Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – Tripeptides – e.g. – tripeptide thyroliberin – etc.
Reexamination Certificate
1998-08-19
2001-06-19
Celsa, Bennett (Department: 1614)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
Tripeptides, e.g., tripeptide thyroliberin , etc.
C530S333000, C562S561000, C562S562000, C562S564000
Reexamination Certificate
active
06248866
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to novel hypusine derivatives useful as reagents for synthesizing peptides containing hypusine.
BACKGROUND OF THE INVENTION
Description of the Prior Art
Hypusine [N
&egr;
-(4-amino-2-hydroxybutyl)lysine], 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, one at position 2 and one at position 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 “eIF-5A” (formerly called eIF-4D or IF-M
2
BX; the nomenclature for initiation factors having been revised) [Cooper et al.,
Proc. Natl. Acad. Sci. USA,
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, eIF-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 eIF-5A protein from human red blood cells and elucidated the amino acid sequence surrounding the single hypusine residue, as Thr-Gly-Hpu-His-Gly-His-Ala-Lys. Furthermore, and most interesting, because of the potential application to the control of HIV replication [Bevec et al.,
Proc. Natl. Acad. Sci. USA,
Vol. 91, pages 10829-10833 (1994); and Ruhl et al.
J. Cell Biol.,
Vol. 123, pages 1309-1320 (1994)], the synthesis of eIF-5A analogues is of great therapeutic significance.
Since hypusine is specific to eIF-5A, antibodies derived from hypusine-containing peptides could be used to quantitate the levels of eIF-5A directly and with high specificity. Interest in developing an antibody assay of eIF-5A to investigate the physiological role of this important initiation factor prompted 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;
-carbobenzoxy-(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.
Since there exists synthetic methodology for accessing hypusine itself, it would also be desirable to have a selectively protected hypusine reagent that could be used to incorporate this unusual amino acid into selected peptides.
It is an object of the present invention to provide such novel hypusine reagents, as well as methods for their synthesis.
SUMMARY OF THE INVENTION
These and other objects are realized by the present invention, one embodiment of which comprises a derivative of hypusine useful as a reagent for synthesizing peptides containing hypusine, the derivative having the formula:
wherein: Q
1
and Q
2
may be the same or different and are amino protective groups; Q
3
is an amino protective group which is orthogonal to Q
1
and Q
2
; and Z is a hydroxy protective group.
An additional embodiment of the invention relates to a method for synthesizing a hypusine reagent as defined above comprising:
a. providing an ester of N
&egr;
-, N
&agr;
-diprotected L-lysine, the ester having the formula:
wherein prot and prot′ are N-protective groups which are mutually orthogonal and R is the residue of an esterifying alcohol which is orthogonal with respect to prot and prot′,
b. removing prot from N
&egr;
of (a) to produce a compound of the formula:
c. converting (b) to a compound of the formula:
d. converting (c) a chlorohydrin of the formula:
e. displacing the Cl group of (d) with CN to produce a nitrile of the formula:
f. debenzylating the N
&egr;
group and converting the CN group of (e) to an amine group to produce an amino alcohol of the formula:
g. acylating the free amino groups of (f) to provide a di-N-protected N
&egr;
-protected L-lysine ester of the formula:
h. removing R and prot′ from (g) to produce a compound of the formula:
and
i. acylating the free amino group and protecting the OH group to produce the hypusine derivative (1).
A further embodiment of the invention relates to an alternate method for synthesizing the hypusine reagent, wherein:
j. converting the compound of formula (b) to a chlorohydrin of the formula:
k. converting the compound of formula (j) to an N
&egr;
-protected chlorohydrin of the formula:
l. displacing the Cl group of (k) with CN to produce a nitrile of the formula:
m. deprotecting the N
&egr;
group and converting the CN group of (I) to an amine group to produce the amino alcohol of the formula (f), followed by steps g, h, and i as above produces the hypusine derivative (1).
REFERENCES:
Abbruzzese et al., “Inhibition of deoxyhypusine hydroxylase by polyamines and by a deoxyhypusine peptide,”Biochim, Biophys, Acta, pp. 997:248-255 (1989).
Bergeron, et al., “Development of a Hypusine Reagent For Peptide Synthesis”,J.Org.Chem., vol. 62, No. 10, pp.3285-3290 (1997).
Bergeron, et al., “Total Syntheses of (+) Hypusine and Its (2S,9S)-Diastereomer”,J.Org.Chem., vol. 58, No. 24, pp.6804-6806 (1993).
Bergmann, et al., “Uber ein all-gemeines Verfahren der Peptid-Synthese”,Ber.Dtsch., vol. 65, pp.1192-1201 (1932).
Bernady, et al., “Prostaglandins and Congeners.20. Synthesis of Prostaglandins via Conjugate Addition of Lithium trans-1-Alkenyltrialkylalanate Reagents. A Novel Reagent for Conjugate 1,4-Additions”,J.Org.Chem., vol.44, pp.1438-1447 (1979).
Beyermann, et al., “Rapid Continuous Peptide Synthesis via FMOC Amino Acid Chloride Coupling and 4-(Aminomethyl)piperidine Deblocking”,J.Org.Chem., vol. 55, pp.721-728 (1990).
Borch, et al., “The Cyanohydridoborate Anion as a Selective Reducing Agent”,J.Am.Chem.Soc., vol. 93, pp.2897-2904 (1971).
Mehta, et al., “Improved Efficiency and Selectivity in Peptide Synthesis: Use of Triethylsilane as a Carbocation Scavenger in Deprotection of t-Butyl Esters and t-Butoxycarbonyl-Protected Sites”,Tetrahedron Lett., vol.33, pp.5441-5444 (1992).
Stewart, et al., “Protection of the Hydroxyl Group in Peptide Synthesis”, inThe Peptides, Gross, et al., eds., Academic Press, New York, vol.3, pp.169-201 (1981).
Tarbell, et al., “New Method to Prepare N-t-Butoxycarbonyl Derivatives and the Corresponding Sulfur Analogs from di-t-Butyl Dicarbonate or di-t-Butyl Dithiol Dicarbonates and Amino Acids”,Proc.Natl.Acad,Sci,USA, vol.69, pp.730-732 (1972).
Wang, et al., “Cleavage and Deprotection of Peptides on MBHA-Resin With Hydrogen Bromide”,Int.J.Peptide&Protein Res., vol.40, pp.344-349 (1992).
Celsa Bennett
Hamilton Brook Smith & Reynolds P.C.
University of Florida Research Foundation Incorporated
LandOfFree
Hypusine reagent for peptide synthesis does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Hypusine reagent for peptide synthesis, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Hypusine reagent for peptide synthesis will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2488873