Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – Synthesis of peptides
Reexamination Certificate
2000-03-06
2002-12-10
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
Synthesis of peptides
C530S334000, C530S335000, C530S336000, C530S337000, C530S338000, C530S328000, C530S329000, C514S015800, C514S016700, C514S017400
Reexamination Certificate
active
06492491
ABSTRACT:
BACKGROUND OF THE INVENTION
Hypusine [N
&egr;
-(4-amino-2-hydroxybutyl)lysine], or [2S,9R)-2,11-diamino-9-hydroxy-7-azaundecanoic acid], an unusual naturally occurring amino acid, having the structure.
was first isolated from bovine brain extracts by Shiba et al. in 1971
[Biochim. Biophys. Acta
. Vol. 244, pages 523-531 (1971)]. The molecule has two chiral centers at positions 2 and 9, each of which can be classified R or S by the Cahn-Ingold-Prelog method. The (2S,9R) diastereomer (B), formed as a post-translational modification
of lysine, has been shown to occur on a precursor protein of the eukaryotic initiation factor 5A (formerly called elF-4D) [Cooper et al.
Proc. Natl. Acad. Sci. U.S.A
., Vol. 80, pages 1854-1857 (1983); and Safer,
Eur. J Biochem
., Vol. 186, pages 1-3 (1989)]. This initiation factor 5A is unique in that it is the only known cellular protein that contains the amino acid hypusine (Hpu). In the mid-1970's elF-5A was shown to stimulate ribosomal subunit joining and to enhance 80 S-bound Met-t-RNA, reactivity with puromycin [Anderson et al.,
FEBS Lett
., Vol. 76, pages 1-10 (1977); and Kemper et al.,
J. Biol. Chem
., Vol. 251, pages 5551-5557 (1976)]. Later, in 1983, Cooper et al., supra, suggested that a hypusine-modified protein serves as an important initiation factor in all growing eukaryotic cells. In 1986, Park et al.,
J. Biol. Chem
., Vol. 261, pages 14515-14519 (1986)] isolated the elF-5A protein from human red blood cells and elucidated the amino acid sequences surrounding the single hypusine residue, as Thr-Gly-Hpu-His-Gly-His-Ala-Lys (SEQ ID NO: 1). Furthermore, and most interesting because of the potential application to the control of HIV replication [Bevec et al.,
J. Proc. Natl. Acad. Sci. U.S.A
., Vol. 91, pages 10829-10833 (1994); and Ruhl et al.,
J. Cell Biol
., Vol. 123, pages 1309-1320 (1994)], the synthesis of elF-5A analogues are of great therapeutic significance.
Since hypusine is specific to elF-5A, antibodies derived from hypusine-containing peptides could be used to quantitate the levels of elF-5A directly and with high specificity. Interest in developing an antibody assay of elF-5A to investigate the physiological role of this important initiation factor prompter total synthesis of hypusine and its (2S,9R)-diastereomer [Bergeron et al.,
J. Org. Chem
., Vol. 58, pages 6804-6806 (1993)]. The key step in the synthesis involved the N
&egr;
-alkylation of N
&egr;
-benzyl-N
&agr;
-carbobenzyloxy-(L)-lysine benzyl ester with (R)- or (S)- epichlorohydrin to give the respective (2S,9R)- and (2S,9S)-chlorohydrins. Subsequent displacement of the respective chlorides by cyanide ion provided the protected hypusine skeletons. The final step, hydrogenation over PtO
2
in AcOH, followed by neutralization and re-acidification, yielded the respective (2S,9S)- and (2S,9R)-hypusine dihydrochlorides. A comparison of the reported hypusine optical rotation with that of the synthetic (2S, 9R)-hypusine B confirmed the stereochemical integrity of both chiral centers throughout the synthesis.
Synthetic methodology for accessing hypusine itself exists and it was desirable to have a selectively-protected hypusine reagent which could be used to incorporate this unusual amino acid into selected peptides. Copending application Ser. No.: 08/962,300, filed Oct. 31, 1997 entitled “Hypusine Reagent for Peptide Synthesis,” now U.S. Pat. No. 5,973,113 the entire contents and disclosure of which are incorporated herein by reference, describes a selectively protected hypusine reagent useful for incorporating hypusine into peptides, as well as methods for preparation of the hypusine reagent.
It is an object of the present invention to provide novel hypusine-containing peptides, as well as methods for their synthesis utilizing the above-described hypusine reagent.
SUMMARY OF THE INVENTION
The above and other objects are realized by the present invention, one embodiment of which relates to novel peptides containing the hypusine moiety.
More specifically, the present invention relates to novel hypusine-containing peptides synthesized utilizing the hypusine reagent:
wherein: Q
1
, Q
2
and Q
3
may be the same or different and are amino protective groups, provided that Q
3
is orthogonal to Q
1
and Q
2
; and
Z is a hydroxy protective group.
A further embodiment of the invention relates to compounds of structure (2)
S—Hpu—T (2)
which may be synthesized using hypusine reagent (1), wherein Hpu is the hypusine amino acid residue, S and T are each independently peptide residues from zero to about 12 amino acids in length. Compounds of the invention find utility in the study of biochemical processes involving hypusine.
Another embodiment of the invention concerns improved methods of peptide synthesis wherein the above-described hypusine reagent is employed to prepare novel hypusine-containing peptides.
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Bergeron, R.J. et.al., “Total Syntheses of (+)-Hypusine and Its (2S,9S)-Diastereomer,”J. Org. Chem. 58:6804-6806 (1993).
Martinez, et al., “On the Use of Carboxamidomethyl Esters (CAM Esters) in the Synthesis of Model Peptides. Scope and Limitations,”Tetrahedron, vol. 41(4):739-743 (1985).
Martinez, J. et al., “Carboxamidomethyl Esters (CAM Esters) as Carboxyl Protecting Group,”Tetrahedron Lett., vol. 24(47):5219-5222 (1983).
Shioiri, T. et al., “Diphenylphosphoryl Azide. A New Convenient Reagent for a Modified Curtius Reaction and for the Peptide Synthesis,”J. Am. Chem. Soc., 94(17):6203-6205 (1972).
Wang, S.S. et al. “Cleavage and deprotection of peptides on MBGA-resin with hydrogen bromide,”Int. J. Peptide Protein Res., 40:344-349 (1992).
Yamashiro, D. et al., “The Use of N&agr;, Nim-Bis (tert-butyloxycarbonyl) Histidine and N&agr;, -2-(p-Biphenylyl) Isopropyloxycarbonyl-Nim-tert-butyloxy-carbonylhistidine in the Solid-Phase Synthesis of Histidine-Containing Peptides,”J. Am. Chem. Soc., vol. 94(8):
Hamilton Brook Smith & Reynolds
Low Christopher S. F.
Lukton David
University of Florida Research Foundation
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