Water soluble multi-biotin-containing compounds

Abstract

Water-soluble discrete multi-biotin-containing compounds with at least three (3) biotin moieties are disclosed. The water-soluble biotin-containing compounds may additionally comprise one or more moieties that confer resistance to cleavage by biotinidase or that is cleavable in vitro or in vivo. The discrete multi-biotin-containing compounds may include a reactive moiety that provides a site for reaction with yet another moiety, such as a targeting, diagnostic or therapeutic functional moiety. Biotinylation reagents comprising water-soluble linker moieties are also disclosed and may additionally comprise a biotinidase protective group. Methods for amplifying the number of sites for binding biotin-binding proteins at a selected target using multi-biotin compounds also are disclosed.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation-in-part of application Ser. No. 091324,267, filed Jun. 2, 1999, which was a continuation-in-part of application Ser. No. 08 / 798,413 filed Feb. 7, 1997, now abandoned, which claims priority from U.S. Provisional Patent Application No. 60 / 011,321, filed Feb. 8, 1996.FIELD OF THE INVENTION [0002] The present invention relates generally to discrete multi-biotin-containing compounds, biotinylation reagents, and methods for synthesizing such compounds and reagents. More specifically, the present invention relates to discrete biotin-containing compounds with at least three (3) biotin moieties, and to biotinylation reagents suitable for in vitro and in vivo applications which biotinylation reagents are soluble in aqueous media and preferably resistant to biotinidase. The biotin-containing compounds additionally may comprise constituents that confer other functionalities, such as conjugation sites for diagnosti...

AI Technical Summary

Benefits of technology

[0015] One aspect of the present invention is directed to discrete multi-biotin-containing compounds that are useful in vivo to amplify a signal or increase the amount of material bound to a site. As used herein, the term “discrete” means a singular molecular entity and not a mixture of molecular entities (such as those resulting from a random polymerization reaction or a random labeling reaction). For example, the amount of radioactivity presented to a cancer cell that has been labeled with an antibody can be greatly increased through the use of multi-biotin-containing compounds according to the invention.

[0018] In structure (A), P is a biotinidase protective group that blocks biotinidase activity. The “biotinidase protective group”, P, reduces the effectiveness of biotinidase to cleave the biotin moiety from the molecule of the invention. P can be one or more of α-amino acids, N-methyl moieties, α-alkyl moieties, or aryl moieties.

Problems solved by technology

One disadvantage of using biotin derivatives for many of these applications is their generally low solubility in aqueous media.

Insolubility of biotin derivatives and biotinylation reagents in aqueous solutions is particularly problematic for in vivo applications where organic solvents cannot be used.

In addition to causing solubility problems, the lipophilic nature of the biotin derivatives with spacer molecules of low water solubility may cause them to associate with blood components, rendering their biological half-life in in vivo applications longer than desired.

Prior investigators have attached water solubilizing moieties to biotin and to compounds containing two biotin moieties, however, the prior art has failed to disclose or suggest biotin-containing compounds that comprise at least three (3) biotin moieties which are resistant to the enzyme biotinidase.

However, these patents only teach biotin-containing compounds of up to two (2) biotin moieties.

These patents also report that the disclosed adducts are not stable in serum due to the presence of biotinidase.

Poor in vivo stability therefore limits the use of such conjugates in therapeutic applications.

This patent, however, does not suggest water-soluble linker moieties of 6 to 50 atoms in length and biotinidase blocking groups in a discrete multi-biotin-containing compound.

Such random biotin-containing compounds have a very limited utility in diagnostic and therapeutic applications.

Further, this patent does not address the need for a biotinidase blocking moiety.

These probes are not discrete molecular entities and do not address the need for resistance to biotinidase activity, nor do they suggest the use of water-soluble linker moieties of 6 to 50 atoms in length.

Images