β-Casomorphins: Substitution of phenylalanine with β-homo phenylalanine increases the μ-type opioid receptor affinity

Article abstract of DOI:10.1016/S0960-894X(00)00187-6

Two analogues of bovine β-casomorphin-7 and β-casomorphin-5 containing a β-homo phenylalanine in substitution of the phenylalanine in position 3 were synthesised and tested for their μ-opioid receptor affinity. The modification enhanced the μ receptor affinity 5-fold in the case of modified β-CM-7 and 2-fold for modified β-CM-5 when compared to the natural peptides. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00187-6

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1185±1188
ꢀ-Casomorphins: Substitution of Phenylalanine with ꢀ-homo
Phenylalanine Increases the ꢁ-Type Opioid Receptor Anity
Luigi Longobardo, a,b,* Dominique Melck, c Rosa Siciliano, b Antonello Santini, a,b
Vincenzo Di Marzo c and Giancarlo Cammarota b
a
Á
Á
Dipartimento di Scienza degli Alimenti, Universita di Napoli ``Federico II'', Via Universita 100, 80055 Portici, (Napoli) Italy
bIstituto di Scienze dell'Alimentazione, CNR, via Roma 52, Avellino, 83100, Italy
cIstituto per la Chimica di Molecole di Interesse Biologico, Via Toiano 6, 80072, Arco Felice, Italy
Received 23 December 1999; accepted 20 March 2000
AbstractÐTwo analogues of bovine b-casomorphin-7 and b-casomorphin-5 containing a b-homo phenylalanine in substitution of
the phenylalanine in position 3 were synthesised and tested for their m-opioid receptor anity. The modi®cation enhanced the m
receptor anity 5-fold in the case of modi®ed b-CM-7 and 2-fold for modi®ed b-CM-5 when compared to the natural peptides.
# 2000 Elsevier Science Ltd. All rights reserved.
In addition to its well-known nutritive role, milk also
releases chemical signals to the recipient organism. In
this context, a number of peptides derived from milk
may act as opioid receptor ligands. While casoxins and
lactoferroxins display opioid antagonistic properties,
other peptides such as b-casomorphin-7, H-Tyr-Pro-Phe-
Pro-Gly-Pro-Ile-OH (b-CM-7) and b-casomorphin-5 H-
Tyr-Pro-Phe-Pro-Gly-OH, (b-CM-5) behave like opioid
receptor agonists.1 Most of the information available to
date concerns b-CMs. These peptides can be released by
enzymatic hydrolysis of b-casein in the gut of both
adults and newborns, and may elicit opioid e€ects by
acting as hormone-like substances. Recently, it has also
been reported that b-CM-5 stimulates neurite out-
growth in a mouse neuroblastoma cell line, suggesting
that b-CM-5 may play a role as a neurite elongation
factor during the suckling period.2 Several synthetic b-
CM derivatives have been shown to be highly speci®c
and potent m-type opioid receptor ligands, and have
been frequently used as pharmacological tools in opioid
research. In particular, the e€ect on m- and d-opioid-like
properties of the aromatic amino acid substitution3 in
the position-3 of some cyclic b-CM-5 analogues and the
modi®cation in the Tyr1-Phe3 domain has been found to
play a crucial role for biological activity.4,5
potentially active biological compounds. These novel
amino acids have been the object of several studies7 in
which the focus was mainly on the structure and phar-
macological properties compared to the natural a-amino
acids counterparts. b-Peptides, made entirely with b-
amino acids, have been so far synthesised8 in order to
understand what properties can be acquired following
b-amino acid insertion.
The availability of several b-homologues of protein amino
acids by a new, fast and ecient procedure,6 developed to
prepare b-homo amino acids in any protected form by
direct homologation of their corresponding N-protected
a-amino acid, allowed us to prepare b-homo analogues
of bioactive b-CMs.
We have synthesised and tested, in a m-type opioid
receptor binding assay, two new analogues of b-CM-7
and b-CM-5, containing the natural amino acid pheny-
lalanine substituted by the b-homo counterpart, H-Tyr-
Pro-bhPhe-Pro-Gly-Pro-Ile-OH and H-Tyr-Pro-bhPhe-
Pro-Gly-OH.
With this type of substitution, the sequence Tyr1-Phe,3
commonly found in b-CMs as well as in other opioid
peptides, such as dermophin and deltorphin, contain an
extra methylene group in the direction of the carbonyl of
the phenylalanine residue. The natural and modi®ed
peptides were tested and compared for their capability to
displace the potent radioligand m-opioid receptor agonist
[3H]-DAMGO9 from rat brain membrane preparations.
In recent years, b-homo amino acids (previously repor-
ted as homo-b6) have raised considerable interest as
*Corresponding author. Tel.:+39-825-34052; fax:+39-825-781585;
e-mail: luilongo@unina.it
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00187-6

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L. Longobardo et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1185±1188
Synthesis
The heptapeptide analogue was assembled via C-elon-
gation of 4, Boc-bhPhe-Pro-OMe, by coupling it with
the C-protected tripeptide 7, H-Gly-Pro-Ile-OMe, fol-
lowed by N-elongation via the coupling of the resulting
pentapeptide with the N-protected dipeptide 9, Boc-
Tyr(tBu)-Pro-OH. The C-protected tripeptide 7, H-Gly-
Pro-Ile-OMe was synthesized step-wise beginning with the
coupling of Boc-Pro-OH with H-Ile-OMe. After the usual
work-up, the product 5, Boc-Pro-Ile-OMe, was N-depro-
tected with 50% of TFA in CH2Cl2 for 300, and then used
in the reaction with Boc-Gly-OH. The subsequent N-
deprotection of the new coupling product 6, Boc-Gly-
Pro-Ile-OMe, resulted in fragment 7 (Scheme 2).
The modi®ed peptides were prepared by solution methods
using the t-Boc and methyl ester protection, dicyclohexyl-
carbodiimide and 1-hydroxybenzotriazole as coupling
agents and N-methymorpholine as the base. As for the
natural peptides, commercially available ones were used.
The b-homo-(S)-phenylalanine was prepared with a new
homologation procedure,6 with a slight modi®cation in
the hydrolytic steps involved in the reported method.
The ®nal acid hydrolysis of the N-Boc-protected b-
amino nitriles 1 into free b-homo-amino acid was actually
performed as a three-step, one pot reaction in which,
after the removal of the Boc protection, the nitrile was
converted into the methyl ester. The latter was then
hydrolyzed by the addition of water.10
The C-deprotection of dipeptide 4 and its coupling with
fragment 7 yielded the full protected pentapeptide 8
(Scheme 3). The pentapeptide 8 was N-deprotected and
used in the coupling reaction with the N-protected dipep-
tide Boc-Tyr(tBu)-Pro-OH 9, based on the reaction of the
commercially available N-Boc-Tyr(tBu)-OH with proline
methyl ester followed by ester hydrolysis. The coupling
produced the fully protected heptapeptide 10. To obtain
free heptapeptides, ester hydrolysis at the isoleucine
Subsequently, the free b-homo-phenylalanine 2 obtained
in this way was reprotected with a Boc group to yield 3.
The latter compound was used in the coupling reaction
with proline methyl ester in THF, which provided the
Boc dipeptide methyl ester 4 (in 96% yield), as illustrated
in Scheme 1.
Scheme 1. Synthesis of Boc-bhPhe-Pro-OMe.
ꢀ
0
.
Scheme 2. Synthesis of C-protected tripeptide 7: (a) HCl Ile-OMe, NMM, DCC/HOBt, THF, 0 to rt, 3 h; (b) 50% TFA in CH2Cl2, rt, 30 (c) Boc-
Gly-OH, NMM, DCC/HOBt, THF, 0ꢀ to rt, 3 h.
Scheme 3. Synthesis of the heptapeptide analogue: (a) 1 M LiOH (aq), MeOH, rt, 1 h; (b) 7, NMM, DCC/HOBt, THF, 0ꢀ to rt, 3 h. (c): HCl Pro-
.
OMe, NMM, DCC/HOBt, THF, 0ꢀ to rt, 3 h; (d) 50% TFA in CH2Cl2 rt, 300; (e) 9, NMM, DCC/HOBt, THF, 0ꢀ to rt, 3 h; (f) 1 M LiOH (aq),
MeOH, rt, 24 h; (g) 50% TFA in CH2Cl2, rt, 1 h.

L. Longobardo et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1185±1188
1187
residue of 10 was performed and a subsequent removing
of the acid-labile protecting group, with 50% TFA in
CH2Cl2 for 1 h produced the free heptapeptide 11. The
HPLC analysis established that the ester hydrolysis of
10 was completed in 24 h, and showed that hydrolysis
occurred without racemization.
Table 1. Analytical data for natural and modi®ed (b-CM-7) and (b-
CM-5)
Peptide
Retention
time11 (min)
Molecular
Weight12
Experimental Theoretical
Boc-bhPhe-Pro-OMe
Tyr-Pro-Phe-Pro-Gly
Tyr-Pro-bhPhe-Pro-Gly
Tyr-Pro-Phe-Pro-Gly-Pro-Ile
Tyr-Pro-bhPhe-Pro-Gly-Pro-Ile
Ð
390.3
579.3
593.3
789.5
803.4
390.23
579.25
593.25
789.38
803.38
The pentapeptide analogue was assembled via N-elonga-
tion of 4 by its coupling with 9, followed by the coupling
with glycine methyl ester (Scheme 4). The modi®ed penta-
peptide, H-Tyr-Pro-bhPhe-Pro-Gly-OH, was synthesised
in a similar way to the heptapeptide 11, but the key dipep-
tide 4 was ®rst deprotected at the Boc group and then
coupled with the N-protected dipeptide 9, thereby produ-
cing the full protected tetrapeptide 12. The ester hydrolysis
of 12 and a new coupling with H-Gly-OMe yielded the
protected pentapeptide 13. Ultimately, the removal of
the protecting group yielded the free pentapeptide 14
(Scheme 4).
17.73
18.21
21.10
21.68
Receptor Binding Assay
The natural b-casomorphins are m-selective opioid ago-
nist with moderate potency and they exhibit only low d-
and almost no k-receptor anities.13 The binding a-
nities of the natural and modi®ed peptides for m-recep-
tors were determined by measuring the displacement of
[3H]-DAMGO from rat brain membrane binding
sites.14,15 Incubations were performed for 90 min at 25 ꢀC
with 1 nM labeled ligand in a ®nal volume of 1 mL.
Scheme 5 reports the overall synthetic pathway of
[bhPhe]3-b-CM-7 and [bhPhe]3-b-CM-5.
The purity and the chemical structure of the synthesised
peptides were veri®ed by RP-HPLC followed by electro-
spray mass spectrometry analyses. Results are summarised
in Table 1. The peptides were more than 95% pure and
there was a excellent agreement between the expected
and the experimental mass values, thus con®rming the
putative peptide structures.
IC50 values were obtained from log dose-displacement
curves, and Ki values were calculated from the IC50
values by means of Chang and Pruso€ equation,16 by
using the value of 0.53Æ0.06 nM for the dissociation
constant of [3H]-DAMGO (Bmax 120,7Æ3,67 fmol/mg).
The results are shown in Table 2.
Scheme 4. Synthesis of the pentapeptide analogue: (a) 50% TFA in CH2Cl2, rt, 300; (b) 9, NMM, DCC/HOBt, THF, 0ꢀ to rt, 3 h; (c) 1 M LiOH (aq)
ꢀ
.
in MeOH, 1 h; (d) HCl Gly-OMe, NMM, DCC/HOBt, THF, 0 to rt, 3 h; (e) 50% TFA in CH2Cl2, rt, 1 h.
Scheme 5. [bhPhe]3-b-Casomorphins synthesis.

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L. Longobardo et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1185±1188
Table 2. IC50 and Ki values for natural and modi®ed b-casomorphins
References and Notes
Peptides
IC50 (mM)
Ki (mM)
1. Teschemacher, H.; Koch, G.; Brantl, V. Biopolymers 1997,
43, 99.
2. Sakaguchi, M.; Murayama, K.; Satoh, M.; Takeuchi, M.;
Matsumura, E. Neurosci. Lett. 1998, 251, 97.
3. Schmidt, R.; Wilkes, B. C.; Chung, N.; Lemieux, C.; Schil-
ler, P. W. Int. J. Pept. Protein Res. 1996, 48, 411.
4. Kostetsky, P. V.; Arkhipova, S. F. Biochemistry (Mosc.)
1999, 64, 1005.
b-CM-7
20.0Æ2.5
4.0Æ1.5
4.5Æ1.2
2.0Æ0.8
7.54Æ0.89
1.44Æ0.54
1.62Æ0.43
0.72Æ0.20
[bhPhe]3-b-CM-7
b-CM-5
[bhPhe]3-b-CM-5
5. Brandt, W.; Stoldt, M.; Schinke, H. J. Computer Aided Mol.
Des. 1996, 10, 201.
6. Caputo, R.; Cassano, E.; Longobardo, L.; Palumbo, G.
Tetrahedron 1995, 51, 12337.
7. Enantioselective Synthesis of b-Amino Acids; Juaristi E.
Ed.; Wiley-VCH. 1997.
8. Abele, S.; Vogtli, K.; Seebach, D. Helv. Chim. Acta 1999,
82, 1539 and literature cited therein.
The evaluation of m-receptor binding activity of the
modi®ed b-CMs showed that the synthetic analogues
not only retained binding activity but were even more
potent: 5-fold in the case of modi®ed b-CM-7 and 2-fold
for modi®ed b-CM-5 in comparison with the natural
peptides.
9. Kosterlitz, H. W.; Paterson, S. J. Br. J. Pharmacol. 1980, 73,
299.
10. Longobardo, L.; Cassano, E.; Cammarota, G.; Melk, D.
Manuscript in preparation.
These results suggest that also non cyclic analogues of
bioactive peptides can be used in the search for new
opioid ligands, although the two analogues presented in
this paper are less potent at m-opioid receptors than cyc-
lic analogues of b-CM-5 (e.g., the Tyr-c[-d-Orn-2-Nal-d-
Pro-Gly-]) which contain, however, more substantial
modi®cations3 with respect to the natural peptide. Fur-
ther studies are in progress in our laboratory in order to
evaluate if the anities at d-and k-opioid receptor and the
agonist activity of the modi®ed peptides is di€erent from
the natural ones. However, the results presented here
clearly indicate that a slight chemical modi®cation, a-
CH2- group in the phenylalanine, induces an appreciable
variation in the m-type binding anities in the studied b-
CMs peptides. In the case of b-casomorphin, the b-
homo amino acid containing analogues may represent a
useful pharmacological tool, particularly in considera-
tion of the enhanced stability to enzymatic hydrolysis of
the b±homo residues.17
11. Analyses of the synthetic peptides were carried out by RP-
HPLC on a Vydac C18 column (250Â4.6 mm, 5 mm) using a
Waters HPLC System (Datasystem Millenium, HPLC pumps
Waters 510, Detector Waters 486). Eluents were: 0.1% tri-
¯uoroacetic acid (solvent A) and 0.07% tri¯uoroacetic acid in
95% acetonitrile (solvent B). The elution was performed by
means of a linear gradient from 10 to 60% solvent B over 25min
at a ¯ow rate of 1 mL/min. The elution was monitored at 220 nm.
12. HPLC fractions were submitted to electrospray mass spec-
trometry (ESMS) analysis using a platform single quadrupole
mass spectrometer (Micromass). Samples were dissolved in 1%
acetic acid in 50% acetonitrile and 2±10mL were injected into the
mass spectrometer at a ¯ow rate of 10mL/min. The quadrupole
was scanned from m/z 300 to 1600 at 10 s/scan and the spectra
were acquired and elaborated using the MassLynx software. All
mass values are reported as monoisotopic masses.
13. Brantl, V.; Teschemacher, H.; Blasig, J.; Henschen, A.;
Lottspeich, F. Life Sci. 1981, 28, 1903.
14. Srucs, M.; Belkeva, M.; Simon, J.; Benyhe, S.; Toth, G.; Hepp,
J.; Wolemamn, M.; Medzihardzhy, K. Life Sci. 1987, 41, 177.
15. Oktem, H. A.; Varga, E.; Hepp, J.; Medzihardzhy, K.;
Lajtha, A.; Borsodi, A. Life Sci. 1991, 48, 1757.
16. Cheng, Y. C.; Pruso€, W. H. Biochem. Pharmacol. 1973,
22, 3099.
17. Seebach, D.; Overhand, M.; Kuhnle, F. N. M.; Martinoni,
B.; Oberer, L.; Hommel, U.; Widmer, H. Helv. Chim. Acta
1996, 79, 913.
Acknowledgements
This work was supported by the Italian National Council
for Research (CNR). The authors are grateful to Professor
Francesco Addeo for critical discussion and revision of the
manuscript.