Analogues of neuropeptide Y containing β-aminocyclopropane carboxylic acids are the shortest linear peptides that are selective for the Y1 receptor

Article abstract of DOI:10.1002/anie.200390078

Unique, conformationally restricted β-amino acids are a constituent of truncated linear peptides such as 1, which have been obtained for the first time; these species show high and selective affinity toward the Y₁ receptor. The high affinity of

Full text of DOI:10.1002/anie.200390078

Communications
Conformationally Restricted Peptides
Experimental Section
The antimony thin films were synthesized in ultra-high vacuum (base
pressure: 4 î 10ꢀ11 mbar) by evaporation of Sb4 molecules from a
resistance-heated effusive oven (temperature: 3308C; deposition
rate: 0.1 nmsꢀ1; Sb: 99.9999%, Johnson-Matthey). The layer thick-
ness is given in monolayers of Sb atoms. The MoS2 substrates were
prepared by cleavage according to the procedure given in reference
[13]. The AuSb2 surface alloy was prepared starting from Au(111).[14]
On this substrate the surface alloy AuSb2 with the (100) orientation
forms spontaneously, if less than one monolayer of antimony is
deposited at room temperature.[15] The beetle-type STM[16] is located
in an analysis chamber directly attached to the preparation cham-
ber.[17] To increase image contrast, the STM data were electronically
differentiated directly during acquisition. Therefore the STM images
appear as if they are illuminated from the side.
Analogues of Neuropeptide Y Containing
b-Aminocyclopropane Carboxylic Acids are the
Shortest Linear Peptides That Are Selective for
the Y1 Receptor**
Norman Koglin, Chiara Zorn, Raphael Beumer,
Chiara Cabrele, Christian Bubert, Norbert Sewald,
Oliver Reiser,* and Annette G. Beck-Sickinger*
Dedicated to Professor Peter Welzel
on the occasion of his 65th birthday.
Received: April 8, 2002
Revised: October 11, 2002 [Z19057]
Neuropeptide Y (NPY) is one of the most abundant neuro-
peptides in the mammalian central nervous system. It consists
of 36 amino acids and has an amide group at the C terminus.
To date NPY is the strongest known stimulator of food intake
in rat and mice models. Other important biological functions
are vasoconstriction in the periphery, regulation of behavior
and modulation of pain and epileptic seizures.[1]
In mammals, the activities of NPYare mediated by at least
three different G-protein-coupled receptors (Y1, Y2, and Y5).
Because NPY shows sub-nanomolar affinity towards all of
them, it is still difficult to distinguish the physiological roles of
each receptor in vivo. To address this problem, the knowledge
of the particular bioactive conformation at each receptor is
indispensable for the further development of subtype-selec-
tive ligands. Substitutions of single amino acids have revealed
that especially the highly conserved C-terminal part of NPY,
with its two positively charged arginine side chains in
positions 33 and 35 and the tyrosine amide in position 36,
plays a crucial role during the recognition process by the
respective receptor. Because of its flexibility, no defined
structure could be assigned to this important part of the
molecule to date. It is assumed that different secondary
structure motifs of the C terminus are responsible for
receptor subtype selectivity.
[1] J. Donohue, The structures of the elements, Wiley-Interscience,
New York, 1974.
[2] J. M¸hlbach, P. Pfau, E. Recknagel, K. Sattler, Surf. Sci. 1981,
106, 18.
[3] A. F. Hollemann, N. Wiberg, Lehrbuch der Anorganischen
Chemie, 101. ed., Walter de Gruyter, Berlin, 1995.
[4] H. Sontag, R. Weber, Chem. Phys. 1982, 70, 23.
[5] V. Kumar, Phys. Rev. B 1993, 48, 8470.
[6] Y. W. Mo, Phys. Rev. Lett. 1992, 69, 3643.
[7] J. Cohen, J. Appl. Phys. 1954, 25, 798; M. Hashimoto, K.
Umezawa, R. Murayama, Thin Solid Films 1990, 188, 95; A.
Hoareau, J. X. Hu, P. Jensen, P. Melinon, M. Treilleux, B.
Cabaud, Thin Solid Films 1992, 209, 161; H. Murmann, Z. Phys.
1929, 54, 741; J. A. Prins, Nature 1933, 131, 760; H. Levinstein, J.
Appl. Phys. 1949, 20, 306.
[8] H. Richter, H. Berckhemer, G. Breitling, Z. Naturforsch. A 1952,
9, 236.
[9] B. Kaiser, T. M. Bernhardt, B. Stegemann, J. Opitz, K. Rade-
mann, Nucl. Instrum. Methods Phys. Res. Sect. B 1999, 157, 155;
T. M. Bernhardt, B. Kaiser, K. Rademann, Phys. Chem. Chem.
Phys. 2002, 4, 1192.
[10] P. Lamparter, S. Steeb, W. Knoll, Z. Naturforsch. A 1976, 31, 90.
[11] J. Eiduss, R. Kalendarev, A. Rodionov, A. Sazonov, G. Chik-
vaidze, Phys. Status Solidi B 1996, 193, 3.
[12] G. Fuchs, P. Melinon, F. Santos Aires, M. Treilleux, B. Cabaud,
A. Hoareau, Phys. Rev. B 1991, 44, 3926.
[13] J. G. Kushmerick, S. A. Kandel, P. Han, J. A. Johnson, P. S. Weiss,
J. Phys. Chem. B 2000, 104, 2980.
[14] J. A. DeRose, T. Thundat, L. A. Nagahara, S. M. Lindsay, Surf.
Sci. 1991, 256, 102.
[*] Prof. Dr. O. Reiser, Dr. C. Zorn, Dr. R. Beumer, Dr. C. Bubert
Institut f¸r Organische Chemie
Universit‰t Regensburg
Universit‰tsstrasse 31, 93040 Regensburg (Germany)
Fax: (þ49)941-9434-121
[15] B. Stegemann, T. M. Bernhardt, B. Kaiser, K. Rademann, Surf.
Sci. 2002, 511, 153.
[16] K. Besocke, Surf. Sci. 1987, 181, 145.
[17] B. Kaiser, T. M. Bernhardt, K. Rademann, Nucl. Instrum.
Methods Phys. Res. Sect. B 1997, 125, 223; T. M. Bernhardt,
Dissertation thesis, Humboldt-University, Berlin, 1997.
E-mail: oliver.reiser@chemie.uni-regensburg.de
Prof. Dr. A. G. Beck-Sickinger, Dipl.-Biochem. N. Koglin,
Dr. C. Cabrele
Institut f¸r Biochemie
Universit‰t Leipzig
Talstrasse 33, 04103 Leipzig (Germany)
Fax: (þ49)341-9736-998
E-mail: beck-sickinger@uni-leipzig.de
Prof. Dr. N. Sewald
Institut f¸r Organische und Bioorganische Chemie
Universit‰t Bielefeld
Postfach 100131, 33501 Bielefeld (Germany)
[**] This work was supported by the Deutsche Forschungsgemeinschaft
(BE1264/3-1 and RE948-4/1) and the Fonds der Chemischen
Industrie and through generous chemical gifts from BASF AG, Bayer
AG, and Degussa AG.
202
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Angewandte
Chemie
Table 1: Sequences and affinities of shortened b-ACC-containing NPY analogues at the Y receptors. The
affinities are expressed as Ki values [nm].[a] For more details see text.
It has been shown that substitutions close
to these major C-terminal positions allow a
distinction to be made between some recep-
tor subtypes. For example, the introduction
of a proline residue in position 34 of the
NPY sequence significantly reduced the
Y2 receptor affinity and maintained the af-
finity toward the Y1 and Y5 receptors.[2] The
introduction of the turn-inducing Ala Aib
sequence in positions 31 and 32, respectively,
led to a Y5-selective analogue.[3] Whereas the
influence of the Ala Aib substitution on the
peptidic structure could be further elucidat-
[c]
Sequences[b]
Y1
Y2
Y5
D[V]R
RHYINLITRQRY-NH2
RHYINLITR~RY-NH2
RHYINLITR!RY-NH2
RHYINLI~RQRY-NH2
RHYINLI!RQRY-NH2
RHYINLI~R~RY-NH2
RHYINLI!R~RY-NH2
>1000
21[d]
>1000
724
>1000
>1000
>1000
617
17.5
5.6
3.7
1.5
1.6
2.9
3.5
3.3
2.2
2.0
5.1
1.4
9.9
4
5
6
7
8
9
10
11
12
13
37(ꢁ20)
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
50(ꢁ10)
>150
>1000
118
~
RHYINLR R~RY-NH2
29(ꢁ13)
RHYINLR!R!RY-NH2
1000
235
>1000
>1000
>1000
>1000
>1000
~
NLR R~RY-NH2
RHYINLITRPRY-NH2
RHYINLIPRPRY-NH2
RHYINLITRbRY-NH2
>1000
>1000
>1000
ed by NMR spectroscopic investigations, the 14
15
proline substitution at position 34 did not
sufficiently restrain the C terminus to enable
~
!
For explanations of and , see Scheme 1; b¼b-homoglutamine; other substitutions are marked in
the structural requirements of NPY binding bold. [a] Competitive binding assays were performed as described previously using 3H-NPY (Amersham)
as the radioligand.[3] The Ki values were determined by using the Cheng Prusoff equation with Kd values
at other receptor subtypes to be deduced.[4]
Accordingly, novel approaches are required
for the identification of the bioactive con-
formation at the Y1 receptor.
To rigidify the peptide backbone and to
induce or to stabilize distinct secondary
3
for H-NPY of 0.18 nm for the Y1 receptor and 2.4 nm for the Y5 receptor. [b] All peptides were N-
terminally acetylated, except for peptides 6 and 7. [c] D[V]R [10ꢀ3 degcm2 dmolꢀ1] is the difference of the
mean-residue molar ellipticity values [V]R at 222 nm, obtained from the CD spectra in buffer and in the
presence of 30% TFE. Peptide concentrations were 30 mm. [d] Ki value previously published.[8]
structure motifs, analogues of the C-terminal NPY sequence
with new conformationally restricted building blocks were
investigated. We expected that the incorporation of such
constraints would be especially effective at positions 32 and
34, which are in direct proximity to the most important amino
acids Arg33 and Arg35. By using this strategy, we identified
b-aminocyclopropane carboxylic acids (b-ACC) as novel
constricted building blocks that are highly effective for the
stabilization of secondary structures in peptides, and more-
over, complement known constrained a-amino acids such as
proline or a-aminoisobutyric acid (Aib) in their properties.
The incorporation of b-ACCs into peptides, however,
turned out to be challenging. Because of the 1,2-donor-
acceptor substituted cyclopropane structure, b-ACCs are
unstable in the N-unprotected form, which necessitates
special coupling techniques to incorporate them into pep-
tides.[5] The b-amino acid 1 (Scheme 1) can be readily
synthesized in diastereo- and enantiomerically pure form
starting from N-Boc-pyrrole.[6] We synthesized the prerequi-
site dipeptides 2 and 3, which then were used as dipeptide
building blocks in solid-phase peptide synthesis. The b-ACC-
containing dipeptides were introduced into the growing
peptide chain by a manual coupling step after TBTU activa-
tion[7] and further elongation by automated Fmoc/tert-butyl
coupling procedures.[3] The resulting peptides were analyzed
by HPLC and MALDI-MS, which confirmed the intact
cyclopropyl ring system. The conformational properties of
the C-terminal NPY analogues containing these b-ACC
dipeptides were investigated by circular dichroism (CD)
spectroscopy, and their affinity to the Y receptors was probed
by competition binding assays with living cells that selectively
express one receptor subtype (compounds 4 15, Table 1).
From previous studies it is known that the full-length
molecule of NPY is necessary for binding to the Y1 receptor.
N-terminally truncated analogues, such as NPY(13 36) or
NPY(18 36) bind only to the Y2 receptor, whereas NPY(2
36) is recognized by Y2 and Y5.[2b] However, in contrast to the
unsubstituted C-terminal fragment NPY(25 36)[8] we ob-
tained b-ACC-containing NPY analogues, which bind to the
Y1 receptor (peptides 4, 8, and 10, Table 1) with high affinity
and selectivity.
The affinity of the b-ACC-containing NPY analogues is
strongly dependent on the position of the substitution and the
absolute configuration of the b-ACC derivative. The intro-
~
duction of a b-ACC-moiety with the ( )-configuration in
Scheme 1. The synthesis of b-ACC-containing dipeptides for facile in-
troduction into peptide sequences: a) 1) HCl(g)/EtOAc; 2) Fmoc-
Arg(Pmc)-OH, EDC, pyridine, 88 97%; b) Pd/C, 1,4-cyclohexadiene
(5 equiv), MeOH, 77 88%. Bn¼benzyl, Boc¼tert-butoxycarbonyl,
EDC¼1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride,
Fmoc¼9-fluorenylmethyloxycarbonyl, Pmc¼2,2,5,7,8-pentamethylchro-
man-6-sulfonyl.
position 34 was crucial for binding to the Y1 receptor.
Accordingly, only a single b-ACC substitution with this
configuration in position 34 (peptide 4) was sufficient to
evoke nanomolar biological affinity at the Y1 receptor and
!
high selectivity, whereas a ( )-configured b-ACC-residue in
position 34 (peptide 5) remained biologically inactive at all
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Communications
reported herein.[9] Accordingly it can be assumed
that b-ACC substitutions cause a similarly con-
strained arrangement of the critical side chains in
linear peptides to that which can otherwise be
obtained by simple cyclization. It is remarkable that
similar NPY analogues containing the natural con-
formationally restricted amino acid proline are
biologically inactive at all investigated receptors
(peptides 13 and 14). Similarly, the introduction of
the b-amino acid b-homoglutamine[10] (b) into posi-
tion 34 yielded also only an inactive peptide 15.
Accordingly, the observed affinities at the Y1 recep-
OH
CO2Me
CO2Me
O
H
O
O
H
H
Ac-Arg-His-Tyr-Ile-Asn-Leu
N
H
N
N
N
N
H
CONH2
NH
NH2
N
H
H
O
O
NH
N
H
N
H
NH2
~
Scheme 2. A C-terminal NPY analogue (peptide 8) containing two ( )-configured b-ACC resi-
dues.
tor are exclusively evoked by a substitution with the
~
conformationally constricted ( )-b-ACC derivative in posi-
tion 34 or in combination with a similarly configured b-ACC
residue in position 32.
For the unsubstituted C-terminal fragment, conforma-
tional studies by circular dichroism (CD) showed the typical
CD spectrum of a random peptide in buffer and also a high a-
helical content in the presence of the a-helix-promoting
additive 2,2,2-trifluoroethanol (TFE), whereas the b-ACC-
containing analogues behaved completely differently (Fig-
ure 1). A higher content of structured peptide with a
minimum around 206 nm was found and a significantly
reduced tendency of a-helix formation was observed in the
presence of TFE, as shown by the D[V]R values in Table 1.
The D[V]R values are obtained by subtracting the [V]R values
at 222 nm in the presence of TFE from those obtained in
buffer. From these studies it can be concluded that b-ACC
substitutions stabilize distinct backbone conformations and
thereby significantly reduce the tendency towards a-helix
formation in helix-promoting environments such as TFE or
membranes.
Thus the b-ACC disubstituted and truncated NPY ana-
logues 8 and 10 are the most rigidified peptides but have still
the structural requirements for biological activity at the
Y1 receptor; to date such reqirements can neither be induced
with natural amino acids nor with analogous b-amino acids.
These peptides are therefore most suitable for further
structural investigations and for the development of new lead
structures in pharmaceutical chemistry. Further studies are
currently under investigation to gain more insights into the
peptide structure and the influence of the configuration of the
cyclopropane ring system on the secondary structure. Ac-
cordingly, we could show that b-ACC residues are extremely
useful for the rigidification of peptides. The application of this
concept to other systems could lead to new types of
peptidomimetics.
Figure 1. CD-spectra of the unsubstituted C-terminal NPY fragment
NPY(25 36) in 20 mm phosphate buffer (pH 7.0; ) and in the pres-
ence of 30% TFE (––), in comparison with the b-ACC disubstituted
* * * *
*
*
analogue (peptide 8) in buffer (
) and in 30% TFE (
), respec-
tively. Peptide concentrations were 30 mm.
investigated receptors. Peptides 6 and 7, which contain a b-
ACC residue at position 32, were biologically inactive in both
configurations. Combination of the two b-ACC-substitutions
~
with the same ( )-configuration of the cyclopropane ring in
positions 32 and 34 did not improve affinity at the Y1 receptor
(peptides 8 and 10, Scheme 2), but led to more rigid peptides
such as can be seen in Figure 1. In peptide 10, even the
substitution of an Ile by an Arg residue in position 31 is
tolerated at the Y1 receptor, if it is combined with the
~
bioactive ( )-configuration of the b-ACC residues in posi-
tions 32 and 34. However, peptide 10 is slightly less selective,
as its affinity at the Y5 receptor increased by approximately
five fold. This increase could arise from a new ionic
interaction of the Arg31 side chain with the Y5 receptor. If
!
the opposite ( )-configuration of the b-ACC residue is used
in position 32 and 34, the disubstituted peptide 11 will remain
!
biologically inactive. Peptide 9, with the ( )-configured b-
~
Received: July 5, 2002 [Z19670]
ACC residue in position 32 but with a bioactive ( )-b-ACC
derivative in position 34, shows reduced affinity to the
Y1 receptor. Interestingly, the further shortened b-ACC-
~
[1] a) C. Cabrele, A. G. Beck-Sickinger, J. Pept. Sci. 2000, 6, 97
122; b) A. Inui, Trends Pharmacol. Sci. 1999, 20, 43 46.
disubstituted octapeptide 12, with two ( )-configured b-
ACCs, still shows moderate affinity. Accordingly, these four
peptides (4, 8, 10, and 12) are the first and only linear NPY
segments with affinity and selectivity at the Y1 receptor.
A recently reported disulfide-bridged, cyclic, C-terminal
NPY analogue shows similar affinities at the Y1 receptor to
those of the linear b-ACC-substituted peptides 4, 8, and 10
[2] a) J. Fuhlendorff, U. Gether, L. Aakerlund, N. Langeland-
Johansen, H. Th˘gersen, S. G. Melberg, U. Bang Olsen, O.
Thastrup, Proc. Natl. Acad. Sci. USA 1990, 87, 182 186; b) C.
Gerald, M. W. Walker, L. Criscione, E. L. Gustafson, C. Batzl-
Hartmann, K. E. Smith, P. Vaysse, M.M. Durkin, T. M. Laz,
D. L. Linemeyer, A. O. Schaffhauser, S. Whitebread, K. G.
204
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Angewandte
Chemie
Hofbauer, R. I. Taber, T. A. Branchek, R. L. Weinshank, Nature
1996, 382, 168 171.
[3] C. Cabrele, M. Langer, R. Bader, H. A. Wieland, H. N. Doods,
O. Zerbe, A. Beck-Sickinger, J. Biol. Chem. 2000, 275, 36043
36048.
[4] A. Khiat, M. Labelle, Y. Boulanger, J. Pept. Res. 1998, 51, 317
322.
[5] a) C. Bubert, C. Cabrele, O. Reiser, Synlett 1997, 827 829; b) C.
Zorn, F. Gnad, S. Salmen, T. Herpin, O. Reiser,Tetrahedron Lett.
2001, 42, 7049 7053.
[6] R. Beumer, C. Bubert, C. Cabrele, O. Vielhauer, M. Pietzsch, O.
Reiser, J. Org. Chem. 2000, 65, 8960 8969.
[7] TBTU: 1H-Benzotriazolium 1-[bis(dimethylamino)methylene]-
tetrafluoroborate-3-oxide;
b-ACC-containing
dipeptide
(1 equiv), TBTU (1.5 equiv), N,N’-diisopropylethylamine
(3 equiv) in 0.5m N-hydroxybenzotriazole in N,N-dimethylform-
amide; reaction time 1 h; double coupling.
Figure 1. Structurally authentic terminal borylene complexes that have
been obtained by salt elimination reactions.
[8] B. Rist, O. Zerbe, N. Ingenhoven, L. Scapozza, C. Peers, P. F. T.
Vaughan, R. L. McDonald, H. A. Wieland, A. G. Beck-Sick-
inger, FEBS Lett. 1996, 394, 169 173.
[9] Y. Takebayashi, H. Koga, J. Togami, A. Inui, H. Kurihara, K.
Koshiya, T. Furuya, A. Tanaka, K. Murase, J. Pept. Res. 2000, 56,
409 415.
however, comprise boron in higher coordination numbers
than two.[3] Most of these borylene complexes (Figure 1) were
obtained by salt elimination reactions from dianionic
carbonylmetallates and suitable dihaloboranes. Although this
method of preparation was initially very successful, it appears
to be limited to the use of the homoleptic carbonylmetallates
Na2[Fe(CO)4] and Na2[M(CO)5] (M ¼ Cr, W), despite the
rather broad availability of dianionic transition metal com-
plexes and corresponding synthetic equivalents.[4] Thus,
combination of terminal borylenes with any coligand other
than carbonyl is precluded as yet, although such complexes
with various ligands are of significant interest.[5]
Recently, we reported on the photochemically induced
intermetallic borylene transfer as a novel and potentially
useful way of synthesizing both bridged and terminal
borylene complexes.[2b] However, as far as the latter were
concerned at that time, this route provided only an alternative
[10] A. M¸ller, C. Vogt, N. Sewald, Synthesis 1998, 837 841.
Borylene Half-Sandwich Complexes
5
¼ ¼
[(h -C5H5)(OC)3V B N(SiMe3)2:
A Half-Sandwich Complex with a
Terminal Borylene Ligand**
Holger Braunschweig,* Miriam Colling,
Chunhua Hu, and Krzysztof Radacki
¼ ¼
access to the chromium complex [(OC)5Cr B N(SiMe3)2],
Dedicated to Professor Thomas P. Fehlner
on the occasion of his 65th birthday
which we obtained somewhat earlier by conventional salt
elimination.[2a] Further exploitation of this method has now
led to the synthesis and full characterization of [(h5-
¼ ¼
In contrast to the rather well-developed chemistry of bridged
C5H5)(OC)3V B N(SiMe3)2] (5), the first half-sandwich
complex with a terminal borylene ligand.
[1]
ꢀ
ꢀ
borylene complexes LxM B(R) MLx their terminal coun-
¼ ꢀ
terparts LxM B R are still exceptionally rare and restricted
The title compound 5 was formed upon irradiation of
to five structurally authentic examples[2]–two of which,
[(OC)5Cr B N(SiMe3)2] (2) in the presence of [(h5-
¼ ¼
C5H5)V(CO)4] for four days at ꢀ308C in toluene according
to Equation (1). Compound 5 was separated by fractional
crystallization and isolated in 42% yield as a dark yellow
[*] Prof. Dr. H. Braunschweig,+ Dr. K. Radacki
Department of Chemistry
Imperial College
London SW7 2AY (UK)
E-mail: h.braunschweig@mail.uni-wuerzburg.de
Dr. M. Colling, Dr. C. Hu
Institut f¸r Anorganische Chemie
Technische Hochschule
52056 Aachen (Germany)
[+] New address:
Institut f¸r Anorganische Chemie der Universit‰t W¸rzburg
Am Hubland, 97074 W¸rzburg (Germany)
Fax: (þ49)0931-8884623
crystalline material, which is readily soluble in all common
aliphatic and aromatic solvents and only modestly air- and
moisture sensitive.
Single crystals of 5 suitable for X-ray structural analysis
were grown from hexane solutions at ꢀ808C. The compound
crystallizes in the space group P21/c and the molecule adopts a
[**] This work was supported by the Deutsche Forschungsgemeinschaft,
the Fonds der Chemischen Industrie, EPSRC, and the Royal Society.
We are grateful to the Rechenzentrum of Aachen University of
Technology (RWTH) for generously placing computation time at our
disposal.
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