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WO1998042730A1 - Substrats utilises dans des milieux aqueux et organiques, et procedes associes de preparation et d'utilisation - Google Patents

Substrats utilises dans des milieux aqueux et organiques, et procedes associes de preparation et d'utilisation Download PDF

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Publication number
WO1998042730A1
WO1998042730A1 PCT/US1998/006065 US9806065W WO9842730A1 WO 1998042730 A1 WO1998042730 A1 WO 1998042730A1 US 9806065 W US9806065 W US 9806065W WO 9842730 A1 WO9842730 A1 WO 9842730A1
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WO
WIPO (PCT)
Prior art keywords
hydrophilic
procedure
organic
support
compatible
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.)
Ceased
Application number
PCT/US1998/006065
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English (en)
Inventor
Ronald N. Zuckermann
Fred E. Cohen
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.)
Novartis Vaccines and Diagnostics Inc
Original Assignee
Chiron Corp
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 Chiron Corp filed Critical Chiron Corp
Priority to AU68712/98A priority Critical patent/AU6871298A/en
Publication of WO1998042730A1 publication Critical patent/WO1998042730A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C07K1/042—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers characterised by the nature of the carrier
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C07K1/047—Simultaneous synthesis of different peptide species; Peptide libraries

Definitions

  • the initial support-bound "monomer” is generally used as a building-block in a multi-step synthesis procedure, conducted in a nonaqueous medium, to form a complete ligand, such as in the synthesis of oligopeptides, oligopeptoids, oligonucleotides, and the like.
  • aqueous medium as used herein is used to refer to a liquid medium containing less than about 50 vol.% organic solvents, preferably less than 25 vol.% organic solvents, more preferably less than about 20 vol.% organic solvents, still more preferably less than about 15 vol.% organic solvents, and most preferably less than about 10 vol.% organic solvents.
  • the substrate When the functionalized substrate is in the "first state,” then, the subset of hydrophilic sites is protected and the remaining sites are unprotected; the substrate is then hydrophobic and may be used in processes requiring organic reagents and solvents, such as synthesizing organic ligands on the substrate at the unprotected sites.
  • the functionalized substrate When the functionalized substrate is in the "second state,” the formerly protected subset of hydrophilic sites is no longer protected, and the substrate, generally having organic materials such as oligomeric ligands bound thereto, is again hydrophilic.
  • the organic materials bound to the support may be complete ligands that were covalently attached, or, oligomeric or other types of ligands that were synthesized on the support using solid-phase organic synthetic techniques.
  • a protecting group which binds to two out of the three hydroxyl moieties thus "ties up" two out of the three hydroxyl moieties, while leaving the third hydroxyl group unprotected and thus capable of use in chemical reactions as desired, e.g., to synthesize oligomeric compounds thereon, to bind complete ligands, or the like.
  • Alternative, but substantially equivalent, materials can be used as well, wherein individual groups of hydroxyl moieties (or other hydrophilic moieties) are provided and somewhat isolated from each other, at least to the extent that a single protecting reagent can bind the majority but not all of the unprotected hydroxyl groups.
  • the Trisacryl® surface shown in Figure 2 can be replaced with a support-bound monosaccharide moiety similarly bound to the substrate through a linker, such as an amide.
  • fraction of hydrophilic sites which are to be protected can readily be controlled by adjusting the quantity of protecting reagent used to correspond to the fraction of hydrophilic sites to be protected.
  • ligands include oligopeptides. oligonucleotides, oligosaccharides. oligomers of peptide mimetics such as oligopeptoids, and the like. Oligopeptides are particularly useful ligands herein as they provide for a direct approach to affinity chromatography, and single amino acids can be used as well.
  • the protected hydrophilic groups are then deprotected using cleavage reagents appropriate to the selected protecting groups.
  • the substrate surface is once again rendered hydrophilic, but now has the aforementioned ligands bound thereto.
  • the substrate may be used in any process which involves the use of support-bound ligands in an aqueous medium, e.g., screening and separation processes, wherein a specific support-bound ligand of interest is isolated and identified, or wherein the support-bound ligands are used to bind a component of interest in a sample, which may then be isolated and identified.
  • acid e.g., trifluoroacetic acid, or "TFA," as shown in Figure 2
  • TFA trifluoroacetic acid
  • Boc t- butyloxycarbonyl
  • Fmoc 9-fluorenylmethoxycarbonyl
  • benzyloxycarbonyl Cbz
  • /?-toluenesulfonyl Tos
  • 2,4-dinitrophenyl benzyl, biphenylisopropyloxycarboxycarbonyl, cyclohexyl, isopropyl, acetyl, o- nitrophenylsulfonyl, and the like.
  • a mixture of support-bound oligopeptides is synthesized using a solid-phase synthesis technique such as described above, the support-bound oligopeptides are then screened, in an aqueous medium, to identify a particular oligopeptide of interest, and that oligopeptide is then resynthesized on the support, using organic reagents and solvents as explained above.
  • the method of using the novel substrates to prepare and use a combinatorial library involves the same general procedures outlined above with respect to partial protection of the hydrophilic surface to render the substrate hydrophobic, synthesis of support-bound ligands using organic reagents and solvents, followed by deprotection and use of the support-bound ligands in processes requiring an aqueous medium.
  • a plurality of reaction vessels is provided each containing a substrate as described herein.
  • the novel substrates are not limited to use in combinatorial chemistry, but find use in a wide variety of contexts, including separation and removal of a component of interest in a sample, i.e., by binding that component to the support-bound ligands (e.g., with respect to oligonucleotides, this step could involve hybridization and the use of labeled oligomers), electrophoresis, and spectroscopic detection.
  • Figure 3 illustrates use of the present substrate in a different context. As may be seen in the Figure, the same agarose substrate, present in all of the reaction vessels, is used for equimolar mixture synthesis, and can then be used, directly, in affinity chromatographic and electrophoretic screening.
  • the solid phase synthesis is carried out in an organic medium, after partial protection of surface hydrophilic groups to render the substrate hydrophobic, with the latter steps, chromatography and electrophoresis, carried out in an aqueous medium after removal of the protecting groups, such that the surface is in regenerated hydrophilic form.
  • combinatorial processes and chromatographic applications represent important fields of use for the present invention.
  • the invention is useful in at least the following contexts: (i) in affinity chromatography with the generation of receptor/molecule specific non-biological ligands on the substrate using solid-phase organic chemistry; and (ii) in the generation of a family of non-biological ligands on a support to create more general purpose derivatized supports for chromatographic separation of biological and non-biological molecules.
  • combinatorial processes coupling combinatorial synthesis and combinatorial library deconvolution strategies, using a single solid support, is also an important application of the invention In other processes as well, the invention simplifies synthesis and subsequent use of support-bound materials.
  • Trisacryl GF-LB (100 mL settled volume) was washed with water (2 x 300 mL), ethanol (2 x 100 ml) and ether (3 x 300 mL). The resin was air-dried for 30 min. on a vacuum manifold, and dried overnight in a drying pistol over phosphorus pentoxide at 50 C. The resin was then suspended in a solution of dry tetrahydrofuran (THF, 250 mL) containing 2, 2-dimethoxy propane (30 mL, 250 mmol) and p-toluenesulfonic acid (3 g). The suspension was agitated at room temperature overnight under nitrogen. The resin was then drained and washed with THF (3 x 300 mL) and dried in vacuo to afford acetonide-protected resin.
  • THF dry tetrahydrofuran
  • the acetonide protected resin was suspended in dry THF (250 mL) to which sodium hydride was added (200 mmol, 4.8g). The suspension was mixed for 10 min. which was followed by the addition of chloroacetic acid. The mixture was heated to reflux for 6 hr., after which the resin was washed with THF (3 x 300 mL). neutralized with 0.1 N acetic acid in methanol, followed by washing with dichloromethane (2 x 300 mL).
  • the carboxymethylated resin was then coupled to a diamine linker by suspending the resin in dichloromethane (250 mL) containing 2,2'-(ethylene-dioxyl)bis(ethylamine) (19 g, 125 mmol) and diisopropyl-carbodiimide (23 ml, 150 mmol).
  • dichloromethane 250 mL
  • 2,2'-(ethylene-dioxyl)bis(ethylamine) (19 g, 125 mmol) and diisopropyl-carbodiimide 23 ml, 150 mmol.
  • the reaction mixture was mixed at room temperature for 6 hr., followed by washing with dichloromethane (3 x 300 mL), and drying in vacuo to yield the amino-derivatized protected resin.
  • the amino-derivatized protected resin prepared in Example 1 (200 mg) was suspended in dimethylformamide (3 mL), and drained. A peptoid ligand was synthesized.
  • the resin was acylated by adding to the resin 830 ⁇ L of 0.6 M bromoacetic acid in DMF, followed by 200 ⁇ L of 3.2 M DIC in DMF. This solution was agitated for 30 minutes at room temperature and then drained. This step was then repeated a second time.
  • the resin was then washed with DMF (2 x 2 mL) and DMSO (1 x 2 mL). To the washed resin was added 1.0 mL of a 1 M solution of benzylamine in DMSO.
  • the amino-derivatized resin can also be used for the synthesis of any organic compounds requiring an amine handle.
  • the resin can also be used for the synthesis of combinatorial libraries by using the resin-splitting method.
  • Example 3 Deprotection of ligand-support:

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention porte sur de nouveaux substrats fonctionnalisés ayant, dans un premier état, une surface hydrophile, et dans un second état, une surface hydrophobe de sorte que le substrat puisse être utilisé dans des milieux aqueux ou organiques. La surface du substrat contient une pluralité de sites hydrophiles pouvant être facilement protégés ou déprotégés. Généralement, en utilisation, une fraction des sites est protégée, les autres étant laissés disponibles pour participer à des processus synthétiques organiques devant être effectués au moyen de réactifs et solvants organiques, p.ex., une synthèse organique en phase solide de ligands pouvant être ou non oligomères. Après synthèse, les sites hydrophiles protégés sont déprotégés, régénérant la surface du substrat sous forme hydrophile, en vue d'être utilisés avec des réactifs aqueux, p.ex. dans des procédures de détection et/ou de séparation effectuées en milieux aqueux.
PCT/US1998/006065 1997-03-21 1998-03-20 Substrats utilises dans des milieux aqueux et organiques, et procedes associes de preparation et d'utilisation Ceased WO1998042730A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU68712/98A AU6871298A (en) 1997-03-21 1998-03-20 Substrates useful in both aqueous and organic media, and associated methods of preparation and use

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US82819597A 1997-03-21 1997-03-21
US08/828,195 1997-03-21

Publications (1)

Publication Number Publication Date
WO1998042730A1 true WO1998042730A1 (fr) 1998-10-01

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AU (1) AU6871298A (fr)
WO (1) WO1998042730A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001033230A3 (fr) * 1999-11-02 2002-01-10 Chiron Corp Purification et affichage differentiel de composants d'echantillons biologiques
WO2003056337A1 (fr) * 2001-12-21 2003-07-10 Biacore Ab Immobilisation d'agglutinants
US6783929B1 (en) 1999-11-02 2004-08-31 Chiron Corporation Biological sample component purification and differential display
US7148058B2 (en) 2000-06-05 2006-12-12 Chiron Corporation Protein microarrays on mirrored surfaces for performing proteomic analyses
US7153682B2 (en) 2000-06-05 2006-12-26 Chiron Corporation Microarrays on mirrored substrates for performing proteomic analyses
CN115698714A (zh) * 2020-03-26 2023-02-03 德累斯顿工业大学 用于自动化按需生物分子阵列合成的新方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994027719A1 (fr) * 1993-05-27 1994-12-08 Protogene Laboratories, Inc. Procede et appareil pour realiser une serie de reactions chimiques sur une surface de support

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994027719A1 (fr) * 1993-05-27 1994-12-08 Protogene Laboratories, Inc. Procede et appareil pour realiser une serie de reactions chimiques sur une surface de support

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, vol. 102, no. 6, 11 February 1985, Columbus, Ohio, US; abstract no. 46635, R ARSHADY: "A new synthetic approach for the preparation of polymer supports based on beaded copolymers of styren and 2,4,5-trichlorophenyl acrylate: synthesis and swelling behavior off poly(styrene-co-acrylamide) resins" XP002074985 *
CHEMICAL ABSTRACTS, vol. 123, no. 13, 25 September 1995, Columbus, Ohio, US; abstract no. 170151, XP002074984 *
L WINTHER ET AL.: "Hydrophlic film supports for use in peptide synthesis and assays", INNOVAT. PERSPECT. SOLID PHASE SYNTH. COLLECT. PAP., INT. SYMP., 3RD, 1993, pages 705 - 706 *
MAKROMOL. CHEM., vol. 185, no. 11, 1984, pages 2387 - 2400 *
R ARSHADY & F FALLAH: "Amphiphilic gels for peptide synthesis", JOURNAL OF POLYMER SCIENCE, POLYMER CHEMISTRY EDITION., vol. 30, no. 8, 1992, NEW YORK US, pages 1705 - 1716, XP000286978 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001033230A3 (fr) * 1999-11-02 2002-01-10 Chiron Corp Purification et affichage differentiel de composants d'echantillons biologiques
US6783929B1 (en) 1999-11-02 2004-08-31 Chiron Corporation Biological sample component purification and differential display
US7148058B2 (en) 2000-06-05 2006-12-12 Chiron Corporation Protein microarrays on mirrored surfaces for performing proteomic analyses
US7153682B2 (en) 2000-06-05 2006-12-26 Chiron Corporation Microarrays on mirrored substrates for performing proteomic analyses
WO2003056337A1 (fr) * 2001-12-21 2003-07-10 Biacore Ab Immobilisation d'agglutinants
CN115698714A (zh) * 2020-03-26 2023-02-03 德累斯顿工业大学 用于自动化按需生物分子阵列合成的新方法

Also Published As

Publication number Publication date
AU6871298A (en) 1998-10-20

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