Improving metabolic stability by glycosylation: Bifunctional peptide derivatives that are opioid receptor agonists and neurokinin 1 receptor antagonists

Article abstract of DOI:10.1021/jm900473p

In order to obtain a metabolically more stable analgesic peptide derivative, O-β-glycosylated serine (Ser(Glc)) was introduced into TY027 (Tyr-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH-3′,5′-Bzl(CF₃) ₂) which was a previously reported bifunct

Full text of DOI:10.1021/jm900473p

5164 J. Med. Chem. 2009, 52, 5164–5175
DOI: 10.1021/jm900473p
Improving Metabolic Stability by Glycosylation: Bifunctional Peptide Derivatives That Are Opioid
Receptor Agonists and Neurokinin 1 Receptor Antagonists
Takashi Yamamoto,† Padma Nair,† Neil E. Jacobsen,† Josef Vagner,† Vinod Kulkarni,† Peg Davis,‡ Shou-wu Ma,‡
Edita Navratilova,‡ Henry I. Yamamura,‡ Todd W. Vanderah,‡ Frank Porreca,‡ Josephine Lai,‡ and Victor J. Hruby*,†
†Department of Chemistry, University of Arizona, 1306 E. University Boulevard, Tucson, Arizona 85721, and ‡Department of Pharmacology,
University of Arizona, 1501 N. Campbell Avenue, Tucson, Arizona 85724
Received December 19, 2008
In order to obtain a metabolically more stable analgesic peptide derivative, O-β-glycosylated serine
(Ser(Glc)) was introduced into TY027 (Tyr-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH-30,50-Bzl(CF3)2)
which was a previously reported bifunctional compound with δ/μ opioid agonist and neurokinin-1
receptor antagonist activities and with a half-life of 4.8 h in rat plasma. Incorporation of Ser(Glc) into
various positions of TY027 gave analogues with variable bioactivities. Analogue 6 (Tyr-D-Ala-Gly-Phe-
Nle-Pro-Leu-Ser(Glc)-Trp-NH-30,50-Bzl(CF3)2) was found to have effective bifunctional activities with
a well-defined conformation with two β-turns based on the NMR conformational analysis in the
presence of DPC micelles. In addition, 6 showed significant improvement in its metabolic stability
(70 ( 9% of 6 was intact after 24 h incubation in rat plasma). This improved metabolic stability, along
with its effective and δ selective bifunctional activities, suggests that 6 could be an interesting research
tool and possibly a promising candidate as a novel analgesic drug.
Introduction
effective potentials, as well as their low toxicities, endogenous
peptides have been rarely used in clinical treatment of pain
primarily because of their poor metabolic stabilities, which
limits the delivery of peripherally administered peptides into
the site of action. Moreover, the blood-brain barrier (BBB),
which is a protective barrier of the CNS, possesses membrane-
bound oxidative enzymes and peptidases that degrade per-
ipherally administered peptide drugs before they reach the
CNS.1 Therefore, a peptide drug possessing good metabolic
stability together with an effective therapeutic potential
should be a promising candidate for novel types of drugs.
To date, several studies have shown that the glycosylation of
a peptide can provide a significant increase in stability and other
biological properties,2-5 although in some cases the introduc-
tion of hydrophilicity into these molecules can result in a
decrease or even loss of bioactivities.6,7 Other reports have
shown that the glycosylation of short peptides can change tissue
distribution patterns8-10 and enhance peptide-membrane
interactions11 compared to the original nonglycosylated
sequences. In fact, we have shown that the introduction of a
glycosylated residue into enkephalin derivatives leads to the
enhanced biodistribution of a peptide into the brain, giving
better analgesic efficacies compared to the nonglycosylated
enkephalins.5,12-15 Glycosylation can also modify the mole-
cular structure of a synthetic peptide in both the aqueous phase
and membrane-like environments16,17 leading to a change in the
molecular stucture that has significant effects on the pharmaco-
logical and metabolic properties of bioactive peptides.18
Endogenous peptides play important roles in the main-
tenance of homeostasis as hormones and neurotransmitters in
both the peripheral and the central nerve system (CNSa).
Indeed, the biochemical degradation of endogenous peptides
appears to be an aspect in their roles as signal transmitters,
since in many cases protease degradation changes them into
an inactive form after the signal has been appropriately
transferred. However, the rapid degradation of peptides is
undesirable for analgesic drugs, since generally it is desirable
that the effects should last for a longer period in order to
maintain effective therapeutic potency. Thus, despite their
*To whom correspondence should be addressed. Phone: (520) 621-6332.
Fax: (520)-621-8407. E-mail: hruby@email.arizona.edu.
a Abbreviations: BBB, blood-brain barrier; Boc, tert-butyloxycarbonyl;
BSA, bovine serum albumin; Cl-HOBt, 1-hydroxy-6-chlorobenzotriazole;
CHO, Chinese hamster ovary; CNS, central nervous system; DAMGO, [D-
Ala2, NMePhe4, Gly5-ol]enkephalin; DCM, dichloromethane; DIEA, diiso-
propylethylamine; DOR, δ opioid receptor; DPC, dodecylphosphocholine;
DPDPE, c[D-Pen2, D-Pen5]enkephalin; DQF-COSY, double quantum
filtered correlation; FMPB-AM, 4-(4-formyl-3-methoxyphenoxy)butyryl-
aminomethyl; Fmoc, fluorenylmethoxycarbonyl; GPI, guinea pig ileum;
GTPγS, guanosine 50-(γ-thio)triphosphate; HCTU, 1H-benzotriazolium-
1-[bis(dimethylamino)methylene]-5-chlorohexafluorophosphate-(1-)3-oxide;
HRMS, high-resolution mass spectrometry; LMMP, longitudinal muscle
with myenteric plexus; MOR, μ opioid receptor; MVD, mouse vas deferens;
NK1, neurokinin-1; NOE, nuclear Overhauser enhancement; NOESY,
nuclear Overhauser enhancement spectroscopy; rMD, restrained molecular
dynamics; rmsd, root-mean-square deviation; ROESY, rotating frame Over-
hauser effect spectroscopy; RP-HPLC, reverse phase high-performance liquid
chromatography; Ser(Glc), O-β-glucosylated serine; SPPS, solid phase pep-
tide synthesis; TFA, trifluoroacetic acid; TIPS-OTf, tirisopropylsilyl trifluoro-
methanesulfonate; TMOF, trimethyl orthoformate; TNBS, 2,4,6-trinitroben-
zene sulfonic acid; TOCSY, total correlation spectra; Trp-NH-3,5-Bzl(CF3)2,
30,50-(bistrifluoromethyl)benzylamide of tryptophan. Abbreviations used
for amino acids and designation of peptides follow the rules of the IUPAC-
IUB Commission of Biochemical Nomenclature in J. Biol. Chem. 1972, 247,
977-983.
In the present study, we report the design, synthesis, and
biological evaluation for a series of glycosylated peptide
derivatives to examine the effect of glycosylation on metabolic
stability, bioactivity, and pseudomembrane-bound confor-
mation for targeting analgesic peptides in comparison with
the nonglycosylated derivatives. The peptide design was based
r
pubs.acs.org/jmc
Published on Web 07/22/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5165
conducted to clarify the specific interactions of the glycopep-
tides with model cell membranes.15,20,38
Results and Discussion
Peptide Synthesis. The glycopeptide derivatives 3-6 were
synthesized using the reductive amination technique on a
4-(4-formyl-3-methoxyphenoxy)butyryl AM (FMPB-AM)
resin, which is a common solid support for carboxamides,39
using the NR-Fmoc solid-phase peptide synthesis (SPPS)
strategy. First, an excess of 30,50-bis-trifluoromethylbenzy-
lamine was introduced on the resin in the presence of NaBH-
(OAc)3 and trimethyl orthoformate (TMOF) to convert the
aldehyde moiety of FMPB-AM resin to the corresponding
secondary amine. The resulting resin-bound 30,50-bistrifluor-
omethylbenzylamine was reacted with NR-Fmoc-Trp(Nin-
Boc)-OH using HCTU in the presence of 2,6-lutidine, fol-
lowed by treatment with 20% piperidine to remove the NR-
Fmoc protecting group. Couplings of the following pro-
tected amino acids were carried out with standard in situ
activating reagents HCTU, in the presence of DIEA, to
generate Cl-HOBt esters. Fmoc-Ser(O-β-D-Glc(OAc)4)-
OH,40 Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Nle-OH,
Fmoc-Phe-OH, Fmoc-Gly-OH, and Boc-Tyr(tBu)-OH were
used for respective coupling as protected amino acids. The
removal of the acetyl protecting groups on glucose was
Figure 1. Sequences of opioid and NK1 receptor peptides.
on our developing bifunctional concept in which the molecule
has both an opioid agonist pharmacophore at the N-terminal
portion and a NK1 antagonist pharmacophore at the C-
terminus (Figure 1).19-21 This concept was based on the
important observations that the simultaneous administration
of opioid agonist and NK1 antagonist provided significant
benefits of enhanced antinociceptive potency in acute pain
states and in preventing opioid-induced tolerance in chronic
preclinical trials.22-26 Therefore, the developing bifunctional
compounds should be expected to show enhanced analgesic
effects without showing the undesirable adverse effects and thus
be a drug candidate for pain control.19-21 For the best bioac-
tivity profile for the δ/μ opioid receptor ligands, a compound
that is a δ opioid receptor preferring agonist may possess
potential clinical benefits compared to the ones for a μ opioid
receptor agonist in terms of the greater relief of neuropathic
pain,27 reduced respiratory depression,28 and constipation29 as
well as a minimal potential for the development of physical
dependence.30 Thus, a new generation of opioid agonist with
selectivity and efficacy for δ opioid receptors over μ opioid
receptors should display reduced toxicity and abuse potential
coincident with therapeutic use. In fact, our lead bifunctional
compounds, TY005 (Tyr1-D-Ala2-Gly3-Phe4-Met5-Pro6-Leu7-
Trp8-O-30,50-Bzl(CF3)2)31 and TY027 (1: Tyr1-D-Ala2-Gly3-
Phe4-Met5-Pro6-Leu7-Trp8-NH-30,50-Bzl(CF3)2),20,32 have been
shown to reverse neuropathic pain without producing opioid-
induced tolerance, which validates our hypothesis that opioid
agonist/NK1 antagonist bifunctional ligands would be as
effective in treating neuropathic pain.
In our present drug design, Nle, which is a bioisoster of Met
with high resistance to oxidation,33-35 was first introduced
into the fifth position of 1 to provide 2, which could be
considered as a biological and physicochemical isoster of 1
with improved metabolic stability (Figure 1). Glycosylation
was made on 2 with O-β-glucosylated serine (Ser(Glc)) which
was incorporated into residues 5, 6, 7, or 8 of 2 gave four novel
carbohydrate derivatives 3-6 (Figure 1). The biological ac-
tivities of the synthesized bifunctional glycopeptides (3-6)
were extensively evaluated using our well-established radioli-
gand binding assays, GTPγS binding assays, and isolated
tissue-based functional assays using guinea pig ileum (GPI)
and mouse isolated vas deferens (MVD) tissues.19-21 The
metabolic stability of the synthesized derivatives was tested by
incubation in rat plasma at 37 °C. Since understanding
membrane-bound structures of ligands and ligand-membrane
interactions is indispensable for further insight into their
diverse biological behaviors,20 the NMR structures of synthe-
sized glycopeptides 3-6 were obtained using distance and j
dihedral angle constraint information in membrane-mimick-
ing DPC micelles20,36,37 to evaluate the biological and con-
formational effect of site-specific glycosylation. Additional
NMR studies using the paramagnetic agent Mn2þ were
accomplished with 80% H2NNH2 H2O in methanol,15 and
3
then the resin was treated with TFA/TIPS-OTf/thioanisole
(9:2:1, v/v) to obtain the corresponding crude glycopeptide
derivative, which was treated with 2 N NH4F solution
followed by triethylamine. The glycopeptide derivative was
purified on a C-18 reversed phase silica gel column followed
by RP-HPLC purification (>98% purity). The final purified
peptides were characterized by analytical HPLC, 1H NMR,
1
HRMS, and TLC. H NMR studies showed cis/trans iso-
merization at the Pro6 residue in some peptides. The ratios of
the cis/trans isomers and their assignments are available in
the Supporting Information. Compound 2 was synthesized
as previously described.20
Biological Activities. The initial biological evaluations
were performed on the bifunctional peptide 2, in which
Met5 of 1 was substituted by Nle, to confirm their bioiso-
sterism. As expected, almost all the bioactivities of 2
were within or close to the experimental error range of 1
(Tables 1-3). Although the only major difference was found
in the Emax values at δ-opioid receptors in the GTPγS
binding assays (60% and 121% for 1 and 2, respectively),
these two compounds had very similar bioactivities. Thus,
the biologically active conformation of 2 was expected to be
comparable to that of 1. As also expected, 2 degraded more
slowly in rat plasma than 1 did (Figure 2). Because of this
improvement in terms of metabolic stability, carbohydrate
residues were incorporated into the sequence of 2.
The substitution of the fifth position in the sequence of 2 by
Ser(Glc) (3) resulted in a large decrease in binding affinities at
both δ and μ opioid receptors with only 4.4-fold δ-selectivity
(Ki=59 and 260 nM, respectively; Table 1). This trend in the
opioid selectivity was maintained in the GTPγS binding assays
and also in the isolated tissue bioassays using the MVD and
GPI, suggesting the importance of the fifth position for both
μ and δ opioid agonist activities (Tables 2 and 3).19 Next, the
Ser(Glc) was substituted for Pro at the sixth position (4), which
was reported to be an important residue for affinity at the NK1
receptors41 but also influenced the opioid receptors as well.

5166 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Yamamoto et al.
Table 1. Binding Affinities of Bifunctional Peptide Derivatives at δ/μ Opioid Receptors and NK1 Receptors
hDOR,a [3H]DPDPEb rMOR,a [3H]DAMGOc
hNK1,d [3H]-substance Pe rNK1,d [3H]-substance Pf
g
g
g
g
compd
1h
log IC50
Ki (nM)
log IC50
Ki (nM) Ki(μ)/Ki(δ)
log IC50
Ki (nM)
log IC50
Ki (nM) Ki(hNK1)/Ki(rNK1)
-8.84 ( 0.07 0.66
-8.67 ( 0.05 1.0
-6.91 ( 0.09 59
-7.12 ( 0.09 36
-8.10 ( 0.05 3.7
-8.83 ( 0.06 0.77
2.6
-7.44 ( 0.05 16
-7.17 ( 0.07 32
-6.12 ( 0.15 260
-5.15 ( 0.20 3400
-7.77 ( 0.07 8.0
-7.22 ( 0.09 30
1.4
24
32
-10.91 ( 0.10 0.0065
-11.57 ( 0.59 0.0028
-12.21 ( 0.61 0.0011
-8.38 ( 0.07
-9.75 ( 0.30
-9.93 ( 0.05
-7.61 ( 0.03 7.3
-7.68 ( 0.03 6.8
-8.35 ( 0.02 1.5
-7.14 ( 0.03 23
-7.30 ( 0.03 14
-7.02 ( 0.05 34
1100
2400
5600
13
2
3
4.4
94
4
1.8
0.077
0.052
5
2.2
39
180
650
6
biphalini
DPDPEj
DPDPEk
L-732, 138
0.54
380
370
1.6
10
610
3700
-8.83 ( 0.02
0.73
-6.40 ( 0.03 130
180
a Competition analyses were carried out using membrane preparations from transfected HN9.10 cells that constitutively expressed the δ and μ opioid
receptors, respectively. b Kd =0.45 ( 0.1 nM. c Kd =0.50 ( 0.1 nM. d Competition analyses were carried out using membrane preparations from
transfected CHO cells that constitutively expressed rat or human NK1 receptors. e Kd=0.16 ( 0.03 nM. f Kd=0.40 ( 0.17 nM. g The log IC50
(
standard error are expressed as logarithmic values determined from the nonlinear regression analysis of data collected from at least two independent
experiments performed in duplicate. The Ki values are calculated using the Cheng and Prusoff equation to correct for the concentration of the
radioligand used in the assay. h Reference 20. i Reference 61. j References 20 and 62. k Reference 63.
Table 2. Opioid Agonist Functional Activities in [35S]GTPγS Binding Assays
hDORa
rMORa
b
b
compd
1e
log EC50
EC50 (nM)c
Emax (%)d
log EC50
EC50 (nM)c
Emax (%)d
-8.07 ( 0.11
-8.30 ( 0.09
-7.29 ( 0.12
-7.56 ( 0.14
-8.10 ( 0.08
-7.56 ( 0.27
-8.95 ( 0.17
-8.80 ( 0.25
8.6
5.0
52
60 ( 2
120 ( 3
49 ( 2
130 ( 3
64 ( 2
49 ( 3
83
-8.16 ( 0.17
-7.74 ( 0.14
-6.76 ( 0.31
-6.42 ( 0.19
-7.75 ( 0.23
-8.21 ( 0.24
7.0
18
51 ( 3
66 ( 3
24 ( 3
81 ( 6
38 ( 3
50 ( 3
2
3
180
380
18
4
51
5
7.9
28
6
6.2
biphalin
DPDPE
DAMGO
1.1
1.6
69
-7.44 ( 0.19
37
150
a Expressed from HN9.10 cell. b The log EC50 ( standard error are logarithmic values determined from the nonlinear regression analysis of data
collected from at least two independent experiments performed in duplicate. c Antilogarithmic value of the respective EC50. d [total bound - basal]/[basal -
nonspecific] ꢀ 100. e Reference 20.
Table 3. Functional Assay Results for Bifunctional Peptide Derivative
Ligands at Opioid and Substance P Receptors
substance P
antagonist
GPI
opioid agonist
MVD (δ)
GPI (μ)
compd IC50 (nM)a Emax (%)b IC50 (nM)a Emax (%)b Ke (nM)c
1d
2
15 ( 2.0
14 ( 1.6
110 ( 21 100 ( 0
18 ( 4.9
13 ( 5.8
17 ( 4.3
100 ( 0
100 ( 0
490 ( 29
460 ( 160 100 ( 0
1900 ( 470 100 ( 0
250 ( 48
520 ( 56
670 ( 134 93.7 ( 4.1 8.4 ( 1.0
92.5 ( 3.1 10 ( 2.1
10 ( 2.6
2.8 ( 0.73
18 ( 6.0
3
4
100 ( 0
100 ( 0
100 ( 0
100 ( 0
5
95.0 ( 2.9 1.8 ( 0.30
6
biphalin 2.7 ( 1.5
DPDPEe 4.1
DPDPEf 2.5
L-732,138
a Concentration at 50% inhibition of muscle contraction at electri-
cally stimulated isolated tissues (n=4). b The δ and μ opioid agonist
efficacies (Emax values) of tested compounds were calculated using
DPDPE and PL-017 as standards (Emax=100%) for MVD and GPI
assays, respectively. c Inhibitory activity against the substance P induced
muscle contraction in the presence of 1 μM naloxone. Ke: concentration
of antagonist needed to inhibit substance P to half its activity (n=4).
d Reference 20. e Reference 62. f Reference 63.
8.8 ( 0.3
7300
2720
Figure 2. Comparison of the in vitro metabolic stability for 1 (open
circle), 2 (open triangle), 5 (cross), and 6 (filled circle) incubated in
rat plasma at 37 °C. Calculated half-lives of peptide derivatives
(T1/2) were 4.8 h for 1 and >6 h for 2, 5, and 6. 70 ( 9% of 6 was
found intact after 24 h of incubation. The samples were tested in
three independent experiments (n=3), and the mean values were
used for the analysis with the SD. Statistical significance was
determined by Kruskal-Wallis test followed by Tukey’s test.
Asterisks denote significant differences (/, p<0.05; //, p<0.01;
///, p<0.001).
250 ( 87
The affinity of 4 at the δ opioid receptor was reduced (Ki =
36 nM) with very low affinity for the μ opioid receptor
(Ki=3400 nM). GTPγS binding assays also gave decreased
activities at both the δ and μ opioid receptors (EC50 = 51
and 380 nM, respectively) with high Emax values (134% and
81% for δ and μ opioid receptors, respectively). Interestingly,
and different from the binding assay results for the receptors,
the IC50 value of 4 in the MVD tissue assay showed almost
the same potency as 1 and 2, with better activity in the
GPI assay (18 and 250 nM for MVD and GPI assays,
respectively). This might be due to differences of efficacy in
the isolated tissue.

Article
Replacement with Ser(Glc) at the seventh position
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5167
pig NK1 receptor than to the rat or mouse NK1, and some
NK1 antagonists have large species differences consistent
with the reported homology.42,43 However, our results sug-
gest that the known species difference does not give a
sufficient explanation in the case of these bifunctional pep-
tides, independent of the presence or absence of a glycosy-
lated residue. These significant differences may suggest that
these peptides have affinities for guinea pig NK1 receptor
similar to those for the rNK1 receptor.
(compound 5) yielded a good result for radioligand binding
assays at both the δ and μ opioid receptors (Ki = 3.7 and
8.0 nM, respectively; Table 1) relative to those of 3 and 4.
The binding affinity of 5 at the μ opioid receptor was 3.8-fold
higher than that of 2, whereas its affinity at the δ receptor
was 3.7 times lower, leading to a ligand with only modest
δ-selectivity (2.2-fold). This selectivity was maintained in the
GTPγS binding assays, but its IC50 in the MVD tissue was
13 nM with 40-fold selectivity over that in the GPI assay
(Tables 2 and 3). As a result, the glycopeptide with Ser(Glc)7
(5) had the same levels of functional activities compared to
those of 2 in the isolated tissues and gave the best affinities
and activities among the three glycosylated octapeptides
3-5. It should be noted that the direct modifications of
4 and 5 were made within the NK1 antagonist pharma-
cophores which are located in the C-terminal half of these
peptides, and thus 2, 4, and 5 had the same sequence for the
opioid pharmacophore. However, the binding affinities of
4 and 5 at the opioid receptors, especially the affinity of 4 at
the μ opioid receptors, showed significant changes from
those of 2. This change may be due to a glycosylation induced
conformational change in the N-terminal halves where the
opioid pharmacophore was incorporated, or a direct steric
effect of the introduced sugar moiety could make some
contribution to the binding. Finally, the insertion of Ser(Glc)
between Leu7 and Trp8 in the sequence of 2 produced 6 which
showed increased opioid affinities at δ receptors, resulting in
the best affinity and δ-selectivity among the synthesized
glycopeptides 3-6 (Ki=0.77 and 30 nM for δ and μ opioid
receptors, respectively). The Ki values of 6 for δ and μ opioid
receptors were within or close to the error range of the
corresponding values of 2, suggesting that the insertion of
Ser(Glc)8 influenced the three-dimensional conformation of
the opioid pharmacophore less than for 3-5. Compound 6
showed 49% agonist efficacy at the δ opioid receptor com-
pared to the standard compound (DPDPE), and the ob-
served highly δ-selective binding affinity was consistent with
the results in the isolated tissue-based assays (IC50=17 and
670 nM in the MVD and GPI assays, respectively). Thus, 6
could be regarded as an effective opioid agonist with the
highest δ-selectivity among 1-6.
The Pro6 was reported to play an important role for hNK1
affinity,41,44 and thus, the replacement of Pro6 with Ser(Glc)
resulted in a ligand with 640 times less affinity at the hNK1
receptor compared to that of 2 (4; Ki=1.8 nM). According
to a reported modeling study,42 the Trp-derived NK1
antagonist L-732,138 (Ac-Trp-O-30,50-Bzl(CF3)2) binds at
the transmembrane domain of hNK1 receptor with a great
deal of tolerance for substitution of its acetyl group, which
was directed toward the extracellular region. Although a
large binding tolerance was expected, the Ser(Glc)6 incor-
poration in 4 might induce changes in the three-dimensional
structure. 4 also had the lowest Ke value in the GPI tissue
among the glycopeptides 3-6 (18 nM, Table 3). As for
the species difference between the hNK1 and rNK1 recep-
tors, all the synthesized glycopeptide derivatives 3-6 showed
higher affinities at the hNK1 than at the rNK1 receptor
(Table 1). It should be noted that 4 showed only a 13-fold
difference between species, whereas the other glycopeptide
derivatives (3, 5, and 6) showed at least 180 times better
affinities for the hNK1 receptors than for the rNK1, suggest-
ing the importance of Pro6 for the ligand at the hNK1
receptors. 4 also had the smallest species difference between
human and guinea pig NK1 receptors, implying that the
incorporation of Ser(Glc)6 or the removal of Pro6 has an
important effect on the species differences in binding at the
NK1 receptors.
Considering both the opioid agonist and NK1 antagonist
activities, glycosylation at the position next to the Trp
showed the best results (5 and 6) with good opioid affinities
and activities together with effective potency at the NK1
receptors. Thus, the metabolic stabilities of 5 and 6 were
evaluated to estimate the effect of glycosylation and to
compare with those of the peptides possessing no glycosy-
lated residue (1 and 2) by incubation in rat plasma at 37 °C
(Figure 2). The degradation curve of 5 was improved from
that of 1 but was almost equivalent to that of 2, implying that
glucose introduction at Ser7 had a negligible effect on
recognition by degrading enzymes (Figure 2). Interestingly,
the Ser(Glc)8 insertion (6) resulted in a large improvement:
70% of 6 was still found intact after 24 h incubation.
Apparently the Ser(Glc)8 plays an important role in the
enzymatic degradation due to the masking of the cleavage
site by the large carbohydrate portion. Another possibility is
that the observed improved metabolic stability of compound
6 can be attributed to the existence of Leu7 and Trp9 next to
the sterically hindered Ser(Glc); the sequence of Leu7-Ser-
(Glc)8-Trp9 in compound 6 has three bulky side chains that
should repel each other to form a rather fixed conformation
at the C-terminus. This effect may be due to the insertion of
large sugar portion in the appropriate position of the pep-
tide, and this conformational change should make some
contribution to metabolic stability as well. Because of this
improved metabolic stability together with the excellent and
δ-selective bifunctional activities, 6 could be considered as
the best derivative among 3-6.
The binding affinities (Ki values) at the hNK1 receptors of
5 and 6, whose residues next to Trp were glycosylated, were
good (77 and 52 pM, respectively; Table 1) but less than those
of 1 and 2. The affinities of 5 and 6 at the rNK1 receptor also
were less than those of 1 and 2. These biological shifts were
reasonable, since the sterically hindered glucose moiety
could interact spatially with the neighboring Trp, which is
a critical pharmacophore for binding to the NK1 receptors.41
However, the activities of 5 and 6 against substance P
stimulation in the GPI tissue assay were higher than those
of 1 and 2 (Ke=1.8 and 8.4 nM for 5 and 6, respectively),
while 3, which had the Ser(Glc) at the fifth position far from
Trp8, showed 10-fold higher affinity for the hNK1 receptor
(Ki=1.1 pM) than 2. The affinity of 3 at the rNK1 receptor
was 5600-fold lower than the Ki value at the hNK1 receptor
(Ki=1.5 nM), and its activity in the GPI was consistent with
the binding affinity at the rNK1 receptors. The general
explanation of such a difference in the NK1 antagonist
activities between different species has been provided pre-
viously by the known sequence differences between rat,
human, and guinea pig NK1 receptors: it is well-known that
the human NK1 receptor has higher homology to the guinea

5168 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Yamamoto et al.
Figure 3. Diagram of HN-HR coupling constants, NOE connectivities, and HR chemical shift index (CSI) for (A) 3, (B) 4, (C) 5, and (D) 6.
The HR CSI was calculated using the random-coil values reported by Andersen et al.64,65 The residue Bzl stands for the respective C-terminal
moieties.
1
Conformational Analysis Based on H NMR Studies in
Membrane-Mimicking Circumstances. As discussed pre-
viously,20 the membrane-bound conformation of peptides has
attracted interest recently, since the docking event of a ligand
with its receptor, such as a GPCR, with its binding site in the
transmembrane domain (such as opioid receptors and
NK1 receptors), must take place near the membrane.45,46
Hence, the transfer from solvent to cell membrane may be
considered as an important step in a receptor-ligand binding
event and may be accompanied by a conformational change to
a more biologically relevant form, although it may differ from
the binding conformation at the corresponding receptor.12,15
Thus, the NMR structural analyses of 3-6 were performed to
clarify the relationship between biological activity and confor-
mation in a membrane-mimicking environment as a function of
the site-specific glycosylation in their peptide sequence.
respectively) were used for the conformational analysis. The
3
interresidual NOE connectivities and the JHN-HR coupling
constants of all the peptide derivatives are illustrated in
Figure 3.20 Only 3JHN-HR values of more than 8 Hz or less than
6 Hz were used for j dihedral angle constraints,36,37 and thus,
the numbers of applied dihedral angle constraints were 0, 2, 1,
and 3 for 3, 4, 5, and 6, respectively. Therefore, the total
numbers of restraints were 101, 112, 93, and 243, corresponding
to 10.1, 11.2, 9.3, and 22.1 restraints per residue (the C-terminal
moiety and Glc were considered as residues). In the case of the
observation of cis/trans isomers at the Pro6 residue, only the
major isomer derived cross-peaks are considered in the struc-
tural calculation process, and all other amide bonds are fixed in
the trans configuration (the detailed data are found in the
Supporting Information).20
The 20 lowest-energy structures from restrained molecular
dynamics (rMD) calculations48,49 were used for analysis of
the glycopeptide derivatives 3-6 (Table 4). The superim-
posed images of the 20 structures are shown in Figure 4. The
ensembles of structures for the glycopeptide derivatives 3-6
have small numbers of violations for total NOE restraints,
maximum NOE distances, and j dihedral angles. The re-
straint energies also were reasonably small (0.79, 0.68, 0.87,
and 1.22 kcal mol-1 for 3, 4, 5, and 6, respectively). Since 2
showed similar biological activities as 1, the sequence with
Nle5 could be considered as equivalent to the one with Met5
in terms of the bioactive peptide conformation. Thus, the
conformation of 1 will be used as the standard for compar-
ison with the glycopeptide derivatives 3-6 throughout this
discussion.
Two-dimensional 1H NMR studies including TOCSY,
DQF-COSY, and ROESY (for 3, 4, and 5) or NOESY (for
6) in pH 4.5 buffer (45 mM CD3CO2Na/HCl, 1 mM NaN3,
90% H2O/10% D2O) with a 40-fold excess of perdeuterated
DPC micelles were performed on all four glycopeptide
derivatives 3-6. Nuclear Overhauser effects (NOEs) were
measured using either 2D NOESY (optimal mixing time
450 ms) or 2D ROESY (150 ms), depending upon which
method gave the larger number of cross-peaks. The NMR
structure of 1 in the presence of DPC micelles was previously
reported.20 DPC is a widely used lipid-like surfactant to
determine the solution NMR structures of membrane-bound
proteins and peptides20,36,37 and forms micelles above the
critical micelle concentration.47 All 1H chemical shift assign-
ments of 3-6 are listed in the Supporting Information.
Conformational Calculations. A large number of nonredun-
dant NOE restraints (101, 110, 92, and 240 for 3, 4, 5 and 6,
The calculated structures of 3, which has a Ser(Glc) in the
fifth position of the sequence in place of Met in 1, showed
larger rmsd values, especially for alignment on residues 1-4

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5169
˚
Table 4. Atomic rmsd Values (A) for the Final 19 Conformers Compared to the Most Stable Conformer of Bifunctional Peptide Derivatives
1a
3
4
5
6
Backbone Atoms (N, CR, C0)
calcd on whole molecule
calcd only on 1-4 residues
calcd only on 5-8 residues
1.14 ( 0.43
1.05 ( 0.63
0.45 ( 0.38
2.04 ( 0.40
1.83 ( 0.43
0.48 ( 0.39
2.92 ( 0.67
1.38 ( 0.26
0.94 ( 0.22
0.93 ( 0.27
0.60 ( 0.33
0.56 ( 0.32
0.68 ( 0.30
0.13 ( 0.11
0.47 ( 0.23b
All Non-Hydrogen Atoms
calcd on whole molecule
calcd only on 1-4 residues
calcd only on 5-8 residues and C-terminus
2.09 ( 0.64
2.16 ( 0.98
1.02 ( 0.25
3.35 ( 0.86
2.41 ( 0.62
0.99 ( 0.60
4.44 ( 1.04
2.92 ( 0.54
1.56 ( 0.51
1.68 ( 0.44
0.78 ( 0.63
0.88 ( 0.35
2.01 ( 0.79
0.31 ( 0.40
0.57 ( 0.32b
a Reference 20. b Calculated on five to nine residues and C-terminus.
Figure 4. Ensembles of the best 20 calculated structures in 40-fold DPC micelle/pH 4.5 buffer for 1,20 3, 4, 5, and 6, respectively, with the lowest
restraint energy, (a) aligned on backbone atoms of residues 5-8. The aligned structures are illustrated with the C-terminal benzyl moiety
(purple) and glucose (orange). (b) The most stable conformers are shown with all heavy atoms (C, N, and O).
Table 5. Number of β-Turn Structural Elements and the Distance between R Carbons of ith and (i þ 3)rd Residuesa
1b
3
4
5
6
no. of
β-turns
no. of
β-turns
no. of
β-turns
no. of
β-turns
no. of
β-turns
˚
distance (A)
˚
distance (A)
˚
distance (A)
˚
distance (A)
˚
distance (A)
residues
C1R-C4R
C2R-C5R
C3R-C6R
C4R-C7R
C5R-C8R
C6R-Bzl
3
7.86 ( 1.21
4.95 ( 0.71
1
8.57 ( 0.62
6.99 ( 1.08
7.39 ( 1.24
3
8.08 ( 1.13
7.00 ( 1.05
9
6.91 ( 0.41
0
20
0
11
8
6
0
20
0
4.98 ( 0.33
0
0
0
0
17
17
17
5.37 ( 0.74
5.46 ( 0.60
5.50 ( 0.36
20
0
3.98 ( 0.23
6.56 ( 0.56
0
0
19
20
11
5.79 ( 0.39
6.96 ( 0.57
5
20
7.05 ( 0.16
5.21 ( 1.02
6.32 ( 0.43
18
a Out of the best 20 calculated structures. The distance is the mean distance between two R carbons ( standard deviation (SD). The sequences with less
than 7 A distance between R carbons of ith and (i þ 3)rd residues without helical structure were considered as a β-turn.50 Bzl stands for the benzyl moiety
˚
at the C-terminus. b Reference 20.
(1.83 for backbone atoms and 2.41 for all heavy atoms;
Table 4), than 1. The rmsd values for the entire molecule also
were increased from those of 1 mostly because of the poorly
defined opioid pharmacophore at the N-terminus. Its rmsd
values with respect to residues 5-8 were small, however, and
nearly the same as those of 1. In the original definition, a
structure in which the CR of the ith residue and the CR in the
As a result, glycosylation at residue 5 induced conforma-
tional changes to give a relatively less structured N-terminus
(opioid pharmacophore) and an ordered C-terminus with
rather structured β-turn structures (NK1 pharmacophore) in
the pseudomembrane circumstances. Interestingly, 3 had
decreased affinities for the opioid pharmacophore, whereas
the NK1 binding affinities of 3 were improved from those of
1 and 2 (Table 1).
˚
(i þ 3)rd residue are located less than 7 A apart is considered
a β-turn.50 3 had two β-turns common to 1 at residues 2-5
When the site of glycosylation was shifted to residue 6 (4),
the number of β-turn elements in the N-terminal half got
smaller, whereas the distances between two CR atoms for
and 6-9 (Table 5). It is noted that the distance between the
5
8
˚
CR of Ser and the CR of Trp was also within 7 A, suggesting
a different alignment of the same turn at the C-terminus.
˚
residues 4-7, 5-8, and 6-9 were less than 7 A, indicating an

5170 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Table 6. Hydrogen Bond Observed in the NMR Structures of 3-6a
Yamamoto et al.
molecule no.b
donor
acceptor distance (A)
angle (deg)d
c
˚
1e
7
5
9
Gly3 HN Tyr1 O
Trp8 HN Met5 O
Bzl9 HN f Pro6 O
2.05 ( 0.11
2.04 ( 0.02
2.16 ( 0.11
137.8 ( 8.1
132.3 ( 1.1
158.5 ( 1.9
3
10
7
Phe4 HN DAla2 O 2.06 ( 0.09
144.1 ( 5.4
142.8 ( 7.2
132.1 ( 6.5
151.4 ( 7.6
127.3 ( 3.1
Gly3 HN Tyr1 O
Leu7 HN Ser5 O
Trp8 HN Ser5 O
2.03 ( 0.08
1.96 ( 0.05
2.19 ( 0.12
2.11 ( 0.02
16
13
5
Glc HO4
Glc O6
4
8
Nle5 HN Gly3 O
Bzl9 HN f Nle5 O
2.08 ( 0.16
1.97 ( 0.12
2.30 ( 0.12
2.33 ( 0.05
2.19 ( 0.01
2.12 ( 0.03
138.3 ( 10.7
168.6 ( 9.5
132.8 ( 4.2
150.6 ( 10.9
130.1 ( 0.3
129.8 ( 1.5
8
7
Bzl9 HN
Glc HO2
Glc H6
Ser6 O
Bzl9 F g
Glc O4
Glc O6
11
6
7
Glc HO4
5
6
6
6
7
7
Trp8 HN Pro6 O
2.15 ( 0.13
2.25 ( 0.13
1.96 ( 0.06
2.28 ( 0.07
144.8 ( 4.1
133.7 ( 6.3
155.1 ( 9.3
128.6 ( 4.5
Glc HO3
Glc H6
Gly3 O
Phe4 O
Glc O1
Glc HO3
13
20
5
Tyr1 HN Nle5 O
1.99 ( 0.04
137.4 ( 3.0
146.6 ( 2.3
129.3 ( 1.7
Phe4 HN DAla2 O 2.22 ( 0.11
Glc HO4
Glc O6
2.22 ( 0.06
a The hydrogen bonds that were observed in more than five structures
are listed. b The number of structures of the final 20 for which the listed
hydrogen bond is observed. c The distance is the mean proton-acceptor
atom distance ((SD) in the structures for which a hydrogen bond is
observed. d The angle is the mean angle ((SD) in the structures for
which a hydrogen bond is observed. e Reference 20. f Amide proton of
C-terminal benzyl moiety. g Fluorine atom of C-terminal benzyl moiety.
Figure 5. Glycosylated Ser (cross) and Gly3 (open circle) were in-
dicated in the Ramachandran j,ψ plots for (A) 3, (B) 4, and (C) 5 for
residues 2-7 and (D) 6 for residues 2-8 of 20 final structures. Ser(Glc)
(cross) and Gly3 (open diamond) were specified. Angular order
parameters54 for j (E) and ψ (F) angles are calculated from the
20 final structures for 1 (open circle),20 3 (filled triangle), 4 (open
diamond), 5 (cross), and 6 (filled circle), respectively. For calculating
the ψ angles of Trp8, the nitrogen atoms of C-terminal benzylamide
were used instead of N (i þ 3), respectively.
apparent turn structure around these regions (Table 5). Since
the distances between the two CR atoms for residues 4-7 and
˚
5-8 in 1 were larger than 7 A, these secondary structural
elements in 4 clearly were induced by the substitution of Pro6 by
Ser(Glc). Generally, Pro is known as a turn-inducing amino
acid; thus, it is interesting that the Ser(Glc), but not the Pro
residue, could yield such a β-turn-rich structure near the
substituted site, possibly due to the large steric effect of the
introduced sugar moiety. Although 4 had a large number of
secondary structural elements in the ensemble of NMR struc-
tures, its structural definition was further decreased from that
of 3 (the rmsd values for the whole molecule of 4 were 2.92 for
the backbone atoms and 4.44 for all heavy atoms; Table 4).
These results imply that Pro6 in 1 had an important contribu-
tion to its conformational stability. In fact, 4 had the least
structured conformation among 3-6 and showed the lowest
binding affinities at almost all the receptors tested (Table 1).
On the other hand, the introduction of Ser(Glc) at the
seventh position (5) led to a more defined structure whose
rmsd values (0.93 and 1.68 for backbone and all heavy
atoms, respectively) were smaller than those of 1, although
the number of NOE restraints for 5 was the smallest among
the tested peptides (Table 4). Interestingly, residues 1-4 had
a rather extended and better-defined conformation than in 1
but residues 5-8 did not, suggesting that the Ser(Glc)7 might
act as an address region for the opioid agonist pharma-
cophore. The rmsd values of 5 were smaller than for 3 and 4,
with β-turn structural elements for residues 4-7 (type I) and
6-9 (type IV) (Table 5).
δ-selectivity over the μ receptor. In this compound, the
structurally fixed β-turn at residues 2-5 was found in its
X-ray crystal structure,51 in the solution NMR structure,52
and in the docking study at the δ opioid receptor.53 On the
basis of these observations, we hypothesized that this β-turn
at residues 2-5 could be a key structure to enhance the
δ-selectivity of enkephalin analogues. In fact, 5, possessing
an extended conformation in the opioid pharmacophore, has
low δ-selectivity in terms of the binding affinities. Hydrogen
bonds between glucose and Gly3 or Phe4 were observed in 5,
implying a direct interaction between Ser(Glc)7 and the
N-terminal half that results in a fixed turn structure about
residues 4-7 (Tables 5 and 6). This interaction might play a
part in the induction of the ordered N-terminal half of 5
that was observed. Collectively, the single substitution of
Ser(Glc) at the 5, 6, or 7 residue of 2 led to diverse structural
differences in their NMR structures in the membrane-mi-
micking environment depending on the site of glycosylation,
but all of these glycopeptides 3-5 showed tandem-β-turn
structures in the NK1 pharmacophores where the sugar was
introduced, with more extended opioid pharmacophores.
Compared to the glycopeptide 5 with Ser(Glc)7, the deri-
vative possessing Ser(Glc)8 (6) showed a further decrease in
rmsd values, especially for backbone atoms (the rmsd value
DPDPE (Tyr-c[D-Pen-Gly-Phe-D-Pen]OH) is a derivative
of enkephalin and widely used opioid agonist with high

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5171
Figure 6. Paramagnetic effects on TOCSY spectra of 6: 6 with DPC micelles (top row) and with 200 μM Mn2þ (bottom). Preserved resonances
(labeled) are in a phase not missed by the phase-specific radical probe (Mn2þ). Spectra were compared from the same noise level. X10 represents
the cross-peaks derived from the corresponding aromatic protons of benzyl moiety.
was only 0.13 for residues 1-4; Table 4), mostly owing to its
structured N-terminus with a well-defined β-turn around
residues 2-5. The better-defined structure of the N-termi-
nus, as well as the smaller rmsd values, in the glycopeptide
derivative 6 compared to 1-5 was also confirmed by the j
and ψ angular order parameters shown in Figure 5E and
Figure 5F.54 It is worth mentioning that although the
carbohydrate residue was inserted in the C-terminus, it led
to a more structured N-terminus even though it is distant
from the glycosylated position. Although the rmsd values of
6 decreased from those of 1, the secondary structural ele-
ments of 6 were the same as those of 1; both of these peptides
had two β-turns, at residues 2-5 and at the C-terminus
(Table 5). Interestingly, 6 had a well-defined β-turn at
residues 2-5 and showed good biological affinities for δ
opioid receptors with the highest (39-fold) selectivity over the
μ receptor among the tested peptides. Indeed, the insertion
of a glycosylated moiety at the eighth residue gave rise to
δ-selective opioid agonist and effective NK1 antagonist
activities, presumably in relation to a structured conforma-
tion possessing two β-turns which were common to those in
1. It is noted that the glucose moiety in 5 and 6 should have a
similar effect of masking their backbones from proteolytic
enzymes, but only 6 showed improved metabolic stability
compared to 2. A possible explanation for this difference
is that the ordered peptide conformation induced by the
Ser(Glc)8 insertion could resist enzymatic cleavage of the
peptide backbone. In fact, compound 6 has three bulky
amino acid residues in a row, Leu7-Ser(Glc)8-Trp9, for which
a number of inter-residual NOE cross-peaks were found
between Ser(Glc)8 and Leu7 (8 NOE cross-peaks) or Trp9
(21 NOE cross-peaks), suggesting a sequence with a rather
fixed conformation at the C-terminus.
thus leading to the fixed C-terminal structures (Table 5).
Gly3 was the other residue with positive j angles; 14, 9, 10,
and 20 j angles for the best 20 structures were found positive
at Gly3 for 3, 4, 5, and 6, respectively (Figure 5A-D). It is
interesting that 4 and 5, with smaller numbers of β-turn
elements in the N-terminal halves, had fewer positive j
angles at Gly3, whereas 3 and 6, which had β-turns around
residues 2-5 in which Gly3 was at the (i þ 1) position,
possessed a larger number of Gly3 with positive j angles.
Thus, these positive j angles in the amino acid residues
might be a driving force to form secondary structural
elements in the backbone and could lead to a more ordered
turn conformation, especially in 6.
Paramagnetic Broadening Studies on 1H NMR. In order to
evaluate the mode of interaction of 6 with the model mem-
brane, further NMR experiments using paramagnetic ions
(Mn2þ) were conducted in the presence of DPC micelles. The
Mn2þ ions cause paramagnetic broadening and loss of
resonance intensity of solvent-exposed protons observed as
cross-peaks in the TOCSY spectra (Figure 6).20 As a result of
Mn2þ addition, all the resonances belonging to the backbone
NH protons in 6 were eliminated except for that of Nle5,
indicating that the backbone of 6 is located at the micelle
surface, with only Nle5 buried inside the micelles. Most of the
cross-peaks resulting from protons in side chains and in the
C-terminus were preserved. It is reasonable that mostly
lipophilic side chains and the C-terminus interact with the
lipophilic core of the micelles. These results for 6 were very
similar to the observations for 1 in the same lipid-like
enviornments.20 Interestingly, cross-peaks related to the
hydrophilic glucose were also preserved after Mn2þ addition,
suggesting that the carbohydrate moiety is located inside the
micelles. Thus, the introduction of a glycosylated residue at
the eighth position did not disturb the strong interaction of
the peptide with membrane-mimicking micelles as observed
in 1, but instead provided a more structured conformation.20
Since both binding sites of opioid and NK1 receptors were
found in or near their transmembrane domains,42,55 the
As observed in the Ramachandran plot, 5 possesses a Ser
residue with positive j and ψ angles in 14 out of the 20 best
structures including the most stable one (Figure 5C). These
positive j angles of Ser7 in 5 with a bulky glucose could help
drive its structure to be a β-turn near the glycosylated site,

5172 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Yamamoto et al.
ligand-membrane interaction should make an important
contribution to the docking of the ligand at these receptors.
mixture of DMF (5 mL) and TMOF (5 mL) with 4 N HCl in 1,
4-dioxane (0.6 mL, 2.40 mmol) was introduced. After the mixture
was shaken overnight, the solution was eluted off and a solution
of NaBH(OAc)3 (508 mg, 2.40 mmol) in 5% AcOH in DMF
(10 mL) was transferred into the reaction vehicle for 2 h. The resin
was washed three times with DMF (15 mL) and three times
with DCM (15 mL) and then with DMF (3ꢀ15 mL). A solution
of 30,50-bistrifluoromethylbenzylamine (650 mg, 2.67 mmol)
in DMF/TMOF=1: 1 (10 mL) with 4 N HCl in 1,4-dioxane
(0.6 mL, 2.40 mmol) was prepared again, then added to the
reaction vehicle for 2 h. The solution was filtered off, and the resin
was treated withNaBH(OAc)3 (508 mg, 2.40 mmol) in 5%AcOH
in DMF (10 mL). After the mixture was shaken for 1 h, the
resin was washed with DMF (3ꢀ15 mL), DCM (3ꢀ15 mL), and
DMF (3ꢀ15 mL). Fmoc-Trp(Boc)-OH (1.47 g, 2.79 mmol)
and HCTU (1.15 g, 2.79 mmol) were dissolved in 15 mL of
DMF, and then 2,6-lutidine (536 mg, 5.00 mmol) was added. The
coupling mixture was transferred into the syringe with the resin
and shaken overnight. The coupling was repeated for 2 h, and the
unreacted amino groups were capped using acetic anhydride
(2 mL) and pyridine (2 mL) in DCM (15 mL) for 30 min. Then
the resin was once again washed with DMF (3 ꢀ 15 mL), DCM
(3 ꢀ 15 mL), and DMF (3ꢀ15 mL). The NR-Fmoc protecting
groupwas removedby20% piperidine inDMF(20mL, 1 ꢀ 2 min
and 1 ꢀ 20 min). The deprotected resin was washed with DMF
(3 ꢀ 15mL), DCM (3 ꢀ 15 mL), and then DMF (3 ꢀ 15 mL). The
protected amino acid (3 equiv) and HCTU (2.9 equiv) were
dissolved in 15 mL of DMF. Then DIEA (6 equiv) was added.
Fmoc-Ser(O-β-D-Glc(OAc)4)-OH,40 Fmoc-Leu-OH, Fmoc-Pro-
OH, Fmoc-Nle-OH, Fmoc-Phe-OH, Fmoc-Gly-OH Fmoc-D-
Ala-OH, andBoc-Tyr(tBu)-OH were usedfor respective coupling
as protective amino acids. The coupling mixture was transferred
into the reaction vehicle and then shaken for 1.5 h. All the other
amino acids were sequentially coupled using the procedure
described above, using the TNBS test or chloranil test to check
the extent of coupling. In the case of a positive test result, the
coupling was repeated until a negative test result was obtained.
The resulting batch of resin-bound protected glycopeptide was
carefully washed with DMF (3 ꢀ 15 mL), DCM (3 ꢀ 15 mL),
DMF (3ꢀ15 mL), and DCM (3ꢀ 15 mL) and dried under
reduced pressure. After the glycopeptide was assembled on resin,
the acetyl protecting groups of glucose moiety ware removed with
Conclusion
In order to obtain a novel type of analgesic compound with
good metabolic stability, Ser(Glc) was introduced into the 5,
6, 7, and 8 positions in the sequence of 2. Among the
synthesized derivatives, 6, in which Ser(Glc) was inserted
between two bulky residues Leu and Trp in the sequence of
2, showed improved metabolic stability compared to 2 in rat
plasma. NMR structural analysis in the presence of DPC
micelles indicated that this insertion of the sugar moiety into 6
led to a well-defined conformation with two β-turns at the
residues2-5andthe C-terminus, bothofwhich werecommon
to the previously observed conformation of 1. In fact, the
glycopeptide derivative 6 showed strong interactions with the
model-membrane as previously observed for the nonglycosy-
lated derivative 1. The well-defined conformation and the
existence of a large sugar portion in the sequence of 6 might
play a part in its not being recognized by degrading enzymes.
6 also showed picomolar-level affinity for the hNK1 receptor
and partial but effective agonist activity at the opioid recep-
tors with improved δ-selectivity compared to that of 1.
Because of the improved metabolic stability, along with the
bifunctional activities, 6 could be considered as a valuable
research tool and possibly a promising candidate for a novel
analgesic drug. The importance of these studies is that the site
of glycosylation and the introduced steric repulsion with the
neighboring residues are likely critical factors for the con-
formational and secondary structural elements of the peptide,
both of which should have a significant contribution to
biological recognition at a variety of proteins, including
opioid receptors, NK1 receptors, and several peptide-degrad-
ing proteases.
Experimental Section
Materials. All amino acid derivatives, coupling reagents, and
resins were purchased from EMD Biosciences (Madison, WI),
Bachem (Torrance, CA), SynPep (Dublin, CA), and Chem
Impex International (Wood Dale, IL). The 4-(4-formyl-3-meth-
oxyphenoxy)butyrylaminomethyl (FMPB-AM) resin was ac-
quired from EMD Biosciences. Perdeuterated DPC was
purchased from C/D/N Isotopes (Quebec, Canada). ACS grade
organic solvents were purchased from VWR Scientific (West
Chester, PA), and other reagents were obtained from Sigma-
Aldrich (St. Louis, MO) and used as obtained. The polypropy-
lene reaction vessels (syringes with frits) were purchased from
Torviq (Niles, MI). Myo-[2-3H(N)]inositol, [tyrosyl-3,5-3H-
(N)]D-Ala2-Mephe4-glyol5-enkephalin (DAMGO), [tyrosyl-
80% H2NNH2 H2O in CH3OH (20 mL, 1 ꢀ 30 min and 1ꢀ1 h).
3
The resin was washed three times with EtOAc (15 mL) and three
times with DCM/CH3OH=1: 1 (15 mL), then with DCM (3 ꢀ
15 mL). The cleavage of glycopeptide from the solid support was
performed with the 3 mL of TFA/TIPS-OTf/ thioanisole (9:2:1,
v/v) for 45 min. The obtained crude glycopeptide was treated with
chilled dry ether (45 mL) to give a dark oil. After centrifuge, the
supernatant was decanted off, and then the crude glycopeptide
was precipitated out with chilled dry ether (45 mL). The obtained
yellow solid was dried under reduced pressure. The CH3CN
(5 mL) was added to the precipitated crude glycopeptide, and
the solution was treated with 2 N NH4F (2 mL) for 30 min. The
solution was adjusted to pH 8.0 with triethylamine and stirred for
30 min at 0 °C. Then H2O (20 mL) was added. CH3CN was
carefully titrated until the crude glycopeptide solution became
clear which was subsequently passed through a C-18 reversed-
phase silica gel column (Sep-Pak C18 cartridge, 5 g, Waters,
Milford, MA), and the column was carefully washed by H2O
(40 mL). The glycopeptide absorbed on the column were eluted
with CH3CN (40 mL). The obtained solution was evaporated,
and the residue was dissolved inH2O/CH3CN=1:1 (5 mL) for the
purification on Waters Delta Prep 4000 RP-HPLC system
equipped with Waters XTerra C-18 column (19 mm ꢀ 250 mm,
10 μm, a linear gradient of 33-53%, 33-53%, 35-52%, and
32-52% CH3CN/0.1% TFA H2O in 35 min for 3, 4, 5, or 6,
respectively, at a flow rate of 15.0 mL/min) followed by lyophiliz-
ing. The final pure glycopeptides (>98%) were characterized by
analytical HPLC, 1H NMR, HRMS, and TLC as previously
2,6-3H(N)](2-D-penicillamine,
5-D-penicillamine)enkephalin
(DPDPE), [3H]-substance P, and [35S]guanosine 50-(γ-thio)tri-
phosphate (GTPγS) were purchased from Perkin-Elmer
(Wellesley, MA). Bovine serum albumin (BSA), protease inhi-
bitors, Tris, and other buffer reagents were obtained from Sigma
(St. Louis, MO). Culture medium, penicillin/ streptomycin, and
fetal calf serum (FCS) were purchased from Invitrogen
(Carlsbad, CA).
Peptide Synthesis. The glycopeptides (3, 4, 5, and 6) were
synthesized by using reductive amination of 3,5-bistrifluoro-
methylbenzylamine on FMPB-AM resin followed by the NR-
Fmoc solid-phase methodology. The FMPB-AM resin (675 mg,
0.50 mmol/g) was placed into a 50mL polypropylene syringe with
the frit on the bottom and swollen in DMF (20 mL) for 1 h. The
resin was washed with DMF (3 ꢀ 15 mL), and then a solution of
30,50-bistrifluoromethylbenzylamine (650 mg, 2.67 mmol) in the

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5173
described (available in the Supporting Information).19-21 1H
NMR studies showed cis/trans isomerization about the X-Pro6.
The ratios of two amide rotamers and their assignments also are
available in the Supporting Information. The lyophilized product
was a white amorphous solid.
efficacies on the same cell membrane. The isolated tissue-based
functional assays also were used to evaluate opioid agonist activ-
ities in the GPI (δ) and MVD (μ). For the affinity at the human
NK1 (hNK1) receptors, binding assays utilized membranes from
transfected CHO cells that stably express hNK1 receptors, using
[3H]-substance P as the standard radioligand. The binding assay at
the rat NK1 (rNK1) receptors also were performed using trans-
fected CHO cells that stably express rNK1 receptors. To evaluate
antagonistic activities against substance P stimulation, isolated
tissue bioassays using GPI were performed in the presence of
naloxone to block any opioid activities.
In Vitro Stability of Peptide Derivatives in Rat Plasma56.
Stock solution of compounds (50 mg/mL in DMSO) were
diluted 1000-fold into rat plasma (lot 24927, Pel-Freez Biologi-
cals, Rogers, AK) to give an incubation concentration of 50 μg/mL.
All samples were incubated at 37 °C, and 200 μL of aliquots
were withdrawn at 1 min, 10 min, 30 min, 1 h, 2 h, 4 h, 6 h, and
24 h (only for 6). Then 300 μL of acetonitrile was added and the
proteins were removed by centrifugation. The supernatant was
analyzed for the amount of remaining parent compound by
HPLC (Hewlett-Packard 1090m with Vydac 218TP104 C-18
Acknowledgment. The work was supported by grants from
the USDHS, National Institute on Drug Abuse (Grants
DA-13449 and DA-06284). We thank Dr. Guangxin Lin for
kind assistance and advice with the NMR measurements,
Professor Robin Polt for discussions on glycosylated peptides,
Magdalena Kaczmarska for culturing cells, the University of
Arizona Mass Spectrometry Facility for the mass spectra
measurements, and Hajime Koganei for helpful scientific
discussions. We express appreciation to Margie Colie and
Brigid Blazek for assistance with the manuscript.
˚
column; 4.6 mm ꢀ250 mm, 10 μm, 300 A). The samples were
tested in three independent experiments (n=3), and the mean
values were used for the analysis with the SD.
NMR Spectroscopy in DPC Amphipathic Media and Confor-
mational Structure Determination. All the conformational deter-
minations were performed by the same methods as previously
described,20,36,37 based on the NMR spectra using a Bruker
DRX600 600 MHz spectrometer.
Briefly, the samples were prepared by dissolving the peptide
(4-6 mM) in 0.5 mL of 45 mM sodium acetate-d3 buffer (pH 4.5)
containing 40 equiv of dodecylphosphocholine-d38 and 1 mM
sodium azide (90% H2O/10% D2O) followed by sonication for
5 min. Two-dimensional double quantum filtered correlation
spectroscopy (DQF-COSY), nuclear Overhauser enhancement
spectroscopy57 (NOESY, mixing time=450 ms), rotating frame
Overhauser effect spectroscopy (ROESY, mixing time=150 ms),
and total correlation spectroscopy58 (TOCSY, MLEV-17 mixing
time=62.2 ms, spin-lock field=8.33 kHz) were acquired using
Supporting Information Available: HR-MS, TLC, HPLC,
1
and H NMR data of the peptides 2-6; metabolic stability of
peptides 3 and 4 in rat plasma. This material is available free of
charge via the Internet at http://pubs.acs.org.
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