On the terminal homologation of physiologically active peptides as a means of increasing stability in human serum - Neurotensin, opiorphin, B27-KK10 epitope, NPY

Article abstract of DOI:10.1002/cbdv.201100093

The terminal homologation by CH₂ insertion into the peptides mentioned in the title is described. This involves replacement of the N-terminal amino acid residue by a β²- and of the C-terminal amino acid residue by a β³-homo-amino acid moiety (β²hXaa and β³hXaa, resp.; Fig.1). In this way, the structure of the peptide chain from the N-terminal to the C-terminal stereogenic center is identical, and the modified peptide is protected against cleavage by exopeptidases (Figs.2 and 3). Neurotensin (NT; 1) and its C-terminal fragment NT(8-13) are ligands of the G-protein-coupled receptors (GPCR) NT1, NT2, NT3, and NT analogs are promising tools to be used in cancer diagnostics and therapy. The affinities of homologated NT analogs, 2b-2e, for NT1 and NT2 receptors were determined by using cell homogenates and tumor tissues (Table.1); in the latter experiments, the affinities for the NT1 receptor are more or less the same as those of NT (0.5-1.3 vs. 0.6nM). At the same time, one of the homologated NT analogs, 2c, survives in human plasma for 7 days at 37° (Fig.6). An NMR analysis of NT(8-13) (Tables.2 and 4, and Fig.8) reveals that this N-terminal NT fragment folds to a turn in CD₃OH. - In the case of the human analgesic opiorphin (3a), a pentapeptide, and of the HIV-derived B27-KK10 (4a), a decapeptide, terminal homologation (→3b and 4b, resp.) led to a 7- and 70-fold half-life increase in plasma (Fig.9). With N-terminally homologated NPY, 5c, we were not able to determine serum stability; the peptide consisting of 36 amino acid residues is subject to cleavage by endopetidases. Three of the homologated compounds, 2b, 2c, and 5c, were shown to be agonists (Fig.7 and 11). A comparison of terminal homologation with other stability-increasing terminal modifications of peptides is performed (Fig.5), and possible applications of the neurotensin analogs, described herein, are discussed. Copyright

Full text of DOI:10.1002/cbdv.201100093

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
711
On the Terminal Homologation of Physiologically Active Peptides as a Means
of Increasing Stability in Human Serum – Neurotensin, Opiorphin, B27-KK10
Epitope, NPY
by Dieter Seebach*, Aneta Lukaszuk1), Krystyna Patora-Komisarska1),
Dominika Podwysocka1), James Gardiner2), and Marc-Olivier Ebert
Laboratorium fꢀr Organische Chemie, Departement fꢀr Chemie und Angewandte Biowissenschaften,
ETH-Zꢀrich, Hçnggerberg, Wolfgang-Pauli-Strasse 10, CH-8093 Zꢀrich
(phone: þ41-44-632-2990; fax: þ41-44-632-1144; e-mail: seebach@org.chem.ethz.ch)
and
Jean Claude Reubi*, Renzo Cescato, and Beatrice Waser
Division of Cell Biology and Experimental Cancer Research, Institute of Pathology, University of Berne,
Murtenstrasse 31, P. O. Box 62, CH-3010 Bern (phone: þ41316323242; fax: þ41316328999;
e-mail: reubi@pathology.unibe.ch)
and
Peter Gmeiner* and Harald Hꢀbner
Department of Chemistry and Pharmacy, Emil Fischer Center, Friedrich Alexander University,
Schuhstraße 19, D-91052 Erlangen (phone: þ4991318529383; fax: þ4991318522585;
e-mail: peter.gmeiner@medchem.uni-erlangen.de)
and
Catherine Rougeot*
´
Institut Pasteur – Unite de Biochimie Structurale et Cellulaire/URA2185 – CNRS, 25 rue du Docteur
Roux, F-75724 Paris (phone: þ33140613445; fax: þ33145688399;
e-mail: catherine.rougeot@pasteur.fr)
Dedicated to Professor Gilbert Stork on the occasion of his 90th birthday
The terminal homologation by CH2 insertion into the peptides mentioned in the title is described.
This involves replacement of the N-terminal amino acid residue by a b2- and of the C-terminal amino acid
residue by a b3-homo-amino acid moiety (b2hXaa and b3hXaa, resp.; Fig. 1). In this way, the structure of
the peptide chain from the N-terminal to the C-terminal stereogenic center is identical, and the modified
peptide is protected against cleavage by exopeptidases (Figs. 2 and 3). Neurotensin (NT; 1) and its C-
terminal fragment NT(8–13) are ligands of the G-protein-coupled receptors (GPCR) NT1, NT2, NT3,
and NT analogs are promising tools to be used in cancer diagnostics and therapy. The affinities of
homologated NTanalogs, 2b–2e, for NT1 and NT2 receptors were determined by using cell homogenates
and tumor tissues (Table 1); in the latter experiments, the affinities for the NT1 receptor are more or less
1
)
Postdoctoral fellows, financed by Swiss National Science Foundation (200020-117586, 200020-
126693) and by Novartis Pharma AG, Basel.
Postdoctoral Fellow, financed by the New Zealand Foundation for Research Science and Technology
(No. SWISS0401).
2
)
ꢁ 2011 Verlag Helvetica Chimica Acta AG, Zꢀrich

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
the same as those of NT (0.5–1.3 vs. 0.6 nm). At the same time, one of the homologated NT analogs, 2c,
survives in human plasma for 7 days at 378 (Fig. 6). An NMR analysis of NT(8–13) (Tables 2 and 4, and
Fig. 8) reveals that this N-terminal NT fragment folds to a turn in CD3OH. – In the case of the human
analgesic opiorphin (3a), a pentapeptide, and of the HIV-derived B27-KK10 (4a), a decapeptide,
terminal homologation (! 3b and 4b, resp.) led to a 7- and 70-fold half-life increase in plasma (Fig. 9).
With N-terminally homologated NPY, 5c, we were not able to determine serum stability; the peptide
consisting of 36 amino acid residues is subject to cleavage by endopetidases. Three of the homologated
compounds, 2b, 2c, and 5c, were shown to be agonists (Fig. 7 and 11). A comparison of terminal
homologation with other stability-increasing terminal modifications of peptides is performed (Fig. 5),
and possible applications of the neurotensin analogs, described herein, are discussed.
1. Introduction. – There is a myriad of physiologically active peptides, which
function as neurotransmitters, neuromodulators, hormones, ligands on MHC proteins
and on proteins involved in cell adhesion, antibiotics, etc. A review article published in
2005 [1] is entitled: ꢂOver One Hundred Peptide-Activated G Protein-Coupled
Receptors Recognize Ligands with Turn Structuresꢃ, with these ligands all being
peptides! In spite of this paramount importance of peptides in the chemistry of living
organisms, they have traditionally been considered not suitable as drugs: they are
metabolically unstable, they have poor gastrointestinal and bloodꢀbrain barrier
penetration, and they are rapidly excreted through liver, kidney, and/or feces.
Therefore, non-peptidic compounds, peptidomimetics, have been sought for decades
that serve as agonists (ꢂsubstratesꢃ) or antagonists (ꢂinhibitorsꢃ) of enzymes. This area
has been reviewed in numerous articles and monographs3)4) of which we can cite only
a few herein [2][4–12].
2. b-Peptidic Peptidomimetics. – In 1996, we have entered [13–17] a new area of
peptide chemistry by inserting a CH2 group in each and every amino acid (Fig. 1,a) of
peptides to produce b-peptides with proteinogenic side chains (Fig. 1). Like their
natural counterparts, b-peptides exist as strands, sheets, turns, or helices, but the
secondary structures are observed with as few as four-residue chain lengths. The results
are described in comprehensive review articles covering various aspects of the
chemistry, structure, and biological properties of b-peptides [18–34]. Perhaps the most
notable property of the b-peptides with proteinogenic side chains is their stability
against peptide-cleaving [13][15][35–40] and peptide-metabolizing [39–41] enzymes,
14
the ꢂacid testꢃ having been the injection of C-labelled b-peptides into rats5), with
essentially no detectable degradation or metabolism in seven days. Only certain
environmental microorganisms have been found to contain enzymes, which are able to
cleave b-peptides; the four b-peptidases discovered, so far, belong to the Ntn family of
hydrolases, and their natural substrates are unknown [43–51]6). Not only peptides
3
)
)
)
)
The most comprehensive one is the HoubenꢀWeyl volume E22c [2].
4
5
6
See also the special issue ꢀPeptidomimeticsꢁ in Acc. Chem. Res. [3].
Mass-spectroscopic imaging with mice and unlabelled peptides confirmed these results [42].
An extensive crystal-structure investigation of b-aminopeptidases and their complexes with b-
lactams, including point mutations, will be published shortly (collaboration with T. Heck, B.
Geueke, H.-P. E. Kohler, A. Reimer (EAWAG, Dꢀbendorf), R. Rentsch (EMPA, Dꢀbendorf), and
T. Merz, M. G. Grꢀtter (Universitꢄt Zꢀrich)).

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
713
consisting entirely of b-amino acid residues but also mixed b/a-peptides (Fig. 1,b) are
enzymatically stable [38][41][52], even if they contain a short a-peptide segment
embedded in an otherwise b-peptidic sequence [38]7)8). This proteolytic stability
becomes almost obvious, when the cleavage mechanism of proteases is considered
(Fig. 2).
Fig. 1. Molecular formulae of a- and b-amino acids (a) and of a b-peptide containing an internal a-
peptidic segment (b). The R groups are the proteinogenic side chains in our work [18–26], but can, of
course, also be other groups, including carbocyclic (R1ꢀR2 ¼ꢀ(CH2)nꢀ) or heterocyclic derivatives (see
[19][22][27–33]. The ꢂstarredꢃ C-atoms are stereogenic centers (with R1=R2 in b2,2 and b3,3) and can
have (R)- or (S)-configuration. For the preferred configurations for peptide mimicking, see Fig. 3.
An N- or C-terminal b-amino acid residue will not fit into the binding pocket of an
exopeptidase, and the strand conformation of a b-peptide cannot possibly bind to the
active sites of endopeptidases9)!
The structural differences and the resulting differences of folded conformations
(turns, helices) of a- and b-peptides prevent mimicking a-peptidic structures and
physiological properties by peptides consisting entirely of b-amino acid residues, except
in special cases10). The question arose, whether partial replacement of a-amino acid
residues by backbone-expanded w-amino acid residues in proteins and in peptides is
possible without loss of structure and/or functions. This was, indeed, realized with
proteins, in which b-amino acid residues were incorporated in a loop (ꢂrandom-coilꢃ)
7
)
b2-Oligoazapeptides (from 2-hydrazino carboxylic acids) were also shown to be stable against
peptidolytic cleavage [53].
An N-linked glycosylated b-peptide resists cleavage by glucoamidase A [54].
ꢂProteases Universally Recognize Beta Strands In Their Active Sitesꢃ [6].
See a review article entitled ꢂb-Peptidic Peptidomimeticsꢃ [25].
8
9
)
)
)
10

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
Fig. 2. Schematic presentations of a-peptidic N- and C-terminal amino acid residues (a), and of an a-
peptidic chain (b) in the binding pockets of exo- and endopeptidases, respectively. For the importance of
strand structures in proteolysis, see Footnote 9. The cleavage sites are indicated by red dotted lines. The
strand structures (b and c) of a- and b-peptides are fundamentally different: not only is there an
additional CH2 group in each residue (larger distance between the side chains R and between the amide
moieties), but the chain is polar (all C¼O bonds ꢂupꢃ, all NꢀH bonds ꢂdownꢃ) and non-symmetrically
substituted (all side chains on the same face of the backbone) in b-peptides [22][24] (b-peptides
consisting of b3-amino acid residues of alternating l and d absolute configuration have strand structures
with side chains pointing alternatively in opposite directions: hitherto unpublished results obtained in
our laboratory).
section (of a hemoglobin cleavage site [55])11), in a turn sub-structure (of a
ribonuclease A [56])12), or in an entire amphipathic helix (of interleukin hIL-8
[57])13). With short peptides, replacement of a- by w-, i.e., b-, g-, or d-amino acid
residues, is likely to destroy or significantly perturb the backbone conformation
11
)
The proteolytic cleavage was totally prevented by certain replacements of a- by b-amino acid
residues.
ꢂProtein Prosthesisꢃ.
A ꢂchimeric proteinꢃ.
12
13
)
)

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
715
(strand, turn, helix), except when conducted in a carefully designed manner14
)
15), or in
cases where anchoring by polar side chains at certain distances is decisive for binding
(cf. to major histocompatibility complex, MHC, proteins [65–75]). Several groups have
used what is called a b-amino acid scan, in which each amino acid residue of a
physiologically active peptide was individually replaced by the corresponding b-amino
acid moiety [69–78]. Depending on the particular peptide, this type of a ꢂshotgun
approachꢃ may lead to analogs, which are still active and, at the same time, generally
more stable towards proteolytic cleavage.
3. Terminal Homologation of Four Peptides. – It is not surprizing that replacement
of both terminal a- by b-amino acid residues, i.e., the terminal homologation of a
peptide, can produce peptidomimetics without loss of activity and with highly enhanced
proteolytic stability (Fig. 3): i) the structures of the backbones of the peptide and its
analog are superimposable from the stereogenic center bearing the N-terminal side
chain all the way to the C-atom bearing the C-terminal side chain, and ii) the termini
Fig. 3. Replacement of individual amino acid residues of an oligopeptide A by b3hXaa building blocks to
produce an ꢀoligoꢁ number of analogs B, bis-homologation to a peptidomimetic C, and molecular formulae
(Fischer projection) of the corresponding amino acids. The ꢂboxedꢃ sections of the peptides A and C are
identical. The systematic CIP designation of the sense of chirality (R/S) is not useful in discussions of this
type, since homochiral (definition of Lord Calvin) amino acid residues may have opposite designators,
depending on the structure of their side chains R (cf. Footnote 20).
14
)
)
For a comprehensive review of the work in this area by Balaram and co-workers, see [58]. For
computational contributions, see, e.g., [59–62].
For examples of secondary and tertiary structures formed by peptides featuring repeating patterns
of a- and b-amino acid residues (ꢂheterogeneous backbonesꢃ), see [29][63][64]. Helix secondary
structures of this type of mixed a/b-peptides are better peptidomimetics than those of all-b-peptides.
15

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
Fig. 4. Terminal homologation of Angiotensin IV. Angiotensin IV, a metabolite of angiotensin II in the
reninꢀangiotensin system, retains completely its inhibition in a biological test and is proteolytically
stable after terminal homologation [76][77].
are protected against cleavage by exopeptidases (cf. Fig 2,a). A dramatic demon-
stration with the terminal homologation of Angiotensin IV was provided by Lukaszuk
et al. [76][77] (Fig. 4). Some other methods of terminal peptide modifications for
increased proteolytic stability are compiled in Fig. 5.
For the success of mimicking structure and function of peptides A by terminally
homologated analogs C (Fig. 3), the following aspects have to be considered. i) Since
the structure of the backbone between the terminally inserted CH2 groups is identical,
the conformation of the chain is expected not to change dramatically. ii) There is no
protection against endopeptidases, i.e., the longer the peptide chain is, the larger its
ꢂnaturalꢃ proteolytic vulnerability will be, irrespective of the protection against
exopeptidases. iii) The degree to which the homologation will influence the affinity,
i.e., the binding to a receptor, will depend upon the interaction contribution of the
terminal region(s) of the peptide where the distances and geometrical relationships
between the charged groups NHþ3 and CO2ꢀ, and their respective neighboring H-bond
acceptor (C¼O) and H-bond donor (NꢀH) have been changed.
We have synthesized several homologated analogs of physiologically active
peptides, determined their half-lives in human plasma, and compared some of their

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
717
Fig. 5. Some common methods of terminal peptide modification with protection against the action of
exopepidases. a) N-Terminal methylation and amide groups at C-terminus; b) terminal incorporation of
d-amino acid residues; c) ꢂpeptoidationꢃ of the termini; d) reduction of the C-terminal C¼O group to a
primary alcohol group; e) acylation of the N-terminus; f) pyroGlu, a natural N-terminal protecting group
against aminopeptidases (see neurotensin, Sect. 3,a, below); g) N-terminus with reduced terminal amino
acid, prepared by reductive amination of the corresponding amino aldehyde with the peptide lacking the
terminal amino acid). For specific examples, see, e.g., [2][4][9][78–80] and refs. cit. therein.
activities with those of the natural counterparts and of previously reported b-peptidic
peptidomimetics.
We chose the following peptides for our investigation of terminal homologation
effects: neurotensin (1) and neurotensin(8–13) (2a), opiorphin (3a), the HIV-derived
peptide (epitope) B27-KK10 (4a), and neuropeptide Y (NPY; 5a), which are
commercially available. The only one of these, which we synthesized, is opiorphin.
The C-terminal homologation of the selected peptides was achieved by incorporation
of a b3-amino acid; most16) b3-amino acids Fmoc-b3hXaa(PG)-OH are now commer-
cially available. The Fmoc- or Boc-b2-homo-amino acids for the N-terminal modifi-
cation were, on the other hand, prepared as previously reported by us17).
16
)
)
Except for Xaa¼Cys, Sec, His.
For a review describing the various methods of preparing enantiomerically pure b2-homo-amino
acid derivatives, see [26]. Some b2-homo-amino acids are now also commercially available, e.g.,
b2hVal, b2hSer, b2hPro, and b2hPhe.
17

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
3.1. Neurotensin. – a) Physiological Activities. Neurotensin (NT; 1) [81] is a
tridecapeptide (ligand), which binds with its C-terminal sequence 8–13 (i.e., 2a)18) to
the G-protein-coupled receptor (GPCR) family NT1, NT2, and NT3 [83]. Neurotensin
(1) and its receptors are involved in cancer growth. Surface plasmon resonance (SRP)
analysis, and investigations with resonance energy transfer (RET) and NMR
18
)
According to structureꢀfunction investigations, the C-terminal hexapeptide fragment NT(8–13)
(2a) is sufficient to elicit binding to and activation of the neurotensin receptor [82].

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
719
techniques have recently shed light upon the mechanism of binding between the
neurotransmitter NT (1) and its receptor NT1, found in mammalian gastrointestinal,
cardiovascular, and central nervous systems [84–86]. NT Analogs have been designed
for imaging tumors in animals and humans, and NT antagonists have been shown to
inhibit growth of engrafted tumors. An authoritative review article covering the
literature up to 2005 [87] and a condensed overview19) are recommended for details.
b) Homologated Derivatives of 2a. Neurotensin(8–13) analogs, 2d and 2e, which
contain only one terminal b-amino acid residue have been described in [78]. We have
now prepared the doubly homologated analogs 2b and 2c, bearing (R)- or (S)-b2hArg
on the N-terminus and (S)-b3hLeu on the C-terminus. The stability of the analog 2c,
together with that of the native NT (1) and NT(8–13) (2a), was investigated in human
plasma. Human plasma contains an extensive array of proteins [88], including many
enzymes, especially proteases [89]. Plasma endopeptidases digest proteins at specific
residues (e.g., Arg or Lys by thrombin), releasing fragments, which can be further
digested by exopeptidases, cutting either or both C- and N-terminal residues [90–92].
All our degradation assays were performed at 378, the samples were withdrawn at
different times, whereupon the serum proteins were precipitated by addition of EtOH.
The supernatant was analyzed using RP-HPLC. The half-lives were estimated based on
the peak-area counts. The native peptides NT (1) and NT(8–13) (2a) have half-lives of
4.32ꢁ2.26 h and 17.26ꢁ1.2 min, respectively (Fig. 6). In contrast, the homologated
analog 2c remains intact for over 7 days (Fig. 6).
The affinity of the two terminally homologated analogs 2b and 2c for the human
NT1 receptor was investigated with membrane homogenates from CHO cells stably
19
)
See Sect. 4.37 in review [1] on ligand/GPCR recognition.

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
Fig. 6. Stability of peptides 1, 2a, and 2c in human plasma. The stability was determined at 378. The
samples were withdrawn at different times and analyzed by RP-HPLC. Each point is the average of three
measurements.
expressing the human NT1 and the radioligand [3H]NT. NT2 Receptor binding was
determined using preparations from HEK 293 cells transiently expressing the human
NT2 and [3H]NT(8–13) [93]. All results were compared with the reference NT (1) and
hexapeptide 2a [78]. The ability to displace [3H]NT and [3H]NT(8–13) from the
receptor was lower for both doubly homologated compounds compared to the native
NT(8–13) (2a; Table 1). Moreover, the affinity depends upon the configuration of the
N-terminal residue: the Ki value of the (R)-analog 2b is twice as that of the (S)-analog
2c. This is the expected result, considering the fact that l- or (R)-b2hArg is homochiral
with l- or (S)-Arg, while (S)-b2hArg is heterochiral (cf. Fig. 3)20).
The analogs 2b, 2c, 2d, and 2e were, in addition, tested for their NT1 receptor-
binding profile using NT1 receptor-positive human tumor tissues (colon adenocarci-
nomas and hemangiopericytoma) [95][96] and compared with the binding affinity of
neurotensin (1). The binding affinities of the analogs are listed in Table 1. The (R)-
analog (2b) is two times more potent than the (S)-diastereoisomer 2c, and the affinity
of 2b is comparable to that of 1. Moreover, we tested the two terminally homologated
compounds 2b and 2c functionally in a calcium mobilization assay using HT-29 cells
20
)
As in Fig. 3, we use Lord Kelvinꢃs definition of homo- and heterochiral similarity. For an excellent
discussion ꢀTracing the Origins and Evolution of Chirality and Handedness in Chemical Languageꢁ,
see [94].

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
721
Table 1. Receptor (human NT1 and NT2) Affinity Profiles of Neurotensin and Its Analogs
No. Compound
Ki(hNT1)a) [nm] Ki(hNT2)b) [nm] IC50(hNT1)c)
(cell homogenates)
(tumor tissues)
2d H-b3hArg-Arg-Pro-Tyr-Ile-Leu-OH [78]
2e H-Arg-Arg-Pro-Tyr-Ile-b3hLeu-OH [78]
0.23ꢁ0.047d)
0.61ꢁ0.13d)
0.53ꢁ0.26
0.55ꢁ0.10
0.73ꢁ0.04
1.26ꢁ0.28
n.d.
8.4ꢁ1.8d)
25ꢁ0.0e)
12ꢁ2.8e)
28ꢁ7.1e)
1.2ꢁ0.17d)
4.9ꢁ0.37d)
2b H-(R)-b2hArg-Arg-Pro-Tyr-Ile-b3hLeu-OH 6.1ꢁ2.0e)
2c H-(S)-b2hArg-Arg-Pro-Tyr-Ile-b3hLeu-OH 11ꢁ0.0e)
2a Neurotensin(8–13)
0.24ꢁ0.024d)
1
Neurotensin
0.59ꢁ0.16d)
0.15
a) hNT1-Stably transfected in CHO cells and the radioligand [3H]NT. b) hNT2-Transiently transfected in
HEK293 cells and the radioligand [3H]NT(8–13). c) Human tumor tissues (colon adenocarcinoma and
hemangiopericytoma); IC50 in nm (meanꢁSEM); n¼3, these values refer to the NT1 receptor. d) Ki
Values derived from 3–16 experiments are given as meanꢁSEM. e) Ki Values derived from two
experiments are given as meanꢁSD.
endogenously expressing the NT1 receptor in order to determine their agonistic
behavior. The dose response curves are shown in Fig. 7. Both analogs exhibit an
agonistic behavior in this type of assay.
Surprisingly, the singly homologated compounds 2d and 2e, with N-terminal (S)-
b3hArg and C-terminal (S)-b3hLeu, respectively [78], have affinities similar to those of
the doubly homologated compounds 2b and 2c in the experiments conducted with
tumor tissues. On the other hand, in the membrane homogenates the N-terminal
b3hArg in 2d causes no decrease of binding affinity, whereas all the other modifications,
2b, 2c, and 2e, actually do (Table 1).
Fig. 7. Functional assay for NT analogs. Intracellular calcium mobilization induced in HT-29 cells by
various neurotensin subtype 1 receptor analogs. HT-29 Cells were loaded with the Fluo-4 dye as
described in the Exper. Part. Cells were then analyzed for calcium mobilization in response of increasing
~
&
*
concentrations between 0.1 nm and 1 mm of either 2a ( ), 2b ( ), or 2c ( ). Maximum calcium
mobilization response was obtained by treating the cells with ionomycin. Results are shown as
percentage of maximum calcium mobilization response induced by ionomycin.

722
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
c) NMR Investigations of NT(8–13) (2a) and of the Homologated Derivative 2c.
Neurotensin fragment 2a and the homologated compound 2c were investigated by
NMR spectroscopy: 1H- and 13C-resonance assignments and 3J(HN,H(a)) (or
3J(HN,H(b2)) in the case of Leu13 in 2c) coupling constants are listed in Tables 2 and
3, respectively. An overlay of the HSQC spectra of both compounds together with a
Table 2. 1H- and 13C-NMR Chemical Shifts (d in ppm rel. to TMS) and 3J(HN,H(a)) Coupling Constants
[Hz] of 2a in CD3OH at 258
AA
AAa) HN
H(a) H(b)
H(g)
1.72, 1.66
1.75
1.99
H(d)
3.21
3.20
3.82, 3.63
7.04
H(e)
7.60
7.57
–
6.69
–
–
3J(HN,H(a))
Arg8 R1
Arg9 R2
Pro10 P3
Tyr11 Y4
Ile12
Leu13 L6
–
8.75
–
4.03
4.59
4.41
4.55
4.25
4.40
1.92
1.85, 1.76
2.15, 1.95
3.05, 2.87
1.82
6.4
8.01
7.83
8.31
–
7.6
8.6
7.9
I5
1.52, 1.14, 0.95b)
1.70
0.88
0.96, 0.92c)
1.64
CO
C(a) C(b)
C(g)
C(d)
C(e)
C(z)
Arg8 R1
Arg9 R2
Pro10 P3
Tyr11 Y4
170.4 53.9
172.4 52.9
174.1 61.7
173.4 56.4
173.6 59.1
175.9 52.4
29.8
29.1
30.5
37.8
38.7
41.7
25.0
25.9
26.0
128.9
25.9, 15.9d)
26.1
42.0
42.2
48.8
–
–
–
159.1
159.1
–
131.5
11.4
116.4 157.5
–
–
Ile12
Leu13 L6
I5
–
–
23.5, 22.1e)
a) Amino acid designation in Fig. 8. b) H(g1a), H(g1b), H(g2). No stererospecific assignment at C(g1).
c) H(d1), H(d2) (no stereospecific assignment). d) C(g1), C(g2). e) C(d1), C(d2) (no stereospecific
assignment).
Table 3. 1H- and 13C-NMR Chemical Shifts (d in ppm rel. to TMS) and 3J(HN,H(a)) Coupling Constants
[Hz] of 2c in CD3OH at 258
AA
HN
–
H(a) H(b)
H(b2)
H(g)
1.61, 1.56
1.74
2.01
H(d)
3.18
3.20
3.81, 3.63
7.04
H(e) 3J(HN,H(a))
b2hArg8
Arg9
2.80 1.69, 1.55 3.09, 3.04
7.55
8.69 4.53 1.84, 1.75
–
8.12 4.48 3.06, 2.89
7.73 4.16 1.76
–
–
–
–
7.46
–
5.9
Pro10
4.42 2.16, 1.91
Tyr11
–
6.69
–
–
7.6
Ile12
1.49, 1.12, 0.89a)
1.60
0.88
8.7
b3Leu13
7.95 2.44 1.47, 1.31 4.32
CO C(a) C(b)
C(b2)d)
b2hArg8 175.2 44.7 28.0
0.91, 0.90b)
8.9c)
C(g)
C(d)
C(e) C(z)
43.0
–
–
–
–
27.5
26.0
26.1
129.1
25.9, 15.9e)
26.1
42.3
42.3
48.8
131.3
11.4
23.7, 22.5f)
–
–
–
159.1
159.1
–
Arg9
Pro10
Tyr11
Ile12
173.3 53.7 28.5
174.2 61.8 30.6
173.2 56.7 37.5
172.7 59.4 38.6
116.4 157.5
–
–
–
–
b3Leu13 175.1 40.9 44.7
46.3
a) H(g1a), H(g1b), H(g2); no stererospecific assignment at C(g1). b) H(d1), H(d2) (no stereospecific
f
assignment). e) C(g1), C(g2). ) C(d1), C(d2) (no stereospecific assignment). d) C(b2) is the backbone
c
atom in b-position to the C¼O group. ) 3J(HN,H(b2)).

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
723
detail of their 1H-NMR spectra displaying the region of the amide protons are shown in
Fig. 8. A second set of signals in the spectra of both compounds is attributed to the cis-
isomer of Pro10. The cis/trans ratio is 1:5 for 2a and 1:4 for 2c (the trans-isomer is
identified based on the strong ROESY cross-peak between Arg9 H(a) and Pro10 H(d)).
Table 4 lists distances determined for 2a and 2c that are typically used to estimate the
secondary-structure content of peptides [97].
Table 4. Selected HꢀH Distances [ꢅ] Derived from Cross-Peak Intensities in the ROESY Spectra of 2a
and 2c in CD3OH
i, j
2a
2c
d(H(a)i,HNj)
d(HNi,HNj)
d(H(a)i,HNj)
d(HNi,HNj)
1, 2
2, 3
3, 4
4, 5
5, 6
3, 5
2.4
2.2
2.1
2.2
2.1
3.9
2.2
2.3
2.2
3.3
3.7
3.3
1
Nieto et al. have reported the H-NMR resonance assignments for neurotensin(1–
13) together with the assignment of the amide H-atoms in the neurotensin fragments
1
(1–8) and (8–13) (aqueous solution, 178) [98]. Xu and Deber have reported the H-
resonance assignments for neurotensin(8–13) (2a) in aqueous solution, CD3OH, and a
micelle environment [99]. To the best of our knowledge, 13C-resonance assignments of
neurotensin or neurotensin fragments have not been reported previously.
In aqueous solution, Nieto et al. observed a cis/trans ratio for Pro10 of 1:4 at pH 7.8,
while Xu and Deber observed a ratio of 1:5 at pH 4. These values are in good
agreement with our measurements in CD3OH. Interestingly, however, Xu and Deber
could not detect any cis-isomer in the full-length peptide in CD3OH.
According to the previously cited authors [98][99], neurotensin exists as a random
coil both in H2O and CD3OH. More specifically, from the absence of backbone
NHi,NHiþ1 cross-peaks Xu and Deber concluded that neither a-helix nor b-turn is
present. In contrast to this, in the ROESY spectrum of 2a the two potentially
observable NHi,NHiþ1 cross-peaks were present. They correspond to a distance of 3.3
and 3.7 ꢅ, respectively. Additionally, strong H(a)iNHiþ1 cross-peaks between all
sequential pairs of residues (except Lys9 and Pro10) were observed. Extended regions of
both of the mentioned connectivities point to conformational averaging between the ar
and the b region of f,y-space [97]. For 2a, however, we also observed a weak but
clearly visible cross-peak between H(a)10 and HN12. Such a cross-peak together with
the one between HN11 and HN12 is indicative for a b-turn [97]. The additional strong
cross-peak between H(a)10 and HN11 points to a type II b-turn [100]. In such a
conformation, we would expect values for 3J(HN,H(a)) in the order of 5 Hz at position
3 of the turn [101]. Instead, the coupling constant of Tyr11, which lies at that position, is
7.6 Hz. This is close to the value of 8–10 Hz usually found in b-strands [97]. One has to
keep in mind that, if several conformers are present in solution, the diagnostic value of
coupling constants is low. Since the distance between H(a) at position i and HN at

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
725
position iþ2 of an unfolded or extended peptide chain is >6 ꢅ, the observation of a
NOE between such H-atoms is a valid clue that a relatively high population of turn
conformations is present [102].
This finding is interesting in the light of a standing controversy about the
conformation of neurotensin in its bound state. A molecular-modeling study by Pang
et al. [103] points to a type I turn conformation of the segment 8–12, while the solid-
state NMR structure of neurotensin in the receptor shows an extended conformation
[104]. The latter is in agreement with a model of Barroso et al. [105] in which the N-
terminal region of neurotensin also adopts a linear conformation. The occurrence of an
extended binding conformation is intriguing, because it contradicts the general
assumption that GPCRs recognize turn conformations [1].
We must emphasize that the present NMR studies have been carried out in CD3OH,
which tends to stabilize turn conformations21). Also, the cross-peak between H(a)10
and HN12 is not present in the case of 2c.
3.2. Human Opiorphin (QRFSR; 3a) was discovered by one of our groups five years
ago [107]; it is a physiological inhibitor of two enkephalin-inactivating zinc
ectopeptidases, one being an exopeptidase (human aminopeptidase-N (hAP-N)), the
other an endopeptidase (human neutral endopeptidase (hNEP)). Opiorphin (3a) has
1
21
)
A H-NMR investigation of the full-length neurotensin (1) in a membrane-mimetic environment
led to the conclusion that the C-terminus folds to a b-turn [106].

726
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
potent analgesic activity and is as effective as morphine in various pain models. In mice
and rats, this pentapeptide was found to be non-addictive and to have no drug-tolerance
effects, so that therapeutic applications are considered, possibly after ꢂchemical
optimizationꢃ [108][109].
We homologated opiorphin (3a) on its termini (! 3b), which reduced the
degradation rate in human plasma, prolonging the half-life of the analog ca. sevenfold:
t1/2 1.44ꢁ2.42 h for 3b, compared to 12.15ꢁ2.46 min for 3a (Fig. 9). The inhibitory
potency towards the two membrane-anchored ectoenzymes, NEP (specific endopepti-
dase and carboxypeptidase activities) and AP-N, was tested, by using selective
fluorescence-based enzyme assays. The inhibition potency of the terminally homo-
logated opiorphin, 3b, was reduced by a factor of 10 (Fig. 10 and Table 5). This might
implicate that, even though the positions of the side chains remain intact, the relocation
of the terminal carboxy and/or amino groups has an impact on binding to the
enkephalin-inactivating Zn ectopeptidases, i.e., the interaction contribution(s) of the
termini are more important than in the case of neurotensin.
Fig. 9. Stability of peptides 3a and 3b in human plasma at 378. For details, see caption of Fig. 6 and Exper.
Part.
Table 5. Inhibition of Human hAP-N and hNEP Activities by the Terminally Homologated Opiorphin,
3b. The values for opiorphin (3a) are taken from [107].
No.
Compound
hAP-N
hNEP-endoP
hNEP-CDP
IC50 ꢁSD [mm]
IC50 ꢁSD [mm]
IC50 ꢁSD [mm]
3a
3b
H-Gln-Arg-Phe-Ser-Arg-OH
H-b2hGln-Arg-Phe-Ser-b3hArg-OH
8.1ꢁ0.1
29ꢁ1
33ꢁ6
75ꢁ7
>100
>100
(382ꢁ266 by
(281ꢁ162 by
extrapolation)
extrapolation)
3.3. B27-KK10 (4a) is an HIV-derived decapeptide (KRWIILGLNK), which binds
to the class I major histocompatibility complex (MHC) HLA-B27 and triggers T-cell
responses [110]22). The binding affinity is mainly caused by the anchoring-group side
chains of Arg2 and Lys10, and is hardly affected by replacements of the intermittent
structural moiety, as shown in the case of other HLA-B27-binding peptides (cf.
22
)
In biology and biochemistry, such peptides are also called antigens, antigenic determinants, or
epitopes (see textbooks).

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
727
Fig. 10. Inhibitory activity of peptides 3a (left) and 3b (right) on human NEP and AP-N ectopeptidases
using 96-well fluorimetry-based enzyme assays
autoimmune-disease-related ones [65–68][111]). On the other hand, the response of
the immune system depends upon the particular amino acid sequence or other
fragments between the two anchoring groups.
The incorporation of the terminal b-amino acid residues led to peptide 4b which
was still binding to HLA-B27 but had a significantly improved stability in human
plasma: the homologated analog 4b resisted degradation ca. 70-times longer than the
native B27-KK10 (4a) (3.55ꢁ0.14 h vs. 3.14ꢁ1.28 min)23).
3.4. Human Neuropeptide Y (hNPY, 5a) is a 36-amino acid peptide neuro-
transmitter and has been associated with a number of physiological processes in the
brain, including the regulation of energy balance, memory and learning, and
epilepsy24). Like neurotensin, NPY operates on GPCRs.
23
)
)
Details will be published elsewhere.
24
For a review with numerous references, see Neuropeptide Y – Wikipedia (Discovery, The role in
food intake, in obesity, in anorexia nervosa, and correlation with stress and diet). For the binding
structures of hNPY and the related peptide hPPY (5b) to the receptor, see Sect. 4.36 in [1].

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
The synthesized analog 5c of hNPY was homologated only on the N-terminus. The
determination of the half-life of hNPY (5a) and its analog 5c in human plasma turned
out to be very difficult, if not impossible. We were challenged by the separation of 5a
and 5c from products of degradation and/or metabolism in the RP-HPLC analysis.
Thus, we can only make the statement that there did not seem to be much difference
between the hNPY and the N-terminally homologated hNPY 5c in this test, confirming
the expectation that terminal homologation of a longer peptide may not turn out to be
useful; such a peptide still offers many sites of attack by endopeptidases and
metabolizing enzymes of the serum.

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
729
The analog 5c was tested in receptor autoradiography experiments for NPY Y1 and
Y2 receptor-binding affinities on SK-N-MC [112][113] cells endogenously expressing
the Y1 subtype, and SH-SY5Y [114] cells endogenously expressing the Y2 subtype,
respectively; the affinities were then compared with those of the reference compound
hPYY (5b), a close relative of NPY [1]. The mean IC50 valuesꢁSEM of three
independent pharmacological displacement experiments are listed in Table 6 showing
that the analog 5c has affinities for the receptors Y1 and Y2 that are comparable to those
of hPYY (5b). Moreover, we tested analog 5c functionally in an adenylate cyclase-
activity assay using SK-N-MC cells endogenously expressing the NPY Y1 receptor in
order to determine its agonistic behavior. The doseꢀresponse curves are shown in
Fig. 11. The analog 5c exhibits an agonistic behavior in this type of assay.
Fig. 11. Functional assay for NPY analogs. Forskolin-stimulated intracellular cAMP accumulation
induced in SK-N-MC cells by (Leu31,Pro34)-PYY (LP-PYY) or 5c. The cAMP assay was performed as
described in the Exper. Part. SK-N-MC Cells were treated with increasing concentrations between
&
*
0.01 nm and 1 mm of the reference compound LP-PYY ( ) or 5c ( ). Results are shown as percentage of
forskolin-stimulated cAMP accumulation.
Table 6. NPY Y1 and Y2 Receptor Affinity Profiles of hPYY (5b) and of the NPY Analog 5c. The IC50
values are given as a mean of three experimentsꢁSEM.
No.
NPY(Y1) (SK-N-MC cells)
NPY(Y) (SH-SY5Y cells)
IC50 ꢁSEM [nm]
IC50 ꢁSEM [nm]
5b
5c
0.97ꢁ0.22 (3)
0.23ꢁ0.07 (3)
2.4ꢁ1.2 (3)
0.57ꢁ0.32 (3)
4. Discussion, Conclusions, and Outlook. – There are several surprising aspects of
the results described herein. i) After angiotensin IV, neurotensin (NT) is a second
example of terminal peptide homologation with a high degree of retention of function;
furthermore the NT(8–13) analog 2c shows a unique, essentially complete stability in
human serum. ii) In contrast to previous NMR investigations, our NMR analysis of

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
NT(8–13) (2a) led to the conclusion that it folds to a turn secondary structure in
MeOH solution and thus might not be an exception to the rule that GPCR proteins
generally recognize ligands with turn structures [1]19). iii) The comparative NMR
analysis of NT(8–13) (2a) and its analog 2c (with the ꢂwrongꢃ N-terminal config-
uration!) showed that this homologation has led to a more flexible backbone – and this
was accompanied by a decreased affinity of 2c in all three tests (Table 1). iv) In the case
of opiorphin (3a), terminal homologation to 3b led to a modest sevenfold increase of
stability in serum, combined with an up to tenfold decrease of inhibition potency. v)
While the central part of the hexapeptide NT(8–13) (2a) was obviously not touched by
cleaving or metabolizing enzymes of serum, this was not the case with the pentapeptide
opiorphin (3a) and the decapeptide B27-KK10 (4a), the terminally homologated
analogs of which slowly disappeared from a serum solution. vi) No reliable data could
be obtained with the N-terminally homologated NPY, the native form of which is
known to loose the two N-terminal amino acid residues, with formation of a 34-amino
acid peptide of modified physiological activity [115].
In conclusion, the terminal homologation of peptides may turn out to be an
alternative to other terminal peptide modifications (Fig. 5) for improving stability even
in vivo. We are in the process of seeking collaborations with biomedical research groups
to study metal-complexing DOTA derivatives [116] of terminally homologated
peptides (for possible diagnostic and therapeutic applications) and to conduct
experiments with transgenic animals (for immune suppression and activation).
Experimental Part
General. Abbreviations. Abz: 2-Aminobenzoyl, Boc: (tert-butoxy)carbonyl, DMAP: 4-(dimethyl-
amino)pyridine, DnpOH: 2,4-dinitrophenyl, EDDnp: (2,4-dinitrophenyl)ethylenediamine, Fmoc: [(9H-
fluoren-9-yl)methoxy]carbonyl, FRET: Fçrster resonance-energy transfer, h.v.: 0.1–0.01 Torr. HATU:
O-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate, HBTU: O-(benzotria-
zol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate, HOBt: 1-hydroxy-1H-benzotriazole,
MeIm: methylimidazole, MSNT: 1-(mesitylene-2-sulfonyl)-3-nitro-1,2,4-triazole, NMP: N-methylpyrro-
lidine, PBS: phosphate buffered saline, TFA: trifluoroacetic acid, TIPS: (i-Pr)3SiH, TNBS: 2,4,6-
trinitrobenzenesulfonic acid, Mca: N-(7-methoxycoumarin-4-yl)-acetyl.
Optical rotation: Perkin-Elmer 241 polarimeter (d¼10 cm, 1-ml cell). High-resolution (HR) MS:
IonSpecUltima 4.7-T-FT Ion Cyclotron Resonance (ICR; HR-MALDI, in 2.5-dihydroxybenzoic acid
matrix) spectrometer. For further details, see the specific procedures below.
1. Peptide Synthesis. Amino acids were purchased from Novabiochem or Bachem. Fmoc-
b3hXaa(PG)-OH were obtained from Fluka. Fmoc-b2-homo-amino acids: (R)- and (S)-Fmoc-
b2hArg(Boc)2-OH [117]25), (R)-Fmoc-b2hLys(Boc)-OH [118], (R)-Fmoc-b2hGln(Trt)-OH [119], (R)-
Fmoc-b2hTyr(t-Bu)-OH [120] were prepared as described previously. Rink amide resin and 2-chlorotrityl
chloride resin were purchased from Novabiochem. The natural peptides NT (1) and NT(8–13) (2a) were
obtained from Bachem, all other reagents were purchased from Fluka or Sigma.
1.1. Manual Solid-Phase Peptide Synthesis (SPPS). All peptides were synthesized in an appropriate
glass reactor or syringe by Fmoc methodology [121].
Loading of the 2-Chlorotrityl Chloride Resin (GP 1). The amino acid (3 equiv. rel. to the resin) and 6
equiv. of EtN(i-Pr)2 were dissolved in CH2Cl2 (10 ml per 1 g of the resin), containing, if necessary, a small
amount of DMF to facilitate dissolution of the amino acid. The 2-chlorotrityl chloride resin was pre-
swollen in CH2Cl2 for 1 h, and, after that, the soln. containing the amino acid was added, and the resin was
We thank Novartis for the sample of (S)-Fmoc-b2hArg(Boc)2-OH.
25
)

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
731
shaken for 30–120 min. Then, the resin was washed 3ꢂ with CH2Cl2/MeOH/EtN(i-Pr)2 17:2 :1), then
2ꢂDMF and 3ꢂCH2Cl2. Deprotection of the first amino acid was prolonged (2ꢂ30 min).
Loading of Wang Resin (GP 2). To a soln. of the Fmoc-protected b-amino acid (3 equiv.), in dry
CH2Cl2 (5 ml) under Ar, were added MeIm (2.25 equiv.) and MSNT (3 equiv.). The mixture was stirred
for 2 min and then added to the pre-swollen resin (CH2Cl2 for 1 h), and the suspension was mixed by Ar
bubbling for 4 h. The resin was then filtered, washed with CH2Cl2 (4 ml, 6ꢂ1 min), and dried under h.v.
overnight.
Standard Coupling Procedure (GP 3). Three equiv. of amino acid and 6 equiv. of EtN(i-Pr)2 were
mixed and added to the resin, followed by addition of 3 equiv. of HATU. The soln. was added to the resin
and shaken for 1–2 h or overnight. The completion of the reaction was monitored by TNBS test [122].
Capping of Free Amino Groups (GP 4). The peptideꢀresin was covered with DMF (20 ml/g resin),
and unreacted amino groups were capped using Ac2O (10 equiv.) and DMAP (0.1 equiv.) dissolved in
DMF (0.1 ml/mmol Ac2O) for 1–2 h under Ar bubbling. The resin was then washed with DMF (20 ml/g
resin, 5ꢂ1 min) and CH2Cl2 (20 ml/g resin, 5ꢂ1 min).
Deprotection (GP 5). The Fmoc protecting group was removed by a soln. of 20% piperidine in DMF
(2ꢂ5 min).
Cleavage from the Resin (GP 6). The peptides were cleaved from the resin using TFA/Et3SiH/H2O
9.5 :2.5 :2.5 (ca. 2 ml/0.1 g of resin). Cleavage was performed during 2 h, then the resin was filtered off,
and the filtrate was evaporated. To the oily residue, Et2O was added to precipitate the peptide. The
resulting solid was filtered off and washed with Et2O. The crude product was purified by prep. RP-HPLC
and the collected fractions were lyophilized. The final products were obtained as a TFA salts.
Prep. Reversed-Phase (RP)-HPLC. Prep. HPLC was performed on a Merck/Hitachi HPLC system
(LaChrome; pump type, L-7150; UV detector, L-7400; interface, D-7000; HPLC manager, D-7000),
using a Supelco Discovery BIO Wide Poreꢂ C18 column (25 cmꢂ21.2 mm, 10 mm, Sigma-Aldrich) or
Nucleosil 100-5 C18 column (25 cmꢂ21 mm; Macherey-Nagel). HPLC was performed with a gradient of
0.1% TFA in H2O (A), and 0.08% TFA in MeCN (B), at a flow rate of 10 ml/min with the UV detection
at 220 nm.
Anal. RP-HPLC. Performed on a Merck/Hitachi HPLC system (LaChrome; pump type, L-6200; UV
detector, L-4000; interface, D-6000; HPLC manager, D-7000) using a Supelco Discovery BIOWide Poreꢂ
C18 column (25 cmꢂ4.6 mm, 5 mm, Sigma). HPLC was performed with a gradient of 0.1% TFA in H2O
(A) and 0.08% TFA in MeCN (B), at a flow rate of 1 ml/min with the UV detection at 220 nm.
NMR Spectra. Spectra of 10 mg of 2a and 20 mg of 2c in ca. 700 ml of CD3OH were recorded on
Bruker Avance II and Avance III 600 MHz spectrometers at 258. Resonance assignments were
accomplished using DQF-COSY, TOCSY, HSQC and HMBC spectra. Inter-nuclear distances were
calculated based on the cross-peak intensities in ROESY spectra with a mixing time of 300 ms (cross-
peak volumes between geminal H-atoms at Pro10 C(d), Tyr11 C(b), and Ile12 C(g) corresponding to a
distance of ca. 1.8 ꢅ were used for calibration). All spectra were recorded with presaturation of the
solvent signal.
H-(R)-b2hArg-Arg-Pro-Tyr-Ile-b3hLeu-OH (2b). 2-Chlorotrityl chloride resin (75 mg, 1.1 mmol/g,
0.075 mmol) was esterified with Fmoc-b3-hLeu-OH (56 mg, 0.15 mmol) according to GP 1, followed by
Fmoc deprotection (GP 5). Subsequently the peptide chain was elongated, respectively, with Fmoc-Ile-
OH (59 mg, 0.30 mmol), Fmoc-Tyr(tBu)-OH (70 mg, 0.15 mmol), Fmoc-Pro-OH (51 mg, 0.15 mmol),
Fmoc-Arg(Pbf)-OH (98 mg, 0.15 mmol), and finally Fmoc-(R)-b2hArg(Boc)2-OH (55 mg, 0.09 mmol).
After the last deprotection (GP 5), the peptide was cleaved from the resin (GP 6) to give 62 mg of crude
peptide. The peptide was purified by prep. RP-HPLC (5–50% B in 50 min, Nucleosil 100-5 C18 column)
to give 4 mg of 2b. White solid. Anal. RP-HPLC (0–34% B in 20 min, Nucleosil 100-5 C18 column, tR
11.81). LC/MS: 845.8 ([MþH]þ , C40H68N12O8þ ; calc. 844.52776).
H-(S)-b2hArg-Arg-Pro-Tyr-Ile-b3hLeu-OH (2c). 2-Chlorotrityl chloride resin (150 mg, 1.1 mmol/g,
0.15 mmol) was esterified with Fmoc-b3-hLeu-OH (111 mg, 0.30 mmol) according to GP 1, followed by
Fmoc deprotection (GP 5). Subsequently the peptide chain was elongated, respectively, with Fmoc-Ile-
OH (117 mg, 0.30 mmol), Fmoc-Tyr(tBu)-OH (139 mg, 0.30 mmol), Fmoc-Pro-OH (102 mg,
0.30 mmol), Fmoc-Arg(Pbf)-OH (196 mg, 0.30 mmol), and finally Fmoc-(S)-b2hArg(Boc)2-OH

732
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
(111 mg, 0.18 mmol). After the last deprotection (GP 5), the peptide was cleaved from the resin (GP 6)
to give 190 mg of crude peptide. The peptide was purified by prep. RP-HPLC (5–50% B in 50 min,
Nucleosil 100-5 C18 column) to give 45.8 mg of 2c. White solid. Anal. RP-HPLC (0–34% B in 20 min,
Supelco Discovery BIO Wide Poreꢂ C18 column, tR 18.78). LC/MS: 845.8 ([MþH]þ , C40H68N12Oþ8 ; calc.
844.52776).
H-Gln-Arg-Phe-Ser-Arg-OH (¼Opiorphin; 3a) [123]. 2-Chlorotrityl chloride resin (500 mg,
1.1 mmol/g, 0.5 mmol) was esterified with Fmoc-Arg(Pbf)-OH (655 mg, 1 mmol) according to GP 1,
followed by Fmoc deprotection (GP 5). Subsequently the peptide chain was elongated, respectively, with
Fmoc-Ser(tBu)-OH (387 mg, 1 mmol), Fmoc-Phe-OH (319 mg, 1 mmol), Fmoc-Arg(Pbf)-OH (655 mg,
1 mmol), and Fmoc-Gln(Trt)-OH (617 mg, 1 mmol). After the last deprotection (GP 5), the peptide was
cleaved from the resin (GP 6) to give 192 mg of crude peptide. The peptide was purified by prep. RP-
HPLC (0–34% B in 20 min, Supelco Discovery BIO Wide Poreꢂ C18 column) to give 23.4 mg of 3a. White
solid. Anal. RP-HPLC (0–34% B in 20 min, Supelco Discovery BIOWide Poreꢂ C18 column, tR 9.40). LC/
MS: 693.2 ([MþH]þ , C29H48N12O8þ ; calc. 692.3718).
H-b2hGln-Arg-Phe-Ser-b3hArg-OH (3b). Wang resin (100 mg, 1.1 mmol/g, 0.11 mmol) was esteri-
fied with Fmoc-b3hArg(Pmc)-OH (224 mg, 0.33 mmol) according to GP 2. After capping (GP 4) and
Fmoc-deprotection (GP 5), the peptideꢀresin was coupled, respectively, with Fmoc-Ser(tBu)-OH
(102 mg, 264 mmol), Fmoc-Phe-OH (102 mg, 264 mmol), Fmoc-Arg(Pbf)-OH (172 mg, 264 mmol), and
Fmoc-(R)-b2hGln(Trt)-OH26) (130 mg, 208 mmol) according to GP 3. After final Fmoc deprotection
(GP 5), the peptide was cleaved from the resin (GP 6) to give 74 mg of crude peptide. A portion (50 mg)
was purified by RP-HPLC (3–50% B in 30 min, Nucleosil 100-5 C18 column) to give 15.1 mg of 3b. White
solid. Anal. RP-HPLC (0–34% B in 20 min, Supelco Discovery BIO Wide Poreꢂ C18 column, tR 11.01).
MALDI-MS: 743.3 (12, [MþNa]þ ), 722.3 (40) 721.3 (100, [MþH]þ , C31H53N12Oþ8 ; calc. 721.4109).
1.2. Automated Solid-Phase Peptide Synthesis (GP 7). The chain assembly was performed with an
Applied Biosystems ABI433A coupled to a Perkin-Elmer UV/VIS detector, according to the standard
SPPS protocol using the FastMocꢂ chemistry. For each coupling reaction, 4 equiv. of Fmoc-protected
amino acid and 4 equiv. of HBTU/HOBt (0.45m in DMF) and EtN(i-Pr)2 (2m in NMP) were used.
Deprotection was performed in a soln. of 20% piperidine in NMP, monitored by UV (repeated if
needed).
H-(R)-b2h-Tyr-Pro-Ser-Lys-Pro-Asp-Asn-Pro-Gly-Glu-Asp-Ala-Pro-Ala-Glu-Asp-Met-Ala-Arg-
Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-NH2 (5c). Rink amide resin
(450 mg, 0.55 mmol/g, 0.25 mmol) was used for the peptide synthesis. The peptide synthesis was
performed as described in GP 7. Additionally, two pseudoprolines were used for Ile31-Thr32, Fmoc-Ile-
Thr(Psi(Me,Me)pro)-OH, and Tyr21-Ser22, Fmoc-Tyr(tBu)-Ser(Psi(Me,Me)pro)-OH. After completion
of the reaction, and removing the Fmoc group on N-terminus of Pro, the resin was washed with CH2Cl2
and DMF. Final coupling of Boc-(R)-b2hTyr(tBu)-OH was carried out manually (0.1 g of resin,
0.05 mmol) with 2 equiv. of the Boc-protected amino acid (35.1 mg, 0.1 mmol) activated with 1.9 equiv. of
HATU (36.2 mg, 0.095 mmol) and 6 equiv. of EtN(i-Pr)2 (51.3 ml, 0.3 mmol) in 5 ml of dry DMF. The
coupling reactions were carried out for 4 h under vigorous agitation, and completion of the coupling
reaction was monitored by the Kaiser test [124] for primary amines. The final cleavage from the resin was
performed according to GP 6 for 4 h at r.t. The resulting crude material was purified by prep. PR-HPLC
(5–60% B in 40 min, Nucleosil 100-5 C18 column) to give the fully deprotected linear peptide as a white
powder (75 mg). Anal. RP-HPLC (3–50% B in 30 min, Supelco Discovery BIO Wide Poreꢂ C18 column,
tR 24.42 min.). HR-MS: 4284.1043 (C190H288N55O57Sþ ; calc. 4284.1043).
2. Stability Experiments in Human Plasma. Reagents and Solns. The peptide stock soln. was prepared
by dissolving 1 mg of the peptide in 1 ml of 1ꢂPBS buffer (pH 7.42). The d-Trp stock soln. (used as
internal standard for the HPLC integration) was obtained by dissolving 0.01 mg of d-Trp in 1 ml 1ꢂ PBS
buffer (pH 7.42). The solns. were kept in a freezer at ꢀ208.
26
)
Optical rotation of the (R)-enantiomer was measured: [a]D ¼ þ7.4 (c¼0.95, CHCl3). (S)-
Enantiomer: [a]D ¼ ꢀ10 [119].

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
733
Human blood was collected in the 35-ml heparinized tubes. The blood was transferred into a 50-ml
sterile FalconTM tubes under sterile conditions, and centrifuged at 1200g for 10 min at 48. The supernatant
was removed and stored in a freezer at ꢀ20827).
Metabolic-Stability Measurements. To a stock soln. of peptide (100 ml) and d-Trp (100 ml), freshly
refrozen plasma (50 ml) was added, and the mixture was incubated at 378. At different times, 50 ml of the
mixture was taken and supplemented with EtOH (100 ml), then centrifuged at 2500g for 5 min at r.t. An
aliquot of the supernatant (50 ml) was subjected to anal. RP-HPLC (0–34% B in 20 min, Supelco
Discovery BIO Wide Poreꢂ C18 column).
Data Collection and Calculations. The calculations were based on peak-area counts from the HPLC
analysis. The triplicate values were expressed as percent of the remaining peptide compared to the initial
time point 0 min (recalculated according to the peak-area counts of the internal standard, d-Trp). The
half-lives, as well as the graphic interpretations were accomplished with GraphPad Prism 5.0.
Degradation of the peptide was fitted with the one phase decay model: Y¼(Y0 ꢀPlateau)ꢂexp
(ꢀ KꢂX)þPlateau, where Y0 is the Y value when X (time) is zero (expressed in the same units as Y),
plateau is the Y value at infinite times (expressed in the same units as Y), K is the rate constant
(expressed in reciprocal of the X axis time units, if X is in min, then K is expressed in minꢀ1). The half-life
is in the time units of the X axis, and it is computed as ln(2)/K.
3. Receptor-Binding Experiments [93]. Reagents. The hNTS1 and hNTS2 cDNA were purchased
from the UMR cDNA Resource Center, subcloned into a pcDNA3.1(þ) eukaryotic expression vector.
To create a cell line stably expressing hNTS1, the pcDNA3.1 hNTS1 expression vector was used to clone
CHO FRT cells (Invitrogene, D-Darmstadt). Cells were grown in DMEM-F12 supplemented with 10%
fetal bovine serum (FBS), 2 mm l-glutamine, 1% Pen-Strep (all from Invitrogene), and 400 mg/ml
hygromycin B (Calbiochem Merck Chemicals, Nottongham, UK).
Experiments. For receptor-binding experiments with NTS2, HEK 293 cells were maintained in
DMEM-F12 supplemented with 10% FBS, 2 mm l-glutamine, and 1% Pen-Strep, and kept in a humid
atmosphere at 378 and 5% CO2. Transient transfection was performed according to the calcium
phosphate precipitation method [125], when 30 mg of pcDNA3.1 hNTS2 were added to each 15-cm dish,
cells were grown for 48 h, harvested and worked up as described in [126]. Receptor-binding data were
determined according to protocols as described in [127]. In detail, hNTR1 binding was measured using
homogenates of membranes from CHO cells stably expressing human NTR1 at a final concentration of
4 mg/well and the radioligand [3H]Neurotensin (specific activity 116 Ci/mmol; PerkinꢀElmer, D-Rodgau)
at a concentration of 0.5 nm [128]. Specific binding of the radioligand was determined at a KD value of
0.59ꢁ0.16 nm and a Bmax value of 1150–7290 fmol/mg protein. Unspecific binding was determined in the
presence of 10 mm neurotensin. NTS2 binding was conducted by using homogenates of membranes from
HEK 293. Membranes were incubated at a final concentration of 6 mg/well together with 0.5 nm of
[3H]NT(8–13) (specific activity 136 Ci/mmol; custom synthesis of [Leu-3H]NT(8–13) by GE Health-
care, D-Freiburg) at a KD value of 1.2ꢁ0.17 nm and a Bmax value of 325–2560 fmol/mg protein.
Unspecific binding was determined in the presence of 10 mm NT(8–13) (2a), and the protein
concentration was generally established by the method of Lowry et al. using bovine serum albumin as
standard [129].
Data Collection and Calculations. Data analysis of the competition curves from the radioligand-
binding experiments was accomplished by nonlinear regression analysis using the algorithms in
GraphPad Prism (GraphPad Software, San Diego, CA). EC50 Values derived from the resulting
doseꢀresponse curves were transformed into the corresponding Ki values by utilizing the equation of
Cheng and Prusoff [130].
4. Neurotensin and NPY Receptor Autoradiography and Functional Assays. Reagents. All reagents
were of the best grade available and were purchased from commercial suppliers. The adenylate cyclase
activity kit was from PerkinꢀElmer (Waltham, MA, USA), and the Fluo-4NW calcium assay kit was from
Molecular Probes, Inc. (Eugene, OR, USA).
Cell Lines. The neuroepithelioma cell line SK-N-MC endogenously expressing the NPY Y1 receptor
was obtained from ATCC (HTB-10; LGC Standards, Teddington, Middlesex, UK), and cultured at 378
27
)
The human serum was provided by a healthy donor of the University Hospital Geneva.

734
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
and 5% CO2 in MEM (minimum essential medium) with GlutaMax I and supplemented with 10% FBS,
100 U/ml penicillin and 100 mg/ml streptomycin, 1 mm MEM sodium pyruvate, and MEM non-essential
amino acids (1ꢂ). The neuroblastoma cell line SH-SY5S endogenously expressing the NPY Y2 receptor,
kindly provided by Dr. Paolo Paganetti (Novartis, CH-Basel), was cultured at 378 and 5% CO2 in MEM/
Hamꢃs F12 with GlutaMax I and supplemented with 10% FBS, 100 U/ml penicillin, and 100 mg/ml
streptomycin, 1 mM MEM sodium pyruvate, and MEM non-essential amino acids (1ꢂ). The human
colorectal adenocarcinoma cell line HT-29 endogenously expressing the NT1 receptor was cultured at 378
and 5% CO2 in McCoyꢃs medium with GlutaMax I, supplemented with 10% FBS, 100 U/ml penicillin,
and 100 mg/ml streptomycin. All culture reagents were from Gibco BRL, Life Technologies, (Grand
Island, NY).
Receptor Autoradiography for NT Analogs. The binding affinities of the compounds to be tested
were determined on 20-mm thick cryostat (Microm HM 500, D-Walldorf) sections of resected biopsy
samples of human neurotensin receptor-positive tumor tissues (colon adenocarcinoma and hemangio-
pericytoma) using [125I-Tyr3]NT (2000 Ci/mmol; Anawa, CH-Wangen) as radioligand. Consecutive
sample sections were incubated for 1 h at 48 with the radioligand in 50 mm Tris·HCl (pH 7.6, 0.2% BSA,
5 mm MgCl2, 10 mM bacitracin) in the presence of increasing concentrations of either NT (1) or the
analogs to be tested. Incubation was terminated by washing the sections in ice-cold buffer (50 mm Tris·
HCl, pH 7.6). Sections were subsequently dried by a stream of cold air and then exposed to Kodak films
Biomax MRꢂ for 7 d. The signal density was quantitatively assessed using tissue standards for iodinated
compounds (Amersham, Aylesbury, UK) and a computer-assisted image processing system (Analysis
Imaging System, Interfocus, D-Mering).
Receptor Autoradiography for NPY Analogs. Binding affinities of the compounds were assessed
using sections of cell membrane pellets of SK-N-MC cells for NPY Y1 or SH-SY5S cells for Y2, as
described in [112][131][132]. Briefly, membrane pellets were prepared and stored at ꢀ808, and receptor
autoradiography was performed on 20-mm thick cryostat (Microm HM 500, D-Walldorf) sections of the
membrane pellets, mounted on microscope slides and stored at ꢀ208 as previously described for other
cell lines and receptors [133]. The slides were preincubated in KrebsꢀRinger soln. (NaCl, 119 mm; KCl,
3.2 mm; KH2PO4, 1.19 mm; MgSO4, 1.19 mm; NaHCO3, 25 mm; CaCl2, 2.53 mm; d-glucose, 10 mm;
pH 7.4) for 60 min at r.t. Then, they were incubated for 120 min in the incubation soln. containing the
KrebsꢀRinger soln., 0.1% BSA, 0.05% bacitracin, and 10,000 cpm/100 ml of the [125I]-labeled radioligand
human PYY (hPYY; 2000 Ci/mmol; Anawa, CH-Wangen). Membrane pellet sections were incubated
with [125I]PYY with increasing concentrations ranging from 0.1 up to 1000 nm of non-labeled PYY, as
control, or with the compounds to be tested. After the incubation, the slides were washed two times for
5 min and then rinsed four times in ice-cold preincubation soln. After drying, the slides were exposed to
Kodak films Biomax MRꢂ for 7 d. The signal density was quantitatively assessed using tissue standards
for iodinated compounds (Amersham, Aylesbury, UK) and a computer-assisted image processing system
(Analysis Imaging System, Interfocus, D-Mering).
Intracellular Calcium Mobilization Assay for NT Analogs. Intracellular calcium release was
measured in HT-29 cells using the Fluo-4NW Calcium Assay kit as described for other cell lines in
[133][134]. In brief, HT-29 cells were seeded (10,000 cells per well) in 96-well plates and cultured for 2 d
at 378 and 5% CO2 in culturing medium. At the day of the experiment, the cells were washed with assay
buffer (1ꢂHBSS, 20 mm HEPES) containing 2.5 mm probenecid, and then loaded with 100 ml/well Fluo-
4 dye in assay buffer containing 2.5 mm probenecid for 30 min at 378 and 5% CO2, and then for further
30 min at r.t. For measurement of the intracellular calcium release, the cells were transferred to a
SpectraMax M2e (Molecular Devices, Sunnyvale, CA). Intracellular calcium release was recorded in a
kinetic experiment for 60 s at r.t., monitoring fluorescence emission at 520 nm (with lex 485 nm) in the
presence of the analogs to be tested. Maximum fluorescence (Fmax) was measured after adding
ionomycin. Baseline (control) measurements were taken for untreated cells. Data are shown as %Fmax
obtained with ionomycin as reported in [133]. All experiments were repeated at least three times in
triplicate.
Adenylate Cyclase Assay for NPY Analogs. Adenylate cyclase activity was determined in SK-N-MC
cells using the adenylate cyclase activation flashplate assay (SMP004) from PerkinꢀElmer (Waltham,
MA, USA) as described for other cell lines in [134]. SK-N-MC Cells were seeded in 96-well culture plates

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
735
at 25,000 cells/well and cultured for 2 d at 378 and 5% CO2. Culture medium was then removed from the
wells, and fresh medium (100 ml) containing 0.5 mm 3-isobutyl-1-methylxanthine (IBMX) was added to
each well. Cells were incubated for 30 min at 378. Medium was then removed and replaced with fresh
medium containing 0.5 mm IBMX, with or without 10 mm forskolin and various concentrations of the
peptides to be analyzed. Cells were incubated for 30 min at 378. After removal of the medium, cells were
lysed, and cAMP accumulation was determined using the SMP004 kit from PerkinꢀElmer according to
the instructions of the manufacturers.
5. Measurement of Ectopeptidase Activities by Using 96-Well Fluorimetric Assays. Reagents.
Recombinant human NEP and recombinant human AP-N (devoid of their respective N-terminal
cytosol and transmembrane segment) were purchased from R&D Systems, and used as pure source of
peptidases. Abz-dR-G-L-EDDnp FRET-peptide that is an internally quenched fluorescent substrate
specific for NEP-EndoPeptidase activity was synthesized by Thermo-Fisher Scientific (Germany). Abz-
R-G-F-K-DnpOH FRET-peptide that is an internally quenched fluorescent substrate specific for NEP-
CarboxyDiPeptidase activity was synthesized by Thermo-Fisher Scientific (Germany). Mca-R-P-P-G-F-
S-A-F-K-(Dnp)-OH FRET-peptide (Mca-BK2) that is an intramolecularly quenched fluorogenic
peptide structurally related to bradykinin, which is a selective substrate for measuring NEP and ECE
activity, was purchased from R&D Systems. l-Alanine-Mca (Ala-Mca), a fluorogenic substrate for
measuring aminopeptidase activity was purchased from Sigma.
Measurement of NEP-Endopeptidase Activity Using FRET-Specific Peptide Substrate Abz-dR-G-L-
EDDnp. Using black half-area 96-well microplate, the standard reaction mixture consisted of enzyme in
100 mm Tris·HCl, pH 7, containing 200 mm NaCl (100 ml final volume). The substrate was added after
preincubation for 10 min at 288, and the kinetics of appearance of the fluorescent signal (RFU) was
directly analyzed for 40 min at 288 by using a fluorimeter microplate reader (monochromator Infinite
200-Tecan) at 320 and 420 nm excitation and emission wavelengths, resp.
Measurement of NEP-CarboxyDiPeptidase Activity Using FRET-Specific Peptide Substrate Abz-R-
G-F-K-DnpOH. The standard reaction mixture consisted of enzyme in 100 mm Tris·HCl pH 6.5
containing 50 mm NaCl (100 ml final volume). The substrate was added after preincubation for 10 min,
and the kinetics of appearance of the fluorescent signal (RFU) was directly analyzed for 40 min at 288 at
320 nm excitation and 420 nm emission wavelengths. The Mca-BK2 was submitted to hydrolysis by
rhNEP under the same exper. conditions as described below.
Measurement of AP-N-Ectopeptidase Activity Using Ala-Mca Substrate. The standard reaction
mixture consisted of enzyme in 100 mm Tris·HCl, pH 7.0 (100 ml final volume). The Ala-Mca substrate
was added after preincubation for 10 min at 288, and the kinetics of appearance of the signal was
monitored for 40 min at 288 by using the fluorimeter reader at 380-nm excitation and 460 nm emission
wavelengths [115][135].
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