Invented by Klaus M. Hahn, Alexei Toutchkine, Scripps Research Institute
The Scripps Research Institute invention works as follows
The invention provides peptide synons with protected functional groups that allow attachment of desired moieties (e.g. Functional molecules or probes. These synthons can be used to prepare peptide conjugates. They also provide methods for synthon and conjugate preparation, including the identification of the best probe attachment site. The biosensors include environmentally sensitive dyes which can be used to locate biomolecules in living cells. These dyes can detect chemical and physiological changes as the cell moves, metabolizes and reacts to its environment. Polypeptide biosensors can be used to detect GTP activation in Rho GTPase proteins. The biosensor binds GTP activated Rho GTPase proteins to emit a signal with a different wavelength, intensity, or lifetime than when it is not bound. Fluorophores with improved attachment and detection properties are available that respond to environmental changes. They can be used in living cells or in vitro.
Background for Labeled proteins, peptides and antibodies as well as processes and intermediates that are useful in their preparation
Modified proteins and peptides are useful biophysical tools for studying biological phenomena in vitro as well as in vivo. They can also be used in the identification of new drugs or therapeutic agents. Quantitative live cell imaging with fluorescent proteins and/or peptides has revolutionized the study of cell biology. A new development in this area is the creation of protein and peptide biosensors that exhibit altered fluorescence properties in response changes in their environment, oligomeric status, conformation upon binding, structure or direct ligandbinding. The spatial and temporal detections of biochemical reactions within living cells can be made possible by appropriately labeled fluorescent biomolecules. Giuliano, K. A., and co-authors, Annu. Rev. Biophys. Biomol. Struct. 1995, 24:405-434; Day, R. N. Mol. Endocrinol. 1998, 12:1410-9; Adams, S. R., et al., Nature 1991, 349:694; Miyawaski, A., et al., Nature 1997, 388:882-7; Hahn, K., et al., Nature 1992, 359:736; Hahn, K. M., et al., J. Biol. Chem. 1990, 265 :20335. Richieri, G. V. et al. Mol. Cell. Biochem. 1999, 192:87-94.
Processe for site-specific modification in polypeptides have also been described. These include chemically selective labeling of solution (Brinkley), M. Bioconjugate Chemical 1992, 3:2-13) as well as resin bound peptides. (Hackeng T., et.al., J. Biol. Chem. Submitted; introduction of ketone amino acid through synthetic procedures (Rose K. J. Am. Chem. Soc. 1994, 116:30-33; King, T. P., et al., Biochemistry,1986, 25:5774-5779; Rose, K., et al., Bioconjugate Chem. 1996, 7:552-556; Marcaurelle, L. A., Bertozzi, C. R. Tett. Lett. 1998, 39.7279-7282. Wahl, F., Mutter and M. Tett. Lett. 1996, 37.6861-6864); molecular biology techniques (Cornish V. W. et al, J. Am. Chem. Soc. 1996, 118:8150).
Each of these methods is useful for producing a specific class of labeled biosensors or polypeptides, but they all have limitations that limit their general use. Because of the presence of other nucleophiles, labeling natural amino acid side-chains in solutions is often difficult. Unnatural amino acids such as those that bear ketones must be used for selective labeling. This requires the creation of dye constructs and other amino acids that are not commercially available.
Currently, there are two major hurdles to developing fluorescent biosensors: (1) the difficulty in placing the dye in the polypeptide at the right site and (2) the difficulty in determining the optimal site for placement (Giuliano K. A. et al. Annu. Rev. Biophys. Biomol. Struct. 1995, 24:405-434). For optimal responses to protein structure changes, biophysical probes and solvent-sensitive dyes, they must be precisely placed. This will ensure that the probes are not interfering with biological activity. The requirement for site-specific incorporation (within a protein) of two dyes has been a significant limitation in fluorescence resonance energy transport (FRET). These problems can be solved by total chemical synthesis of proteins (Wilken J., Kent S. B. H. Curr. Op. Biotechnology. 1998, 9:412; Kent, S. B. H. Ann. Rev. Biochem. 1988, 57, 957-989; Dawson, P. E., et al., Science 1994, 266:776-779; Muir, T. W., et al., Proc. Natl. Acad. Sci. 1998, 95.6705-6710; Cotton, G. J. et al. J. Am. Chem. Soc. 1999, 121:1100-1101). Many biophysical probes that are suitable for fluorescent biosensors and other purposes are not stable under the conditions of peptide synthesis. It has been hard to obtain site-specific incorporation.
Labeling with hydrophobic dyes like thionine and methylene blue can pose a problem because these dyes tend to autoaggregate in an aqueous solution at high levels. See, J. Am. Chem. Soc. 63, 69 (1941). These aggregates can cause a shift in the absorption spectrum as well as a decrease in fluorescence. The formation of aggregates in merocyanines and cyanines is also believed to cause a decrease in fluorescence (J. Phys. Chem. 69, 1894 (1965)). This aggregation can cause problems with the conjugation of fluorescent dyes to other molecules, such as proteins. After the dyes have been conjugated, aggregation can be exacerbated by merocyanines or cyanines. For example, Waggoner et al. After conjugation of cyaninisothiocyanate and an antibody, a phenomenon called aggregation has been observed (Cytometry 10, 11-19 (1989). The fluorescence of a conjugate of a cyanin fluorescent color and an anti-HCG antibodies (molar ratio=1.7), is less than that of the unbound cyanin (see U.S. Patent. No. No. Biochem. 217, 197-204 (1994)). Cyanines are stable, cheap, easy to conjugate with other molecules, and small enough to recognize small molecules. However, their fluorescence does not change in response to environmental factors such as solvent polarity. These problems can be eliminated by new dyes.
There is a current need for new fluorescent dyes or peptide synthons with protected functional groups that can selectively be modified to include one or more functional molecules (e.g. After peptide synthesis, a fluorescent label is applied. It is also important to have biophysical probes attached at precise locations and simple methods for making these labeled proteins or antibodies. These labeled proteins, peptides and antibodies in vivo are also required using simpler methods.
The present invention is a highly efficient way to attach biophysical probes and other molecules to unprotected proteins after chemical synthesis. This method uses amino acids with one or more protected aminooxy group, which can either be added during solid-phase peptide synthesis or can be combined through post-expression steps. The protected aminooxy group can be removed after peptide synthesis and then reacted with an electrophilic agent to produce a modified (e.g. a labeled) peptide. Electrophiles such as lysine and cysteine react selectively with the aminooxy group. an activated carboxylic ester such as an N-hydroxy-succinimide ester) in the presence of other nucleophilic groups including cysteine, lysine and amino groups.
Thus, selective peptide modification (e.g. After synthesis with commercially available, and/or chemically sensitive molecules (e.g. probes). This method is compatible with the synthesis C?-thioester-containing peptides, amide-forming and other steps required for the synthesis proteins by total chemical synthesis (or expressed protein ligation). Parallel peptide synthesis allows for the introduction of an aminooxy-containing amino acid into multiple sites to create a polypeptide family. Each member has a specific labeled site. Parallel synthesis allows for the creation of optimized biosensors and other modified polypeptides by combinatorial screening different attachment sites. This will allow for maximum response and minimal perturbation to desired biological activity.
Thus, site-specific modification (e.g. The labeling (or labeling) of peptides has been made possible by a simple and efficient method that allows for high yield, selectivity, compatibility, and both solid-phase and C?-thioester-peptide recombinant syntheses.
Accordingly, this invention provides a synthetic intermediary (i.e. A synthon is useful for the preparation of modified peptides. It is a compound with formula (I).
Peptides containing one or more aminooxy group are useful synthetic intermediates that can also be modified to produce related peptides with altered biological, chemical or physical properties such as a peptide linked with a fluorescent label. The invention also allows for a peptide to have one or more of the following (e.g. 1, 2, 3 or 4 aminooxy groups, provided that the peptide does not contain glutathione. The invention also allows for a peptide to have one or more of the following (e.g. 1, 2, 3 or 4 secondary aminooxy groups.
The invention provides intermediates and methods that permit site-specific modification after synthesis. Functional molecules can be linked to a specific peptide to create a peptide combination with altered biological, chemical, and physical properties. Functional molecules, such as peptides, polynucleotides and therapeutic agents (e.g. For example, functional molecules (e.g.
Thus, the invention also provides an inventive compound of formula (III).
Processes for making synthons of invention, as well as polypeptides, antibodies and protein conjugates of invention, are further embodiments. They are illustrated in the Examples below.
The invention also includes a method of preparing a protein conjugate consisting of a functional molecule and a propeptide. This involves reacting one or more aminooxy group peptides with an electrophilic moiety to create the peptide conjugate.
The present invention also provides environment-sensing colors that can be easily conjugated to proteins and other molecules, without any problems such as fluorescence quenching or aggregation. The present dyes possess bright fluorescence and environmentally-sensitive fluorescence changes suitable for use in living cells. These dyes are more sensitive than cyanine dyes and can be used to make biosensors that report on many aspects of protein behavior or other molecules. The distribution of charged or hydrophobic residues can be affected by protein behaviors, including conformational changes, phosphorylation states, ligand interaction and other post-translational modifications. These changes can be reported by dyes with changes in fluorescence.
The present invention overcomes the disadvantages of the available environmentally-sensitive fluorescent dyes. Fluorescence levels of the present fluorescent probes are high before and after conjugation with other molecules, such as proteins and antibodies. These fluorescence changes can be used for many purposes including in-vivo and in vitro studies of protein behavior.
The invention presents new fluorescent dyes that are easily used by anyone skilled in the art. Any available method can link the dyes to any useful molecule. One embodiment of fluorescent dyes is linked to peptides and polypeptides using the methods described herein. These dyes have the following structure (“IV”).
R9 and R10 are charged chains that enhance water solubility (i.e. “R9 and R10 are chains carrying charged groups to enhance water solubility (i.e. A reactive group is a functional element that is chemically reactive (or can be made chemically responsive) with functional groups commonly found in biological materials or functional groups that are easily converted into chemically reactive derivatives by well-known methods. The charged and reactive groups can be separated by haloacetamide(?NH?) in one embodiment. (C?O)?CH2?X), where X is Cl, Br or I. Alternatively, the charged and reactive groups are separately amine, maleimide, isocyanato (?N?C?O), isothiocyanato(?N?C?S), acyl halide, succinimidyl ester, or sulfosuccinimidyl ester. Another embodiment uses the reactive and charged groups as carboxylic acids (COOH) or derivatives thereof. A suitable derivative of a carboxylic acids is an alkali, alkaline earth metal salt. Alternately, the charged or reactive groups can be reactive derivatives (?COORx) of a carboxylic acids. The reactive group Rx activates the carbonyl of?COORx towards nucleophilic displacement. Rx, in particular, is any group that activates carbonyl toward nucleophilic displacement but not being incorporated into final displacement product. Examples of COORx include naphtol and ester of phenol that has been substituted with at least one strong electron withdrawing or carboxylic acid activated by carbodiimide or acyl chloride or succinimidyl, or sulfosuccinimidyl esters. Other charged and reactive groups include, but are not limited to, sulfonyl halids, sulfonyl azides or alcohols.
The invention further provides a method for identifying the optimal position to place a functional mole on a protein with a peptide backbone that has a known activity. This involves making a series peptide concoctions, each one having the same amino acids sequence and the same functional mole, and then observing which functional group location is not significantly affecting the peptide’s activity.
The invention also provides a method for identifying the optimal position to place a functional molecular in a protein with known activity and an identified segment for attachment. This involves creating a series peptide conjugates, each with the same amino acid sequence as the identified segment, and replacing each identified segment in a series proteins with a peptide combination selected from the series. Each protein will then have a unique functional group, and it is monitored that the location of the functional group does not significantly interfere with the protein’s activity
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