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WO1997014814A1 - Procede de codage binaire destine a etre utilise en chimie combinatoire - Google Patents

Procede de codage binaire destine a etre utilise en chimie combinatoire Download PDF

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Publication number
WO1997014814A1
WO1997014814A1 PCT/US1996/016718 US9616718W WO9714814A1 WO 1997014814 A1 WO1997014814 A1 WO 1997014814A1 US 9616718 W US9616718 W US 9616718W WO 9714814 A1 WO9714814 A1 WO 9714814A1
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WO
WIPO (PCT)
Prior art keywords
nmr
bead
resin
technique
labels
Prior art date
Application number
PCT/US1996/016718
Other languages
English (en)
Inventor
Ravi S. Garigipati
Susanta K. Sarkar
Original Assignee
Smithkline Beecham Corporation
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 Smithkline Beecham Corporation filed Critical Smithkline Beecham Corporation
Priority to JP9516037A priority Critical patent/JP2000500565A/ja
Priority to EP96936686A priority patent/EP0856067A4/fr
Publication of WO1997014814A1 publication Critical patent/WO1997014814A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B70/00—Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or bar codes
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/14—Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
    • C40B50/16—Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support involving encoding steps
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/005—Beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/0054—Means for coding or tagging the apparatus or the reagents
    • B01J2219/00572—Chemical means

Definitions

  • Binary coding has been established as a viable technique in encoding complex combinatorial libraries (Ohlmeyer, et. al., J. Am. Chem. Soc, 1995, 117, 5588). Even though this tagging technique is versatile, the tags have to be released from the bead before they can be identified and the ligand history deciphered.
  • the present invention provides for a technique which is an efficent method to decipher the contents of a combinatorial library.
  • This technique is utilized for the selective identification of a chemical compound still bound to a single bead of a solid-phase synthesis resin.
  • the tag, ⁇ C and/or ⁇ $N is stable to all the reaction conditions imposed upon it in building the library, and is also stable to the conditions to cleave the ligand of interest (i.e. the individual components) of the library.
  • This technique therefore, provides a means whereby the identity of a ligand of interest can be decrypted either before or after it's release.
  • the detection of the labeled substrate is preferably by (multidimensional) isotope-filtered ⁇ H NMR (HMQC).
  • FIGURE 1 shows a ⁇ C labeled substrate bound to a Wang resin.
  • FIGURE 2 (a) shows a 500-Mhz 13 C-filtered H NMR spectrum of the compound in figure 1 coupled to the resing, obtained on a single bead using the decoulper coil of a standard commercially-available heteronuclear Nano-NMR probe.
  • FIGURE 2(b) shows the corresponding unfiltered ⁇ H NMR spectrum from Figure 2(a).
  • FIGURE 3 shows the contour plot of the 2 dimensional IH 13C HMQC spectrum obtained on the same single bead sample as those above.
  • the present invention provides for the novel application of this high resolution NMR technique to decipher the contents of a combinatorial library, i.e. to detect material bound to a single (lOOum) bead of resin.
  • l3 C and/or ⁇ N labeled compound is selectively detected using isotope-filtered *H NMR (HMQC)(Bax, et al., S. J. Magn. Reson. 1986, 67, 565-569) in a two-coil high- resolution magic-angle spinning (MAS) probe (Barbara, T. J. Magn. Reson., Ser. A. 109, 265-269 (1994)).
  • HMQC isotope-filtered *H NMR
  • MAS magic-angle spinning
  • reporter tags that have been used include nucleotides (which were cleaved, amplified by PCR, and sequenced), (see Brenner et al., supra; and Needels et al., M. A. Proc. Natl. Acad. Sci. USA 1993, 90, 10700-10704) peptides (sequenced by Edman degradation) (Kerr et al., J. Am. Chem. Soc.
  • NMR spectroscopy is routinely used to analyze samples that have been cleaved from SPS resins
  • the NMR spectra of resin-bound samples consist of very broad lines, even if the resin is swollen by the addition of solvents. While relatively low-resolution *H and ⁇ C NMR spectra have been reported, (See Giralt, E et al., Tetrahedron 1984, 40, 4141-4152; Albericio, et al., J. Org. Chem. 1989, 54, 360-366; and Mazure et al., C. R. Acad. Sci., Ser.
  • This invention is based upon a technique of coding either suitable SPS components or the compounds themselves with , 3 C/ 15 N labels.
  • the coding method is binary. It is recognized however, that individual uniquely labeled beads could be detected and identified for a combinatorial library.
  • the detection of the labelled substrate is preferably by (multi-dimensional) isotope-filtered *H NMR (HMQC) with Magic Angle Spinning (MAS).
  • HMQC multi-dimensional isotope-filtered *H NMR
  • MAS Magic Angle Spinning
  • This combination is now known as Nano NMR probe technology.
  • This technique utilizes the ability to selectively observe a proton attached to a 13 C/ 15 N label by inverse detection (Muller, L. J. Am. Chem. Soc, 1979, 707 , 4481 ; Bax et al., J. Mag. Reson. , 1986, 67, 565). T 2 , T 3 etc.
  • Ar* has a unique 13 C / 15 N label
  • Tags can be identified and ligand identity deciphered before or after the bioassay ligand
  • Indirect detection of protons attached to carbons and/or nitrogens labeled with 13 C- and/or 15 N-, by isotope filtered NMR, allows one to detect the proton resonances selectively by suppressing all the signals from the protons attached to unlabeled carbons and/or nitrogens (Bax, et al., S. J. Magn. Reson. 1986, 67, 565- 569; Bax, et al., j. Magn. Reson., 1983, 55, 301-315,).
  • This technique is extensively applied for structure determination of biological macromolecules like proteins and nucleic acids. (G.M.Clore and A. M. Gonenborn, NMR of Proteins, CRC Press, Ann Arbor, 1993).
  • the present invention is the novel application of this theory to use in combinatorial chemistry applications, and even more so its novel use on a single bead.
  • the amount of material attached to a single bead in combinatorial chemistry libraries are of the order of about 0.1 to 1 nmole. It is a significant challenge to detect such a small amount of material in the presence of large background signals from the resin, impurities and from the solvent.
  • ⁇ 3 C and/or ⁇ N-labels are attached as a tag in combinatorial libraries and then using suitable NMR techniques, such as isotope-filtered H NMR (HMQC) under magic angle spinning conditions (MAS) (Barbara et al., J. Magn. Reson., Ser. A, 1994, 109, 265-269; and Garroway, A., J. Magn.
  • tags can be inco ⁇ orated along with the ongoing library synthesis.
  • the tags can be used as a binary code.
  • the tagging label can be inco ⁇ orated into the ligand itself.
  • tags A, B and C will have unique ! H and 13 C chemical shifts (Spectroscopic methods in Organic Chemistry, Wiilams & Fleming, McGraw- Hill, 1966) which can be easily monitored using the proposed isotope filtered technique. As noted, this technique can also be used when the label ( 13 C / 15 N ) is inco ⁇ orated into the ligand itself.
  • This experiment can be conducted on a 2 channel NMR spectrometer, with inverse detection and magic angle spinning capability, operating at a proton frequency of 300 -750 MHz.
  • a dual probe IH observe, 13 C, 15 N decoupling
  • magic angle spinning is required for this pu ⁇ ose.
  • Another aspect of the present invention is the ability to utilize either a number of different 13 C/ ⁇ N labels in a sample, (i.e., to encode the bead in the conventional binary way) or to utilize the site of the Cl N labels to encode additional information.
  • the encoding would occur without adding any additional, and potentially disruptive chemical structures to the resin, without designing any complicated mutually compatible parallel synthetic methodologies, and since the detection would occur nondestructively this allows the sample to be left intact and still bound to the resin.
  • An added feature of this novel technique would then allow the sample to be subject to further chemical transformation or analysis, if so desired. This technique allows for synthesis of uniformly labeled molecules and their subsequent analysis by multidimensional isotope filtered NMR experiments.
  • FIG. 2a shows a 500- MHz 13c-filtered iH NMR spectrum of compound 1 coupled to Wang resin, which was obtained on a single bead using the decoupler coil of a standard commercially-available Heteronuclear Nano «NMRTM probe.
  • the single bead was suspended in 30 ⁇ L of dichloromethane- ⁇ in a standard (40 ⁇ L) Nanoprobe cell and spun at the magic angle (54.7°) at ca. 2 KHz.
  • This proton spectrum was acquired using a one-dimensional version of the HMQC sequence --j-- without 13c decoupling but with presaturation ( 11 Hz field strength for 1 s) of the residual protonated dichloromethane resonance at 5.32 ppm.
  • HMQC spectrum obtained on the same single-bead sample.
  • An f i cross section at the f2 frequency of the 13CH30- protons is also shown, indicating both the high sensitivity and the excellent suppression of the protons attached to l ⁇ C.
  • the 17.5 hr experiment used broadband (3.5 kHz) 3c GARP-1 decoupling3 during the 128 msec acquisition time, a 1.13 sec presaturation delay, 512 scans for each of the 49 hypercomplex ti-datapoints, and 5 kHz and 8 kHz spectral widths, and cosine and 10 Hz exponential weightings, in ti and -2, respectively.
  • Nanoprobe sample cell restricts 100% of the sample (40 ⁇ L maximum) within the active region of the receiver coil to ensure that even a single bead would always be detected.
  • Compound 1 was coupled to 100-200 mesh Merrifield resin obtained from Calbiochem was functionalized according tothe procedure noted in Wang, S. S. J. Am. Chem. Soc. 1973, 95, 1328-1333.
  • the beads selected for NMR were measured by a light microscope to have a 100 ⁇ m diameter.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Structural Engineering (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention se rapporte à une technique de marquage d'un ligand, ou de composés liés de manière covalente à une résine de synthèse en phase solide, avec une étiquette contenant 13C et/ou 15N. La détection de ce marquage par un spectre RMN 1H haute résolution filtré par des isotopes, et par des dispositifs de sondage de spin selon un angle magique ('Magic Angle Spinning') permet d'obtenir une technique générale, non destructive, qui laisse l'échantillon, fixé à la résine, intact et disponible pour d'autres transformations chimiques. La limite de détection (∫ 800 pmol) de ce système est suffisante pour pouvoir utiliser la résonance magnétique nucléaire (RMN) dans le déchiffrage du contenu d'une banque chimique combinatoire 'grain unique, composé unique'.
PCT/US1996/016718 1995-10-19 1996-10-18 Procede de codage binaire destine a etre utilise en chimie combinatoire WO1997014814A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP9516037A JP2000500565A (ja) 1995-10-19 1996-10-18 コンビナトリアルケミストリーにて用いるための２元的コード化方法
EP96936686A EP0856067A4 (fr) 1995-10-19 1996-10-18 Procede de codage binaire destine a etre utilise en chimie combinatoire

Applications Claiming Priority (2)

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US562395P 1995-10-19 1995-10-19
US60/005,623 1995-10-19

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WO1997014814A1 true WO1997014814A1 (fr) 1997-04-24

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997037953A1 (fr) * 1996-04-08 1997-10-16 Glaxo Group Ltd. Codage et analyse quantitative de banques combinatoires fondes sur la masse
WO2000062074A1 (fr) * 1999-04-09 2000-10-19 Abbott Laboratories Utilisation d'imagerie par resonance magnetique 13c pour la detection de liaisons
JP2002524739A (ja) * 1998-09-03 2002-08-06 トレリス バイオインフォマティックス， インコーポレイテッド 複数色の標識
DE10117274A1 (de) * 2001-04-06 2002-10-17 Hte Ag The High Throughput Exp Verfahren zur Analyse und Archivierung von Materialien
US6541203B2 (en) 1998-11-23 2003-04-01 President And Fellows Of Harvard College Detecting structural or synthetic information about chemical compounds
US6576472B1 (en) 2000-07-26 2003-06-10 Smithkline Beecham Corporation Chemical constructs for solution phase chemistry
US6584411B1 (en) 2000-07-26 2003-06-24 Smithkline Beecham Corporation Methods to facilitate the calculation of yields of reaction products
US7183059B2 (en) 1998-03-23 2007-02-27 President And Fellows Of Harvard College Synthesis of compounds and libraries of compounds
US8963546B2 (en) 2011-03-09 2015-02-24 Bruker Biospin Ag Method for NMR spectroscopy
DE102013223891A1 (de) * 2013-11-22 2015-05-28 Joanneum Research Forschungsgesellschaft Mbh Kartuschendepositionssammler

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5565324A (en) * 1992-10-01 1996-10-15 The Trustees Of Columbia University In The City Of New York Complex combinatorial chemical libraries encoded with tags

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5565324A (en) * 1992-10-01 1996-10-15 The Trustees Of Columbia University In The City Of New York Complex combinatorial chemical libraries encoded with tags

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BIOPOLYMERS (PEPTIDE SCIENCE), 1995, Vol. 37, LEBL et al., "One-Bead-One-Structure Combinatorial Libraries", pages 177-198. *
J. ORG. CHEM., 1994, Vol. 59, LOOK et al., "Methods for Combinatorial Organic Synthesis: The Use of Fast 13C NMR Analysis for Gel Phase Reaction Monitoring", pages 7588-7590. *
See also references of EP0856067A4 *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997037953A1 (fr) * 1996-04-08 1997-10-16 Glaxo Group Ltd. Codage et analyse quantitative de banques combinatoires fondes sur la masse
US6475807B1 (en) 1996-04-08 2002-11-05 Smithkline Beecham Corporation Mass-based encoding and qualitative analysis of combinatorial libraries
US7183059B2 (en) 1998-03-23 2007-02-27 President And Fellows Of Harvard College Synthesis of compounds and libraries of compounds
JP2002524739A (ja) * 1998-09-03 2002-08-06 トレリス バイオインフォマティックス， インコーポレイテッド 複数色の標識
US6541203B2 (en) 1998-11-23 2003-04-01 President And Fellows Of Harvard College Detecting structural or synthetic information about chemical compounds
WO2000062074A1 (fr) * 1999-04-09 2000-10-19 Abbott Laboratories Utilisation d'imagerie par resonance magnetique 13c pour la detection de liaisons
AU776165B2 (en) * 1999-04-09 2004-08-26 Abbvie Inc. Use of 13C-NMR to detect binding
US6576472B1 (en) 2000-07-26 2003-06-10 Smithkline Beecham Corporation Chemical constructs for solution phase chemistry
US6584411B1 (en) 2000-07-26 2003-06-24 Smithkline Beecham Corporation Methods to facilitate the calculation of yields of reaction products
DE10117274A1 (de) * 2001-04-06 2002-10-17 Hte Ag The High Throughput Exp Verfahren zur Analyse und Archivierung von Materialien
DE10117274B4 (de) * 2001-04-06 2005-03-03 Hte Ag The High Throughput Experimentation Company Verfahren zur Analyse und Archivierung von wenigstens einer Materialbibliothek
US8963546B2 (en) 2011-03-09 2015-02-24 Bruker Biospin Ag Method for NMR spectroscopy
DE102013223891A1 (de) * 2013-11-22 2015-05-28 Joanneum Research Forschungsgesellschaft Mbh Kartuschendepositionssammler
DE102013223891B4 (de) * 2013-11-22 2015-09-17 Joanneum Research Forschungsgesellschaft Mbh Kartuschendepositionssammler und Verfahren zur Eintragsmessung atmospärischer Stoffe

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Publication number Publication date
EP0856067A4 (fr) 2005-09-21
EP0856067A1 (fr) 1998-08-05
JP2000500565A (ja) 2000-01-18

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