Derivatized biotin compounds and methods of use

Details Derivatized biotin compounds and methods of use Derivatized biotin compounds and methods of use

2001-04-06

2003-04-29

McKane, Joseph K. (Department: 1626)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Phosphorus containing other than solely as part of an...

C514S387000, C548S113000, C548S303700

Reexamination Certificate

active

06555528

ABSTRACT:

This invention pertains to derivatized biotin compounds, and to methods of using those compounds.
Biotin (also known as vitamin H) is an essential growth factor found in all animals, plants, fungi, and bacteria. It is found, for example, bound to proteins or polypeptides in the liver, pancreas, kidney, milk, and in yeasts. Biotin is a cofactor for a group of enzymes that catalyze carboxylation reactions, transcarboxylation reactions, and decarboxylation reactions. The reactions catalyzed by biotin-dependent enzymes are involved in several essential metabolic pathways, including gluconeogenesis, fatty acid synthesis, and amino acid catabolism. All biotin-dependent carboxylases catalyze their respective reactions via the two step reaction shown below:
Enzyme-biotin+Mg
2+
+ATP+HCO
3
−
⇄Enzyme-biotin-CO
2
−
+Mg
2+
+ADP+Pi  (1)
Enzyme-biotin-CO
2
−
+acceptor⇄acceptor-CO
2
−
+Enzyme-biotin  (2)
R. Guchhait et al.,
J. Biol. Chem
. vol. 249, pp. 6646-6656 (1974) showed that in the first partial reaction shown above, biotin is carboxylated on the 1′-N. Since bicarbonate is the source of CO
2
for all biotin-dependent carboxylases, carboxylation of the 1′-N of biotin is accomplished by activating bicarbonate through phosphorylation with ATP to form a carboxyphosphate intermediate. The carboxyl group is then transferred from the carboxyphosphate intermediate to biotin to form carboxybiotin.
In the field of biotechnology, biotin has become an important reagent in methods to label, detect, and purify proteins and nucleic acids. These methods are based on the remarkable affinity between biotin and the proteins avidin and streptavidin. The dissociation constant of biotin from avidin or streptavidin is about 10
−15
M, one of the strongest known interactions between a protein and a ligand. While all parts of the biotin molecule contribute to this tight binding, hydrogen bonding donation by the ureido nitrogens is the major contributor.
Q. Han et al., “Synthesis of (+)-Biotin Derivatives as HIV-1 Protease Inhibitors,”
Bioorg
. &
Med. Chem
., vol. 6, pp. 1371-1374 (1996) reported the synthesis of several bis-N-alkylated biotin derivatives, and their activity against HIV-1 protease. All biotin derivatives reported in this paper were substituted on both urea nitrogens. There was no suggestion to selectively substitute biotin at one of the nitrogen atoms only, nor was there any suggestion of how such a selective synthesis might be performed.
Up to 25% diacylated biotin products have previously been reported following acylation of biotin methyl ester with (1) methylchloroformate or (2) trifluoroacetic anhydride. See (1) J. Knappe et al.,
Biochemishe Zeitschrift
, vol. 335, pp. 168-176 (1961); and (2) A. Berkessel et al.,
Bioorganic Chem
., vol. 14, pp. 249-261 (1986); respectively.
P. Tipton et al., “Catalytic Mechanism of Biotin Carboxylase: Steady-State Kinetic Investigations,”
Biochemistry
, vol. 27, pp. 4317-4325 (1988) reported that no biotin analog that had been tested had inhibited biotin carboxylase (at page 4322).
The ureido ring of biotin is of great importance in binding to avidin, and the 1′-N of that ring is directly involved in biotin's role as a carboxytransfer intermediate. However, there have been relatively few studies involving functionalization of the 1′-N of biotin. Most prior research on derivatizing biotin has involved acylation chemistry at the carboxylic acid terminus of the pendant alkyl chain.
We have discovered a method to functionalize biotin at the 1′-N, selectively and with high efficiency. The resulting 1′-N-substituted biotin has an electrophilic “handle” that is amenable to reaction with a wide variety of nucleophiles to generate a new family of biotin analogs. Such nucleophiles might include, for example, acetyl coenzyme A, benzyl alcohol, benzoic acid, aniline, or other nucleophiles known in the art.
This synthesis is more selective for functionalization at the 1′-N of biotin than have been any known prior syntheses. Compounds prepared from the 1′-N-substituted biotin are useful in inhibiting various enzymatic reactions, including the inhibition of HIV protease.
As initial examples, we have synthesized compounds
1
and
3
(see FIGS.
1
and
2
). Compound
1
is formally derived from phosphonoacetic acid coupled to the 1′-N of biotin. The synthesis of Compound
3
was highly efficient and selective: a 98% yield with essentially 100% selectivity, in a synthesis conducted on the multi-gram scale. The synthesis of Compound
1
had an overall yield of 35%, and a 100% selectivity.
We have shown that compound
1
acts as a stable analog of the carboxyphosphate intermediate in naturally-occurring biotin-mediated CO
2
transfer. Compound
1
inhibits the activity of the biotin carboxylase component of the enzyme acetyl CoA carboxylase. This is the first reported biotin-derived inhibitor of biotin carboxylase.
The synthesis of Compound
3
may readily be scaled up to perform large-scale, selective acylations of biotin to form Compound
1
or other end products.


REFERENCES:
patent: 6242610 (2001-06-01), Strongin et al.
Amspacher, D. et al., “Studies on the Catalytic Mechanism of Biotin,” Abstract, Steenbock Symposium (University of Wisconsin, Madison, May 28-31, 1998).
Amspacher, D. et al., “Synthesis of a Reaction Intermediate Analog of Biotin-Dependent Carboxylases via a Selective Derivatization of Biotin,” manuscript submitted to Organic Letters (1999).
Amspacher, D. et al., “Synthesis and Characterization of a Slow-Binding Inhibitor of Biotin Carboxylase,” pp. 131-138 in P. Frey et al. (eds.), Enzymatic Mechanisms (1999).
Amspacher, D. et al., “The Synthesis of a Slow-Binding Inhibitor of Biotin Carboxylase via a Selective Derivatization of Biotin,” Poster presented at 216th American Chemical Society National Meeting (Boston, Aug. 23-27, 1998).
Amspacher, D. et al., “The Synthesis of a Slow-Binding Inhibitor of Biotin Carboxylase via a Selective Derivatization of Biotin,” Newsletter and Abstracts, 216th American Chemical Society National Meeting (Boston, Aug. 23-27, 1998).
Berkessel, A. et al., “On the Structures of Some Adducts of Biotin with Electrophiles . . . ,” Biorganic Chem., vol. 14, pp. 249-261 (1986).
Blanchard, C. et al., “Inhibition of Biotin Carboxylase by a Reaction Intermediate Analog: Implications for the Kinetic Mechanism” pp. 1-17 (unpublished manuscript, 1999).
Blanchard, C. et al., “Mutations at Four Active Site Residues of Biotin Carboxylase Abolish Substrate-Induced Synergism by Biotin,” Biochemistry, vol. 38, pp. 3393-3400 (1999).
Guchhait, R. et al., J. Biol. Chem. vol. 249, pp. 6646-6656 (1974).
Han, Q. et al., “Synthesis of (+)-Biotin Derivatives as HIV-1 Protease Inhibitors,” Bioorg. & Med. Chem., vol. 6, pp. 1371-1374 (1996).
Knappe, J. et al., Biochemishe Zeitschrift, vol. 335, pp. 168-176 (1961).
Tipton, P. et al., “Catalytic Mechanism of Biotin Carboxylase: Steady-State Kinetic Investigations,” Biochemistry, vol. 27, pp. 4317-4325 (1988).
Product No. H-9040 Analytical Data Sheet, BACHEM Bioscience (1999?).

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