Synthesis of a novel paramagnetic amino derivative (epm-5)...

Details Synthesis of a novel paramagnetic amino derivative (epm-5)... Synthesis of a novel paramagnetic amino derivative (epm-5)...

2001-12-21

2004-09-28

McKane, Joseph K. (Department: 1626)

Organic compounds -- part of the class 532-570 series

Organic compounds

Heterocyclic carbon compounds containing a hetero ring...

C548S531000, C530S300000

Reexamination Certificate

active

06797829

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to the chemical synthesis of a new paramagnetic &bgr;-amino acid derivative containing a stable nitroxide radical moiety inserted in its pyrrolidine structure and in which the 9-fluorenylmethyloxycarbonyl group (Fmoc) was chosen as its amine function protecting group. This paramagnetic compound is a new type of spin probe (or spin label) and can be used as alternative report molecule for labeling peptide sequences, other macromolecules and systems where electron paramagnetic resonance spectroscopy (EPR) can be applied. This compound can be used also for other spectroscopic methods such as fluorescence and nuclear magnetic resonance since its paramagnetism may affect the spectra of these methodologies. Due to the presence of both carboxyl and amine groups in its structure, this organic compound may be used for labeling a great variety of other molecules or systems containing reactive functions for these two groups.
BACKGROUND AND SUMMARY OF THE INVENTION
The intermediate for the synthesis of the amino acid derivative of this invention contains the structure 2,2,5,5-tetramethylpyrrolidine-1-oxyl-3-amine-4-carboxylic acid, henceforth denominated as Poac, synthesized more than two decades ago (see, Tetrahedron 491-499 [1965] and Bull. France, 3, 815-817 [1967]). The Poac derivative containing the Fmoc protecting group (2,2,5,5-tetramethylpyrrolidine-1-oxyl-3-(9-fluorenylmethyloxycarbonyl) -amine-4-carboxylic acid is the novel spin probe derivative of the present invention, which also includes the synthesis and use in EPR of this spin probe derivative. This new compound allows the insertion of Poac as a usual amino acid at any position of a peptide sequence, and its denomination will be Fmoc-Poac or EPM-5 in this application. The chemical structure of this paramagnetic molecule is represented in FIG.
1
.
Electron paramagnetic resonance (EPR) [described in
Biological Magnetic Resonance
, Berliner, L. J. and Reuben, J., eds., Plenum Publishing, New York, (1989)], is a modern and very usefull spectroscopic method because it allows the study of paramagnetically labeled macromolecules or biological systems regarding their conformation, mobilities, inter- or intra-molecular interactions, structuring states, and the like. The wide spectrum of EPR application is already detailed in the literature (see,
Free Nitroxyl Radical
, Rozantsev, E. G., Ulrich, H., ed., Plenun Press, London, 1970), where a great variety of spin labels, i.e., chemical compounds which are paramagnetic due to the presence of an unpaired electron in their structure, is listed. They are, therefore, a class of free radicals but are necessarily stable under conditions around normal temperature and physiological pH and also allow several chemical reactions or experiments without affecting their free radical moiety.
Amongst the most commonly used spin labels one can pick out the nitroxide group-containing molecules and where the unpaired electron locates. The most significant progress in the EPR field for labeling of relevant biological structures such as peptides and proteins was achieved with this class of spin probes. Almost two decades ago appeared the first EPR application in the solid phase peptide synthesis methodology [(
The Peptides: Analysis, Synthesis and Biology
, vol. 2, Academic Press, New York, (1980)]. This approach was introduced by our group using, instead, another nitroxide-containing spin label, denominated at that time as Toac (2,2,6,6tetramethylpiperidine-1-oxyl-4-amine-4-carboxylic acid), which protected its amine function with the acid labile tert-butyloxycarbonyl (Boc) group [(see, Braz. J. Med. Biol. Res. 14, 173, (1981) and Biochim. Biophys. Acta, 742, 63, (1983)]. Thus the Boc-Toac spin probe was the first in the literature used to label a peptide sequence as an amino acid. However, due to chemical particularities of the peptide synthesis methodology, it was only possible to couple the Toac group at the peptide N-terminal position. To overcome this shortcoming, an alternative strategy was published by us which finally allowed the insertion of the spin label internally to the peptide sequence [see, J. Am Chem. Soc. 115, 11042 (1993)].
An impressive increase in the application of EPR for peptide chemistry field was further observed in reports investigating peptide conformational properties [v.g J. Am. Chem. Soc. 117, 10555 (1995); FEBS Lett. 375, 239 (1995); Biopolymers 42, 821 (1997)]or of peptidyl-resin solvation (Tetrahedron Lett. 375, 239 [1995]; Biopolymers 42, 821 [1997]). As an amino acid, Toac was introduced in different positions of some biologically active peptides such as angiotensin II and bradykinin, but a partial or total loss of their biological potencies were observed due to the introduction of a non natural compound in their structures. [
Peptides
1996, R. Ramage and R. Epton, eds., Mayflower Scientific Co. p.673 (1998)].
However, we recently described the synthesis of a peptide hormone labeled with Toac where its biological potency was entirely preserved. [e.g., FEBS Lett. 446, 45 (1999)]. This result was obtained with the tridecapeptide &agr;-melanocyte stimulating hormone analogue, owing to its potentialities in a great number of chemical-biological assays (this analogue is paramagnetic, naturally fluorescent and fully active, and was disclosed in commonly owned U.S. patent application Ser. No. 09/935,760, entitled “Paramagnetic And Active Analogue (EPM-2) Of Melanocyte Stimulating Hormone Containing Amino Acid-Type Stable Free Radical”).
In spite of these results, one still remaining shortcoming in the use of Toac in peptide chemistry is the severe difficulty in coupling the subsequent amino acid residue of the peptide sequence during the synthesis. This difficulty seems to be due to the low nucleophilicity of the Toac amine group, whose pKa of 8 (when in free state) decreases to about 5.5 when bound to the N-terminal portion of a peptide chain [v.g Braz J. Med. Biol. Res. 14, 173 (1981) and Biochim. Biophys. Acta. 742, 63 (1983)]. Several recouplings and an increase in the temperature of the coupling reaction are usually necessary to assure complete incorporation of the subsequent amino acid of the desired peptide sequence [J. Am. Chem. Soc. 115, 11042 (1993)].


REFERENCES:
patent: 3966764 (1976-06-01), Goldstein et al.

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