Synthesis and in vitro evaluation of a library of modified endomorphin 1 peptides

Article abstract of DOI:10.1016/j.bmc.2008.04.020

Endomorphin 1 (Endo-1 = Tyr-Pro-Trp-Phe-NH₂), an endogenous opioid with high affinity and selectivity for μ-opioid receptors, mediates acute and neuropathic pain in rodents. To overcome metabolic instability and poor membrane permeability, the N- and C-termini of Endo-1 were modified by lipoamino acids (Laa) and/or sugars, and 2′,6′-dimethyltyrosine (Dmt) replacement of Tyr. Analogues were assessed for μ-opioid receptor affinity, inhibition of cAMP accumulation, enzymatic stability, and permeability across Caco-2 cell monolayers. C-Terminus modification decreased receptor affinity, while N-terminus C8-Laa improved stability and permeability with slight change in receptor affinity. Dmt provided a promising lead compound: [C8Laa-Dmt[1]]-Endo-1 is nine times more stable (t_1/2 = 43.5 min), >8-fold more permeable in Caco-2 cell monolayers, and exhibits 140-fold greater μ-opioid receptor affinity (K_iμ = 0.08 nM).

Full text of DOI:10.1016/j.bmc.2008.04.020

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 6286–6296
Synthesis and in vitro evaluation of a library of modified
endomorphin 1 peptides
Yasuko Koda,a Mark Del Borgo,b Susanne T. Wessling,b Lawrence H. Lazarus,c
Yoshio Okada,d Istvan Totha,b and Joanne T. Blanchfieldb,*
aSchool of Pharmacy, University of Queensland, St. Lucia, Brisbane, Qld 4072, Australia
bSchool of Molecular and Microbial Sciences, University of Queensland, St. Lucia, 4072, Australia
cLaboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences,
Research Triangle Park, NC 27709, USA
dDepartment of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Nishi-ku, Kobe 651-2180, Japan
Received 31 January 2008; revised 7 April 2008; accepted 10 April 2008
Available online 15 April 2008
Abstract—Endomorphin 1 (Endo-1 = Tyr-Pro-Trp-Phe-NH2), an endogenous opioid with high affinity and selectivity for l-opioid
receptors, mediates acute and neuropathic pain in rodents. To overcome metabolic instability and poor membrane permeability, the
N- and C-termini of Endo-1 were modified by lipoamino acids (Laa) and/or sugars, and 20,60-dimethyltyrosine (Dmt) replacement of
Tyr. Analogues were assessed for l-opioid receptor affinity, inhibition of cAMP accumulation, enzymatic stability, and permeability
across Caco-2 cell monolayers. C-Terminus modification decreased receptor affinity, while N-terminus C8-Laa improved stability
and permeability with slight change in receptor affinity. Dmt provided a promising lead compound: [C8Laa-Dmt[1]]-Endo-1 is nine
times more stable (t1/2 = 43.5 min), >8-fold more permeable in Caco-2 cell monolayers, and exhibits 140-fold greater l-opioid
receptor affinity (Kil = 0.08 nM).
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
fer from very poor metabolic stability, with the half-lives
for both peptides in the order of minutes13,14 and low
membrane permeability. In order for these peptides to ful-
fil their potential as pharmaceutically relevant pain-mod-
ulating compounds, their pharmacokinetics must be
improved. In this study we have chemically modified the
termini of endomorphin 1 to investigate systematically
how these changes can affect stability, permeability, and
biological activity. Structure–activity studies on the end-
omorphins have revealed the importance of the Tyr1
and Trp3 residues to receptor binding15,16 and Pro2 to
the correct structural orientation of the peptide.17,18 Our
modifications, therefore, concentrated on the addition
of lipidic and/or carbohydrate units to eithertermini while
maintaining the native peptide sequence.
The opioid peptides are a family of endogenous neuro-
chemical messengers that exhibit high affinity for one or
more of the known opioid receptors. The opioid receptor
system mediates an array of physiological processes
including the respiratory, cardiovascular and gastrointes-
tinal systems, immune responses, neuroendocrine func-
tion, reward, stress, and pain perception and response.1
Two important members of the opioid peptide family
are endomorphin 1 (Tyr-Pro-Trp-Phe-NH2) and endo-
morphin 2 (Tyr-Pro-Phe-Phe-NH2) which are unique
among the opioid peptides as they exhibit high affinity
and extraordinary selectivity for the l-opioid recep-
tor2,3—the receptor most associated with the alleviation
of pain in the CNS.4 Endomorphin 1 and 2 are being
extensively investigated as potential analgesic agents,5–
12 however, like all small peptides, the endomorphins suf-
Increasing the lipophilicity of a compound is a well-rec-
ognized strategy for increasing its passive diffusion
across plasma membranes.19,20 While the methodologies
used to achieve the lipidation are many and varied,21,22
the lipidic groups in this study were introduced as lipoa-
mino acid (Laa) residues, containing either 8, 12 or 18
carbons. We have previously shown that the incorpora-
Keywords: Endomorphin 1; Opioid peptides; Lipoamino acids; Lipo-
saccharides; Drug delivery; Oral peptide delivery.
*
Corresponding author. Tel.: +61 7 3365 3622; fax: +61 7 3365
4299; e-mail: j.blanchfield@uq.edu.au
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.04.020

Y. Koda et al. / Bioorg. Med. Chem. 16 (2008) 6286–6296
6287
tion of Laas into short peptides can increase their meta-
bolic stability and passive diffusion across biological
barriers.23–26 For the majority of the analogues synthe-
sized in this study, the lipoamino acids were introduced
as a racemic mixture of both the L- and D-stereoisomers.
There is evidence, however, that the diasteriomers of
Laa-conjugated peptides can behave differently in vivo,
particularly in terms of their stability to enzymatic deg-
radation.24,27,28 One of the more promising Laa conju-
gates in this study was therefore synthesized
enantiomerically pure and both diastereomers (3 and
4) assessed individually.
pure form and these building blocks were Boc-protected
using a slightly modified procedure of that employed for
the racemic form. It is imperative that the pH of the
reaction mixture is carefully controlled between 4 and
8 in the case of the optically pure compounds to prevent
racemisation. A figure showing RP-HPLC traces of
racemic C8Laa-Endo-1 (2) and each of the diastereo-
merically pure compounds 3 and 4 is supplied in the
Supplementary information and demonstrates that there
is no evidence of racemisation of the Laa during the pro-
tection or coupling reactions.
Compounds 13 and 14 incorporated 2,6-dimethyltyro-
sine (Dmt) in place of the Tyr1 residue of endomorphin
1. The incorporation of Dmt into opioid peptides was
first demonstrated to enhance their receptor binding
and potency by Hansen et al. in 1992.34 Recent studies
have shown that replacement of the Tyr1 with Dmt
can increase the in vitro receptor binding affinity and
the in vivo analgesic activity of endo-135 and endo-
2.36,37 Novel peptide mimetics bearing the Dmt moiety
have also exhibited extremely high l-opioid binding
affinity.38–40 Dmt was therefore incorporated into two
of the peptides in our library, 13 and 14. Synthesis of
N-Boc-20,60-dimethyl-L-tyrosine was achieved using a 5
step literature reported pathway from 3,5-dimethylphe-
nol.41 Compounds 13 and 14 were synthesised using
solution-phase peptide synthetic techniques. Each cou-
pling and purification gave approximately 65% yield un-
til the Boc-C8Laa coupling. This reaction was observed
to generate some impurities by TLC, and this compound
was purified by repeated flash chromatography resulting
in a reduced yield for 14 of 7.2%.
The benefits of glycosylation to the bioavailability of
opioid and other neuroactive peptides have been well
established in recent years.29–32 The integration of these
polyhydroxylated, biologically compatible units can
serve to alter water solubility and are useful in conjunc-
tion with lipidation as a method of customizing the
physicochemical properties of a compound to optimize
its bioavailability. The synthetic lipo- and glycol-
endo-1 analogues assessed in this study are detailed in
Figure 1.
2. Results and discussion
2.1. Chemistry
Solid-phase peptide synthesis techniques were used to
synthesize compounds 1–12. All peptides were purified
to a single peak under two different analytical RP-
HPLC conditions and provided the expected [M+H]+
peak in electrospray mass spectrometry. A summary of
these data is provided in the Supplementary informa-
tion. All the compounds were eventually obtained as
white or off-white solids on lyophilisation from water
with the glycosylated compounds exhibiting significant
hydroscopic properties. The purified yields of the pep-
tides varied from 51.8% for native Endo-1 (1) to a low
3% for the N-terminus glycosylated, C-terminus lipidat-
ed derivative 12. The yield of the N-glucose succinate
derivative 7 was also low (9.8%) and this appeared to
be the result of degradation of the sugar unit during
TFA cleavage of the peptide from the resin. The C-ter-
minus glycosylated peptides also showed some degrada-
tion but less than the N-terminus glyco-derivatives. The
analogues bearing only lipoamino acids were observed
to give much higher mass return on cleavage from the
resin and provide a cleaner crude peptide but suffered
from some loss during HPLC purification due to their
poor water solubility and propensity to adsorb onto re-
verse-phase columns.
The analogues synthesised were initially examined with
respect to their physicochemical properties. Compound
6, the C18Laa conjugate, was found to be completely
insoluble in water or buffered solutions, even with the
addition of DMSO. This analogue was not pursued fur-
ther in the study as it was clear that the use of long lipid-
chain auxiliary groups is not appropriate for short pep-
tides. The C12Laa analogues 5 and 9 were also sparingly
soluble in water but could be solubilised by the addition
of DMSO; however, in both the stability and permeabil-
ity assays evidence suggested that these compounds pre-
cipitated out of solution and were difficult to detect
reliably using LC–MS due to these solubility issues.
We were not able, therefore, to obtain reproducible sta-
bility data for compounds 5 or 9.
2.2. Stability and permeability
The in vitro stability and permeability of the endomor-
phin analogues were assessed using Caco-2 cells. The
Caco-2 cell line, derived from human colorectal carci-
noma cells, is now routinely used as an in vitro model
of the GI tract and to obtain an initial prediction of oral
bioavailability of a compound,42–45 and also as a preli-
minary blood–brain barrier (BBB) permeability screen-
ing tool.46–49
In the bulk of these studies, the lipoamino acids were
incorporated into the peptides as racemates and gave
rise to two diastereomeric peptides that were not sepa-
rated before the in vitro assays. Our previous studies
have shown that peptides bearing the L-lipoamino acids
can be more susceptible to degradation than the D-dia-
stereomers.24,27,28 Enzymatic resolution of 2-amino-
D,L-octanoic acid (C8Laa) using acylase I from Aspergil-
lus melleus33 provided both enantiomers of this Laa in
The stability of each of the analogues was assessed in
a solution of digestive enzymes produced by homogen-

6288
Y. Koda et al. / Bioorg. Med. Chem. 16 (2008) 6286–6296
O
H
N
H
N
O
O
N
H
R2
H
N
O
R1
R3
N
R3
HN
OH
Compound
1 (Endo 1)
R1
H
R2
H
R3
H
O
H2N
(H2C)
2 - 6
H
H
n
2
3
4
5
6
n = 5
H
H
H
H
H
H
H
H
H
H
n = 5 D enantiomer
n = 5 L enantiomer
n = 9
n = 15
OH
O
O
H
HO
HO
7
8
H
H
H
N
OH
O
O
NH2
(H2C)
5
O
NH2
9
H
H
H
(H2C)
O
9
OH
HO
HO
OH
CONH2
10
11
O
OH
H2N
OH
CONH2
O
(H2C)
5
O
OH
O
O
H
N
HO
HO
NH2
12
13
14
H
OH
(H2C)
5
O
H
H
CH3
CH3
O
H2N
H
(H2C)
5
Figure 1. The lipo- glyco- and liposaccharide analogues of endomorphin 1 synthesised and evaluated in this study.
ising fully differentiated Caco-2 cells and removing the
cell debris by centrifugation. The half-life of each
analogue in the presence of the Caco-2 cell homoge-
nate is presented in Table 1 and the degradation
profiles are available graphically in the Supplementary
information.
Endo-1 (1) degraded rapidly with a half-life of between 5
and 9 min and a similar degradation profile was ob-
served for the C-terminus modified Endo-1 analogues
(8 and 10). In contrast, modifications of both the N-
and C-termini (compounds 11 and 12) significantly
improved stability in Caco-2 cell homogenates. The

Y. Koda et al. / Bioorg. Med. Chem. 16 (2008) 6286–6296
6289
Table 1. The half-life of each endo-1 analogue in an homogenate of
Caco-2 cells and their apparent permeability (Papp cm/s) through
Caco-2 cell monolayers
chain length of Laas decreased water solubility. While
5 appeared to be in solution at the start of the experi-
ment, it could be observed precipitating out in the apical
chamber adhering to the plastic assay plate. It is also
possible that this lipophilic compound became associ-
ated with the cell membranes.50 As a result, 5 showed
very low permeability with little detected in the apical
solution following the assay. Analogue 7, with glucose
succinate conjugated at the N-terminus, exhibited a sim-
ilar Papp to Endo-1 (Table 1). Clearly, the hydrophilicity
provided by glycosylation inhibited passive diffusion
across the lipophilic membranes, and the compound
was not transported by carbohydrate transporters
across the monolayers.
Compound
t1/2 (min)
Papp (·10ꢀ7, cm/s)
1
2
5
4.47 0.989
35.4 1.15
ND
23.22
23.76
23.76
ND
38
3
4
ND
5
12.4 4.74
5.45 1.02
8.23 1.29
4.16 0.399
No permeability
16.2 1.89
11.4 1.32
7
8
3.5
9
N.D
4.3
10
11
12
75
33
The C-terminus modified analogues, 8, 9 and 10, exhib-
ited low permeability with 10 being undetected in the
basolateral chamber over the course of the experiment.
These results were, in part, due to the susceptibility to
enzymatic metabolism demonstrated in the previous
experiments. Samples taken from the apical chamber
after the assay showed particularly low concentrations
of these analogues. Compounds with both termini con-
jugated with a combination of Laa and carbohydrate
units (11 and 12) exhibited higher permeability with
Apparent Permeability (Papp) values ꢁ3-fold higher
than Endo-1, but still lower than the N-terminal Laa
conjugate 2.
Dmt analogues
1
2
5
47
0.196 0.017
2.47 0.30
13
14
13.5
43.5
5.04 0.704
5.96 0.679
The data quantified by LC/MS and each point is expressed as a mean
value SD (n = 4). ND, not determined.
half-lives of 11 and 12 were measured at 75 and 33 min,
respectively, and these liposaccharide analogues were
readily soluble in aqueous buffer. N-Terminus modified
Endo-1 analogues (2 and 7) also exhibited improved
enzymatic stability profiles with the half-lives of 23
and 38 minutes respectively. The enantiomerically pure
analogues 3 and 4 were also examined and found to
have identical degradation profiles, which were also
identical to that of the racemic mixture 2. In the case
of N-terminal modified endomorphin analogues, the ste-
reochemistry of the Laa appears to have no effect on the
rate of degradation of the compound. The substitution
of Dmt for Tyr in Endo-1 (13) resulted in a slight
improvement in enzymatic stability, with a half-life of
13.5 min and this was again improved by conjugation
of C8Laa to the N-terminus with analogue 14 exhibiting
a t1/2 = 43.5 min.
The actual numerical Papp values obtained in the Caco-2
cell permeability assay depended greatly on various fac-
tors, such as passage number, age of the cells, TEER
values, and the media and growth conditions used.51–53
Therefore, the technique is best used in a comparative
manner with all the compounds assessed in the same as-
say under the same conditions.52,53 The Dmt analogues
were examined in a different experiment to the other
compounds; the native peptide 1 and the most promis-
ing compound from the previous experiment, 2, were
run with the Dmt compounds to allow direct compari-
son (Table 1). Replacing the Tyr residue of Endo-1 with
Dmt (13 and 14) resulted in a slight increase in lipophil-
icity and a corresponding increase in permeability of the
compounds compared to 1 and 2. The permeability re-
sults clearly showed that, as in the case of the stability
assays, N-terminal lipidation was the most effective
method of improving the bioavailability of endomor-
phin 1.
As the modifications made to the endomorphin se-
quence are modular in nature, it is feasible that the
Laa or sugar groups may be cleaved off in the process
of metabolism, releasing the native peptide. Therefore,
in addition to monitoring the enzyme-treated solutions
for each of the original peptides, the levels of Endo-1
were also measured. None of the analogues produced
significant amounts of the parent peptide on enzymatic
metabolism; therefore, none of the constructs produced
acted as prodrug structures.
2.3. Receptor binding and activity
While improvement to the bioavailability of a peptide is
essential in the development of useful peptide-based
pharmaceutics, this cannot be achieved without consid-
eration of the effect these changes have on the biological
function of the peptide. In the case of the endomorphin
1 analogues, the opioid receptor-binding affinity and
their ability to inhibit the production of cAMP within
a stimulated cell were assessed using the human neuro-
blastoma cell line SH-SY5Y, which endogenously ex-
presses l- and d-opioid receptors.54,55 The results of
the assays using intact SH-SY5Y cells are summarised
in Table 2.
Caco-2 cell monolayers were used to examine the
in vitro permeability of the endomorphin analogues.
Native peptide, Endo-1 (1), displayed low permeability
through Caco-2 cell monolayers with Papp 4.47 ·
10ꢀ7 cm/s. The N-terminus modified analogue 2 exhib-
ited significant permeability with Papp 3.54 · 10ꢀ6 cm/s,
an ꢁ8-fold increase in permeability (Table 1). The
C12Laa conjugate (5) was also expected to exhibit in-
creased permeability; however, the increase in alkyl-

6290
Y. Koda et al. / Bioorg. Med. Chem. 16 (2008) 6286–6296
Table 2. Binding-affinity values of endomorphin 1 analogues deter-
mined by competitive displacement of [3H]DAMGO from whole SH-
SY5Y cells
(2 and 5) decreased opioid receptor affinity. The glucose
moieties would be highly solvated in an aqueous solu-
tion and might represent a sterically demanding group
at the terminus. These data therefore indicate that, while
N-terminus is more amenable to functionalisation than
the C-terminus, there are steric constraints to which
groups can be accommodated without severely altering
receptor affinity.
Compound
Kil (nM)
1
2
1.11 0.79
2.15 1.18
2.92 0.12
3.02 0.14
15.4 7.20
115 42.1
121 52.1
151 66.1
148 67.2
266 122
3
4
5
7
The most promising result was that the Dmt-bearing
analogues, 13 and 14, exhibited pM affinity for the l-
opioid receptor, 20-times higher than the native peptide.
This is consistent with the reports by Okada et al., which
showed that incorporating Dmt into endomorphins 1
and 2 significantly increases l-opioid receptor affinity
and analgesic activity assays in vivo.35,39,60 It is clear
that the presence of a C8Laa residue on the N-terminus
had little effect on the tight binding of the Dmt-peptide.
8
10
11
12
13
14
0.059 0.01
0.081 0.03
Kil values determined by seven concentrations ranging from 10 pM to
1 lM performed in triplicate. Data are means SEM.
It is widely known that the treatment of SH-SY5Y cells
with forskolin causes an accumulation of cAMP within
the cell.61 One of the physiological consequences of ago-
nist binding to the l-opioid receptor inhibits cAMP pro-
duction.61 The peptide analogues were initially treated
at a single concentration (100 nM) to establish whether
these compounds were agonists or antagonists: all of
the analogues tested showed some agonist activity and
the extent to which they inhibited the accumulation of
cAMP is presented in Table 4.
The opioid receptor-binding affinity was also assessed
for a small number of the analogues using a competitive
displacement assay with prepared rat brain membranes
and the data are presented in Table 3. This assay allow-
ing for the assessment of both the l- and d-opioid recep-
tor-binding affinity was performed to initially ensure
that remarkable selectivity of endomorphin for the l-
receptor was maintained upon substitution of the ter-
mini, though this was not a major focus of this study:
it was performed to validate the affinity results obtained
using the SH-SY5Y assay and there was excellent agree-
ment between the two results.
Analogues that showed the highest affinity (1, 2, 13, and
14) were then further examined by means of a concen-
tration–response curve (Fig. 2). Inhibition of cAMP
production was found to plateau between 70% and
75% for all four analogues (Fig. 2). Lipoamino acid-con-
jugated analogues and their parent peptides exhibited
similar concentration–response curves. The calculated
IC50 values of cAMP levels for 1 and 2 were 14 and
26 nM, respectively, indicating a small drop in potency
for the Laa-conjugated analogue (Table 4). In compari-
son, the Dmt-bearing analogues 13 and 14 both exhib-
ited a leftward shift in dose–response curve, and
showed essentially equivalent subnanomolar IC50 values
(Table 5). It is clear that the introduction of Dmt into
the endomorphin sequence greatly enhanced not only
the l-opioid receptor affinity, but also the agonist activ-
ity of the peptide demonstrated by the inhibition of
cAMP.
The receptor affinity results show that compounds mod-
ified only at the C-terminus (8 and 10) showing a
marked decrease in affinity, as did those modified at
both termini (11 and 12). It is clear that additions to
the C-terminus have a significant detrimental effect on
receptor-binding of this peptide suggesting that the free
amide group may be an important contact point in the
binding complex. Chaturvedi et al. also showed that
glycosylation of the C-terminus of endomorphin 1
caused a greater decrease in affinity than glycosylation
of the N-terminus56 and a structure–activity study sug-
gested that the C-terminal Phe conformation is essential
for binding affinity for the l-opioid receptor.57–59
The N-terminus modified compounds (2–7) exhibited
binding affinities close to that of the native peptide. Glu-
cose succinate-conjugated analogue (7) exhibited a much
lower receptor affinity than the Laa conjugates and
increasing the length of the alkyl side chain of the Laas
3. Conclusions
From this study on the effects of the functionalisation of
endomorphin 1 we have gained insight into some viable
strategies that could be employed to convert this simple
opioid to a pharmaceutically relevant analgesic. Our re-
sults clearly indicated that attachment of auxiliary
groups to the C-terminus of the peptide not only de-
stroys much of the receptor affinity, but also offers little
protection against degradation and hence no improve-
ment to the bioavailability of the peptide. Attachment
of simple glucose units to either terminus did not pro-
vide significant improvement to epithelial permeability
Table 3. Binding affinity values of endo-1 analogues determined by
competitive displacement of [3H]DAMGO from prepared rat brain
membranes, values are expressed as means SE n = 3–4
Compound
Kil (nM)
Kid (nM)
d/l
1
2
1.15 0.16
2.86 0.61
14.8 1.6
1280 134
1760 215
836 129
6320 900
5.0 0.6
1110
614
56
5
7
13
115 7.3
0.067 0.02
55
75

Y. Koda et al. / Bioorg. Med. Chem. 16 (2008) 6286–6296
6291
Table 4. The level of inhibition of cAMP production in stimulated SH-
SY5Y cells was induced by treatment with a 100 nM solution of the
test compound
From this series of simple lipid or sugar modifications to
endomorphin 1 and the replacement of Tyr1 by Dmt, we
have generated a pentapeptide, [C8Laa-DMT1]-Endo1
(14), that exhibited t1/2 = 43.5 min in a solution of diges-
tive enzymes, which was >8 times that of the parent pep-
tide as well as permeability of 30 times that of
endomorphin 1. Remarkably, this stable, permeable
peptide exhibits potent in vitro agonist affinity with a
Kil = 0.08 nM, which is 140-fold greater than the parent
peptide and a subnanomolar inhibition of cAMP pro-
duction. Compound 14, therefore, represents a potential
new lead peptide in the journey towards endomorphin 1-
based pharmaceutics.
Compound
cAMP level (fmol)
Inhibition level
of cAMP (%)
Forskolin alone
Non-stimulated cells
1211 111
24 13.8
—
100
1
462 75.1
395 64.6
516 83.5
60.1 93.2
994 145.8
805 206.4
748 183.6
159 58.6
59.8 3.9
65.4 3.35
55.41 4.29
48.39 4.84
15.94 7.5
31.54 10.7
36.25 9.53
84.29 3.04
2
5
6
8
10
11
14
SH-SY5Y cells were stimulated by treatment with 30 lM solution of
forskolin in each assay except in blank. Data presented are mean
values (n = 3) SEM from three experiments in triplicate.
4. Experimental
4.1. General
Dimethylformamide and trifluoroacetic acid (TFA) of
peptide synthesis grade were purchased from Auspep
(Parkville, Australia). HPLC-grade acetonitrile was
purchased from Labscan Asia Co. Ltd (Bangkok,
Thailand). Fmoc-protected amino acids and Rink
amide MBHA resin (100–200 mesh, 0.78 mmol/g load-
ing) were obtained from Novabiochem (Melbourne,
Australia) and Reanal Finechem (Budapest, Hungary).
Piperidine was purchased from Auspep, Melbourne
Victoria. All other chemicals were purchased from
the Aldrich Chemical company unless otherwise sta-
ted. Absorbance measurements were preformed on a
Varian Cary 50 Bio UV–Vis Spectrophotometer. Elec-
trospray ionisation mass spectrometry was performed
on a Perkin-Elmer Sciex API 3000 operating in posi-
90
80
70
60
50
1
40
2
30
13
14
20
10
0
1
tive ion mode and H and 13C NMR spectra were re-
-13
-12
-11
-10
-9
-8
-7
-6
-5
[M] peptide
corded on Bruker Ultra Shield 300, 400 or 500 MHz
Topspin spectrometers using CDCl3 (Aldrich),
CO(CD3)2 (Aldrich), DMSO-d6 (Cambridge Isotope
laboratories, Inc.), or D2O (Cambridge Isotope labo-
ratories, Inc.) as solvents. 13C signals were assigned
with data from distortionless enhancement of polarisa-
tion transfer spectra using a 135-degree decoupler
pulse (DEPT 135). Melting points were determined
using a Yanagimoto micro-melting point apparatus
and thin-layer chromatography (TLC) performed on
Kieselgel G (Merk). Optical rotations were measured
using a Perkin-Elmer 241MC Polarimeter (wavelength
Figure 2. cAMP formation inhibition curve for analogues 1, 2, 13, and
14. All data are expressed as means (n = 3) and SEM of % maximum
inhibition in triplicate. Data are plotted using the one-site competition
functions (PRISM).
Table 5. The IC50 (nM) from full dose–response curve of cAMP
formation inhibition
Compound
IC50 (nM)
1
2
14.0 6.75
26.3 14.5
0.43 0.19
0.32 0.16
k
Na = 589).
13
14
4.1.1. Lipoamino acid synthesis. 2-Amino-D,L-octanoic
acid, 2-amino-D,L-dodecanoic acid, and 2-amino-D,
L-octadecanoic acid were synthesised from diethyl
acetamido malonate and the appropriate alkyl bro-
mide using published procedures.26,62 The analogues
bearing N-terminal Laas were synthesised using tert-
butoxycarbonyl (Boc) protected Laas. Boc protection
was achieved using published procedures and the spec-
tral data for these compounds matched the reported
data.26,62,63 For those analogues where the Laa residue
was at the C-terminus or within the sequence, the
amine group was protected using the 2-acetyldimedone
(Dde) protecting group which was attached using the
literature procedures.64–66
Data are from three experiments conducted in triplicate.
of the compound; furthermore, there is no evidence of
sugar transporter involvement and the steric crowding
associated with the sugar unit decreased receptor bind-
ing. The introduction of short-chain lipoamino acids
to the N-terminus of the peptide provided significant
protection to metabolic enzymes and increased the pas-
sive diffusion of the peptide across Caco-2 cell monolay-
ers, while having minimal effect on receptor affinity or
agonist activity. The length of the lipid chain accommo-
dated depended heavily on the water solubility of the
resulting compound.

6292
Y. Koda et al. / Bioorg. Med. Chem. 16 (2008) 6286–6296
4.2. Resolution of 2-amino-D,L-octanoic acid
washes was 6. The EtOAc layer was dried (MgSO4),
and the solvent was removed in vacuo. The product
was dissolved in 20% acetonitrile in water, and lyophi-
lised to a white solid.
4.2.1. D,L-2-Chloroacetamido-octanoic acid. The hydro-
chloride salt of 2-amino-D,L-octanoic acid (4.0 g,
25.2 mmol), chloroacetyl chloride (4.12 mL, 37.8 mmol),
and pyridine (2.03 mL, 25.2 mmol) were dissolved in
dioxane (2.32 mL, 27.8 mmol) and ethyl acetate
(180 mL) and the solution was heated under refluxing
conditions for 5 h. After cooling to room temperature,
the reaction mixture was washed with 10% citric acid
in water (2 · 50 mL). The organic solvent was dried
(MgSO4), removed in vacuo, and the resulting residue
lyophilised from 20% acetonitrile in water. The isolated
white powder was identified as chloroacetamido-a-2-
aminooctanoic acid.33
4.2.4. tert-Butoxycarbonyl-L-aminooctanoic acid. Yield
0.105 g, 64.5%, MS [M+H]+ m/z: 260 ([M+H]+ of
25
D
TLC Rf = 0.48 (CHCl3/MeOH/AcOH; 90:8:2 v/v,
C13H25NO4 requires 260), ½aꢂ +18.9, (c 1, CHCl3),
1
0.02% ninhydrin in ethanol dip). H NMR (400 MHz,
CDCl3) d 5.06 (d, 1H, NH), 4.25 (q, 1H, aCH), 1.81
(m, 1H, b-CHH), 1.63 (m, 1H, b-CHH), 1.41 (s, 9H,
C(CH3)3), 1.25 (m, 8H, CH2), 0.84 (t, 3H, J = 6.2
CH3). 13C NMR (100 MHz, CDCl3) 176.94 (COOH),
155.66 (CONH), 80.21 (C), 53.32 (CH), 32.21 (CH2),
31.48 (CH2), 28.76 (CH2), 28.23 (CH3), 25.17 (CH2),
22.45 (CH2), and 13.96 (CH3).
Yield; 2.0 g, 33.8%, ESMS (m/z); 200.2 [MꢀCl]+
([MꢀCl]+ of C10H18NO3 requires 200.1). 1H NMR
(400 MHz, CDCl3) d 8.65 (br, m, 1H, OH), 7.08 (d,
1H, J = 8.2 Hz NH), 4.58 (q, 1H a-CH), 4.09 (s, 2H,
CH2Cl), 1.90 (m, 1H, b-CHH), 1.75 (m, 1H, b-CHH),
1.25 (br m, 8H, CH2), 0.85 (t, 3H, J = 6.7 CH3). 13C
NMR (100 MHz, CDCl3) d 176.02 (COOH), 166.41
(NCOR), 52.46 (CH), 42.33 (CHCl), 31.86, 31.45,
28.72, 25.00, 22.45 (5 · CH2), and 13.94 (CH3).
4.2.5. tert-Butoxycarbonyl-D-aminooctanoic acid. Yield
0.08 g, 49%, MS [M+H]+ m/z: 260 ([M+H]+ of
25
D
TLC Rf = 0.48 (CHCl3/MeOH/AcOH; 90:8:2 v/v,
C13H25NO4 requires 260), ½aꢂ ꢀ15.8, (c 1, CHCl3),
1
0.02% ninhydrin in ethanol dip). H NMR (400 MHz,
CDCl3) d 5.06 (d, 1H, NH), 4.25 (br q, 1H, aCH),
1.81 (m, 1H, b-CHH), 1.63 (m, 1H, b-CHH), 1.41 (s,
9H, C(CH3)3), 1.25 (m, 8H, CH2), 0.84 (t, 3H, J = 6.2
CH3). 13C NMR (100 MHz, CDCl3) d 176.99 (COOH),
155.56 (CONH), 79.89 (C), 53.43 (CH), 32.43 (CH2),
31.50 (CH2), 28.79 (CH2), 28.24 (CH3), 25.14 (CH2),
22.45 (CH2), and 13.96 (CH3).
4.2.2. Hydrolysis of 2-chloroacetamido-octanoic acid33.
D,L-2-Chloroacetamido-octanoic acid (1.5 g, 6.3 mmol)
was dissolved in distilled water, and the pH of the solu-
tion was adjusted to 7.3 with 2 N lithium hydroxide
(LiOH). Fresh acylase I enzyme (0.7 g) from Aspergillus
melleus was added, and the solution was incubated at
37 ꢁC for 2 h. The first white precipitant was isolated,
and the solution was left to stand under the same condi-
tions for a further 72 h. A second white precipitant was
isolated, and both precipitates were identified as 2-ami-
no-L-octanoic acid. The remaining solution was acidified
(pH 2) with 35% HCl and stirred overnight. The white
precipitant was collected and identified as 2-amino-D-
octanoic acid.
4.2.6.
N-(2,3,4,5-Tetra-O-acetyl-b-D-glucopyranosyl)-
succinate. This building block for N-terminal glycosyl-
ated peptides was synthesised from glucose pentaacetate
via published procedures with the spectral data of the
product being fully consistent with those of the litera-
ture reports.23,63
Synthesis of 2,3,4-tri-O-acetyl-1-azido-1-deoxy-b-D-glu-
copyranuronic acid and immobilisation onto Rink amide
MBHA resin was achieved through a three-step process
from glucuronic acid via the literature procedures.63,67
2-Amino-L-octanoic acid: yield; 0.38 g, 75.8%, MS (m/
z); 160.43 [M+H]+ ([M+H]+ of C8H17NO2 requires
160.13) [a]D; +21.4ꢁ in AcOH (c = 1 g/mL) (literature
[a]D; +23ꢁ in 6 N HCl (c = 1%)).33
Synthesis of N-(tert-Butoxycarbonyl)-20,60-dimethyl-L-
tyrosine (DMT) was achieved using the five-step litera-
ture reported pathway.41
2-Amino-D-octanoic acid: yield; 0.086 g, 17.0%, MS (m/
z); 160.43 ([M+H]+ of C8H17NO2 requires 160.13) [a]D;
ꢀ21.1ꢁ in AcOH (c = 1 g/mL) (literature [a]D; +23.5ꢁ in
6 N HCl (c = 1%)).33
4.2.7. Solid-phase peptide synthesis. All peptides were
assembled on Rink amide MBHA resin (100–200 mesh,
0.78 mmol/g loading) on a 0.5 mmol scale using HBTU/
DIPEA activation and the in situ neutralisation proto-
col.68 The following protected amino acids were used:
Fmoc-Phe, Fmoc-Trp(Boc), Fmoc-Pro, and Fmoc-
Tyr(t-Bu). The efficiency of each amino acid coupling
was determined by the quantitative ninhydrin reaction69
and couplings repeated until an efficiency >99.6% was
achieved. The chloranil test70,71 was performed to deter-
mine the coupling efficiency for amino acid coupled to a
proline residue.
4.2.3. 2-(tert-Butoxycarbonylamino)octanoic acid. Opti-
cally pure 2-aminooctanoic acid (0.1 g, 0.63 mmol) was
dissolved in DIPEA (0.2 mL). Boc-carbonate (0.16 g,
0.75 mmol) was dissolved in dioxane:water (15 mL,
2:1), and added to the DIPEA solution. The mixture
was stirred for 12 h, and the pH maintained at 8–9 with
DIPEA. The solvent was evaporated in vacuo, and the
pH was adjusted to 4 with 10% citric acid in water.
The crude product was extracted with EtOAc
(2 · 30 mL). The extracts were washed with saturated
NaCl in water (5 · 20 mL) until the pH of the aqueous
The N-Dde-protected Laas were coupled to the growing
peptides using standard coupling techniques, with the
reaction allowed to proceed for 1 hour and repeated

Y. Koda et al. / Bioorg. Med. Chem. 16 (2008) 6286–6296
6293
once. The N-Dde protecting group was removed by
treatment with 2% hydrazine hydrate in DMF (2 · 1 h)
followed by efficient rinsing of the resin with DMF as
usual. The removal of O-acetyl groups of glucose succi-
namide and glucuronic acid was carried out after re-
moval of N-terminal protecting groups. The drained
resin-peptide was suspended in 5 mL of 12.5% (v/v)
hydrazine hydrate in methanol and the suspension
mixed for 18 h at room temperature. The resin was then
drained and washed well with DMF before preparing
for cleavage as usual.
100 lL (5 · 104 cells) was added into each well in a 96-well
cell culture plate. DMEM was supplemented with 10%
FBS, 1% of L-glutamine, 1% of non-essential amino acids,
and 1% of 100 U/mL penicillin/streptomycin and this
media was changed every second day for 21–28 days. To
perform the assay, media was removed from the wells,
each well washed with 0.2% EDTA (2 · 100 lL) followed
by washing three times with HBSS containing 25 mM
Hepes (pH 7.4). Finally, 100 lL of this buffer was placed
in each well and the plate cooled on ice. The cells were then
disrupted by a two-second pulse with a Sonics Vibracell
ultrasonic processor set at 40% amplitude with 130w
mode. The 96-well plate was centrifuged at 2000 rpm for
five minutes to precipitate the cell debris. The contents
of the three of the wells were assayed for total protein con-
tent using the Bio-Rad protein assay72 with BSA as a stan-
dard. The total protein content of the homogenate
supernatant was adjusted with the buffer to 0.6–0.9 mg/
mL in a clean 96-well plate.
When construction of the peptide was complete, the resin
was washed with DMF, DCM, and MeOH and dried. The
peptide was cleaved from the resin by treatment with
TFA/water/triisopropylsilane (TIS) (95:2.5:2.5 v/v,
25 mL) for 6 h. The resin was removed by filtration and
washed with TFA. The solvent was removed from the
peptide solution under a stream of nitrogen and the crude
peptide precipitated with cold diethyl ether, collected and
dissolved in 20% acetonitrile and lyophilised.
The compounds to be tested were dissolved in HBSS–
25 mM Hepes buffer to a final concentration of
200 lM stock solution including 1% by total volume
DMSO. At the start of the assay, 100 lL of this solution
was added to each well containing 100 lL of cell homog-
enate. The concentration of test compound at the begin-
ning of the experiment was 100 lM, and the assay was
performed at 37 ꢁC shaking at 400 rpm. Samples
(10 lL) were taken at selected time points (5, 10, 15,
20, 30, 40, 50, 60, and 120 min) and immediately added
to 5 lL of TFA to stop digestion and then diluted with
85 lL of water/DMSO solution so that there was a final
concentration of 10% by volume of DMSO. This addi-
tion of DMSO was shown to improve the resolution
and reproducibility of the LC/MS analysis, particularly
of the lipophilic peptides.
The purification of peptide analogues was achieved by
preparative RP-HPLC on a Waters 600 controller and
pump with a 490E programmable multi-wavelength
detector. The purity of peptide analogues was deter-
mined by analytical RP-HPLC (Shimazu; SCL-10AVP
system controller, FCV-10ALVP pump, and SPD-6A
UV detector), electrospray ionisation MS (ESI-MS; Per-
kin-Elmer Sciex API 3000), and LC/MS (Shimadzu LC-
10AT HPLC, Perkin-Elmer Sciex API 3000). A table
detailing the yields, ESMS analysis, and HPLC reten-
tion time for each analogue is provided in the Supple-
mentary information.
4.2.8. Solution-phase synthesis of analogues 13 and 14.
The DMT-bearing analogues 13 and14 were synthesised
by segment condensation method in solution as de-
scribed in the recent literature reports.35,60
The concentration of the test compound in each sample
was determined by LC/MS analysis using gradient elu-
tion (Shimadzu LC-10AT HPLC), coupled to a triple
quadrupole mass spectrometer (Perkin-Elmer Sciex
API 3000) and operating in selected ion monitoring
(SIM) mode with positive ion electrospray. Each sample
solution (10 lL) was injected into the system incorporat-
ing a 1:10 splitter and an elution gradient from 100%
Solvent A (0.1% formic acid in water) to 90% Solvent
B (0.1% formic acid in 90% acetonitrile/water) over
5.5 min at a flow rate of 0.3 mL/min was used. A C18
column (50 · 2.0 mm, Phenomexꢂ) or CN column
(50 · 2.0 mm, Phenomexꢂ) was used for HPLC. The
concentrations were calculated by comparison with a
standard curve generated and analysed on the same
day as the samples.
4.3. Cell culture
The Caco-2 and SH-SY5Y cell lines were obtained
from the American Type Culture Collection (Rockville,
USA). Caco-2 cells were maintained in T-75 flasks with
Dulbecco’s modified Eagle’s medium (DMEM, Gibco
life technology) supplemented with 10% foetal bovine
serum (FBS), 1% L-glutamine, and 1% non-essential
amino acids, at 95% humidity at 37 ꢁC, in an atmo-
sphere of 5% CO2. The medium was changed every sec-
ond day. When the cells reached 80% of confluence,
they were subcloned using 0.25% trypsin–EDTA.
Passage numbers 55–75 were used in the assays. SH-
SY5Y cells were maintained in DMEM:Hams F12
medium (Gibco life technology) supplemented with
10% FBS, 1% glutamine, 1% non-essential amino acids
and 1000 U/mL Streptomycin/Penicillin (Gibco life
technology). Cells were subcloned in the same manner
as Caco-2 cells. Passage numbers 10–20 were used in
the assays.
4.3.2. Permeability assay. A suspension of Caco-2 cells
in media was adjusted to ꢁ1 · 106 cells/mL and 100 lL
(ꢁ1 · 105 cells) of this suspension was added to the poly-
carbonate cell culture inserts (pore size 0.4 lm · 6.5 mm
diameter, Transwellꢂ). The media used consisted of
DMEM supplemented with 10% FBS, 1% L-glutamine,
1% non-essential amino acids, and 1% 100 U/mL peni-
cillin/streptomycin. The media was changed every sec-
ond day (0.6 mL in the basolateral chamber and
4.3.1. Enzymatic stability assay. A suspension of Caco-2
cells in media was adjusted to ꢁ5 · 105 cells/mL and

6294
Y. Koda et al. / Bioorg. Med. Chem. 16 (2008) 6286–6296
0.1 mL to the apical chamber) and the monolayers were
used between 21 and 28 days after seeding. Permeability
studies were carried out at pH 7.4 and at 37 ꢁC. The
tight junctions and integrity of the monolayers were
monitored by the transepithelial electrical resistance
(TEER, Xcm2) across each monolayer, measured using
the Millicell-ERS epithelial volt-ohmmeter system (Mil-
lipore Corporation). The TEER values of the assayed
monolayers were measured at 1500–4280 Xcm2 with
minor TEER value differences of 500 Xcm2 detected
after the assays. None of the Endo-1 analogues caused
a significant drop in the TEER values during the assay
indicating that they were not toxic to the cells. The
Caco-2 cell monolayer integrity was also monitored by
measuring the permeability of radiolabelled [14C]-D-
mannitol (Sigma; 55 mCi/mmol, 0.09 mCi/mL in 90%
4.3.4. Intact SH-SY5Y cell receptor-binding assay.
SH-SY5Ycells were plated with binding buffer
(50 mM Tris buffer, pH 7.4, 2% BSA) in 24-well
plates and incubated overnight. Media was removed
and cells were washed with PBS before pre-incuba-
tion in 300 lL binding buffer. Binding studies were
performed with 100 lL of appropriate concentrations
of [3H]DAMGO (a selective l-opioid receptor ago-
nist) and 100 lL competitor or blank in binding buf-
fer at 37 ꢁC for 60 min. Competition binding
experiments were performed using 100 pM [3H]DAM-
GO in the absence or presence of increasing concen-
trations of unlabelled peptides. An excess of
unlabelled DAMGO (1 lM) was used to determine
non-specific binding. Following incubation, the cells
were washed with PBS and recovered from culture
plates using 500 lL 1 M NaOH before transfer to
scintillation vials. Liquid scintillation cocktail (Ultima
Gold, Packard, Meriden, USA) was added to these
vials for counting in a liquid scintillation analyser.
Data are expressed as means SEM of % specific
binding of triplicate determinations performed on at
least three independent plates of cells. Data were
plotted using the one-site competition (PRISM,
Graphpad Inc., San Diego, USA).
EtOH in water).
A mannitol solution (1.80 lCi,
32.73 nmol/4 mL) in HBSS–25 mM Hepes buffer was
added to the donor chamber of three wells.
Compounds to be tested were dissolved in HBSS–
25 mM Hepes buffer to a final concentration of
200 lM including 1% by total volume of DMSO. Prior
to beginning the experiments, the monolayers were
washed with pre-warmed HBSS–25 mM Hepes buffer,
and incubated in the buffer for 30 min. At the start of
the experiment, the apical chamber was emptied and
100 lL of test compound solution added.
4.3.5. Inhibition of cAMP accumulation assay. SH-SY5Y
cells endogenously expressing l- and d-opioid recep-
tors were cultured (100 lL) in a 96-well plate and
incubated (usually overnight) until 80% confluent.
Peptide analogues were diluted in DMEM culture
medium containing 30 lM forskolin and applied to
separate wells for agonist determination and co-incu-
bated for antagonist studies. Cells were incubated with
peptides at 37 ꢁC for 30 min, medium was aspirated
and cells were lysed using lysis buffer from a cAMP
Biotrak EIA kit (Amersham, Buckinghamshire, UK).
An anti-cAMP antibody was added to the lysate,
and the mixture incubated at 4 ꢁC for 2 h followed
by the addition of a cAMP peroxidase conjugate,
and incubated at 4 ꢁC for a further hour. A 3,305,50-
tetramethylbenzidine (TMB) substrate provided by
the Biotrak EIA kit was then incubated with cell ly-
sate for 1 h and finally 1 lM sulfuric acid was added
to facilitate a colour change, which is proportional to
the amount of cAMP captured. A cAMP standard
curve from 0 to 3.2 pmol was also generated concur-
rently. Plates were then read in a plate reader at
450 nm and compared to the cAMP standard curve.
All data are compared to the maximum cAMP re-
sponse to forskolin and are represented as
means standard error of three experiments per-
formed in triplicate. All data are plotted using PRISM
(GraphPad, San Diego, CA).
The permeability study for each compound was per-
formed in at least three wells at 37 ꢁC, with shaking at
400 rpm in a Heridolf Titramax. Samples (400 lL) were
taken from the basolateral chamber at regular interval
time points (30, 90, 120, and 150 min), and replaced with
the same volume of buffer. At the end of experiment
(150 min), 50 lL was taken from the apical chamber.
All the collected samples were stored at ꢀ20 ꢁC to pro-
tect against degradation.
The concentration of the peptides in each sample was
quantified using the LC/MS method described above.
The radioactivity of the [14C]-D-mannitol samples was
quantified by liquid scintillation counting (Liquid Scin-
tillation Systems, BECKMAN LS3801, USA). The Papp
for each compound was calculated from the data using
the following formula
Pappðcm=sÞ ¼ dC=dt ꢃ V r=ðA ꢃ C0Þ
dC/dt steady-state rate of change in the chemical con-
centration (M/s) or radiochemical concentra-
tion (dpm/mL s) in the receiver chamber
Vr
A
volume of the receiver chamber (mL)
surface area of the cell monolayers
initial concentration in the donor chamber (M
or dpm/mL)
C0
Acknowledgments
4.3.3. Prepared rat brain membrane receptor-binding
assay. This assay relied on the method of competitive as-
says for l- and d-opioid receptor ligands using prepared
rat brain P2 synaptosomes as described in detail
previously.38
This work was supported in part by Neurotide Pty Ltd
and a Biotechnology Innovation Fund grant, and in
part by the Intramural Research Program of the NIH,
and NIEHS.

Y. Koda et al. / Bioorg. Med. Chem. 16 (2008) 6286–6296
6295
27. Caccetta, R.; Blanchfield, J. T.; Harrison, J.; Toth, I.;
Benson, H. A. E. Int. J. Pep. Res. Ther. 2006, 12, 327.
28. Blanchfield, J. T.; Lew, R. A.; Smith, A. I.; Toth, I.
Bioorg. Med. Chem. Lett. 2005, 15, 1609.
29. Polt, R.; Dhanasekaran, M.; Keyari, C. M. Med. Res. Rev.
2005, 25, 557.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.2008.
04.020.
30. Polt, R.; Palian, M. M. Drugs Future 2001, 26, 561.
31. Egleton, R. D.; Mitchell, S. A.; Huber, J. D.; Janders, J.;
Stropova, D.; Polt, R.; Yamamura, H. I.; Hruby, V. J.;
Davis, T. P. Brain Res. 2000, 881, 37.
32. Egleton, R. D.; Bilsky, E. J.; Tollin, G.; Dhanasekaran,
M.; Lowery, J.; Alves, I.; Davis, P.; Porreca, F.; Yamam-
ura, H. I.; Yeomans, L.; Keyari, C. M.; Polt, R.
Tetrahedron: Asymmetry 2005, 16, 65.
References and notes
1. Janecka, A.; Fichna, J.; Janecki, T. Curr. Top. Med. Chem.
2004, 4, 1.
2. Zadina, J. E.; Hackler, L.; Ge, L.-J.; Kastin, A. Nature
1997, 386, 499.
3. Goldberg, I. E.; Rossi, G. C.; Letchworth, S. R.; Mathis,
J. P.; Ryan-Moro, J.; Leventhal, L.; Su, W.; Emmel, D.;
Bolan, E. A.; Pasternak, G. W. J. Pharmacol. Exp. Ther.
1998, 286, 1007.
4. Gentilucci, L. Curr. Top. Med. Chem. 2004, 4, 19.
5. Stone, L. S.; Fairbanks, C. A.; Laughlin, T. M.; Nguyen,
H. O.; Bushy, T. M.; Wessendorf, M. W.; Wilcox, G. L.
Neuroreport 1997, 8, 3131.
6. Csullog, E.; Joo, G.; Toth, G.; Dobos, D.; Benedek, G.;
Horvath, G. Pain 2001, 94, 31.
7. Horvath, G.; Szikszay, M.; Tomboly, C.; Benedek, G. Life
Sci. 1999, 65, 2635.
8. Li, Z.-H.; Shan, L.-D.; Jiang, Z.-H.; Guo, S.-Y.; Yu, G.-
D.; Hisamitsu, T.; Yin, D. L. Q.-Z. Acta Pharmacol. Sin.
2001, 22, 976.
9. Obara, I.; Przewlocki, R.; Przewlocka, B. Neurosci. Lett.
2004, 360, 85.
33. Birnbaum, S. M.; Fu, S.-C. J.; Greenstein, J. P. J. Biol.
Chem. 1953, 203, 333.
34. Hansen, D. W.; Stapelfeld, A.; Savage, M. A.; Reichman,
M.; Hammond, D. L.; Haaseth, R. C.; Mosberg, H. I. J.
Med. Chem. 1992, 35, 684.
35. Jinsmaa, Y.; Marczak, E.; Fujita, Y.; Shiotani, K.;
Miyazaki, A.; Li, T. Y.; Tsuda, Y.; Ambo, A.; Sasaki,
A.; Bryant, S. D.; Okada, Y.; Lazarus, L. H. Pharmacol.
Biochem. Behav. 2006, 84, 252.
36. Sasaki, Y.; Sasaki, A.; Niizuma, H.; Goto, H.; Ambo, A.
Bioorg. Med. Chem. 2003, 11, 675.
37. Bryant, S. D.; Jinsmaa, Y.; Salvadori, S.; Okada, Y.;
Lazarus, L. H. Biopolymers 2003, 71, 86.
38. Okada, Y.; Tsuda, Y.; Fujita, Y.; Yokoi, T.; Sasaki, Y.;
Ambo, A.; Konishi, R.; Nagata, M.; Salvadori, S.;
Jinsmaa, Y.; Bryant, S. D.; Lazarus, L. H. J. Med. Chem.
2003, 46, 3201.
10. Przewlocka, B.; Mika, J.; Labuz, D.; Toth, G.; Przewlocki,
R. Eur. J. Pharmacol. 1999, 367, 189.
11. Soignier, R. D.; Vaccarino, A. L.; Brennan, A. M.; Kastin,
A. J.; Zadina, J. E. Life Sci. 2000, 67, 907.
12. Zhao, M.; Wang, J.-Y.; Jia, H.; Tang, J.-S. Brain Res.
2006, 1076, 68.
39. Okada, Y.; Fujita, Y.; Motoyama, T.; Tsuda, Y.; Yokoi,
T.; Li, T. Y.; Sasaki, Y.; Ambo, A.; Jinsmaa, Y.; Bryant,
S. D.; Lazarus, L. H. Bioorg. Med. Chem. 2003, 11, 1983.
40. Balboni, G.; Guerrini, R.; Salvadori, S.; Negri, L.;
Giannini, E.; Bryant, S. D.; Jinsmaa, Y.; Lazarus, L. H.
J. Med. Chem. 2005, 48, 8112.
13. Hau, V. S.; Huber, J. D.; Campos, C. R.; Lipkowski, A.
W.; Misicka, A.; Davis, T. P. J. Pharm. Sci. 2002, 91,
2140.
41. Dygos, J. H.; Yonan, E. E.; Scaros, M. G.; Goodmonson,
O. J.; Getman, D. P.; Periana, R. A.; Beck, G. R.
Synthesis (Stuttg) 1992, 741.
14. Tomboly, C.; Peter, A.; Toth, G. Peptides 2002, 23, 1573.
15. Lengyel, I.; Orosz, G.; Biyashev, D.; Kocsis, L.; Al-
Khrasani, M.; Ronai, A.; Tomboly, C.; Furst, Z.; Toth,
G.; Borsodi, A. Biochem. Biophys. Res. Commun. 2002,
290, 153.
42. Artursson, P.; Karlsson, J. Biochem. Biophys. Res. Com-
mun. 1991, 175, 880.
43. Wong, A. K.; Ross, B. P.; Chan, Y. N.; Artursson, P.;
Lazorova, L.; Jones, A.; Toth, I. Eur. J. Pharm. Sci. 2002,
16, 113.
16. Yang, Y. R.; Chiu, T. H.; Chen, C. L. Eur. J. Pharmacol.
1999, 372, 229.
44. He, X.; Sugawara, M.; Kobayashi, M.; Takekuma, Y.;
Miyazaki, K. Int. J. Pharm. 2003, 263, 35.
17. Paterlini, M. G.; Avitabile, F.; Ostrowski, B. G.; Fergu-
son, D. M.; Portoghese, P. S. Biophys. J. 2000, 78, 590.
18. Podlogar, B. L.; Paterlini, M. G.; Ferguson, D. M.; Leo,
G. C.; Demeter, D. A.; Brown, F. K.; Reitz, A. B. FEBS
Lett. 1998, 439, 13.
45. Zornoza, T.; Cano-Cebrian, M. J.; Nalda-Molina, R.;
Guerri, C.; Granero, L.; Polache, A. Eur. J. Pharm. Sci.
2004, 22, 347.
46. Cruciani, G.; Pastor, M.; Guba, W. Eur. J. Pharm. Sci.
2000, 11, S29.
19. Adessi, C.; Soto, C. Curr. Med. Chem. 2002, 9, 963.
20. Blanchfield, J.; Toth, I. Curr. Med. Chem. 2004, 11, 2375.
21. Toth, I.; Malkinson, J. P.; Flinn, N. S.; Drouillat, B.;
Horvath, A.; Erchegyi, J.; Idei, M.; Venetianer, A.;
Artursson, P.; Lazorova, L.; Szende, B.; Keri, G. J.
Med. Chem. 1999, 42, 4010.
22. Ali, M.; Manolios, N. Lett. Pept. Sci. 2001, 8, 289.
23. Kellam, B.; Drouillat, B.; Dekany, G.; Starr, M. S.; Toth,
I. Int. J. Pharm. 1998, 161, 55.
24. Toth, I.; Flinn, N.; Hillery, A.; Gibbons, W. A.; Arturs-
son, P. Int. J. Pharm. 1994, 105, 241.
25. Flinn, N.; Coppard, S.; Toth, I. Int. J. Pharm. 1996, 137,
33.
47. van der Sandt, I. C. J.; Vos, C. M. P.; Nabulsi, L.; Blom-
Roosemalen, M. C. M.; Voorwinden, H. H.; de Boer, A.
G.; Breimer, D. D. Aids 2001, 15, 483.
48. Lohmann, C.; Huwel, S.; Galla, H. J. J. Drug Target.
2002, 10, 263.
49. Garberg, P.; Ball, M.; Borg, N.; Cecchelli, R.; Fenart, L.;
Hurst, R. D.; Lindmark, T.; Mabondzo, A.; Nilsson, J. E.;
Raub, T. J.; Stanimirovic, D.; Terasaki, T.; Oberg, J. O.;
Osterberg, T. Toxicol. In Vitro 2005, 19, 299.
50. Lang, V. B.; Langguth, P.; Ottiger, C.; WunderliAllensp-
ach, H.; Rognan, D.; RothenRutishauser, B.; Perriard, J.
C.; Lang, S.; Biber, J.; Merkle, H. P. J. Pharm. Sci. 1997,
86, 846.
26. Blanchfield, J. T.; Dutton, J. L.; Hogg, R. C.; Galla-
gher, O. P.; Craik, D. J.; Jones, A.; Adams, D. J.;
Lewis, R. J.; Alewood, P. F.; Toth, I. J. Med. Chem.
2003, 46, 1266.
51. Hosoya, K.; Kim, K. J.; Lee, V. H. L. Pharm. Res. 1996,
13, 885.
52. Behrens, I.; Kamm, W.; Dantzig, A. H.; Kissel, T. J.
Pharm. Sci. 2004, 93, 1743.

6296
Y. Koda et al. / Bioorg. Med. Chem. 16 (2008) 6286–6296
53. Sambuy, Y.; Angelis, I.; Ranaldi, G.; Scarino, M. L.;
Stammati, A.; Zucco, F.. Cell Biol. Toxicol. 2005, 21, 1.
54. Kazmi, S. M. I.; Mishra, R. K. Biochem. Biophys. Res.
Commun. 1986, 137, 813.
55. Kazmi, S. M. I.; Mishra, R. K. Mol. Pharmacol. 1987, 32,
109.
63. Blanchfield, J. T.; Toth, I. Modification of Peptides
and Other Drugs Using Lipoamino Acids and
Sugars. In Peptide Synthesis and Applications; Howl,
J., Ed.; Humana Press: Totawa, NJ, 2005; Vol. 298,
p 45.
64. Nash, I. A.; Bycroft, B. W.; Chan, W. C. Tetrahedron Lett.
1996, 37, 2625.
65. Chhabra, S. R.; Hothi, B.; Evans, D. J.; White, P. D.;
Bycroft, B. W.; Chan, W. C. Tetrahedron Lett. 1998, 39,
1603.
66. Wang, F.; Manku, S.; Hall, D. G. Org. Lett. 2000, 2, 1581.
67. Malkinson, J. P.; Falconer, R. A.; Toth, I. J. Org. Chem.
2000, 65, 5249.
68. Alewood, P.; Alewood, D.; Miranda, L.; Love, S.;
Meutermans, W.; Wilson, D. Methods Enzymol. 1997,
289, 14.
56. Chaturvedi, K.; Shahrestanifar, M.; Howells, R. D. Mol.
Brain Res. 2000, 76, 64.
57. Tomboly, C.; Kover, K. E.; Peter, A.; Tourwe, D.;
Biyashev, D.; Benyhe, S.; Borsodi, A.; Al-Khrasani, M.;
Ronai, A. Z.; Toth, G. J. Med. Chem. 2004, 47, 735.
58. Fujita, Y.; Tsuda, Y.; Li, T. Y.; Motoyama, T.; Takah-
ashi, M.; Shimizu, M. J. Med. Chem. 2004, 47, 3591.
59. In, Y.; Minoura, K.; Tomoo, K.; Sasaki, Y.; Lazarus, L.
H.; Okada, Y.; Ishida, T. FEBS J. 2005, 272, 5079.
60. Li, T. Y.; Fujita, Y.; Tsuda, Y.; Miyazaki, A.; Ambo, A.;
Sasaki, Y.; Jinsmaa, Y.; Bryant, S. D.; Lazarus, L. H.;
Okada, Y. J. Med. Chem. 2005, 48, 586.
69. Sarin, V. K.; Kent, S. B. H.; Tam, J. P.; Merrifield, R. B.
Anal. Biochem. 1981, 117, 147.
61. Harrison, C.; McNulty, S.; Smart, D.; Rowbotham, D. J.;
Grandy, D. K.; Devi, L. A.; Lambert, D. G. Br. J.
Pharmacol. 1999, 128, 472.
62. Gibbons, W. A.; Hughes, R. A.; Charalambous, M.;
Christodoulou, M.; Szeto, A.; Aulabaugh, A. E.; Mas-
cagni, P.; Toth, I. Liebigs Ann. Chem. 1990, 1175.
70. Vojkovsky, T. Peptide Res. 1995, 8, 236.
71. Chan, W. C.; White, P. D. Fmoc Solid Phase Peptide
Synthesis: A Practical Approach; Oxford University Press:
Oxford, 2000.
72. Bradford, M. M.; Williams, W. L. Fed. Proc. 1976, 35,
274.