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BIOCHEMISTRY

An advance in the study of peptides

Work of Brazilian group is internationally recognized

Three Brazilian research scientists have won world renown in today’s strategic sector of biochemistry: they have developed techniques to analyze more efficiently the molecular structure of proteins and peptides (comprising amino acids produced synthetically or by the hydrolysis of proteins). Their discoveries, begun in the decade of the eighties, were described in the May 29 issue of the prestigious magazine “Chemical & Engineering News” (pages 54-59), of the American Chemical Society.

The work of Clóvis Nakaie, head of the Biophysics Department of the Federal University of São Paulo (Unifesp), Shirley Schreier, of the Biochemistry Department of the University of São Paulo (USP), and Antônio Cechelli de Mattos Paiva, Professor de Biophysics of Unifesp, facilitates the study of the molecular configuration of proteins and peptides as well as the interaction of these compounds among themselves and with cellular membranes. The results have many applications, among which is the possible creation of therapeutic drugs.

The objective was to perfect the method that uses spin labels (direction of the axis of rotation of the electron). Inserted inside a molecule, these spin labels allow the researcher to obtain data on molecular configuration and interactions between the two, through the spectroscopy technique using the electron paramagnetic resonance method (EPR).

It is a difficult, time consuming and expensive study, above all due to the complexity of the peptides, which are formed by a chain of amino acids. Each amino acid has a lateral chain (r), which hangs from the main chain of the peptide.

The conventional EPR method was to connect a spin label to a chain r to obtain a signal. However, using this method, the spin label remained on the periphery of the main chain, the major focus of the study and the information was masked by the high degree of mobility of the lateral chains, making precise data difficult to obtain.

New spin label
The group’s major breakthrough was to use a spin label that can be linked to the main chain. The spin label is the TOAC (acid 2,2,6,6-tetrametylpiperidine-1-oxyl-4-amino-4-carboxylate), the only molecule known that is at the same time both an amino acid as well as very stable free radical. As an amino acid, it can be introduced into peptides, proteins and other macromolecules, and the fact that it contains a nitrous oxide radical, allows it to function as a spin label.

Being a paramagnetic compound (since it has an unpaired electron), the TOAC allows the EPR spectroscopy to study the structural and dynamic properties of the isolated spin label and the molecule to which it is coupled as well as the effects of changes in the molecule’s environment.

As an example, with the spectra obtained using the EPA method, it is possible to observe changes in the configuration when a compound connects to a biological membrane or model. Thus the connection of spin label allows us to obtain data on the degree and nature of the movement, of the configuration and of the molecular structuring or of the system being investigated.

Challenge overcome
The TOAC has the advantage of being an amino acid and the disadvantage of being a free radical – and therefore exposed to chemical breakdown, according to its environment. This was the major challenge when the idea was developed of linking it to peptide chains through the synthesis methodology and peptide chemistry, techniques which the group has already mastered.

Paiva, a specialist in the chemistry of peptides and Shirley, in whose laboratory the EPR technique is much used, discussed the problem together with the participation of the then post-graduating Nakaie. From these discussions, and the initial promising results, the technique which permitted the incorporation of TOAC in the peptide chain and the study of the peptide configurations has surged, developed in1980 in the Ph.D. thesis of Nakaie.

The advance made by Nakaie consisted in finding a sequence of procedures (or protocols) for chemical research that enabled the TOAC to be linked to the tip of a peptide. Due to its chemical structure and rigidity of its connection to the peptide chain, this spin label supplies more precious data on the configuration of the peptide than those obtained with spin labels used up to then, normally linking only the lateral chains of the amino acids.

Special molecule
Paiva clarifies: “We developed the synthesis and the application of a special molecule that is incorporated in the peptide in a very closely fitting and intimate manner. From this point on, we could deal with peptide sequences, the dynamic of which would be faithfully recorded by an efficient spin label”.

“Up to then”, says research scientist Shirley, “no one had proved that it was possible to use peptide spin labeling with a paramagnetic amino acid such as the TOAC. Additionally, we also showed that the EPR spectrum of the TOAC or the TOAC-peptide was affected by the pH (acid ratio) level, resulting from the ionization of the amino grouping of this spin label. This result left in the air, therefore, the intriguing possibility that this spin label or the spin labeled peptide could act as pH sensory devices in the environment where they are to be found.”

After completing his doctorate, Nakaie continued to follow this line of research and has been responsible for the main advances in the development of strategies for synthesis, labeling and the use of labeled peptides with TOAC and derivatives in the last few years.

Uncommon strategy
“Having overcome the first challenge”, says Nakaie, “ another one which was more complex arose: ideally TOAC being an amino acid, it should be possible for it to be inserted in the internal positions of the peptide sequences as well and not just on the tips. However, for reasons of chemical instability in the synthesis procedure used, this was not possible at the time. In the necessary synthesis strategy for the introduction of the spin label into the middle of the chain, there was an irreversible decomposition in the nitrous oxide group of the TOAC, making it impossible to obtain the EPR spectra.”

Only in the following decade (in 1993), with the advances in the chemistry of peptides, did Nakaie and his post graduate student, Reinaldo Marchetto – today teaching at the Institute of Chemistry of the  São Paulo State University in Araraquara – manage to solve the problem: they combined steps of the two methods of peptide synthesis, introduced an alkaline treatment process at the end stage of the synthesis and thus obtained the first peptide labeled with TOAC in an internal position of the sequence (TOAC7-angiotensin II).

The last barrier was crossed for using TOAC with any amino acid and opening up the possibility of inserting it in any position in the chain, thus enormously widening its potential for use.

“So, we sent the results of our work to an important specialized magazine in Chemistry (“Journal of American Chemical Society”). The article was accepted with a recommendation for urgent publication, thus clearly showing its importance”, comments Shirley.

With funding from FAPESP, Nakaie has carried on with his research to improve the synthesis of peptides in resins and to verify with the TOAC, how these resins behave under different situations. The spin labeling by TOAC allows the synthesis to be closely monitored – work which is very expensive. The monitoring for example, allows changes to be made in the nature of the solvent, choosing phases in which the signal of the spin label indicates greater mobility (or solubility) of the compound, which improves the results.

Nakaie explains: “There are solvents where the grain of resin remains ‘packaged’ and others in which the grain ‘loosens’ to a greater extent, the chains become freer and the reaction more rapid and efficient”. With greater mobility, the peptide synthesis becomes cheaper. These most recent results were the theme of a publication in 1999.

Biological relevance
Proteins and peptides have a major physiological importance. The study of the way they interact with other peptides, proteins, carbohydrates, antibodies and the varied components of the cellular membrane, can give us essential information at the molecular level to understand how hormones, enzymes and other compounds work. This may lead to the development of new therapeutic drugs.

Studies are also being intensified in the direction of relating configuration and biological function of biologically active peptides such as hormones involved in the maintenance of life or what influences the cause of illnesses. Shirley underlines: “The physiological function of a compound is the consequence of its structure and for this reason, these studies are important”.

It is in this context that the research of the scientists is highlighted and recognized in the article in “Chemical & Engineering News”. The article describes the scientists as pioneers in a line of research also followed over the past twenty years by others – particularly the groups led by Claudio Toniolo, of Rome University and G.L. Millhauser, of the University of California.

Repercussion
Besides attracting the attention of American and European scientists, the group’s work – pioneer in the simplification and cheapening of the peptide synthesis – has had repercussions in the private sector: Toronto Research Chemicals of Canada invested in spin labeling and began the production of TOAC.

The only regret of the research scientists is that the technique was never patented in 1993, when the discovery was published in the “Journal of American Chemical Society”.  “At the time, no one talked of patents here. It was something which never entered our heads”, explains Nakaie. However, in the last few years the situation has changed and he has patented the more recent stages of the work.

Profiles:
• Antônio Cechelli de Mattos Paiva graduated in medicine at the Escola Paulista de Medicina (1952) ( São Paulo School of Medicine, where he was head professor of Biophysics (1966-1998). He was also head of Biochemistry at the Institute of Chemistry of USP (1980-1984).
Project: Relations between Structure and Activity of Coupled Receptors to G Proteins: Receptors of Vasomotor Peptides
Investment: R$ 331,886.26 and US$ 130,840.00

• Clóvis Ryuichi Nakaie, graduated in Pharmaceutical Sciences (1974) and has a Ph.D. qualification in Biochemistry (1980) from the Escola Paulista de Medicina, now the Federal University of São Paulo (Unifesp). Since 1978, he has been a professor at Unifesp, where he heads up the Biophysics Department.
Project: Synthesis and Physical Chemistry Studies of Styrene Copolymers as Matrices for Peptide Synthesis and Liquid Column Chromatography
Investment: R$ 65,858.45 and US$ 107,779.00

• Shirley Schreier graduated in Chemistry at USP (1962), where she also became a professor (1965) gained her Ph.D. (1969) and became head of the Biochemistry Department of the Chemistry Institute (1990). She was a visiting research scientist at various centers overseas, notably at the National Research Council of Canada in the seventies.
Project: Local Anesthetics: Synthesis, Structural and Physical-Chemical Properties and Interaction with Model and Biological Membranes
Investment: R$ 206,415.79 and US$ 259,048.91

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