Synthesis, structure elucidation, in vitro biological activity, toxicity, and Caco-2 cell permeability of lipophilic analogues of α-conotoxin MII

Article abstract of DOI:10.1021/jm020426j

The α-conotoxin MII is a two disulfide bridge containing, 16 amino acid long peptide toxin isolated from the marine snail Conus magus. This toxin has been found to be a highly selective and potent inhibitor of neuronal nicotinic acetylcholine receptors (nAChRs) of the subtype α3β2. To improve the bioavailability of this peptide, two lipidic analogues of MII have been synthesized, the first by coupling 2-amino-D,L-dodecanoic acid (Laa) to the N terminus (LaaMII) and the second by replacing Asn5 in the MII sequence with this lipoamino acid (5LaaMII). Both lipidic linear peptides were then oxidized under standard conditions. ¹H NMR shift analysis of these peptides and comparison with the native MII peptide showed that the tertiary structure of the N-conjugated analogue, LaaMII, was consistent with that of the native conotoxin, whereas the 5LaaMII analogue formed the correct disulfide bridges but failed to adopt the native helical tertiary structure. The N terminus conjugate was also found to inhibit nAChRs of the subtype α3β2 with equal potency to the parent peptide, whereas the 5LaaMII analogue showed no inhibitory activity. The active LaaMII analogue was found to exhibit significantly improved permeability across Caco-2 cell monolayers compared to the native MII, and both peptides showed negligible toxicity.

Full text of DOI:10.1021/jm020426j

1266
J . Med. Chem. 2003, 46, 1266-1272
Syn th esis, Str u ctu r e Elu cid a tion , in Vitr o Biologica l Activity, Toxicity, a n d
Ca co-2 Cell P er m ea bility of Lip op h ilic An a logu es of r-Con otoxin MII
J oanne T. Blanchfield,† J ulie L. Dutton,‡,§ Ronald C. Hogg,§ Oliver P. Gallagher,† David J . Craik,‡ Alun J ones,‡
David J . Adams,§ Richard J . Lewis,‡,§ Paul F. Alewood,‡ and Istvan Toth*,†
School of Pharmacy, Institute for Molecular Bioscience, and School of Biomedical Sciences, The University of Queensland,
Brisbane 4072, Australia
Received September 26, 2002
The R-conotoxin MII is a two disulfide bridge containing, 16 amino acid long peptide toxin
isolated from the marine snail Conus magus. This toxin has been found to be a highly selective
and potent inhibitor of neuronal nicotinic acetylcholine receptors (nAChRs) of the subtype R3â2.
To improve the bioavailability of this peptide, two lipidic analogues of MII have been
synthesized, the first by coupling 2-amino-D,L-dodecanoic acid (Laa) to the N terminus (LaaMII)
and the second by replacing Asn5 in the MII sequence with this lipoamino acid (5LaaMII).
1
Both lipidic linear peptides were then oxidized under standard conditions. H NMR shift
analysis of these peptides and comparison with the native MII peptide showed that the tertiary
structure of the N-conjugated analogue, LaaMII, was consistent with that of the native
conotoxin, whereas the 5LaaMII analogue formed the correct disulfide bridges but failed to
adopt the native helical tertiary structure. The N terminus conjugate was also found to inhibit
nAChRs of the subtype R3â2 with equal potency to the parent peptide, whereas the 5LaaMII
analogue showed no inhibitory activity. The active LaaMII analogue was found to exhibit
significantly improved permeability across Caco-2 cell monolayers compared to the native MII,
and both peptides showed negligible toxicity.
In tr od u ction
release of dopamine in the mesolimbic dopamine system,
thus mediating the reinforcing properties of nicotine.11
The R-conotoxins have several characteristics that
recommend them as drug candidates. They are small
peptides that are particularly stable to enzymes and
acidic conditions. The stable, well-defined structure of
the R-conotoxins (subclasses R3/5, R4/7, and R4/3) is
mediated by the formation of usually two, but occasion-
ally three, disulfide bonds in these cysteine-rich pep-
tides.12,13
Predatory marine snails belonging to the genus Conus
use spectacularly complex cocktails of bioactive peptide
toxins to immobilize their prey.1 Many different classes
of conotoxins have been characterized in terms of their
structure and the receptors they target.2 The R-cono-
toxin class antagonizes nicotinic acetylcholine receptors
(nAChRs). The structure of these small peptides (12-
19 residues) is constrained by disulfide bonds that
stabilize the active conformer.
R-Conotoxin MII is a potent and highly selective
competitive antagonist of the R3â2 nAChR. The toxin
was first isolated from Conus magus by Cartier et al.,14
and the three-dimensional solution structure was de-
scribed initially by Shon et al.15 and later by Hill et al.
in their study on the effects of different solvent condi-
tions on the helicity of the peptide.16 R-Conotoxin MII,
a 16 amino acid peptide (Figure 1) belongs to the R4/7
subclass by virtue of the Cys2-Cys8 and Cys3-Cys16
disulfide bonds. The toxin exhibits an IC50 value of 0.5
nM toward Xenopus oocytes expressing R3â2 nAChRs
and is several orders of magnitude less potent toward
other subunit combinations.14 The segments of the
receptor responsible for this exceptional selectivity have
been identified and include sequence segments 121-
181 and 181-195, especially Lys185 and Ile188 on the
R3 subunit, and 1-54, 54-63, and 63-80 on subunit
â2, with particular importance placed on Thr59.17
For any peptide to progress to the clinic, the issues
of bioavailability and membrane permeability must be
addressed. Although MII is relatively stable under
biological conditions, it would not be expected to readily
cross intestinal mucosal membranes or the blood-brain
barrier. Increasing the lipid solubility of a hydrophilic
nAChRs are ligand-gated ion channel receptors ex-
pressed in neurons and skeletal muscle. The receptors
are pentamers assembled from 16 different subunits
designated R1-R9, â1-4, γ, δ, and ꢀ. Neuronal nAChRs
contain only subunits R2-7, R9, and â2-4.3 The diver-
sity this affords nAChRs presents a formidable chal-
lenge to the search for methods of distinguishing the
various subtypes. Fortunately, various toxins from
animal venoms including the R-conotoxins have shown
remarkable selectivity for different subtypes of recep-
tors.4-7 These compounds have been invaluable as
biochemical and pharmacological tools.2,7,8 Neuronal
nAChRs have been implicated in several disease states
including Alzheimer’s, Tourette’s and Parkinson’s dis-
eases and schizophrenia.3,9,10 Those receptors that in-
corporate a â2 subunit have been directly linked to
nicotine addiction by their ability to stimulate the
* Address correspondence to this author at the School of Pharmacy,
The University of Queensland, St. Lucia, QLD, Australia 4072
(telephone +61 7 3365 1386; fax +61 7 3365 1688; e-mail I.Toth@
pharmacy.uq.edu.au).
† School of Pharmacy.
‡ Institute for Molecular Bioscience.
§ School of Biomedical Sciences.
10.1021/jm020426j CCC: $25.00 © 2003 American Chemical Society
Published on Web 03/04/2003

Lipophilic Analogues of R-Conotoxin MII
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 7 1267
6-11 in aqueous solution.15,16 The study of the solvent
accessibility of the residues by Hill et al. led them to
suggest Pro6, Val7, Leu10, Glu11, and Asn14 as likely
candidates for the residues that interact with the
nAChRs.16 Thus, we assumed that the formation of this
helix which presents these residues to the surface of the
peptide is essential for the activity of this peptide.
Syn th esis. The synthesis of active, native MII has
been achieved by oxidation in air of the deprotected
linear peptide in a dilute solution of 0.1 M NH4HCO3
(pH 8).16 Although this method afforded predominately
the correctly folded MII conformer, it was not known if
the presence of non-native groups such as lipoamino
acids on the termini of the linear peptide or within the
sequence would alter the outcome of this oxidation.
The solid phase synthesis of the MII sequence con-
taining 2-amino-D,L-dodecanoic acid on the N terminus
(LaaMII) required no alterations to the standard HBTU
activation protocol used in the synthesis of native MII.
The coupling efficiency of the lipoamino acid coupling
was comparable with those of the natural amino acids.
The synthesis of an alternative lipophilic analogue of
MII, which contained 2-amino-D,L-dodecanoic acid in-
corporated at position 5 of the MII sequence in place of
the Asn residue (5LaaMII, Figure 1), was completed
using the same solid phase synthetic techniques and
purified and oxidized under the same conditions as the
Laa-MII conjugate. Asn-5 was chosen as the residue
to be replaced because it lies outside the important
helical portion of the active molecule but is far enough
removed from the Cys2 and Cys3 residues to minimize
the possible adverse steric effects of the C12 side chain
on the formation of the disulfide bonds. Unfortunately,
to date there are no structure-function relationship
data available for MII; thus, the importance of Asn-5,
or any other residue in the structure, was not known.
LC/MS analysis of the product of the oxidation showed
the required loss of 4 mass units, suggesting the
formation of two disulfide bonds.
F igu r e 1. Sequence of R-conotoxin MII and the C12-Laa
conjugates LaaMII and 5LaaMII. All of the peptides have
amidated C termini.
compound has been established as an important factor
in improving absorption via passive transport across
intestinal mucosal membranes.18 There are numerous
reports of peptides that have been coupled to various
lipid units which have resulted in improved intestinal
delivery characteristics.19 For example, lauric acid (C12)
was attached to the N terminus of thyrotropin-releasing
hormone (TRH), which resulted in significantly in-
creased penetration across the upper small intestine
compared to the parent peptide in an in vitro everted
sac experiment.20 A captoyl-tetragastrin conjugate
produced significantly increased acid secretion in rats
compared to the native tetragastrin.21 We have dem-
onstrated previously that the conjugation of TRH and
luteinizing hormone releasing hormone (LHRH) with
one or two lipoamino acids (Laas) increased the peptides’
half-lives in a homogenate of human intestinal epithelial
cells by ∼30-fold from only 2-5 min to 1-3 h,22 and in
vivo experiments in rats demonstrated a significant
increase in the stability and oral uptake of the peptide
conjugates.23
The use of Laas to introduce lipidic groups into
peptides allows the conjugation of one or more of these
groups via standard peptide-coupling techniques and for
them to be incorporated into the solid phase synthesis
of a peptide. Thus, with a long-term goal of improving
the bioavailability of conotoxins, our study aims to
determine the effect of incorporating a C12 Laa into the
MII sequence on the tertiary structure, in vitro activity,
toxicity, and transepithelial transport of the peptide.
The human intestinal epithelial cell line, Caco-2,
derived from a human colorectal carcinoma has become
an increasingly important tool in drug delivery research
as an in vitro model for intestinal absorption. Caco-2
cells spontaneously differentiate into monolayers of
polarized enterocytes under conventional cell culture
conditions, expressing high levels of several brush
border hydrolases and forming tight cellular junc-
tions.24,25 The permeability of compounds through the
Caco-2 monolayer can be related to the extent of oral
absorption in humans,26,27 and thus it is an important
in vitro assay in assessing drug delivery systems.
The increase in lipophilicity of LaaMII and 5LaaMII
necessitated the use of C4 stationary phase in the HPLC
purification procedures, but this was the only minor
change to the procedures used in the synthesis of the
lipophilic MII conjugates. Both linear peptides were
exposed to the oxidation conditions described above,16
and the folded peptides were examined by NMR.
NMR In vestiga tion . Proton shifts are sensitive to
the local environment of the proton and are therefore
useful indicators of structural change. The NH shifts
(Figure 2A), RH shifts (plotted relative to random coil
RH shifts in Figure 2B), and side-chain H shifts (not
shown) for LaaMII were very similar to those for MII,
indicating that overall there was little structural dif-
ference between the two peptides. Peaks for the three
N-terminal residues were broadened probably as a
result of movement of the lipid chain, with the NH
peaks of Gly1 and Cys2 so broadened that they could
not be identified in the spectra. The only LaaMII H shift
that differed significantly from MII was the RH shift of
Cys3, which is not surprising due to its proximity to the
N-terminal lipid. Small changes in other shifts, such as
the NH of His9 and the RHs of Cys8 and Glu11, may
reflect some subtle structural changes. The negative
deviation of the RH shifts of residues Pro6 to Glu11 of
Resu lts a n d Discu ssion
Investigations of the solution structure of MII have
determined that the peptide is helical over residues

1268 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 7
Blanchfield et al.
F igu r e 3. Differences in âΗ shifts for MII (striped bars) and
5LaaMII (white bars). X represents Asn in MII and the
lipoamino acid in 5LaaMII. Gly residues do not have âHs, and
Val residues have only one âH, so nothing is graphed for Gly1
or Val7.
and then the free thiol groups alkylated by maleimide.
The resultant product ion spectrum from the LC/MS/
MS analysis of the partially reduced and alkylated
mixture revealed the ions at m/z 258 corresponding to
the b2 ion (Gly-Cys2-Mal), m/z 361 corresponding to the
b3 ion (Gly-Cys2-Mal-Cys3), and m/z 448 corresponding
to the b4 ion. The doubly charged ions b152+ (m/z 987.8),
2+
2+
2+
b14 (936.3), b13 (879.8), and b12 (822.8) were also
present with no maleinyl group present on Cys16. These
data indicated that in the oxidized form of 5LaaMII, the
disulfide bonds form between Cys2 and Cys8 and
between Cys3 and Cys16, the same pattern of con-
nectivity that is seen in native MII and in LaaMII.28 It
was clear, therefore, that the presence of the Laa in
position 5 of the peptide sequence did not inhibit the
formation of the correct disulfide connectivities but did
prevent the formation of the required helix tertiary
structure. This large structural change may be the
result of the lipid chain associating with the hydrophobic
residues of the peptide. The lipid may affect Pro6 which,
with the exception of the cysteines, is the only conserved
residue in R-conotoxins and is thought to be vital for
the formation of the helix.
F igu r e 2. (A) Differences between the amide proton chemical
shifts of LaaMII (black bars) or 5LaaMII (white bars) and MII.
No values are given for Gly1 as its NH cannot be seen. Pro6
does not have an NH. The NH of Cys2 in LaaMII could not be
identified in spectra so it could not be compared to MII and is
absent in this figure. X5 represents Asn5 in MII and LaaMII
and represents the lipoamino acid in 5LaaMII. (B) Secondary
R proton chemical shifts of MII (striped bars), LaaMII (black
bars), and 5LaaMII (white bars). Secondary RH shifts were
calculated by subtracting the RH shift for the residue in a
random coil peptide from the actual RH shift measured for
that residue in MII and the MII analogues. The RHs of Gly1
and Cys2 were not identified for LaaMII and are absent in
the graph. The two RHs of Gly1 in MII and in 5LaaMII were
overlapped so only one Gly1 secondary RH shift was plotted
for each of these peptides. 0.29 was subtracted from the
secondary shifts for residue 5 as it precedes a Pro. X5 indicates
Asn5 in LaaMII and MII and 5Laa in 5LaaMII.
Biologica l Activity. The NMR studies of these two
MII analogues showed that the tertiary structure of the
two compounds were significantly different and the in
vitro R3â2 nAChR inhibition activities reflect this result.
Rapid focal application of ACh (500 µM) to dissociated
neurons of the rat parasympathetic ganglia held at -60
mV, in the presence of 100 nM atropine, resulted in a
characteristic biphasic inward current comprising an
initial transient peak, which rapidly decayed to a
steady-state current [Figure 4A(i)]. Native MII and the
Laa-MII conjugate inhibited the ACh-evoked current
in a dose-dependent manner. The Laa-MII conjugate
at a concentration of 4.5 nM reduced the nACh-activated
current to 92 ( 1% (n ) 3) of control, whereas 4.5 nM
native MII reduced the nACh-activated current to 90
( 7% (n ) 3) of the control response. At a concentration
of 22 nM Laa-MII and native MII reduced the ampli-
tude of the nACh-activated current to 57 ( 4% (n ) 4)
and 57% (n ) 2) of control, respectively. Finally, 222
nM Laa-MII and 222 nM native MII reduced the
amplitude to 41% (n ) 2) and 47 ( 4% (n ) 3) of control.
These data corresponded to IC50 values of 12.8 nM for
LaaMII and 10 nM for native MII. 5LaaMII, on the
other hand, had no effect on the nACh-induced current
amplitude at concentrations up to 10 µM (n ) 3).
MII from the RH shifts of similar residues in random
coil peptides, indicative of helical secondary structure
over those residues, was retained in LaaMII (Figure 2B).
Overall the structural analysis showed that addition of
the lipoamino acid to the N terminus of MII caused
minimal change to the structure of the peptide.
Analysis of the NMR spectra of the 5LaaMII conju-
gate showed that its proton shifts differed dramatically
from the proton shifts of MII (Figure 2). Analysis of the
secondary RH shifts of 5LaaMII, that is, the RH shifts
relative to those for residues in random coil peptides,
indicated that it was not as well structured as MII and
did not feature the helical region found in MII (Figure
2B). âH shift differences can also be used to assess how
structured a peptide is as âHs tend to be well separated
in structured peptides. Comparison of the âH shift
differences, shown in Figure 3, confirmed that 5LaaMII
was not as well structured as MII.
To investigate the cause of such a remarkable change
in the tertiary structure of the 5LaaMII peptide, the
precise disulfide connectivities were determined by
partial reduction and alkylation of the oxidized peptide.
In this experiment, the disulfide bonds in the peptide
were partially reduced by tris(2-carboxyethyl)phosphine

Lipophilic Analogues of R-Conotoxin MII
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 7 1269
Ta ble 1. Apparent Permeability Values for Mannitol, MII, and
LaaMII
compound
Papp × 10-6 (cm/s)
mannitol
MII
LaaMII
5.88 ( 0.87
4.97 ( 1.47
20.5 ( 2
F igu r e 5. Graphical representation of the Papp values ob-
served for mannitol, MII, and LaaMII.
Ta ble 2. Percent Hemolysis Observed in Rat Red Blood Cells
When Treated with SDS, MII, or LaaMII
% hemolysis
max
min
100
0
F igu r e 4. Effect of native MII and conjugates on ACh-evoked
currents in parasympathetic neurons. (A) (i) 5LaaMII (1 µM)
has no effect on the peak ACh-evoked current, whereas 1 µM
LaaMII inhibits the current by ∼50%. (ii) In the same neurone
native MII reduces the current by a similar amount to Laa-
MII and the current recovers completely on washout of the
toxin. (B) Bar graph of the relative peak ACh-evoked current
by 5LaaMII, LaaMII, and native MII. Shaded and white
columns represent data for 100 nM and 1 µM, respectively.
Data represent mean ( SEM, n ) 3-4.
SDS, 1 mM
SDS, 2 mM
SDS, 5 mM
MII, 1 mM
MII, 2 mM
MII, 5 mM
LM, 1 mM
LM, 2 mM
LM, 5 mM
12.25
51.54
62.66
0.53
0.83
1.35
1.05
0.45
0.83
of mannitol, indicating minimal permeability across the
monolayer, as would be expected for a peptide of the
size and hydrophilicity of MII. LaaMII, however, exhib-
ited a 4-fold increase in the apparent permeability
compared to that of the parent peptide (Figure 5). This
significant increase in Papp for LaaMII indicated that
this peptide is expected to show significant absorption
through the human gastrointestinal tract.27
These results clearly showed that the presence of an
Laa on the N terminus of MII had no effect on the
potency of the peptide’s inhibition of the R3â2 nAChRs.
Proton chemical shift analysis of the Laa-MII conjugate
also showed that the lipidic group had no effect on the
tertiary structure adopted by the peptide under the
standard MII oxidation conditions. The 5LaaMII ana-
logue, however, showed complete loss of inhibition
toward the nAChRs at the concentrations examined, due
to the undesired tertiary structure adopted during the
oxidation of this peptide.
Ca co-2 Cell P er m ea bility. Caco-2 cell monolayers
are being used in pharmaceutical research with increas-
ing frequency to study the transepithelial transport of
drugs. The ultimate purpose of conjugating MII to
lipoamino acids is to improve its oral bioavailability.
Thus, as an in vitro assessment of the merits of the
strategy, the Caco-2 cell monolayer permeability of MII
and the active analogue LaaMII were measured. As the
integrity and permeability of monolayers can vary
between experiments, a control compound, [14C]manni-
tol, which is known to have negligible oral bioavailabil-
ity, was also studied in the same experiment. The
peptides were radiolabeled by acetylation using tritium-
labeled acetic anhydride, and the amount of peptide
present in the basolateral chamber was quantified by
liquid scintillation. The average (n ) 4) apparent
permeability values (Papp) of the three compounds are
listed in Table 1. The Papp for MII was similar to that
Toxicity. Before in vivo experiments using these
peptides can be initiated, some assessment of their
toxicity is required. The integrity of the Caco-2 cell
monolayer before and after treatment with the peptides,
as measured by the transepithelial electrical resistance
(TEER) across the monolayer, is one indication of
toxicity. Neither MII nor LaaMII showed significant
toxicity to Caco-2 cells under these conditions. As a
further test of the peptides’ toxicity, the effect of each
compound on red blood cells isolated from a male rat
was assessed. This assay measured the extent of
hemolysis of the cells over 30 min. In addition to the
two peptides, sodium dodecyl sulfate (SDS) was also
examined in the experiment. SDS is known to be toxic
to red blood cells at the concentrations used in the assay
and therefore was included as a positive control. The
results of the experiment are detailed in Table 2.
Neither MII nor LaaMII caused appreciable hemolysis
of red blood cells, even at the relatively high concentra-
tions used in the assay.

1270 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 7
Blanchfield et al.
with a solution of 20% mercaptoethanol, 10% DIEA, and 70%
DMF for 30 min with one repetition of this procedure. The
N-terminal Boc group was then removed by treatment with
TFA, and the resin was washed successively with DMF, DCM,
and methanol and then dried under vacuum. The peptide was
cleaved from the resin by treatment with 20 mL of liquid HF,
1 mL of p-cresol, and 1 mL of p-thiocresol at 0 °C for 1 h. After
removal of the HF, the crude product was precipitated and
washed in cold diethyl ether and then dissolved in 50%
acetonitrile. This solution was lyophilized and the crude
peptide purified by preparative RP-HPLC on a Vydac C18 22
× 250 mm column in the case of R-conotoxin MII and a Vydac
C4 22 × 250 mm column in the case of LaaMII and 5LaaMII.
Purification was performed using a linear solvent gradient
from 100% solvent A to 80% solvent B (solvent A ) 0.1%TFA
in water, solvent B ) 80% acetonitrile, 0.1% TFA in water) in
35 min at a flow rate of 5 mL/min. The presence of the desired
peptides in the fractions was detected by ESMS analyzing for
the appearance of the [M + H]+ ion (MII ) m/z 1716, LaaMII
) m/z 1913, and 5LaaMII ) m/z 1799) and the more abundant
[M + 2H]2+ ion (MII ) m/z 858.5, LaaMII ) m/z 957, and
5LaaMII ) m/z 900). The purity of the preparative HPLC
fractions was determined by analytical RP-HPLC using a
Vydac C18 22 × 4.6 mm column with a flow rate of 1 mL/min
and a linear gradient from 100% solvent A to 80% solvent B
over 30 min. The fractions containing pure peptide were
combined and lyophilized to afford the peptides as white solids
(reduced MII yield ) 40%, reduced LaaMII yield ) 24%,
reduced 5LaaMII yield ) 28%).
(c) Oxid a tion . The purified peptides were oxidized by
dissolving in 0.1 M NH4HCO3 to a final peptide concentration
of 20 µM and stirring vigorously at room temperature for 5
days. The oxidized peptides were purified as described above.
[M + H]+: MII ) m/z 1712, LaaMII ) m/z 1909, 5LaaMII )
m/z 1795. [M + 2H]2+: MII ) m/z 856.5, LaaMII ) m/z 955,
5LaaMII ) m/z 898. Oxidized MII yield ) 34%, oxidized
LaaMII yield ) 10%, oxidized 5LaaMII yield ) 67%.
Disu lfid e Con n ectivity 5La a MII. Purified peptide was
dissolved in NH4OAc (pH4) to an approximate concentration
of 0.1 mg/mL. This solution (5 µL) was treated with 2 µL of
tris(2-carboxyethyl)phosphine solution (0.1 M) and the reaction
monitored by LC/MS until ∼50% reduction had occurred.
Maleimide solution (20 µL of 0.1 M) was then added and the
reaction again monitored for the appearance of the dialkylated
peptide ([M + 2H]2+ ) 996.3), at which time the solution was
acidified by the addition of 1 µL of TFA. The resulting mixture
was analyzed using an Applied Biosystems Qstar Pulsar
electrospray Qqtof mass spectrometer running in information-
dependent acquisition mode, MS/MS scanning being switched
on by detection of the [M + 2H]2+ m/z 996.3 ion. Interpretation
of the data was assisted by use of Bioanalyst 1.1 from Applied
Biosystems. The LC component consisted of a 2.1 × 150 mm
Zorbax SB300 C3, 5 µm column using a solvent gradient of
0-40% solvent B over 40 min (solvent A ) 0.1% formic acid
in H2O, solvent B ) 90% acetonitrile, 0.1% formic acid) at a
flow rate of 300 µL/min.
Con clu sion s
The synthesis of two lipophilic analogues of the
R-conotoxin MII was achieved via solid phase methods
followed by oxidation of the linear peptide. The N-
terminal substituted analogue retained both the tertiary
structure and in vitro inhibitory potency of the parent
peptide. The analogue bearing a lipoamino acid at
position 5 in place of an Asn residue in the sequence
failed to adopt the desired tertiary structure and showed
no inhibitory activity toward nAChRs.
The active analogue LaaMII was also found to exhibit
significantly enhanced Caco-2 cell permeability, indicat-
ing improved intestinal absorption, and was also shown
to be nontoxic to both epithelial cells and red blood cells.
These data clearly indicate that LaaMII is an excellent
candidate for future in vivo biodistribution experiments.
Exp er im en ta l Section
Syn th esis. MBHA resin and protected amino acids were
obtained from Novabiochem (Melbourne, Australia). DMF and
TFA of peptide synthesis grade were purchased from Auspep
(Parkville, Australia). HPLC grade acetonitrile was purchased
from Labscan Asia Co. Ltd. (Bangkok, Thailand). Mass
spectrometric measurements were performed using a triple-
quadrupole PE Sciex API 3000 mass spectrometer with posi-
tive ion electrospray (ES). The mobile phase was a mixture of
50% solvent A (0.1% formic acid in water) and 50% solvent B
(0.1% formic acid in 90% acetonitrile/water) at a flow rate of
0.1 mL/min.
(a ) 2-(ter t-Bu toxyca r bon yla m in o)-D,L-d od eca n oic a cid .
Sodium (3.81 g, 166 mmol) was dissolved in ethanol (100 mL)
under nitrogen, and diethyl acetamido malonate (30.00 g, 138
mmol) was added followed by 1-bromodecane (42.76 g, 193
mmol). The solution was refluxed overnight under a nitrogen
atmosphere. Upon cooling, the mixture was poured onto
crushed ice (600 mL) and stirred. The precipitated product was
collected and air-dried. The crude product was refluxed
overnight in a solution of HCl/DMF (9:1, 200 mL). Upon
cooling, the precipitated product was collected, washed with
ice water, and air-dried to afford R-aminododecanoic acid
hydrochloride [37.57 g, 97% MS [M + H]+ m/z 216 ([M + H]+
of C12H25NO2 requires 216)]. 2-Amino-D,L-dodecanoic acid
hydrochloride (24.24 g, 96.3 mmol) was suspended in a solution
of tert-butyl alcohol/water (2:3, 500 mL) and the pH adjusted
to 13 with sodium hydroxide (5 M). Di-tert-butyl dicarbonate
(31.52 g, 144 mmol) in tert-butyl alcohol (50 mL) was added.
The solution was stirred overnight, maintaining the pH at 13.
The mixture was diluted with water (200 mL), and solid citric
acid was added to pH 3. The mixture was extracted with ethyl
acetate (5 × 150 mL), and the combined extracts were dried
(MgSO4) and evaporated to yield a crude product (oil and
crystals). This product was recrystallized from warm aceto-
nitrile to afford R-(tert-butoxycarbonylamino)dodecanoic acid
(21.98 g, 72%): mp 61-63 °C, lit. mp 62-64 °C;29 MS, m/z [M
+ H]+ 316 ([M + H]+ of C17H33NO4 requires 316), 260; 1H NMR
(300 MHz, CDCl3) δ 7.55 (1H, br s, COOH), 5.09 (1H, d, amide
NH), 4.29 (1H, m, R-CH), 1.9-1.5 (2H, m, â-CH2), 1.43 [9H, s,
C(CH3)3], 1.24 (16H, m, 8CH2), 0.86 (3H, t, CH3); 13C NMR
(75 MHz, CDCl3) δ 177.6, 155.6, 80.1, 53.4, 32.4, 31.9, 29.6,
29.5, 29.4, 29.3, 29.2, 28.3, 25.3, 22.7, 14.1.
NMR Exp er im en ts. Samples for NMR experiments con-
tained ∼1.5 mM peptide in 40% C2H3CN/60% 1H2O. NMR
experiments were carried out on Bruker 500 and 750 MHz
NMR spectrometers. These included double quantum filtered
correlation spectroscopy (DQF-COSY),32 total correlation spec-
troscopy (TOCSY) with a mixing time of 80 ms,33 and nuclear
Overhauser effect spectroscopy (NOESY) with mixing times
of 200 and 350 ms.34,35 Spectra were internally referenced to
DSS at 290 K. Additional spectra were acquired at 280, 285,
295, and 300 K for LaaMII to allow assignment of resonances
that were overlapped at 290 K. Spectra for 5LaaMII were also
acquired at 288 and 298 K with the peptide dissolved in 30%
(b) P ep tid e Ch a in Assem bly a n d Clea va ge. All of the
peptides were assembled on p-MBHA resin (100-200 mesh,
0.67 mmol/g loading) using HBTU/DIEA activation and the
in situ neutralization protocol for Boc chemistry.30 The peptides
were synthesized on a 0.5 mmol scale, and the following amino
acid side-chain protection was used: Cys(MeBzl), Ser(Bzl), His-
(DNP), Glu(OcHx). Each residue was coupled for 15 min; the
coupling efficiencies were determined by the quantitative
ninhydrin reaction,31 and recoupling was performed if the
efficiency was <99.2%. Prior to removal of the N-terminal Boc
group, the DNP protecting group was removed by treatment
1
C2H3CN/70% H2O.
Spectra were processed on a Silicon Graphics Indigo work-
station using XWIN-NMR (Bruker).
n ACh R In h ibition Stu d ies. (a ) Cell P r ep a r a tion . Para-
sympathetic neurons were dissociated from neonatal (3-8-day-

Lipophilic Analogues of R-Conotoxin MII
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 7 1271
old) rat intracardiac ganglia as described previously36 and
maintained in culture for 24-48 h.
(c) P er m ea b ilit y Assa y. The assays were performed in
HBSS containing 25 mM HEPES (pH 7.4) in air at 95%
humidity at 37 °C. Prior to the study, the monolayers were
washed in prewarmed HBSS-Hepes for 30 min. At the start
of the experiments 100 µL of the peptide or mannitol solutions
(200 µM) was added to the donor side of the monolayers and
600 µL of HBSS-Hepes without drug was added to the
receiver side. The plates were shaken in a Heifolf Titramax
1000 at 400 rpm at 37 °C during the whole experiment. At
regular intervals (30, 90, and 150 min) the Transwell insert
containing the monolayer was lifted carefully and placed in a
new receiver chamber containing fresh buffer. The entire
contents of each receiver chamber were added to 4 mL of
Wallac OptiPhase HiSafe 3 liquid scintillation cocktail, and
the radioactivity was measured in a liquid scintillation spec-
trometer (Tri-Carb 2700 TR).
(b) Electr op h ysiologica l Recor d in g. Membrane currents
were recorded using the whole-cell recording configuration of
the patch clamp technique. Electrical access to the cell interior
was obtained using the perforated-patch whole-cell recording
configuration.37 A final concentration of 240 µg/mL amphot-
ericin B in 0.4% DMSO was used in the pipet solution. Patch
electrodes were pulled from thin-wall borosilicate glass (Har-
vard Apparatus Ltd., Kent, U.K.) and had resistances of ∼1
MΩ. Access resistances using the perforated patch configura-
tion were routinely 4-8 MΩ before series resistance compen-
sation.
ACh-evoked currents were recorded using an Axopatch 200A
patch clamp amplifier (Axon Instruments Inc., Union City,
CA), filtered at 2 kHz and then digitized at 10 kHz (Digidata
1200A interface, Axon Instruments Inc.) and stored on the
hard disk of a PC for viewing and analysis. Voltage protocols
were applied using pClamp software (version 6.1.2, Axon
Instruments Inc.). Numerical data are presented as the mean
( SEM (n, number of observations).
(c) Solu tion s a n d Rea gen ts. The pipet filling solution for
perforated patch experiments contained 75 mM K2SO4, 55 mM
KCl, 5 mM MgSO4, and 10 mM 4-(2-hydroxyethyl)piperazine-
1-ethanesulfonic acid (HEPES), titrated with N-methyl-D-
glucamine to pH 7.2. The control extracellular solution con-
tained 140 mM NaCl, 3 mM KCl, 2.5 mM CaCl2, 1.2 mM
MgCl2, 7.7 mM glucose, and 10 mM HEPES-NaOH. Acetyl-
choline (500 µM) and atropine (100 nM, to inhibit muscarinic
ACh receptor activation) were applied for a duration of 2 s
using a rapid piezo application system to minimize the rapid
desensitization of the R7 component of the whole-cell ACh-
evoked current.38 Peptides were applied to the extracellular
pipet solution as well as the constant perfusing solution.
Experiments were carried out at 22 °C. The osmolality of all
solutions was monitored with a vapor pressure osmometer
(Westcor 5500) and was in the range of 280-290 mmol/kg. All
chemicals used were of analytical grade. Acetylcholine chloride
and atropine sulfate were supplied by Sigma Chemical Co.
Ca co-2 Cell P er m ea bility Stu d ies. (a ) 3H-La belin g of
P ep tid es. Purified peptide was dissolved in a minimum
volume of DMF, and (3H3CCO)2O (0.5 equiv) was added
followed by DIEA (3 equiv). The solution was allowed to stand
at room temperature overnight. Non-radioactive acetic anhy-
dride (0.5 equiv) was then added and the reaction continued
for 8 h. The volatiles were then removed under a steady stream
of N2 gas and the reaction lyophilized to dryness. The residue
was redissolved in water and lyophilized again to yield a fluffy
white powder. This powder was dissolved in Hank’s balanced
salt solution (HBSS) containing 0.5% DMSO in sufficient
volume to produce a 200 µM solution.
(d ) Deter m in a tion of P er m ea bility Coefficien ts. The
apparent permeability coefficients (Papp, cm s-1) were deter-
mined according to the following equation:
Vr
dC
dt
Papp
)
×
AC0
dC/dt is the steady-state rate of change in the radiochemical
concentration (dpm mL-1 s-1) in the receiver chamber, Vr is
the volume of the receiver chamber (mL), A is the surface area
of the cell monolayers, and C0 is the initial concentration of
the donor chamber (dpm mL-1). Four replicates of each
compound were performed, and the Papp values in Table 1
represent an average of these values.
Hem olysis Assa y. Solutions of MII, LaaMII, and SDS in
phosphate-buffered saline (PBS) with 1% DMSO were pre-
pared (2, 4. and 10 mM). Blood (9 mL) was collected from a
male Sprague-Dawley rat via cardiac puncture and treated
with 1 mL of a 4% sodium citrate solution. The red blood cells
(RBC) were isolated from the whole blood by centrifugation
at 800g for 10 min. The plasma was removed, and the cells
were washed three times with (PBS). After washing, the cells
were resuspended in 10 mL of PBS. The RBC preparation (100
µL) was placed in the required number of wells of a 96-well
tissue culture plate, which was shaken in a Heifolf Titramax
1000 at 400 rpm at 37 °C for 30 min. A 100 µL aliquot of one
of the test compound solutions was then added to each well.
Six wells received only PBS. The plates were incubated for a
further 30 min. Three concentrations of each compound (1, 2,
and 5 mM) were assayed in triplicate. When incubation was
complete, the solutions from three of the wells that received
only PBS were removed. The plate was then centrifuged at
maximum speed for 2 min to pellet any remaining whole cells.
A 20 µL aliquot of the supernatant of each well, or the whole
cell preparation from the three PBS wells removed prior to
centrifugation, was added to 5 mL of Drabkin’s reagent
containing Brij35 detergent. These solutions were then wrapped
in foil and placed in the dark for 30 min. The absorbance of
each sample at 540 nm was then measured. The wells treated
with only PBS that were not centrifuged represented the
maximum hemolysis (100%), whreas the wells treated with
PBS that were centrifuged represented the background or
minimum hemolysis. The percentage of hemolysis caused by
each concentration of compound was calculated using the
average Abs540 in the following equation:
(b) Cell Cu ltu r e. Caco-2 cells were obtained from the
American Type Culture Collection (Rockville, MD). Transwell
polycarbonate inserts were from Costar (Cambridge, MA), and
cell culture reagents were purchased from Gibco-BRL (Grand
Island, NY).
Caco-2 cells were maintained in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% fetal bovine serum
(FBS), 2 mM L-glutamine, and 1% nonessential amino acids
at 95% humidity and 37 °C in an atmosphere of 5% CO2. The
medium was changed every second day. After reaching 80%
confluence, the cells were subcultured using 0.2% EDTA and
0.25% trypsin. Approximately 5 × 105 cells (passage 57) were
seeded onto polycarbonate cell culture inserts (Transwell,
mean pore size ) 0.45 µm, 6.5 mm diameter) and cultivated
in DMEM supplemented with 10% FBS, 2 mM L-glutamine,
1% nonessential amino acids, 100 units/mL penicillin, and 100
µg/mL streptomycin. The cells were allowed to grow and
differentiate for 21 days. The medium was changed every
second day. Transepithelial electrical resistance (TEER) of the
monolayers was measured using the Millicell-ERS system
(Millipore Corp., Bedford, MA) before and after the drug
transport experiments. All of the monolayers used had initial
and final TEER values >400 Ω/cm2.
Abs540 - minAbs540
maxAbs540 - minAbs540
% hemolysis )
¥ 100
Ack n ow led gm en t. This work was supported by the
NH&MRC (Project Grant 102477).
Su p p or tin g In for m a tion Ava ila ble: Tables of the 1H
shifts of MII, LaaMII, and 5LaaMII in 40% C2H3CN/60% 1H2O,
acquired at 290 K and internally referenced to DSS, and
extracts of the product ion mass spectrum of the ES/LC/MS/
MS analysis of the partially reduced and alkylated 5LaaMII

1272 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 7
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