EP1032586A1 - Hypusin enthaltende peptide - Google Patents

Hypusin enthaltende peptide

Info

Publication number
EP1032586A1
EP1032586A1 EP98943259A EP98943259A EP1032586A1 EP 1032586 A1 EP1032586 A1 EP 1032586A1 EP 98943259 A EP98943259 A EP 98943259A EP 98943259 A EP98943259 A EP 98943259A EP 1032586 A1 EP1032586 A1 EP 1032586A1
Authority
EP
European Patent Office
Prior art keywords
peptide
gly
hypusine
thr
hpu
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP98943259A
Other languages
English (en)
French (fr)
Other versions
EP1032586A4 (de
Inventor
Raymond J. Bergeron, Jr.
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Florida
University of Florida Research Foundation Inc
Original Assignee
University of Florida
University of Florida Research Foundation Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Florida, University of Florida Research Foundation Inc filed Critical University of Florida
Publication of EP1032586A1 publication Critical patent/EP1032586A1/de
Publication of EP1032586A4 publication Critical patent/EP1032586A4/de
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/0606—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing heteroatoms not provided for by C07K5/06086 - C07K5/06139, e.g. Ser, Met, Cys, Thr
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06139—Dipeptides with the first amino acid being heterocyclic
    • C07K5/06147—Dipeptides with the first amino acid being heterocyclic and His-amino acid; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/081—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing O or S as heteroatoms, e.g. Cys, Ser
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0815—Tripeptides with the first amino acid being basic
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to novel hypusine-containing peptides.
  • hydroxy-7-azaundecanoic acid an unusual naturally occurring amino acid, having the structure:
  • This initiation factor 5A is unique in that it is the only known cellular protein that contains the amino acid hypusine (Hpu).
  • Hpu amino acid hypusine
  • elF-5A was shown to stimulate ribosomal subunit joining and to enhance 80 S-bound Met-t-RNAj 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)1. Later, in
  • the present invention relates to novel hypusine-containing peptides synthesized utilizing the hypusine reagent:
  • Q ⁇ Q 2 , and Q 3 may be the same or different and are amino protective groups, provided that Q 3 is orthogonal to Qi and Q 2 ; and Z is a hydroxy protective group.
  • a further embodiment of the invention relates to compounds of structure (2)
  • hypusine reagent (1) 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.
  • 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.
  • FIGs. 1 and 2 depict example reaction schemes for preparing peptides of the invention.
  • Figures 1 and 2 correspond to the chemistry described in Examples 1 through 8.
  • novel peptides (2) of the present invention comprise any synthetic peptide that incorporates within its structure the hypusine moiety, which is synthesized according to a method involving the use of the above-described hypusine reagent (1).
  • S and T are peptide residues from zero to about 12 amino acids in length, and preferably, are peptide residues from zero to about six amino acids in length. Most preferably, S and T are peptides residues from zero to about three amino acids in length S and T may vary independently in length and in composition of amino acid residues. The terminal amino acid reside of T may be hydroxylated.
  • Non-limiting examples of peptides of the invention are: L-Ser-L-Thr-L-Ser-L-Lys-L-Thr-Gly-Hpu-L-His-Gly-L-His-L-Ala-L-Lys,
  • peptides of the invention find utility in the study of biochemical processes involving hypusine, such as in the study of transport mechanisms for elF5A.
  • the peptides of the invention may be prepared employing conventional steps of peptide synthesis except that the above-described hypusine reagent (1) is employed to incorporate the hypusine moiety into the peptide chain.
  • Conventional peptide synthesis steps are disclosed, for example, in Moroder et al., "Hormonal Receptors in Digestive Tract Physiology," G. Rosselin et al., eds., Elsevier/North- Holland Biomedical Press, Amsterdam, pages 129-135 (1979); and Moroder et al., Phvsiol. Chem., Vol. 360, pages 787-790 (1979).
  • the synthesis of peptides is generally carried out through the condensation of the carboxyl group of an amino acid, and the amino group of another amino acid, to form a peptide bond.
  • a sequence can be constructed by repeating the condensation of individual amino acid residues in stepwise elongation or, in some cases, by condensation between two pre-formed peptide fragments (fragment condensation).
  • fragment condensation the amino and carboxy groups that are not to participate in the reaction must be blocked with protecting groups which should be readily introduced, be stable to the condensation reactions and be removed selectively from the completed peptide. If a peptide involves amino acids with side chains that may react during condensation, the problem of protection becomes increasingly difficult.
  • Solid phase peptide synthesis involves attachment of a first amino acid to a solid support, such as a resin, followed by sequential addition of subsequent amino acids which results in assembly of the peptide chain on the solid support.
  • Peptides can also be synthesized by related methods involving coupling peptide fragments to solid supports as discussed by Erickson et al., supra, pages 268-269. This technique involves the synthesis of small peptide segments containing a few amino acids, which segments are then coupled to each other using fragment condensation techniques to form larger peptides. Fragment condensation techniques can be combined with standard solid phase techniques wherein small peptides are attached to resins followed by sequential attachment of single amino acids or other peptide segments. Alternatively, sequential attachment of small peptides to single resin-bound amino acids can also be accomplished. The combination of the two approaches provides flexibility to synthetic schemes.
  • the synthesized peptide is then removed from the resin, usually by chemical means such as treatment with hydrofluoric acid (HF).
  • HF hydrofluoric acid
  • the chemical treatment also removes various amino acid and peptide protecting groups, such as CBZ, t-BOC or tosyl, which mask the reactivity of amino acid functional groups during synthesis.
  • the initial attachment to the resin involves the C- terminal amino acid of the peptide to be synthesized, which amino acid is covalently attached to the resin through an ester or amide linkage involving its ⁇ -carboxyl group. Synthesis then proceeds from the C- to the N-terminal. N-terminal to C- terminal peptide synthesis is less frequently used because the chemistry is more difficult and unwanted side reactions are more common.
  • the first amino acid may be covalently attached to the resin, in some cases, through its functional side chain.
  • Initial attachment of an amino acid to the resin by means of the side chain functional group allows the possibility of bi-directional synthesis starting with the attached amino acid. Bi-directional synthesis cannot be performed if the initial amino acid is attached through the ⁇ -COOH or ⁇ -NH 2 group.
  • Side chain functional groups which have been used for attachment to resins include the sulfhydryl group of cysteine, the imidazole group of histidine, the ⁇ -amino group
  • hypusine reagent described herein may be employed to access any hypusine-containing peptide
  • the method of the invention will be illustrated with reference to the following syntheses. It will be understood that any conventional peptide synthesis may be modified to prepare a hypusine-containing peptide by simply utilizing the herein described hypusine reagent at any convenient stage thereof.
  • the hypusine-containing pentapeptide found in eIF-5A capped at its N-terminus with L-Cys i.e., L-Cys-Thr-Gly-Hpu-His-Gly is a typical target peptide.
  • L-Cys which is not contained in the natural peptide, was fixed to the sequence with the idea of being able to covalently link the peptide via a disulfide bond to a larger protein, in order to ultimately generate antibodies.
  • the final hexapeptide 12 was constructed stepwise from the three aforementioned fragments, i.e., 1 , 15 and 18. Hydrogenolysis of the N ⁇ - CBZ group of 17 provided the amine HCI salt (74%) which was condensed with hypusine reagent 1 to give the di-CBZ-THP protected Hpu-His-Gly tripeptide 18 in 85% yield.
  • the hypusine reagent described has been demonstrated to be a highly useful synthon in accessing the elF-5A pentapeptide sequence. While the yields are generally excellent for these kinds of systems, the most notable feature is the flexibility that this methodology offers in synthesizing related elF-5A mimics.
  • the polymer-bound peptide 21 was synthesized using an Applied Biosystems 432A Synthesizer. Amino acid analysis for 21 : Gly 2.09, His 1.03, Thr 0.88. An aliquot of 21 (49 mg, 19.3 ⁇ mol), phenol (250 mg) and pentamethylbenzene (250 mg) were dissolved in degassed TFA (5.0 ml) at 0°C. Saturated HBr in acetic acid solution (0.2 ml), triisopropylsilane (0.1 ml) and 1 , 2-ethanedithiol (0.1 ml) were added under an argon atmosphere. The solution was stirred at room temperature for 1 hour and concentrated under reduced pressure.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
EP98943259A 1997-11-21 1998-08-19 Hypusin enthaltende peptide Withdrawn EP1032586A4 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US97565697A 1997-11-21 1997-11-21
US975656 1997-11-21
PCT/US1998/017221 WO1999026963A1 (en) 1997-11-21 1998-08-19 Hypusine peptides

Publications (2)

Publication Number Publication Date
EP1032586A1 true EP1032586A1 (de) 2000-09-06
EP1032586A4 EP1032586A4 (de) 2000-11-15

Family

ID=25523257

Family Applications (1)

Application Number Title Priority Date Filing Date
EP98943259A Withdrawn EP1032586A4 (de) 1997-11-21 1998-08-19 Hypusin enthaltende peptide

Country Status (5)

Country Link
EP (1) EP1032586A4 (de)
JP (1) JP2001524488A (de)
AU (1) AU9109098A (de)
CA (1) CA2310027A1 (de)
WO (1) WO1999026963A1 (de)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SG176085A1 (en) * 2009-05-15 2011-12-29 Stichting Nl Kanker Inst Lysine compounds and their use in site- and chemoselective modification of peptides and proteins
CN108026180B (zh) 2015-08-28 2022-06-07 豪夫迈·罗氏有限公司 抗羟腐胺赖氨酸抗体及其用途

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5344846A (en) * 1992-12-30 1994-09-06 The United States Of America As Represented By The Department Of Health And Human Services Compositions and methods for inhibiting deoxyhypusine synthase and the growth of cells
US5538897A (en) * 1994-03-14 1996-07-23 University Of Washington Use of mass spectrometry fragmentation patterns of peptides to identify amino acid sequences in databases

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
R.J.BERGERON ET AL.: "Developement of a Hypusine Reagent for Peptide Synthesis" J.ORG.CHEM., vol. 62, no. 10, May 1997 (1997-05), pages 3285-90, XP002914567 *
See also references of WO9926963A1 *

Also Published As

Publication number Publication date
EP1032586A4 (de) 2000-11-15
WO1999026963A1 (en) 1999-06-03
JP2001524488A (ja) 2001-12-04
AU9109098A (en) 1999-06-15
CA2310027A1 (en) 1999-06-03

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