Molecular rod and uses

Details Molecular rod and uses Molecular rod and uses

2001-02-06

2002-06-11

Riley, Jezia (Department: 1656)

Synthetic resins or natural rubbers -- part of the class 520 ser

Synthetic resins

Mixing of two or more solid polymers; mixing of solid...

C435S006120, C435S007100, C435S007200, C526S076000, C526S072000, C525S160000, C525S141000, C525S119000, C525S095000, C525S089000, C525S073000, C525S007000

Reexamination Certificate

active

06403705

ABSTRACT:

This application is a National Stage Application under 35 U.S.C. §371 of PCT/FR99/01207 filed May 21, 1999. This application claims priority under 35 U.S.C. §119 to France 98/06540 filed May 25, 1998, the entire contents of which are herein incorporated by reference.
The present invention relates to molecular rods, to their uses in a method of attaching and/or crystallizing macromolecules, to the resulting products and to the applications of said products in the field of materials and structural biology, especially as biosensors or biomaterials.
Knowledge of the structure of proteins and especially their active sites is essential to an understanding of their mechanism of action. Several methods are available for conducting such studies: X-rays, NMR and electrocrystallography (2D crystallization).
To perform the actual crystallization, the technique of two-dimensional crystallization on a lipid monolayer or film at the air/water interface (E. E. Ugziris et al., Nature, 1983, 301, 125-129) makes it possible to form auto-organized systems of biological macromolecules (crystals) and determine the structures of these molecules by electron microscopic analysis of the crystals obtained.
This method consists in creating a lipid monolayer at an air/liquid interface, the lipids being selected to interact with the proteins, present in the liquid phase, which attach themselves to the lipids and then form an organized network.
Attachment of the proteins to the lipids of the monolayer involves chemical interactions at the polar head of the lipids. These interactions are either nonspecific—in which case the lipids possess charged polar ends giving rise to crystallization by ionic interactions—or specific. In the latter case, the polar head of the lipids carries ligands with a strong affinity for the proteins to be attached.
In particular, it has been possible to show that soluble proteins can crystallize two-dimensionally on charged lipid films or lipid films functionalized by a ligand of the protein studied (B. J. Jap et al., Ultramicroscopy, 1992, 46, 45-84).
More recently, lipids functionalized by metal complexes such as nickel complexes (E. W. Kubalek et al., J. Struct. Biol., 1994, 113, 117-123) have made it possible to crystallize so-called histidine-tagged fusion proteins. These proteins in fact possess a sequence composed of several histidines at their N-terminal or C-terminal end. It has been possible to show that the attachment of such proteins to a nickel-functionalized lipid is due to a strong interaction between the nickel complex and the polyhistidine sequence (C. Vénien-Brian et al., J. Mol. Biol., 1997, 274, 687-692). Such functionalized lipids have enabled crystallization to be achieved, especially in cases where the appropriate ligand was not available.
However, the crystallization of proteins on lipid films has the disadvantage of being relatively random and of being dependent on numerous factors which cannot easily be controlled simultaneously:
The ligand carried by the lipids must be sufficiently accessible to be able to interact with the proteins. This accessibility depends on the length of the spacer arm between the lipid and the ligand: if it is too short, it allows the protein to penetrate inside the lipid layer; if it is too long, it gives the bound protein too great a degree of freedom and increases the incidence of defects in the crystal.
The lipid monolayer must be sufficiently fluid to give the bound protein a sufficient lateral and rotational mobility, thereby enabling the proteins to organize themselves relative to one another and develop intermolecular contacts so as to produce the crystal.
Another difficulty inherent in crystallization on a lipid monolayer concerns the stability of the monolayer; in fact, the stability of the air/liquid interface is difficult to control. In addition, the lipid monolayer must remain stable, not only before but also after attachment of the proteins, in order to allow spatial organization of the proteins.
For the microscopic study, which follows the crystallization step, it is necessary to create a large number of planes because of the planar nature of the structure obtained.
Consequently, the Inventors set out to provide structures, hereafter called molecular rods, which were suitable for attaching and crystallizing biological macromolecules in solution, as well as a method of attaching said biological macromolecules in solution and optionally of inducing their auto-organization, said method satisfying the practical needs better than the 2D crystallization methods used in the prior art.
The present invention relates to molecular rods, characterized in that they have a structure represented by the following general formula I:
in which:
P is a polymer selected from the group comprising polyphenylenes, polyphenylenevinylenes, polyphenyleneethynylenes and polyvinylenes, as illustrated by the formulae below:
in which:
A is a hydrogen atom or one of the following groups: alkyl, OH, O-alkyl, NH
2
, NH-alkyl, CO
2
H, CO
2
-alkyl, CONH
2
, CONH-alkyl;
GpF (functional group) is a group B—R, in which:
B (bonding arm) is selected from C
1
-C
10
hydrocarbon groupings which are optionally substituted by alkyl groups, may or may not have units of unsaturation or polyoxyethylene units and may or may not have phosphate groups in the middle of the chain, such as:
in which:
m is an integer from 1 to 10, and
X is O, NHCO, OCO, COO, CONH, S, CH
2
or NH and constitutes, at the ends of said hydrocarbon groupings, organic coupling groups of the ester, amide, ether or thioether type; and
R is a hydrophilic group selected from positively or negatively charged groups; ligands or analogues of biological macromolecules such as, without implying a limitation, biotin, novobiocin, retinoic acid, steroids or antigens; or organometallic complexes interacting with amino acids or nucleic acids, such as complexes of copper, zinc, nickel, cobalt, chromium, platinum, palladium, iron, ruthenium or osmium with ligands like IDA, NTA, EDTA, bipyridine or terpyridine, said ligands optionally being functionalized by alkyl groups for bonding to E (at X); without implying a limitation, positively or negatively charged groups are understood as meaning ammonium, carboxylate, phosphate or sulfonate groups; the following groups may be mentioned as examples: —N(CH
3
)
3
+
or —CO
2
−
;
n is an integer between 5 and 1000;
p is an integer between 0 and 10; and
E (spacer segment) is a chemical unit whose nature does not disturb the rigid structure of the skeleton formed by P, and is a phenylene, ethynylene or vinylene unit or a combination of these units, such as phenyleneethynylene, as illustrated by the formula below:
in which A is a hydrogen atom or one of the following groups: alkyl, OH, O-alkyl, NH
2
, NH-alkyl, CO
2
H, CO
2
-alkyl, CONH
2
, CONH-alkyl.
Together with GpF and E, the different polymers P as defined above give the following formulae:
In terms of the present invention, alkyl is understood as meaning linear or branched or optionally substituted C
1-C
6
alkyl groups.
The substituents of the C
1
-C
10
hydrocarbon groupings B are selected particularly from C
1-C
6
alkyls.
Polymers whose skeleton has a large number of units of conjugation (polyphenylene, polyphenylenevinylene, polyphenyleneethynylene) have already been described (Angew. Chem. Int. Ed., 1998, vol. 37, pp. 402-428) and are used for their electronic and fluorescent properties in non-linear optics (Macromolecules, 1994, 27, 562-571 and J. Phys. Chem., 1995, 99, 4886-4893).
The polymers according to the present invention are functionalized by groups GpF which, in association with the moiety E, give the molecular rod according to the invention particular properties:
it is linear, rigid and soluble in aqueous media;
it is regularly functionalized by groups with a very strong affinity for biological macromolecules; and
when dissolved with a biological macromolecule, it is particularly suitable for the attachment and/or auto-organization of said macromolecules to/on said rod by molecular

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