WO1992007864A1 - Improved process for the synthesis of oligomers - Google Patents

Improved process for the synthesis of oligomers Download PDF

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Publication number
WO1992007864A1
WO1992007864A1 PCT/US1991/007630 US9107630W WO9207864A1 WO 1992007864 A1 WO1992007864 A1 WO 1992007864A1 US 9107630 W US9107630 W US 9107630W WO 9207864 A1 WO9207864 A1 WO 9207864A1
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Prior art keywords
nucleoside
process according
water
per equivalent
oxygen
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PCT/US1991/007630
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English (en)
French (fr)
Inventor
Robert E. Klem
William B. Marvin
Timothy A. Riley
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Genta Incorporated
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Publication date
Application filed by Genta Incorporated filed Critical Genta Incorporated
Priority to AU88753/91A priority Critical patent/AU669103B2/en
Priority to DK91919675T priority patent/DK0592434T3/da
Priority to JP3518321A priority patent/JPH06509542A/ja
Priority to DE69130331T priority patent/DE69130331T2/de
Priority to EP91919675A priority patent/EP0592434B1/en
Publication of WO1992007864A1 publication Critical patent/WO1992007864A1/en

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    • 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

Definitions

  • the present invention is directed to an improved process of synthesizing oligomers.
  • the purified dimers may be linked together to give a tetramer and so forth.
  • a solid phase method is employed, using a 5'-O-protected nucleoside attached to a solid support.
  • a 5'-O-protected nucleoside is attached to a solid support and an oligomer is synthesized by chain assembly using alternating terminal 5'- deprotection reactions and coupling reactions.
  • excess reagent is added to drive the reaction to completion and unreacted
  • oligomer length is obtained.
  • oligomer is cleaved from the support, protecting groups are removed, and the deprotected oligomer is purified.
  • Suitable solid supports for those synthesis methods include silica gel, controlled pore glass beads (CPG) and polystyrene.
  • Instruments for the solid phase synthesis of oligomers are commercially available.
  • the instructions provided by the manufacturers of these instruments include preferred ratios of reactants and reagents for oligomer synthesis.
  • the coupling step large excesses of monomer and activator of monomer have been recommended.
  • the oxidation step the recommended Page missing at the time of publication
  • nucleoside includes a nucleosidyl moiety or unit and is used interchangeably therewith.
  • nucleotide refers to a subunit of a nucleic acid consisting of a phosphate group, a sugar and a nitrogen containing base.
  • RNA the sugar is ribose.
  • DNA it is a 2-deoxyribose. This term also includes analogs of such subunits.
  • nucleotide multimer refers to a chain of nucleotides linked by internucleoside phosphate linkages, or analogs thereof.
  • oligonucleotide is a nucleotide multimer generally about 3 to about 100 nucleotides in length, but which may be greater than 100 nucleotides in length. They are usually considered to be synthesized from nucleotide monomers.
  • a "deoxyribooligonucleotide” is an oligonucleotide consisting of deoxyribonucleotide monomers.
  • a "polynucleotide” refers to a nucleotide multimer generally about 100 nucleotides or more in length. These are usually of biological origin or are obtained by enzymatic means.
  • a "monomeric unit” refers to a unit of either a nucleotide reagent or a non-nucleotide reagent of the present invention, which the reagent contributes to a polymer.
  • non-nucleotide monomeric unit refers to a monomeric unit which does not significantly participate in hybridization of an oligomer. Such monomeric units must not, for example, participate in any significant hydrogen bonding with a nucleotide, and optionally include groupings capable of
  • oligomer e.g. such as crosslinking alkylation, intercalating and chelating agents.
  • Voligonucleotide/non-nucleotide multimer is a multimer generally of synthetic origin having less than 100 nucleotides, but which may contain in excess of 200 nucleotides and which contains one or more non-nucleotide monomeric units.
  • oligomer refers to oligonucleotides, nonionic oligonucleoside alkyl- and aryl-phosphonate analogs, phosphorothioate analogs of oligonucleotides, phosphoamidate analogs of oligonucleotides, neutral phosphate ester
  • oligonucleotide analogs such as phosphotriesters and other oligonucleotide analogs and modified oligonucleotides, and also includes nucleotide/non-nucleotide polymers.
  • the term also includes nucleotide/non-nucleotide polymers wherein one or more of the phosphorous group linkages between monomeric units has been replaced by a non-phosphorous linkage such as a formacetal linkage, a sulfamate linkage, or a carbamate linkage.
  • alkyl- or aryl-phosphonate oligomer refers to nucleotide/non-nucleotide polymers) having internucleoside (or intermonomer) phosphorus group linkages wherein at least one alkyl- or aryl- phosphonate linkage replaces a phosphodiester linkage.
  • methylphosphonate oligomer refers to nucleotide oligomers (or nucleotide/non-nucleotide polymer) having internucleoside (or intermonomer) phosphorus group linkages wherein at least one methylphosphonate
  • internucleoside linkage replaces a Phosphodiester internucleoside linkage.
  • p in, e.g., as in ApA represents a phosphate diester linkage
  • E in, e.g., as in CpG represents a methylphosphonate linkage.
  • Certain other sequences are depicted without the use of p or p to indicate the type of phosphorus diester linkage.
  • a as in ATC indicates a phosphate diester linkage between the 3'-carbon of A and the 5' carbon of T
  • A, as in ATC or ATC indicates a methylphosphonate linkage between the 3'-carbon of A and the 5'-carbon of T or T.
  • non-adverse conditions describes conditions (of reaction or synthesis) which do not substantially adversely affect the oligomer skeleton and its sugar, and base components, nor the solid support.
  • One skilled in the art can readily identify functionalities, coupling methods, deblocking and deprotection procedures and cleavage conditions which meet these criteria.
  • blocking conditions describes the conditions used to remove the blocking (or protecting) group from the 5'-OH group on a ribose or deoxyribose group.
  • deprotecting conditions describes the conditions used to remove the protecting groups from the nucleoside bases.
  • cap refers to a step in the reaction cycle in which any 5'-hydroxyl groups of the first nucleoside (of a particular reaction cycle) that failed to condense (i.e. react) with the activated coupling group of the second nucleoside of that reaction cycle) are blocked, rendering them unreactive in further reaction cycles.
  • loading refers to the amount of nucelosidyl moiety (or nucleoside) which is coupled or linked (by a linking mnoiety) to a support or the polymeric moiety of a polymeric reagent and is typically expressed in ⁇ moles nucleoside per gram support.
  • support refers to a solid particulate material to which a nucleoside is linked and from which an oligomer can be synthesized. Supports used in synthesizing oligomers are typically substantially inert and nonreactive with the reagents used in the synthesis of oligomers. Page missing at the time of publication
  • the basic solid phase process of forming a phosphorous- containing internucleoside linkage is depicted in Figure 1 and includes four steps: (a) deblocking of the 5'-hydroxyl of the first nucleoside; (b) adding the second nucleoside (or “monomer” in Figure 1) to the reaction mixture having first nucleoside in the presence of an activator under coupling and activating conditions, so that the second nucleoside "couples” or “condenses” with the first nucleoside to give an internucleoside linkage having a trivalent phosphorus group; (c) oxidizing the trivalent phosphorus group to a pentavalent phosphorus group using a low water oxidizer reagent; and (d) capping to block unreacted (or uncoupled) 5'-hydroxyl groups on the first nucleoside.
  • the present invention is directed to a process for the preparation of deoxyribonucleoside phosphate or phosphonate esters of the formula
  • T is a blocking group for a primary hydroxyl group
  • B is a base
  • R is hydroxy, alkyl, aryl, optionally substituted alkoxy, or optionally substituted aryloxy
  • Sp is a support or a nucleoside 5'-p-osphorus ester of the formula
  • a low water oxidizer agent which comprises about 2% or less water and at least about 1 to about 5 equivalents water per equivalent first nucleoside, preferably from about 0.1% to about 0.5% water.
  • This step involves treating the first nucleoside to remove the blocking group to give a 5'-hydroxyl which is capable of reacting with the coupling group of the second nucleoside.
  • the blocking group is preferably di-(p-anisoyl)phenylmethyl ("dimethoxytrityl", “trityl” or “DMT”) which is conveniently acid labile and is removable under non-adverse conditions.
  • a suitable reagent for detritylation is dichloroacetic acid in
  • the effluent solution may be collected after each detritylation and assayed spectrophotometrically to give the coupling efficiency of the preceding reaction sequence.
  • the coupling step is sensitive to moisture.
  • any water must be removed from the first nucleoside- support before the coupling step.
  • the first nucleoside is dried under vacuum, and preferably, an argon atmosphere is introduced, before the activated second nucleoside ("monomer") is introduced.
  • the X 1 substituent of the coupling group preferably comprises a dialkylamino group.
  • Such coupling groups are preferably activated to enhance reaction with the 5'- hydroxyl of the first nucleoside.
  • Tetrazole comprises a suitable activator and acts by protonating the nitrogen atom of the dialkylamino group and also by forming a nucleoside-3'-0- phosphomonotetrazolide via a substitution reaction. Both species will undergo rapid nucleophilic substitution reactions with the 5'-hydroxyl group of the first nucleoside to form an
  • the coupling step may be conducted by mixing an aliquot of second nucleoside (monomer) and an aliquot of tetrazole solution in a dry, argon filled vessel and then injecting the monomer-tetrazole mixture immediately into a vessel containing the deprotected first nucleoside and allowing a reaction time of about three minutes.
  • the coupling mixture activated monomer
  • the support is washed by acetonitrile before proceeding with the oxidizing step.
  • the relatively unstable trivalent phosphorus of the internucleoside linkage is converted into a stable pentavalent phosphorus linkage by treatment with an oxidizer reagent.
  • This reagent conventionally comprises iodine, tetrahydrofuran, 2,6- lutidine and from at least about 2% to about 25% or more water.
  • Oxidizer reagents which comprise relatively large amounts of water have been used for oligomer synthesis and are recommended by manufacturers of DNA synthesizers such as
  • Milligen the manufacturer of Biosearch DNA synthesizers, recommends an oxidizer reagent which comprises 2.3 equivalents iodine (l 2 ) and 716 equivalents water per equivalent of internucleoside linkage (i.e. per equivalent first nucleoside), or about 9% water.
  • oxidizer reagents have at least about 2% water up to about 25% or more water.
  • oxidizer reagents having on the order of from about 0.1% to about 0.5%, water such as reagents in the range of about 0.25% to about 0.18% water (or about 2.2
  • the trivalent phosphorus internucleoside linkage is oxidized to a pentavalent phosphorus internucleoside linkage using a low water oxidizer reagent which comprises less than about 2% water and at least from about 1 to about 5 equivalents water per equivalent first nucleoside.
  • the oxidizer reagent comprises about 100mM to about 200mM oxidizigg agent and at least about 2 to about 5 equivalents oxidizing agent per equivalent first nucleoside.
  • Suitable oxidizing agents include iodine (I 2 ).
  • low water oxidizer reagents which contain significantly lower amounts of water is additionally advantageous because the coupling step is especially water- sensitive, and with use of these low water oxidizer reagents in the oxidizing step, there is less water to be removed before the growing oligomer goes through the coupling step of the next reaction cycle.
  • use of low water oxidizer reagents in a solid phase process may reduce the washing steps needed to dry the support carrying the growing oligomer chain and, thus, reduce the overall amount of solvent or drying agent required to dry the support.
  • the oxidizing step is generally complete in less than one minute using a low water oxidizer reagent as described above (which comprises iodine, water, tetrahydrofuran and 2,6- lutidine).
  • a low water oxidizer reagent as described above (which comprises iodine, water, tetrahydrofuran and 2,6- lutidine).
  • the support carrying the first nucleoside is washed well with acetonitrile, until it and the effluent (washings) are colorless, before the capping reagent is added,
  • the capping step serves to render unreactive any remaining free hydroxyl groups of the first nucleoside which did not react with activated coupling group of the second nucleoside.
  • the capping step ensures that subsequent addition reactions proceed only by propagating chains of the desired nucleoside sequence.
  • uncondensed 5'-hydroxyl groups of the first nucleoside are acetylated by acetic anhydride rendering them inert (or
  • DMAP dimethylaminopyridine
  • acetic anhydride and DMAP solutions may be added concomitantly.
  • Capping is generally complete in about two minutes or less. Excess capping reagent is removed by
  • the use of the capping step by ensuring that previously uncondensed 5'-nydroxyls are not extended, means that in the deblocking step, the amount of DMT released is directly proportional to the coupling efficiency of the reaction cycle and, accordingly, measurement of the amount of DMT released in the deblocking (or detritylation) step may be used to monitor coupling efficiencies.
  • excess capping reagent may help to scavenge residual water remaining on the solid support from the oxidizer reagent.
  • oligomer to give 150 ⁇ moles of that nucleoside is placed in the reactor vessel of a Biosearch 8800 DNA synthesizer.
  • the support is treated with 5 X 14.6 ml aliquots of 2.5% dichloracetic acid in dichloromethane. The bright orange colored solution is collected for later spectrophotometric analysis to determine coupling efficiencies.
  • the support is then washed with 7 x 17.5 ml aliguots of dry acetonitrile.
  • To the support is added 4 ml of a 100 mM solution of phosphonamidite monomer (400 ⁇ moles or 2.7 equivalents, or if the added monomer is T, slightly less may be used, about 2.5 equivalents).
  • tetrazole (1.98 ml, 894 ⁇ moles, 450 mM or 2.24 equivalents with respect to monomer
  • the mixture is allowed to stir for about three minutes, is filtered and then washed with 2 x 2.8 ml acetonitrile.
  • oxidizer 25 g/l I 2 , 0.18% water, 25% 2,6-lutidine, 74.82% tetrohydrofuran
  • the mixture is allowed to stir for about one minute and is then filtered and washed with 4 x 18 ml dry acetonitrile.
  • the material on the support (dinucleotide) is then treated with the concomitant addition of 10 ml CAP A solution (40% acetic anhydride, 60% tetrahydrofuran) and 10 ml CAP B solution (0.625% 4- dimethylaminopyridine in anhydrous pyridine). The mixture is allowed to stir for about one minute and then filtered. The support is washed with 6 x 18 ml acetonitrile.
  • nucleoside monomeric units For addition of additional nucleoside monomeric units to the growing oligomer chain, the above procedure is repeated, using the appropriate nucleoside monomer until the oligomer of designated length and sequence is synthesized.
  • Table I depicts a comparison of reagent ratios conventionally used and those used according to the preferred process of the present invention.
  • Table II lists the coupling efficiencies obtained in synthesizing two methyphosphonate oligomers using this general procedure.
  • the lot of C monomer used in the syntheses reported in Table II was found to have about 2.5% triethylamine.
  • Triethylamine is used in the eluant to purify the monomer and is normally removed by the three subsequent co-evaporations with acetonitrile.
  • the Tetrazole activator is a weak acid and acts as such to activate the monomer and enhance coupling of monomer to support (or oligomer chain). It is believed the residual triethylamine (a base) may have neutralized the tetrazole activator, thereby decreasing coupling. Procedures which normally remove triethylamine in this instance did not adequately remove residual triethylamine.
  • hydroxylated methacrylic polymer beads 100 g, 650 A pore size, 40-90 ⁇ m particle size
  • epoxide groups 812 ⁇ eq/g
  • 1,12-dodecanediamine 100 g
  • 1,4-dioxane 1 liter
  • the mixture was stirred and refluxed for 18 hours.
  • the mixture was then filtered warm and the solids washed with warm dioxane (300 ml).
  • the solid material was washed with dichloromethane and air dried.
  • the solid was suspended in a 2.5% solution of dichloracetic acid in
  • Solid support (2.44 g) derivatized with 5'-0-DMT-N- isobutyryl 3'-0-succinyl deoxycytidine (61.5 ⁇ moles/g) was placed in the reactor vessel of a Biosearch 8800 DNA synthesizer. This solid support was treated with 5 x 14.6 ml aliguots of 2.5% dichloracetic acid in dichloromethane. The bright orange colored solution was collected for later spectrophotometric analysis. The support was then washed with 7 x 17.5 ml aliquots of dry acetonitrile.
  • Cap A solution (10 ml, 40% acetic anhydride, 60% tetrahydrofuran) and Cap B solutions (10 ml, 0.625% 4-dimethylaminopyridine in anhydrous pyridine). This mixture was allowed to stir for 1 minute. The mixture was filtered and the support washed with 6 x 18 ml portion of acetonitrile. At this point the cycle was repeated starting with
  • the synthetic cycle consisted of two programs as follows:
  • Deblocking Removal of the 5'-0-dimethoxytrityl with dichloroacetic acid. Wash the oligomer with dry acetonitrile to prepare for coupling.
  • Oxidizing Oxidize using an oxidizer reagent which comprises 0.1 M I 2 with 0.1 M water in tetrahydrofuran/2,6- lutidine.
  • the programs used with the synthesizer were named MTHL_06 (main) and CPLAWll (coupling) and were obtained from the manufacturer.
  • Oxidizer 0.1 M I 2 in tetrahydrofuran/2,6- lutidine/water (74.82/25/0.18:v/v/v).
  • Wash A acetonitrile containing ⁇ 30 ppm water.
  • Wash B acetonitrile containing ⁇ 30 ppm water.
  • the oligomer was synthesized using a support which comprised controlled pore glass beads derivatized with deoxyguanosine.
  • the average coupling efficiency was 96.7% per cycle based on trityl absorbance observed at 504 nm with a standard deviation of 2.02.
  • the overall yield of oligomer (18 mer) was 56.5%.
  • Table III lists the coupling efficiencies obtained in each reaction cycle in synthesizing this oligomer.
  • Table IV depicts a comparison of coupling efficiencies (C.E.) using oxidizer reagents having water contents ranging from
  • Oligomers were synthesized using either a Biosearch 8750 or 8800 DNA synthesizer. Reaction cycles were carried out as described previously (see, e.g. Examples 3 and 4) using the noted monomer ratio and oxidizer reagent.

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PCT/US1991/007630 1990-10-26 1991-10-21 Improved process for the synthesis of oligomers WO1992007864A1 (en)

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AU88753/91A AU669103B2 (en) 1990-10-26 1991-10-21 Improved process for the synthesis of oligomers
DK91919675T DK0592434T3 (da) 1990-10-26 1991-10-21 Forbedret fremgangsmåde til syntese af oligomerer
JP3518321A JPH06509542A (ja) 1990-10-26 1991-10-21 オリゴマーを合成するための改良法
DE69130331T DE69130331T2 (de) 1990-10-26 1991-10-21 Verbessertes verfahren zur synthese von oligomeren
EP91919675A EP0592434B1 (en) 1990-10-26 1991-10-21 Improved process for the synthesis of oligomers

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WO1993012135A1 (en) 1991-12-12 1993-06-24 Gilead Sciences, Inc. Nuclease stable and binding competent oligomers and methods for their use
WO1994001446A3 (en) * 1992-07-09 1994-03-03 Beckman Instruments Inc Derivatized organic solid support for nucleic acid synthesis
FR2707296A1 (fr) * 1993-07-09 1995-01-13 Genset Sa Procédé de synthèse d'acides nucléiques sur support solide et composés utiles notamment comme support solide dans ledit procédé.
EP0697414A1 (en) 1994-07-19 1996-02-21 Gen-Probe Incorporated Compounds and methods for inhibiting propagation of human immunodeficiency virus
US5629413A (en) * 1993-07-19 1997-05-13 Gen-Probe Incorporated Oligonucleotides with activity against human immunodeficiency virus
US5674995A (en) * 1995-06-07 1997-10-07 Gen-Probe Incorporated Oligonucleotides specific for cytokine signal transducer gp130 mRNA
US5716846A (en) * 1995-06-07 1998-02-10 Gen-Probe Incorporated Method for inhibiting cellular proliferation using antisense oligonucleotides to interleukin-6 receptor mRNA
US5731148A (en) * 1995-06-07 1998-03-24 Gen-Probe Incorporated Adduct protection assay
US5739309A (en) * 1993-07-19 1998-04-14 Gen-Probe Incorporated Enhancement of oligonucleotide inhibition of protein production, cell proliferation and / or multiplication of infectious disease pathogens
US5747470A (en) * 1995-06-07 1998-05-05 Gen-Probe Incorporated Method for inhibiting cellular proliferation using antisense oligonucleotides to gp130 mRNA
US5824475A (en) * 1993-07-19 1998-10-20 Gen-Probe Incorporated Oligonucleotide screening assay using DNA-altering agents and probes with protectable labels
US6025133A (en) * 1996-12-30 2000-02-15 Gen-Probe Incorporated Promoter-sequestered oligonucleoside and method of use
US6074826A (en) * 1995-01-19 2000-06-13 Gen-Probe Incorporated Nucleic acid amplification oligonucleotides and probes to Lyme disease associated Borrelia
US6100027A (en) * 1995-06-07 2000-08-08 Gen-Probe Incorporated Nucleic acid probes and amplification oligonucleotides for Neisseria species
US6146833A (en) * 1997-02-11 2000-11-14 Beckman Coulter, Inc. Polymeric reagents for immobilizing biopolymers
WO2008016988A1 (en) 2006-08-01 2008-02-07 Gen-Probe Incorporated Methods of nonspecific target capture of nucleic acids
WO2014028739A1 (en) 2012-08-15 2014-02-20 Isis Pharmaceuticals, Inc. Method of preparing oligomeric compounds using modified capping protocols
EP2757091A1 (en) 2008-04-01 2014-07-23 Biosearch Technologies, Inc. Stabilized Nucleic Acid Dark Quencher-Fluorophore Probes
WO2016160646A1 (en) * 2015-04-02 2016-10-06 Merck Sharp & Dohme Corp. Process for making phosphoramidate protected nucleoside compounds
WO2018209068A1 (en) 2017-05-11 2018-11-15 Gen-Probe Incorporated Compositions and methods for isolating target nucleic acids
US10301310B2 (en) 2014-05-09 2019-05-28 Biosearch Technologies, Inc. Cosmic quenchers
US10519485B2 (en) 2015-04-15 2019-12-31 Biosearch Technologies, Inc. Dual quencher probes
WO2021097358A1 (en) 2019-11-14 2021-05-20 Gen-Probe Incorporated Compositions and methods for capturing target nucleic acids

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US7435390B2 (en) * 2001-01-26 2008-10-14 Third Wave Technologies, Inc. Nucleic acid synthesizers
US6932943B1 (en) 2001-01-26 2005-08-23 Third Wave Technologies Nucleic acid synthesizers
US10913767B2 (en) * 2010-04-22 2021-02-09 Alnylam Pharmaceuticals, Inc. Oligonucleotides comprising acyclic and abasic nucleosides and analogs

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Cited By (32)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993012135A1 (en) 1991-12-12 1993-06-24 Gilead Sciences, Inc. Nuclease stable and binding competent oligomers and methods for their use
WO1994001446A3 (en) * 1992-07-09 1994-03-03 Beckman Instruments Inc Derivatized organic solid support for nucleic acid synthesis
FR2707296A1 (fr) * 1993-07-09 1995-01-13 Genset Sa Procédé de synthèse d'acides nucléiques sur support solide et composés utiles notamment comme support solide dans ledit procédé.
WO1995001987A1 (fr) * 1993-07-09 1995-01-19 Genset Procede de synthese d'acides nucleiques sur support solide et comoposes utiles notamment comme support solide dans ledit procede
US5739309A (en) * 1993-07-19 1998-04-14 Gen-Probe Incorporated Enhancement of oligonucleotide inhibition of protein production, cell proliferation and / or multiplication of infectious disease pathogens
US5629413A (en) * 1993-07-19 1997-05-13 Gen-Probe Incorporated Oligonucleotides with activity against human immunodeficiency virus
US5919701A (en) * 1993-07-19 1999-07-06 Gen-Probe Incorporated Oligonucleotides with activity against human immunodeficiency virus
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AU669103B2 (en) 1996-05-30
JPH06509542A (ja) 1994-10-27
EP0592434B1 (en) 1998-10-07
DK0592434T3 (da) 1999-06-21
EP0592434A4 (en) 1996-04-24
US5726300A (en) 1998-03-10
ES2124231T3 (es) 1999-02-01
NZ240325A (en) 1993-06-25
TW216423B (GUID-C5D7CC26-194C-43D0-91A1-9AE8C70A9BFF.html) 1993-11-21
CA2094803A1 (en) 1992-04-27
DE69130331T2 (de) 1999-03-04
IL99844A (en) 1995-07-31
AU8875391A (en) 1992-05-26
DE69130331D1 (de) 1998-11-12
EP0592434A1 (en) 1994-04-20
ATE171947T1 (de) 1998-10-15
IE913761A1 (en) 1992-05-22

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