Polyamide chains of precise length

Details Polyamide chains of precise length Polyamide chains of precise length

1999-08-23

2003-04-22

Russel, Jeffrey E. (Department: 1654)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

15 to 23 amino acid residues in defined sequence

C525S420000, C528S335000, C530S327000, C530S328000, C530S345000, C530S409000

Reexamination Certificate

active

06552167

ABSTRACT:

TECHNICAL FIELD
The present invention relates to polyamide chains of precise length (i.e., a precise number of monomer units) and methods for their preparation. More particularly, the present invention relates to methods for chemically modifying target molecules, e.g. macromolecules, particularly biologically important polypeptides, and surfaces (e.g., gold or glass) by means of covalent attachment of polyamide chains of precise length. Even more particularly, the invention relates to polyethylene glycol-based chains of precise length.
BACKGROUND
It is well recognized that the properties of numerous materials such as peptides, polypeptides such as proteins, and bioconjugates, can be enhanced by grafting organic chain-like molecules onto them. Such grafting can increase the usefulness of a material as a linker to connect multiple copies of a structural motif, increase a material's shielding from the immune system, and increase the half-life of a material. Biosensor surfaces may also be enhanced by first grafting organic chain-like molecules onto the surface (usually gold or glass) prior to covalent attachment of biomolecules, for example, the dextran coated sensors sold by Biacore AB (Sweden).
The organic chain-like molecules often used to enhance properties are polyethylene glycol-based or “PEG-based” chains, i.e., chains that are based on the repeating unit —CH
2
CH
2
O—. See for example, Tsutsumi, et al.,
Jpn. J Cancer Res
. 85:9-12 (1994), where an ester of monomethoxy polyethylene glycol was shown to increase the potency of human tumor necrosis factor-&agr;. PEG-based chains are flexible, amphiphilic, non-immunogenic and not susceptible to cleavage by proteolytic enzymes. Preparations of materials that have been modified by PEG or PEG-based chains, have reduced immunogenicity and antigenicity. See for example, Abuchowski, et al.,
Journal of Biological Chemistry
252(11):3578-3581 (1977), where PEG was shown to alter the immunological properties of bovine serum albumin. PEG also serves to increase the molecular size of the material to which it is attached, thereby increasing its biological half-life. These beneficial properties of the PEG-modified materials make them very useful in a variety of therapeutic applications.
The grafting of PEG chains or PEG-based chains onto proteins is known. See for example, Zalipsky, U.S. Pat. No. 5,122,614, which describes PEG that is converted into its N-succinimide carbonate derivative. Also known are PEG chains modified with reactive groups to facilitate grafting onto proteins. See for example, Harris, U.S. Pat. No. 5,739,208, which describes a PEG derivative that is activated with a sulfone moiety for selective attachment to thiol moieties on molecules and surfaces and Harris, et al., U.S. Pat. No. 5,672,662, which discloses active esters of PEG acids that have a single propionic or butanoic acid moiety. This area is extensively reviewed in Zalipsky,
Bioconjugate Chemistry
6:150-165 (1995). Besides use of PEG, Wright, EP 0 605 963 A2 describes linking reagents that contain water soluble polymers that form a hydrazone linkage with an aldehyde group on a protein.
Polyamide chains also are useful as organic chain-like molecules to enhance properties. In addition, acute toxicity screening in rodents suggests that polyamides are neither toxic nor immunogenic (Hai, et al.,
Bioconj. Chem
. 9:645-654 (1998)).
Problems are encountered, however, since state of the art technology does not provide for the synthesis of organic chain-like molecules having a determinable length.
Techniques used to prepare PEG or PEG-based chains, even those of fairly low molecular weight such as 3400 (see for example, Kramer, et al.,
Nature
395:710 (1998)), involve a poorly-controlled polymerization step which leads to preparations having a spread of chain lengths about a mean value, i.e., they involve polymer preparations of —(CH
2
CH
2
O)
m
— where m does not have a discrete value but rather, has a range of values about a mean. This is very evident in mass spectra of PEG chains themselves and of compounds to which PEG chains have been grafted. For example, in Johnson, et al.,
Chemistry
&
Biology
4:939 (1997), PEG chains of nominal relative molecular mass 3400 and 5000, when grafted onto a small peptide, give rise to products with mass ranges of mean ±1000, i.e., a range of 2000 amu. This is a typical result of state of the art methods. When sufficient mass resolution is available, the spectrum shows many signals spaced 44 amu apart. See, for example, Lu, et al.,
Int. J. Peptide Protein Res
. 43:127-138 (1994). This large range in mass corresponds to a corresponding range in chain lengths. Accordingly, products containing such PEG or PEG-based chains are not homogeneous and consist of molecules possessing short, medium and long chains. The situation is worse for compounds possessing two PEG chains as, statistically, they must consist of a mixture of molecules possessing two short chains, a short and a long chain, and two long chains, so the variance in mass is larger than for products which have only one chain. Since chain length affects mass, biological half-life, shielding from the immune system, and spacing of subunits when such a chain is used to link two moieties (as in Johnson, et al., Chemistry & Biology, supra), the biological effect of a compound which possesses one or more conventional PEG chains is that of an average of the effects of the individual species present (those with short chains, those with medium chains and those with long chains) and their relative concentrations (which change with time in principle as the biological half-life is a function of mass for a set of similar compounds). This complex situation is tolerated because PEG is very useful.
Solid phase peptide synthesis yields well-defined polyamides of the repeating unit “—NH—Y—CO—” but requires protected derivatives and deprotection steps. Polyamides of the repeating unit —NH—Y—NH—CO—X—CO—, such as “—NH—(CH
2
)
6
—NH—CO—(CH
2
)
4
—CO—” (e.g. Nylon 66) are made by polymerizing diacids with diamines. This synthesis, along with more recent techniques involving solution polymerization of diacids with diamines, yields a product having a wide range of chain lengths. The absence of protecting groups is largely responsible for the resulting heterogeneity of chain length in these polyamides.
In light of the many potential applications of materials modified with organic chain-like molecules, there is a need in the art for improved chains for use in modifying target macromolecules or materials, such as surfaces. Accordingly, there is a need for a method of producing such polymers, but having a determinable length, by automated solid phase synthesis without the need for protecting groups. In particular, there is a need for PEG-based chains of precise length (i.e., chains containing a —(CH
2
CH
2
O)
m
— group, where m has a single value), as well as methods for constructing such chains. In this manner, the disadvantages inherent in and observed for the currently used PEG chains, both for medical and non-medical uses, can be overcome. The polyamide chains of precise length and methods provided in the present invention meet these needs and others as well.
SUMMARY OF THE INVENTION
The present invention relates to a new class of polymers, polyamide-based chains containing a precise number of repeating monomer units (—NH—Y—NH—CO—X—CO—), which are synthesized by automated solid phase synthesis without the need for protecting groups and where X and Y can be varied independently at each step. Dimers, branched constructions and multimeric molecules which display phage-derived binding peptides, are easily assembled with these precision length polyamide chains, which can usefully replace polypeptides and polyethyleneglycol as molecular spacers.
More particularly, the present invention provides a water soluble organic polyamide-based chain of precise length having a precise number of repeating units, based upon the building up by amide bond formation of a precise number of monomer units, a

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