Chemistry: analytical and immunological testing – Including sample preparation
Reexamination Certificate
2001-08-24
2003-06-24
Carlson, Karen Cochrane (Department: 1653)
Chemistry: analytical and immunological testing
Including sample preparation
C530S412000, C530S300000, C436S173000
Reexamination Certificate
active
06582968
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to the chemical synthesis of a melanocyte stimulating hormone (&agr;-MSH) analogue containing a stable amino acid-type free radical (spin probe or spin label) that maintains entirely the biological activity of the native hormone. This maintenance of &agr;-MSH biological potency and the presence of the paramagnetic spin label in its structure allow this analogue to be studied through electron paramagnetic resonance (also known as EPR or RPE, depending on the language used, English or Portuguese) and contains relevant potentialities for several additional applications in the biochemical-medical fields, related to the investigation of several already known physiological effects of this hormone.
2. Technical Background and Prior Art
The melanocyte stimulating hormone (&agr;-MSH) seems to be involved in several physiologic processes in higher animals (e.g. The Melanotropic Peptides, Vaudry, H & Eberle, N, eds., New York, 1993). Among these processes one may mention the effect upon the fetal growth, behavior, inflammation (e.g. Drugs of the Future, 15, 41[1990]), obesity [Nature 385, 165 (1997)], erectile function, [J. Urol. 160, 389 (1998)], etc. No matter what, the more relevant effect of this hormone considered as a neuroimunemodulator, is related with the skin darkening effect (The Melanotropic Peptides, New York, [1993]).
The skin darkening of mammals and of other animals is basically controlled by the amount of melanin, a biological compound synthesized from the amino acid tyrosine and mediated by the enzyme tyrosinase. The melanin molecules are stored inside granules of cellular structures denominated melanocytes, and it has been observed that the more aggregated these granules in the cells are, the clearer the individual's skin will become. The control of this granular aggregation in the organism is carried out by a compound known as melatonin (N-acetyl-5-methoxy-tryptamine). Contrariwise, darker skin is due to more dispersed melanin-containing granules in the cells, and this dispersion control in the organism is performed by the aforementioned &agr;-MSH, which is a peptide found in the pituitary gland of several animal species, including the human, and its amino acid sequence is already known, as represented below:
(Acetyl-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-CONH
2
)
Therefore, besides the importance of this hormone to the physiological effects already mentioned, a better understanding of the &agr;-MSH effect can be useful, for example, for possible elucidation of the causes or mechanisms of several diseases regarding irregular pigmentation of the skin. And in some lower animals, this peptide hormone is also very important because it allows alterations of the color of the skin as a function of the ambient luminosity, thus facilitating the survival of some species.
The first chemical synthesis of this peptide was made some decades ago (J. Am. Chem. Soc. 83, 2289 [1961]) and recent researches on this important hormone have aimed at clarifying its action mechanism at the cellular membrane level, because its specific receptor was already characterized and found in different tissues and organs (Science 257, 1248 [1992]), including tumor cells (Proc. Natl. Acad. Sci. USA 93, 13715 [1996]). For this, several approaches are applied, among which many are spectroscopic, so that, besides furnishing conformational information of the hormone in solution, they can also supply details on interaction and positioning in synthetic or natural membranes.
Within this context, a more potent &agr;-MSH analogue has for instance already been labeled with fluorescent probes in specific positions of its sequence for further conformational and structural studies and for detection of its cellular receptor (J. Pharm. Sci. 74, 237 [1985]).
Prior to this application, there has not been published an &agr;-MSH analogue containing a paramagnetic compound (spin probe or spin label) that maintains entirely its original biological activity. The usefulness of hormone labeling with this special type of marker molecule has the advantage of, for the first time, facilitating the application of the RPE method already mentioned [Spin Labeling—Theory and Applications, Berliner, L.J., New York, 1989] for the investigation of this important tridecapeptide hormone.
In contrast with other spectroscopic methods, ESR permits the detection of conformational alterations of the hormone either in solution or associated with macrostructures such as membranes, based on spectral data that monitor the degree of motion of the molecule or of the system where the spin probe is bound. In addition, owing to the fluorescent quenching property of the nitroxide function of the spin label (Biochemistry, 20, 1932 [1981]) the ESR method allows a unique alternative approach for conjugation as compared with the conventional fluorescence method.
However, the most important pre-requisite in any strategy of introducing a spin marker in the &agr;-MSH molecule or in any other biological molecule of interest is the need for maintenance of original biological potency. It is not so probable that it is as different from the radioactive labeling of hormone which does not modify its chemical structure, a non-natural compound and with significant size is being inserted in the structure of the native hormone under study. Besides this pre-requisite, it was also necessary that the introduction of the spin probe in the hormone structure should be in such a way as to reflect closely the peptide conformational features. For this reason, spin labels that bind to the hormone through a great amount of chemical bonds (long spin probes) and therefore, with high rotation freedom were not considered to be very appropriate. This is the case in some examples referred to in the literature where a long and flexible marker was used for ESR study of peptides, e.g. Nature 359, 653 (1992). The ideal case would be, therefore, a paramagnetic probe that binds as rigidly as possible to the peptide structure and directly in its skeleton through a peptide bond as usually happens with amino acid residues.
The inventors initiated the use, some decades ago, of an amino acid-type spin probe abbreviated as Toac (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid)—e.g. Bull. Soc. Chim. Fr. 815 (1967) in the peptide chemistry field, and it seemed to fulfill partially these requirements for binding more rigidly to the structure of the hormone. By containing the amine and carboxylic groups in a same carbon of the heterocyclic Toac structure, this spin label can be introduced as an amino acid directly to the peptide backbone.
To make it possible to couple in a peptide sequence through the classic solid phase peptide synthesis methodology, [e.g. Peptides: Analysis, Synthesis and Biology (Barany, G. and Merrifield, R. B. 1980)], the tert-butyloxycarbonyl group was introduced (Boc) in the Toac amino group function, according to Braz. J. Med. Biol. Res. 14, 173 (1981). Due to the lability of the free radical nitroxide group in a strong acid medium present during the peptide synthesis method (trifluoroacetic acid), its introduction was only possible in the N-terminal position of the peptide structure e.g. Biochim. Biophys. Acta, 742, 63 (1983). Later on, this limitation of the use of ESR in peptides was also overcome by the inventors, when using another Toac-amino group protection, the base labile 9-fluorenylmethyloxycarbonyl (Fmoc), in J. Am. Chem. Soc. 115, 11042 (1993). With this protecting group we demonstrated for the first time in the literature a way of introducing the spin probe Toac at any internal positions of the peptide hormone structure, making possible therefore the substitution of any residue of amino acid of its original sequence for this paramagnetic compound. A great variety of examples of application of this strategy were later published, but none of them had repo
Barbosa Simone dos Reis
Cilli Eduardo Maffud
Lamy-Freund Maria Tereza
Nakaie Clóvis Ryuichi
Carlson Karen Cochrane
Conselho Nacional de Desenvolvimento Cientifico e Technologico P
Liu Samuel Wei
Morrison & Foerster / LLP
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