manual SPPS vessel 2-chlorotrityl resin (190 mg,
amino acids and synthesized amino acids were loaded on the 0.15 mmol) was bubbled with argon in CH2Cl2 for 15 min. The
peptide synthesizer as 0.2 M DMF solutions. All amino acid resin was then filtered and transferred to a screw-top vial.
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Diisopropylethyl amine (DIPEA) (0.21 mL, 1.2 mmol) and
1,6-Oas-oxytocin (3c). Peptide was isolated as a single peak
Fmoc-L-Sac-OH (206 mg, 0.6 mmol) were dissolved in CH2Cl2 using C8 RP-HPLC Method A (15 mg, 15%). Retention time
(3 mL) and added to the resin. The resulting slurry was shaken (Method C) = 18.1 min. 1H NMR (D2O, 500 MHz): δ 7.10 (d,
for 2.5 hours, filtered and washed with CH2Cl2 (3 × 10 mL). 2H, J = 8.2 Hz, Tyr Ar–H), 6.81 (d, 2H, J = 8.3 Hz, Tyr Ar–H),
The resin was then capped by bubbling with argon in MeOH– 5.94–5.82 (m, 2H, CHvCH2), 5.35–5.22 (m, 4H, CHvCH2),
DIPEA–CH2Cl2 (10 : 5 : 85) for 15 min. The solution 4.84 (app. t, 1H, J = 5.7 Hz), 4.70 (dd, 1H, J = 7.9, 5.8 Hz), 4.66
was removed by filtration and the resin then washed with DMF (app. t, 1H, J = 7.8 Hz), 4.41 (dd, 1H, J = 8.4, 6.0 Hz), 4.34–4.29
(3 × 10 mL) and CH2Cl2 (3 × 10 mL) and dried under argon.
(m, 1H), 4.24–4.39 (m, 2H), 4.08–4.00 (m, 5H), 3.94–3.75 (m,
7H), 3.72–3.66 (m, 2H), 2.98 (d, 1H, J = 7.6 Hz), 2.80 (dd, 1H,
J = 15.5, 5.5 Hz), 2.72 (dd, 2H, J = 15.4, 8.0 Hz), 2.33 (app. t,
Procedure for loading amino acids onto Wang resin
A 3-necked round bottom flask equipped with a stirring bar 2H, J = 7.8 Hz), 2.30–2.24 (m, 1H), 2.08–1.90 (m, 6H), 1.78–1.58
was flame dried and cooled in a desiccator under argon. The (m, 5H), 1.44–1.36 (m, 1H), 1.14–1.04 (m, 1H), 0.92–0.80 (m,
amino acid (10.0 equiv.) was dissolved in dry CH2Cl2 and 12H). MW calculated for C49H77N12O14 1057.5677, found high reso-
cooled to 0 °C. Diisopropylcarbodiimide (DIC) (5.0 equiv.) was lution (FTICR-ESI-MS) 1057.5675 (M + H)+.
added and the reaction was stirred at 0 °C for 20 min. The
1,6-Sac-oxytocin (3d). Peptide was isolated as a single peak
reaction mixture was then concentrated in vacuo and re- using C8 RP-HPLC Method A (15 mg, 14%). Retention time
dissolved in DMF (10 mL). Wang resin (1.0 equiv.) was added (Method C) = 18.6 min. 1H NMR (D2O, 500 MHz): δ 7.02 (d,
to a manual SPPS vessel and washed with CH2Cl2 (2 × 5 mL) 2H, J = 8.4 Hz, Tyr Ar–H), 6.82 (d, 2H, J = 8.2 Hz, Tyr Ar–H),
and DMF (5 mL). The resin was pre-swollen by bubbling with 5.76–5.86 (m, 2H, CHvCH2), 5.14–5.24 (m, 4H, CHvCH2),
argon in DMF (5 mL) for 1 hour and filtered. The activated 4.78 (t, 1H, J = 7.2 Hz), 4.70–4.64 (m, 2H), 4.41 (dd, 1H, J = 8.3,
anhydride solution was added to the resin followed by 5.5 Hz), 4.34–4.29 (m, 1H), 4.21 (dd, 1H, J = 9.2, 5.4 Hz), 4.15
4-dimethylaminopyridine (DMAP) (0.1 equiv.) and bubbled (t, 1H, J = 6.4 Hz), 4.04 (d, 1H, J = 8.6 Hz), 3.89 (app. q, 2H, J =
with argon for 2 hours. The solvent was removed by filtration 21.3 Hz), 3.80–3.72 (m, 2H), 3.22 (d, 2H, J = 7.1 Hz), 3.18 (d,
and the resin washed with DMF (3 × 5 mL). The resin was then 2H, J = 7.1 Hz), 3.00 (d, 2H, J = 7.7 Hz), 2.97–2.92 (m, 3H),
capped by bubbling with argon in 20% acetic anhydride in 2.83–2.77 (m, 1H), 2.78–2.66 (m, 2H), 2.34 (t, 2H, J = 7.6 Hz),
DMF (10 mL) for 15 min and filtered. The resin was washed 2.31–2.34 (m, 1H), 2.10–1.92 (m, 6H), 1.78–1.58 (m, 5H),
with DMF (3 × 5 mL) and CH2Cl2 (3 × 5 mL) and dried under 1.42–1.36 (m, 1H), 1.14–1.04 (m, 1H), 0.98–0.80 (m, 12H). MW
argon.
calculated for C49H77N12O12S2 1089.5220, found high resolution
1,6-Agl-oxytocin (3a). Peptide was isolated as a single peak (FTICR-ESI-MS) 1089.5217 (M + H)+.
using C8 RP-HPLC Method A (16 mg, 16%). Retention time
Fmoc-1,6-Sac-oxytocin (3d′). Peptide was isolated as a single
(Method C) = 15.8 min. 1H NMR (D2O, 500 MHz): δ 7.10 (d, peak using C8 RP-HPLC Method A (4 mg, 3%). Retention time
2H, J = 8.0 Hz, Tyr Ar-H), 6.81 (d, 2H, J = 8.0 Hz, Tyr Ar-H), (Method C) = 27.2 min. MW calculated for C64H87N12O14S2
5.84–5.62 (m, 2H, CHvCH2), 5.28–5.12 (m, 4H, CHvCH2), 1311.5906, found high resolution (FTICR-ESI-MS) 1311.5918
4.70–4.60 (m, 3H), 4.44–4.38 (m, 1H), 4.33–4.26 (m, 1H), (M + H)+.
4.21–4.19 (m, 1H), 4.08–4.02 (m, 2H), 3.88 (app. q, 2H, J =
Fmoc-1,6-SAgl-oxytocin (3e). Peptide was isolated as a single
14.4 Hz), 3.80–3.72 (m, 1H), 3.70–3.62 (m, 1H), 3.00–2.90 (m, peak using C18 RP-HPLC Method E (4.5 mg, 0.03 mmol scale,
1
2H), 2.78 (dd, 1H, J = 15.7, 5.2 Hz), 2.70 (dd, 1H, J = 15.7, 13%). Retention time (Method C) = 19.4 min. H NMR (D2O,
8.1 Hz), 2.64–2.60 (m, 2H), 2.55–2.50 (m, 1H), 2.43–2.36 (m, 2H), 600 MHz): δ 7.10 (d, 2H, J = 8.7 Hz, Tyr Ar-H), 6.81 (d, 2H, J =
2.32 (t, 1H, J = 7.7 Hz), 2.28–2.23 (m, 2H), 2.08–1.86 (m, 7H), 8.5 Hz, Tyr Ar-H), 5.14–5.24 (m, 4H, CRHvCR′H), 4.68–4.60
1.76–1.56 (m, 6H), 1.46–1.36 (m, 1H), 1.16–1.14 (m, 1H), (m, 2H), 4.41–4.39 (m, 1H), 4.32–4.29 (m, 1H), 4.23–4.20 (m,
0.92–0.80 (m, 12H). MW calculated for C47H73N12O12 997.5465, 1H), 4.07–4.05 (m, 2H), 3.89 (app. q, 2H, J = 15.8 Hz),
found high resolution (FTICR-ESI-MS) 997.5461 (M + H)+.
3.77–3.62 (m, 2H), 3.13–3.08 (m, 2H), 2.97 (d, 2H, J = 7.7 Hz),
1,6-Pgl-oxytocin (3b). Peptide was isolated as a single peak 2.82–2.50 (m, 6H), 2.43–2.40 (m, 1H), 2.35–2.32 (m, 2H),
using C8 RP-HPLC Method A (16 mg, 16%). Retention time 2.28–2.23 (m, 1H), 2.06–1.89 (m, 7H), 1.73–1.59 (m, 4H),
(Method C) = 19.0 min. 1H NMR (D2O, 500 MHz): δ 7.20 (d, 1.45–1.41 (m, 1H), 1.14–1.08 (m, 1H), 0.98–0.80 (m, 12H). MW
2H, J = 8.1 Hz, Tyr Ar-H), 6.90 (d, 2H, J = 8.2 Hz, Tyr Ar-H), calculated for C51H80N12O12S2 1117.5538, found high resolution
6.00–5.82 (m, 2H, CHvCH2), 5.14–5.06 (m, 4H, CHvCH2), (HRMS-ESI-MS) 1117.5533 (M + H)+.
4.77–4.71 (m, 2H), 4.68–4.62 (m, 1H), 4.48 (t, 1H, J = 7.0 Hz),
7,14-Agl-crotalphine (4a). Peptide was isolated as a single
4.42–4.35 (m, 1H), 4.25 (t, 1H, J = 7.3 Hz), 4.12 (d, 1H, J = peak using C18 RP-HPLC Method B (20 mg, 13%). Retention
8.7 Hz), 4.06 (t, 1H, J = 6.4 Hz), 3.96 (app. q, 2H, J = 15.6 Hz), time (Method D) = 20.9 min. 1H NMR (D2O, 600 MHz): δ
3.90–3.82 (m, 1H), 3.75–3.68 (m, 1H), 3.10–2.00 (m, 2H), 2.88 7.37–7.23 (m, 5H, Phe–ArH), 5.84–5.71 (m, 2H, CHvCH2),
(dd, 1H, J = 15.5, 5.7 Hz), 2.80 (dd, 1H, J = 15.5, 8.2 Hz), 2.40 (t, 5.22–5.10 (m, 4H, CHvCH2), 4.73–4.68 (m, 1H), 4.65 (dd, 1H,
2H, J = 7.8 Hz), 2.37–2.31 (m, 1H), 2.28–1.40 (m, 27 H), J = 9.1, 4.9 Hz), 4.46–4.24 (m, 9H), 3.94 (s, 2H), 3.86–3.72 (m,
1.22–1.14 (m, 1H), 1.16–0.87 (m, 12H). MW calculated for 5H), 3.72–3.62 (m, 3H), 3.16 (dd, 1H, J = 13.8, 6.6 Hz), 3.01 (dd,
C51H81N12O12 1053.6078, found high resolution (FTICR-ESI-MS) 1H, J = 13.8, 9.0 Hz), 2.84 (dd, 1H, J = 16.2, 7.8 Hz), 2.75 (dd,
1053.6086 (M + H)+.
1H, J = 15.6, 7.2 Hz), 2.64–1.72 (m, 33H). MW calculated for
636 | Org. Biomol. Chem., 2013, 11, 630–639
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C66H94N17O25 1524.6601, found high resolution (FTICR-ESI-MS) temperature. DMSO (0.02 mL) was added and the reaction
1524.6591 (M + H)+.
stirred for a further 12 hours. The resin was then filtered
7,14-Pgl-crotalphine (4b). Peptide was isolated as a single through a manual SPPS vessel and washed successively with
peak using C18 RP-HPLC Method B (15 mg, 10%). Retention methanol and CH2Cl2. A small amount of resin was cleaved
time (Method D) = 25.3 min. 1H NMR (D2O, 600 MHz): using TFA–TIPS–H2O (95 : 2.5 : 2.5) to confirm cyclization had
δ 7.37–7.23 (m, 5H, Phe–ArH), 5.90–5.80 (m, 2H, CHvCH2), occurred. Fmoc-deprotection was performed by bubbling the
5.08–4.95 (m, 4H, CHvCH2), 4.78–4.60 (m, 3H), 4.45–4.37 (m, resin in a solution of 20% piperidine in DMF (3 × 10 mL ×
4H), 4.30–4.23 (m, 4H), 3.96–3.64 (m, 9H), 3.17 (dd, 1H, J = 5 min) with argon. The resin was washed with DMF (3 ×
13.8, 6.6 Hz), 3.01 (dd, 1H, J = 13.8, 9.0 Hz), 2.86 (dd, 1H, J = 10 mL), DCM (3 × 10 mL) and dried under argon. The peptide
15.6, 6.6 Hz), 2.76 (dd, 1H, J = 15.6, 7.2 Hz), 2.48–1.66 (m, was then cleaved from resin using TFA–TIPS–H2O
35H), 1.50–1.36 (m, 4H). MW calculated for C70H102N17O25 (95 : 2.5 : 2.5), filtered, concentrated in vacuo and precipitated
1580.7227, found high resolution (FTICR-ESI-MS) 1580.7229 with cold ether. The crude peptide was dissolved in water
(M + H)+.
(4 mL) and acetonitrile (2 mL) and purified by RP-HPLC
7,14-Oas-crotalphine (4c). Peptide was isolated as a single (Method E).
peak using C18 RP-HPLC Method B (21 mg, 13%). Retention
General procedure for RCM in the t-BuOH system
time (Method D) = 21.9 min. 1H NMR (D2O, 600 MHz): δ
7.32–7.18 (m, 5H, Phe–ArH), 5.90–5.80 (m, 2H, CHvCH2), Fmoc-1,6-Sac-oxytocin 3d′ (1.0 mg, 0.8 μmol) and MgCl2·6H2O
5.28–5.14 (m, 4H, CHvCH2), 4.72–4.64 (m, 2H), 4.62–4.58 (m, (813 mg, 4.0 mmol) were dissolved in Milli-Q H2O (7.0 mL)
1H), 4.53 (t, 1H, J = 4.2 Hz), 4.41 (t, 1H, J = 5.4 Hz), 4.46–4.36 and stirred at room temperature. A solution of Hoveyda–
(m, 3H), 4.36–4.30 (m, 1H), 4.28–4.26 (m, 1H), 4.20 (dd, 1H, J = Grubbs 2nd Generation Catalyst (0.25 mg, 0.4 μmol) in t-BuOH
9.0, 4.2 Hz), 4.06–3.60 (m, 18H), 3.01 (dd, 1H, J = 13.8, 6.6 Hz), (3.0 mL) was then added and the resulting solution stirred
2.95 (dd, 1H, J = 13.8, 9.0 Hz), 2.81 (dd, 1H, J = 16.2, 6.6 Hz), at 37 °C. The reaction progress was monitored by MALDI
2.73 (dd, 1H, J = 16.2, 7.2 Hz), 2.49–1.70 (m, 27H), 1.67–1.75 and after 3 hours the starting material had been completely
(m, 1H). MW calculated for C68H98N17O27 1584.6813, found consumed. The product was then purified by prep-scale C8
high resolution (FTICR-ESI-MS) 1584.6831 (M + H)+.
RP-HPLC using Method A. The product containing fractions
7,14-Sac-crotalphine (4d). Peptide was isolated as a single were pooled, concentrated and lyophilized to yield cyclized
peak using C18 RP-HPLC Method B (20 mg, 12%). Retention Fmoc-1,6-Sac-oxytocin as a fluffy white powder (0.8 mg, 78%).
time (Method D) = 24.1 min. 1H NMR (D2O, 600 MHz): δ
General procedure for RCM in the micellar system
7.38–7.24 (m, 5H, Phe–ArH), 5.85–5.75 (m, 2H, CHvCH2),
5.21–5.13 (m, 4H, CHvCH2), 4.68–4.66 (m, 1H), 4.54–4.20 (m, Hoveyda–Grubbs 2nd Generation Catalyst (12.5 mg, 20.0 μmol)
5H), 4.39 (t, 1H, J = 6.0 Hz), 4.31 (dd, 2H, J = 4.8, 3.6 Hz), 4.25 was suspended in 8.2 mM SDS (10.0 mL) and stirred for
(dd, 1H, J = 9.6, 4.8 Hz), 3.98–3.66 (m, 10H), 3.19 (dd, 4H, J = 10 min. 1,6-Oas-oxytocin (1.0 mg, 1.0 μmol) and MgCl2·6H2O
18.0, 7.2 Hz), 3.16–3.70 (m, 8H), 2.52–1.74 (m, 27H), 1.80–1.75 (1.0 g, 5.0 mmol) were then added and the resulting green
(m, 1H). MW calculated for C68H98N17O25S2 1616.6356, found micellar solution stirred at 37 °C for 24 hours. The reaction
high resolution (FTICR-ESI-MS) 1616.6342 (M + H)+.
mixture was washed with CH2Cl2 (3 × 10 mL) and the resulting
7,14-SAgl-crotalphine (4e). Peptide was isolated as a single colourless solution was diluted with Milli-Q water (10.0 mL).
peak using C18 RP-HPLC Method B (8 mg, 0.03 mmol scale, The reaction mixture was passed through a prep-scale C8
1
12%). Retention time (Method D) = 25.3 min. H NMR (D2O, RP-HPLC using Method A.
600 MHz): δ 7.36–7.24 (m, 5H, Phe–ArH), 5.63–5.49 (m, 4H,
Cyclized cis-1,6-Agl-oxytocin (3f). The stereochemistry was
RCHvCHR′), 4.71 (t, 1H, J = 6.7 Hz), 4.66 (dd, 1H, J = 9.0, determined to be cis by comparison of the alkene splitting
4.8 Hz), 4.45–4.42 (m, 2H), 4.41–4.37 (m, 2H), 4.34–4.28 (m, 3H), pattern with previously published spectra.7a Peptide was iso-
4.25 (dd, 1H, J = 9.3, 4.7 Hz), 3.92–3.96 (m, 2H), 3.85–3.64 (m, lated as a single peak using C18 RP-HPLC Method E (0.7 mg,
7H), 3.16 (dd, 1H, J = 13.8, 7.0 Hz), 3.09 (dd, 2H, J = 15.3, 0.03 mmol scale, 2.4% overall yield). Retention time (Method
6.9 Hz), 3.01 (dd, 1H, J = 13.8, 9.0 Hz), 2.84 (dd, 1H, J = 15.8, C) = 15.5 min. 1H NMR (D2O, 600 MHz): δ 7.20 (d, 2H, J =
6.1 Hz), 2.76 (dd, 1H, J = 15.8, 7.6 Hz), 2.60–1.90 (m, 35H), 8.4 Hz, Tyr Ar–H), 6.9 (d, 2H, J = 8.4 Hz, Tyr Ar–H), 5.68–5.64
1.79–1.75 (m, 1H). MW calculated for C70H99N17O25S2 820.8225, (m, 1H, RCHvCHR′), 5.60–5.56 (m, 1H, RCHvCHR′),
found high resolution (HRMS-ESI-MS) 820.8222 (M − 2H)−2
.
4.64–4.62 (m, 1H), 4.44–4.42 (m, 2H), 4.31–4.26 (m, 3H),
4.08–4.04 (m, 2H), 3.90 (m, 2H), 3.73–3.60 (m, 2H), 3.17 (dd,
1H, J = 14.4, 6.1 Hz), 3.00 (m, dd, 1H, J = 14.3, 8.6 Hz),
General procedure for RCM on-resin
To a flame-dried 10 mL round-bottomed flask equipped with 2.87–2.71 (m, 6H), 2.53–2.47 (m, 2H), 2.43–2.26 (m, 6H),
a magnetic stirring bar and flushed with argon was added 2.09–1.89 (m, 9H), 1.70–1.60 (m, 5H), 1.27–1.20 (m, 1H),
linear Fmoc-1,6-Sac-oxytocin on rink amide resin (110 mg, 0.94–0.87 (m, 12H). MW calculated for C45H69N12O12 969.5152,
0.03 mmol). The resin was suspended in CH2Cl2 (5 mL) and found high resolution (FTICR-ESI-MS) 969.5144 (M + H)+.
Hoveyda–Grubbs 2nd generation catalyst (4.4 mg, 20 mol%)
Cyclized trans-1,6-Agl-oxytocin (3g). The stereochemistry
was added. The resulting suspension was refluxed with gentle was determined to be trans by comparison of the alkene split-
stirring under argon for 24 hours and cooled to room ting pattern with previously published spectra.7a Peptide was
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isolated as a single peak using C18 RP-HPLC Method E (Method C) = 25.6 min. MW calculated for C62H83N12O14S2
(0.8 mg, 0.03 mmol scale, 2.8% overall yield). Retention time 1283.5588, found high resolution (FTICR-ESI-MS) 1283.5593
(Method C) = 16.0 min. 1H NMR (D2O, 600 MHz): δ 7.21 (d, (M + H)+.
2H, J = 8.5 Hz, Tyr Ar–H), 6.9 (d, 2H, J = 8.4 Hz, Tyr Ar–H), 5.65
1-Pip-6-Pgl-oxytocin (3k). Peptide was isolated as a single
(dt, 1H, J = 15.1, 7.4 Hz, RCHvCHR′), 5.53 (dt, 1H, J = 14.7, peak using C8 RP-HPLC Method A (4 mg, 4%). This was used
7.4 Hz, RCHvCHR′), 4.71–4.68 (m, 1H), 4.64–4.62 (m, 1H), as a standard to confirm the identity of the hydroamination
4.44–4.41 (m, 1H), 4.31–4.28 (m, 1H), 4.13–4.10 (m, 2H), 4.00 side-product by LC MS/MS comparison. Retention time
(d, 1H, J = 6.2 Hz), 3.90 (app. q, 2H, J = 17.6 Hz), 3.73–3.60 (m, (Method C) = 17.4 min. 1H NMR (D2O, 500 MHz): δ 7.10 (d,
2H), 3.14 (dd, 1H, J = 14.4, 7.1 Hz), 3.04–2.95 (m, 2H), 2H, J = 8.3 Hz, Tyr Ar–H), 6.80 (d, 2H, J = 8.3 Hz, Tyr Ar–H),
2.79–2.71 (m, 2H), 2.64–2.59 (m, 1H), 2.55–2.50 (m, 1H), 5.85 (ddt, 1H, J = 17.1, 10.4, 6.6 Hz, CHvCH2), 5.06 (d, 1H, J =
2.45–2.27 (m, 6H), 2.11–1.98 (m, 5H), 1.92–1.85 (m, 2H), 17.3, CHvCHH), 5.00 (d, 1H, J = 10.3, CHvCHH), 4.66 (dd,
1.70–1.60 (m, 5H), 1.39–1.34 (m, 1H), 1.16–1.13 (m, 1H), 1H, J = 7.8, 6.0 Hz), 4.62 (t, 1H, J = 7.8 Hz), 4.58 (dd, 1H, J =
0.94–0.87 (m, 12H). MW calculated for C45H69N12O12 969.5153, 8.9, 4.9 Hz), 4.40 (dd, 1H, J = 8.1, 5.9 Hz), 4.29 (dd, 1H, J = 9.5,
found high resolution (FTICR-ESI-MS) 969.5144 (M + H)+.
4.9 Hz), 4.20 (t, 1H, J = 7.3 Hz), 4.06 (d, 1H, J = 8.6 Hz),
Cyclized 1,6-Pgl-oxytocin (3h). Peptide was isolated as a 3.94–3.82 (m, 4H), 3.80–3.75 (m, 1H), 3.66–3.60 (m, 1H), 3.44
mixture of isomers using C8 RP-HPLC Method A (1.5 mg, (d, 1H, J = 12.7 Hz), 3.02 (td, 1H, J = 12.4, 2.1 Hz), 2.97 (d, 2H,
0.03 mmol scale, 5% overall yield). Retention time (Method C) 7.8 Hz), 2.79 (dd, 1H, J = 15.5, 5.8 Hz), 2.71 (dd, 1H, J = 15.5,
= 16.9 min. 1H NMR (D2O, 600 MHz): δ 7.20 (d, 2H, J = 8.6 Hz, 8.0 Hz), 2.37–2.22 (m, 4H), 2.19–2.13 (m, 1H), 2.11–1.84 (m,
Tyr Ar–H), 6.84 (d, 2H, J = 8.6 Hz, Tyr Ar–H), 5.53–5.49 (m, 1H, 11 H), 1.80–1.56 (m, 10H), 1.51–1.37 (m, 3H), 1.16–1.08 (m, 1H),
RCHvCHR′), 5.45–5.40 (m, 1H, RCHvCHR′), 4.89–5.87 (m, 0.96–0.79 (m, 12H). MW calculated for C50H79N12O12 1039.5935,
1H), 4.52–4.50 (m, 1H), 4.44–4.42 (m, 1H), 4.29–4.27 (m, 1H), found high resolution (FTICR-ESI-MS) 1039.5931 (M + H)+.
4.15–4.13 (m, 1H), 3.97–3.81 (m, 6H), 3.67–3.62 (m, 1H), 3.10
Cyclized 7,14-Pgl-crotalphine (4h). Peptide was isolated as a
(dd, 1H, J = 14.3, 6.3 Hz), 3.04 (dd, 1H, J = 16.8, 4.1 Hz), 2.92 mixture of isomers peak using C18 RP-HPLC Method
(dd, 1H, J = 14.2, 9.0 Hz), 2.78 (dd, 1H, J = 16.2, 10.2 Hz), B. Retention time (Method D) = 20.8 min. MW calculated for
2.42–2.24 (m, 4H), 2.08–1.53 (m, 27 H), 1.50–1.40 (m, 2H), C68H98N17O25 1552.6901, found high resolution (FTICR-ESI-MS)
1.29–1.21 (m, 2H), 0.93–0.87 (m, 12H). MW calculated for 1552.6916 (M + H)+.
C49H77N12O12 1025.5778, found high resolution (FTICR-ESI-MS)
Cyclized 7,14-Oas-crotalphine (4i). Peptide was isolated as a
mixture of isomers using C18 RP-HPLC Method B. Retention
1025.5781 (M + H)+.
Cyclized 1,6-Oas-oxytocin (3i). Peptide was isolated as a time (Method D) = 18.6 min. MW calculated for C66H94N17O27
mixture of isomers using C8 RP-HPLC Method A (2.5 mg, 1556.6500, found high resolution (FTICR-ESI-MS) 1556.6479
0.03 mmol scale, 8% overall yield). Retention time (Method C) (M + H)+.
= 16.6 min. 1H NMR (D2O, 600 MHz): δ 7.20 (d, 2H, J = 8.5 Hz,
Cyclized 7,14-Sac-crotalphine (4j). Peptide was isolated as a
Tyr Ar–H), 6.85 (d, 2H, J = 8.4 Hz, Tyr Ar–H), 5.77–5.70 (m, 2H, mixture of isomers using C18 RP-HPLC Method B. Retention
RCHvCHR′), 4.85–4.80 (m, 2H), 4.46–4.43 (m, 1H), 4.31–4.28 time (Method D) = 17.7 min. 1H NMR (D2O, 500 MHz): δ
(m, 2H), 4.16–3.97 (m, 9H), 3.93–3.69 (m, 9H), 3.15 (dd, 1H, J = 7.37–7.22 (m, 5H, Phe–ArH), 5.63–5.54 (m, 2H, RCHvCHR′),
14.5, 5.6 Hz), 3.00–2.92 (m, 2H), 2.82 (dd, 1H, J = 16.0, 4.55–4.38 (m, 6H), 4.33–4.27 (m, 1H), 4.25 (dd, 1H, J = 9.3,
10.0 Hz), 2.43–2.27 (m, 5H), 2.06–1.91 (m, 6H), 1.68–1.59 (m, 4.7 Hz), 4.05–4.00 (m, 1H), 3.98–3.66 (m, 9H), 3.16 (dd, 2H,
5H), 1.42–1.39 (m, 1H), 1.22–1.15 (m, 1H), 0.94–0.82 (m, 12H). J = 14.1, 6.5 Hz), 3.05–2.75 (m, 7H), 2.50–1.90 (m, 25H), 1.81–
MW calculated for C47H73N12O14 1029.5364, found high 1.75 (m, 1H). MW calculated for C66H94N17O25S2 1588.6043,
resolution (FTICR-ESI-MS) 1029.5360 (M + H)+.
found high resolution (FTICR-ESI-MS) 1588.6053 (M + H)+.
Cyclized 1,6-Sac-oxytocin (3j). Peptide was isolated as a
mixture of isomers using C8 RP-HPLC Method A (2 mg,
0.03 mmol scale, 6%). Retention time (Method C) = 18.0 min.
1H NMR (D2O, 600 MHz): δ 7.21 (d, 2H, J = 8.5 Hz, Tyr Ar–H),
Acknowledgements
6.86 (d, 2H, J = 8.6 Hz, Tyr Ar–H), 5.66–5.60 (m, 2H, We thank Dr Randy Whittal, Jing Zheng and Bela Reiz for their
RCHvCHR′), 4.84–4.80 (m, 1H), 4.46 (dd, 1H, J = 8.6, 5.2 Hz), assistance with LC MS/MS and FTICR-ESI-MS experiments.
4.32 (dd, 1H, J = 10.0, 4.5 Hz), 4.16 (t, 1H, J = 6.1 Hz), 4.12 (dd, These investigations were supported by the Natural Sciences
1H, J = 8.5, 5.4 Hz), 4.03 (d, 1H, J = 6.0 Hz), 3.90 (app. q, 2H, J and Engineering Research Council of Canada (NSERC) and
= 21.5 Hz), 3.82–3.72 (m, 2H), 3.26–3.14 (m, 4H), 3.12–3.08 (m, the Canada Research Chair in Bioorganic and Medicinal
1H), 3.01–2.92 (m, 5H), 2.83–2.76 (m, 2H), 2.41–2.26 (m, 4H), Chemistry (J. C. V.).
2.07–1.89 (m, 8H), 1.87–1.81 (m, 1H), 1.71–1.59 (m, 4H),
1.42–1.35 (m, 1H), 1.21–1.15 (m, 1H), 0.95–0.86 (m, 12H). MW
calculated for C47H73N12O12S2 1061.4907, found high resolution
Notes and references
(FTICR-ESI-MS) 1061.4905 (M + H)+.
Cyclized Fmoc-1,6-Sac-oxytocin (3j′). Peptide was isolated as
mixture of isomers C8 RP-HPLC Method A. Retention time
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