WO2004113553A2 - Procede de production d'amidites pures et d'oligonucleotides - Google Patents

Procede de production d'amidites pures et d'oligonucleotides Download PDF

Info

Publication number
WO2004113553A2
WO2004113553A2 PCT/US2004/018704 US2004018704W WO2004113553A2 WO 2004113553 A2 WO2004113553 A2 WO 2004113553A2 US 2004018704 W US2004018704 W US 2004018704W WO 2004113553 A2 WO2004113553 A2 WO 2004113553A2
Authority
WO
WIPO (PCT)
Prior art keywords
amidite
critical
sample
impurity
independently selected
Prior art date
Application number
PCT/US2004/018704
Other languages
English (en)
Other versions
WO2004113553A8 (fr
WO2004113553A3 (fr
Inventor
Anthony N. Scozzari
Achim H. Krotz
Original Assignee
Isis Pharmaceuticals, 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 Isis Pharmaceuticals, Inc. filed Critical Isis Pharmaceuticals, Inc.
Publication of WO2004113553A2 publication Critical patent/WO2004113553A2/fr
Publication of WO2004113553A8 publication Critical patent/WO2004113553A8/fr
Publication of WO2004113553A3 publication Critical patent/WO2004113553A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • 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
    • 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
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
    • 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
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical

Definitions

  • the present invention relates to oligonucleotide synthesis.
  • the present invention provides methods for making high quality phosphoramidites and oligonucleotides derived therefrom.
  • Oligonucleotides have been used in various biological and biochemical applications. They have been used as primers and probes for the polymerase chain reaction (PCR), as antisense agents used in target validation, drug discovery and development, as ribo2ymes, as aptamers, and as general stimulators of the immune system. As the popularity of oligonucleotides has increased, the need for producing greater sized batches, and greater numbers of small-sized batches, has increased at pace. Additionally, there has been an increasing emphasis on reducing the costs of oligonucleotide synthesis, and on improving the purity and increasing the yield of oligonucleotide products.
  • PCR polymerase chain reaction
  • oligonucleotide synthesis A number of innovations have been introduced to the art of oligonucleotide synthesis. Amongst these innovations have been the development of excellent orthogonal protecting groups, activators, reagents, and synthetic conditions.
  • the oligonucleotides themselves have been subject to a variety of modifications and improvements. Amongst these are chemistries that improve the affinity of an oligonucleotide for a specific target, that improve the stability of an oligonucleotide in vivo, that enhance the pharmacokinetic (PK) and toxicological (Tox) properties of an oligonucleotide, etc. These novel chemistries generally involve a chemical modification to one or more ofthe constituent parts ofthe oligonucleotide.
  • oligonucleotide thus embraces a class of compounds that include naturally- occurring, as well as modified, oligonucleotides. Both naturally-occurring and modified oligonucleotides have proven useful in a variety of settings, and both may be made by similar processes, with appropriate modifications made to account for the specific modifications adopted.
  • a naturally occurring oligonucleotide i.e. a short strand of DNA or RNA may be envisioned as being a member of the following generic formulas, denominated oligo-RNA and oligo-DNA, respectively, below:
  • n is an integer of from 1 to about 100
  • Bx is one of the naturally occurring nucleobases.
  • oligonucleotide occurs as the anion, as the phosphate easily dissociates at neutral pH, and an oligonucleotide will generally occur in solid phase, whether amorphous or crystalline, as a salt.
  • oligonucleotide encompasses each ofthe anionic, salt and free acid forms above.
  • oligonucleotide may be thought of as being an oligomer of m monomeric subunits represented by the following nucleotides:
  • oligonucleotide deoxyribonucleotide wherein each Bx is a nucleobase, wherein the last residue is a nucleoside (i.e. a nucleotide without Hie 3 '-phosphate group).
  • each G ⁇ is 0 or S
  • each G 2 is OH or SH
  • each G 3 is O, S, CH 2 , or NH
  • each R 2 ' is H, OH, O-rg, wherein rg is a removable protecting group, a 2'-substituent, or together with R 4 ' forms a bridge
  • each R 3 ' is H, a substituent, or together with R 4 ' forms a bridge
  • each R t ' is H, a substitutent, together with R 2 ' forms a bridge, together with R forms a bridge, or together with R 5 ' forms a bridge
  • each q is 0 or 1
  • each R 5 ' is H, a substituent, or together with R t ' forms a bridge
  • each G 6 is O, S, CH 2 or
  • oligonucleotides include the solid phase methods first described by Caruthers et al. (See, for example, US Patent No. 5,750,666, incorporated herein by reference, especially columns 3-58, wherein starting materials and general methods of making oligonucleotides, and especially phosphorothioate oligonucleotides, are disclosed, which parts are specifically incorporated herein by reference.) These methods were later improved upon by K ⁇ ster et al. (See, for example, US Patent No.
  • a primer support is prepared by covalently linking a suitable nucleoside to a support (SS) through a linker.
  • SS support
  • LL is a linking group that links the nucleoside to the support via G 3 .
  • the linking group is generally a di-functional group, covalently binds the ultimate 3 '-nucleoside (and thus the nascent oligonucleotide) to the solid support during synthesis, but which is cleaved under conditions orthogonal to the conditions under which the 5 '-protecting group, and if applicable any 2'-protecting group, are removed.
  • T' is a removable protecting group, and the remaining variables have already been defined, and are described in more detail herein.
  • Suitable primer supports may be acquired from Amersham Biosciences under the brand name Primer Support 200TM.
  • the primer support may then be swelled in a suitable solvent, e.g. acetonitrile, and introduced into a column of a suitable solid phase synthesis instrument, such as one of the synthesizers available form Amersham Biosciences, such as an AKTAoligopilotTM, or OligoProcessTM brand DNA/RNA synthesizer.
  • a suitable solvent e.g. acetonitrile
  • a suitable solid phase synthesis instrument such as one of the synthesizers available form Amersham Biosciences, such as an AKTAoligopilotTM, or OligoProcessTM brand DNA/RNA synthesizer.
  • Each of the steps (l)-(4) may be, and generally is, followed by one or more wash steps, whereby a clean solvent is introduced to the column to wash soluble materials from the column, push reagents and/or activators through the column, or both.
  • the steps (l)-(4) are depicted below:
  • T' is selected to be removable under conditions orthogonal to those used to cleave the oligonucleotide from the solid support at the end of synthesis, as well as those used to remove other protecting groups used during synthesis.
  • An art-recognized protecting group for oligonucleotide synthesis is DMT (4,4'-dimethoxytrityl).
  • the DMT group is especially useful as it is removable under weakly acid conditions.
  • an acceptable removal reagent is 3% DCA in a suitable solvent, such as acetonitrile.
  • the wash solvent if used, may conveniently be acetonitrile.
  • the support may be controlled pore glass or a polymeric bead support.
  • Some polymeric supports are disclosed in the following patents: US 6,016,895; US 6,043,353; US 5,391,667 and US 6,300,486, each of which is specifically inco ⁇ orated herein by reference.
  • pg is a phosphorus protecting group, such as a cyanoethyl group.
  • NR Ni R N2 is an amine leaving group, such as diisopropyl amino, and for teaching of suitable activator (e.g. tetrazole).
  • suitable amidites, and methods of manufacturing amidites are set forth in the following patents: US 6,133,438; US 5,646,265; US 6,124,450; US 5,847,106; US 6,001,982; US 5,705,621; US 5,955,600; US 6,160,152; US 6,335,439; US 6,274,725; US 6,329,519, each of which is specifically inco ⁇ orated herein by reference, especially as they relate to manufacture of amidites.
  • Suitable activators are set forth in the Caruther et al. patent and in the K ⁇ ster et al. patent.
  • Especially suitable activators are set forth in the following patents: US 6,031,092 and US 6,476,216, each of which is expressly inco ⁇ orated herein by reference.
  • the oxidant is an oxidizing agent suitable for introducing Gi.
  • Gi oxygen
  • G 2 sulfur
  • the oxidant may also be referred to as a filiation agent or a sulfur-transfer reagent.
  • Suitable thiation agents include the so-called Beaucage reagent, 3H-l,2-benzothiol, phenylacetyl disulfide (also referred to as PADS; see, for example the patents: US 6,114,519 and 6,242,591, each of which is inco ⁇ orated herein by reference) and thiouram disulf ⁇ des (e.g. N,N,N',N'- tetramethylthiouram disulfide, disclosed by US patent No. 5,166,387).
  • the wash may be a suitable solvent, such as acetonitrile.
  • the oxidation step is followed by a capping step, which although not illustrated herein, is an important step for synthesis, as it causes free 5' -OH groups, which did not undergo coupling in step 1, to be blocked from being coupled in subsequent synthetic cycles.
  • Suitable capping reagents are set forth in Caruthers et al., K ⁇ ster et al., and other patents described herein. Suitable capping reagents include a combination of acetic anhydride and N-methylimidazole.
  • Synthetic cycle steps (l)-(4) are repeated (if so desired) n-1 times to produce a support- bound oligonucleotide:
  • the protecting group pg may be removed by a method as described by Caruthers et al. or K ⁇ ster et al., supra.
  • pg is a cyanoethyl group
  • the methodology of K ⁇ ster et al., e.g. reaction with a basic solution is generally suitable for removal of the phosphorus protecting group.
  • TAA frieuiylamine
  • oligonucleotide may be visualized as having the formula:
  • Figures la-n show exemplary syntheses of representative amidite sample impurities useful in the methods ofthe invention.
  • the present invention provides methods of characterizing an amidite sample, the method comprising: identifying at least one critical impurity and a critical impurity signal representative of each of said critical impurities; obtaining an amidite sample signal from an amidite sample comprising a critical impurity signal component representative of said critical impurity in the amidite sample; comparing the critical impurity signal and the critical impurity signal component to determine the amount of said critical impurity in said amidite sample; and either rejecting said amidite sample if the amount of the critical impurity is greater than a predetermined critical impurity threshold, or
  • the present invention provides methods of characterizing an amidite sample, the methods comprising: identifying at least one non-critical impurity and a non-critical impurity signal representative of each of said non-critical impurities; obtaining an amidite sample signal from an amidite sample comprising a non-critical impurity signal component representative of said non-critical impurity in the amidite sample; comparing the non-critical impurity signal and the non-critical impurity signal component to determine the amount of said non-critical impurity in said amidite sample; and either rejecting said amidite sample if the amount of the non-critical impurity is greater than a predetermined non-critical impurity threshold, or
  • the present invention provides methods of characterizing an amidite sample, the methods comprising: identifying' y critical impurities and y critical impurity signals representative of each of the v critical impurities, wherein y is an integer of 2 or more; obtaining an amidite sample signal from an amidite sample comprising y critical impurity signal components representative of the quantity of each of the y critical impurities in the amidite sample; comparing the y critical impurity signals and the y critical impurity signal components to determine a quantity of each of they critical impurities in said amidite sample; summing the quantities of each of y critical impurities in the amidite sample to obtain a sum 7; rejecting said amidite sample if one or more of quantities of y critical impurities is greater than a predetermined critical impurity threshold, or rejecting said amidite sample if the sum Y is greater than a predetermined critical impurity sum threshold; and otherwise accepting said amidite sample as an amidite starting material
  • the foregoing method further comprises: identifying x non-critical impurities and x non-critical impurity signals representative of each of the x non-critical impurities (wherein x is an integer of 2 or more); obtaining an amidite sample signal from an amidite sample comprising x non-critical impurity signal components representative of the quantity of each of the x non-critical impurities in the amidite sample; comparing the x non-critical impurity signals and the x non-critical impurity signal components to determine a quantity of each of the x non-critical impurities in said amidite sample; summing the quantities of each of x non-critical impurities in the amidite sample to obtain a sum X; rejecting said amidite sample if one or more of quantities of x non-critical impurities is greater than a predetermined non-critical impurity threshold, or rejecting said amidite sample if the sum X is greater than a predetermined non-critical impurity sum threshold; and
  • the present invention provides methods of characterizing an amidite sample, the methods comprising: identifying x non-critical impurities and x non-critical impurity signals representative of each of Hie x non-critical impurities, wherein x is an integer of 2 or more; obtaining an amidite sample signal from an amidite sample comprising x non-critical impurity signal components representative of the quantity of each ofthe x non-critical impurities in the amidite sample; comparing the x non-critical impurity signals and the x non-critical impurity signal components to determine a quantity of each of the x non-critical impurities in said amidite sample; summing the quantities of each of x non-critical impurities in the amidite sample to obtain a sum ; rejecting said amidite sample if one or more of quantities of x non-critical impurities is greater than a predetermined non-critical impurity threshold, or rejecting said amidite sample if the sum X is greater than a
  • the present invention provides methods of making an oligonucleotide, the methods comprising: providing the amidite starting material accepted any ofthe foregoing methods; and conducting at least one synthetic cycle in which said nucleoside amidite is coupled to a moiety, optionally in the presence of a suitable activator or solvent, under conditions suitable to form a phosphate diester intermediate.
  • the amidite sample comprises a compound of formula (I):
  • T' is a removable protecting group; and pg is a phosphorous protecting group.
  • critical impurity is selected from compounds of formulas:
  • rg for formulas (e), (j), (k), (1), (o), (q), and (r), is DMT.
  • pg is selected from: methyl, ethyl, cyanoethyl, or 2-cyanopropyl. In some such embodiments, pg is cyanoethyl.
  • -NR NI R N2 is an amine leaving group.
  • each of R M and R N2 is independently selected from C ⁇ -C 6 straight-chained or branched alkyl, or R and R N2 taken together with the nitrogen atom to which they are attached form a heterocycloalkyl or a heterocycloalkenyl ring.
  • each of R N ⁇ and R N2 is isopropyl.
  • the critical impurity signal, the amidite sample signal, and the critical signal component are obtained by nuclear magnetic resonance spectroscopy, liquid chromatography, mass spectrometry, high pressure liquid chromatography, or liquid chromatography/mass spectrometry.
  • the non-critical impurity is selected from a compound of formulas:
  • rg is DMT.
  • pg is selected from methyl, ethyl, cyanoethyl, or 2-cyanopropyl. In some further such embodiments, pg is cyanoethyl.
  • R N ⁇ _ R N2 is an amine leaving group.
  • each R N ⁇ and R N2 is independently selected from C ⁇ -C 10 straight-chained or branched alkyl, or R N ⁇ and R N2 taken together with the nitrogen atom to which they are attached form a heterocycloalkyl or a heterocycloalkenyl ring.
  • R NI and Rr ⁇ are isopropyl.
  • the present invention also provides methods of making oligonucleotides, the methods comprising providing an amidite sample that has been accepted according to the foregoing procedure, and conducting at least one cycle in which said amidite is coupled to a moiety, optionally in the presence of a suitable activator and/or solvent, and under conditions suitable to form a phosphite diester intermediate: wherein n is 0 or an integer and the other variables are as defined above.
  • the moiety to which the amidite is coupled is the moiety (H*G 6 -) in the formula:
  • the present invention also provides novel compounds ofthe following formulae (a) - (r). Such compounds may be used as authentic compounds for identifying critical or non-critical impurity signals representative of an amount of a critical or non-critical impurity, and said critical or non-critical impurity signal representative of an amount of a critical or non-critical impurity may be used in the methods outlined above.
  • compounds of formulae (a), (b), (c), (d), (f), (g), (h), (i), (m), (n), and (p), are non-critical impurities useful in the methods of the present invention.
  • compounds of formulae (), (j), (k), (1), (o), (q), and (r), are critical impurities useful in the methods ofthe present invention.
  • the present invention also provides an amidite starting material that has been validated by one ofthe methods set forth hereinabove.
  • the present invention also provides methods of determining which impurities in an amidite sample are critical and non-critical impurities.
  • the present invention also provides methods of discriminating between amidite samples that are acceptable and non-acceptable for oligonucleotides synthesis.
  • the present invention also provides methods for of oligonucleotide synthesis, the method comprising validating an amidite starting material according to one of the foregoing methods, and carrying out one or more synthesis cycles comprising coupling, oxidizing and capping.
  • One advantage to the present invention is that it provides a method to discriminate between critical and non-critical amidite impurities.
  • the present invention provides further advantages.
  • the present invention provides standards for determining the amount of critical impurities in an amidite sample, and standards for determining the amount of non- critical impurities in an amidite sample.
  • Another advantage of the present invention is that it provides methods of obtaining critical impurity signals that are representative ofthe amounts of critical impurities in an amidite sample.
  • a further advantage of the present invention is that it provides methods of obtaining non-critical impurity signals that are representative ofthe amounts of non-critical impurities in an amidite signal.
  • values representative of critical impurity signals may be stored in a computer-readable format and may be compared to an amidite sample signal by a computer.
  • values representative of non-critical impurity signals may be stored in a computer-readable format and may be compared to an amidite sample signal by a computer.
  • acceptance or rejection criteria may be stored in a computer-readable format.
  • a computer may compare an amidite sample signal to said acceptance and/or rejection criteria, and may provide a human- readable indicator of an amidite sample's acceptance or rejection.
  • the present invention thus provides novel compounds, methods of obtaining and using both critical and non-critical impurity signals, methods of using those critical and non-critical impurity signals to make decisions regarding amidite starting material suitability for oligonucleotide synthesis, and methods of carrying out oligonucleotide synthesis using the product of such decisions.
  • the present invention is concerned with the art of oligonucleotide synthesis, and in particular with providing starting materials of excellent purity, the use of which will provide oligonucleotides having excellent purity.
  • the invention provides, in particular, methods of identifying amidite starting materials having purity profiles suitable for synthesizing oligonucleotides having excellent purity, and in some embodiments purity sufficient to qualify the oligonucleotides for inclusion in drug preparations.
  • an amidite sample is a composition of matter comprising a phosphoramidite.
  • the phosphoramidite is embraced by formula (I):
  • the variables set forth in formula I are as defined above, and as further described below.
  • the amidite sample also may comprise at least one impurity, which, when present in the sample, is said to have an impurity concentration (expressed in suitable units, e.g. ppm, percent (w/w), etc.).
  • the method according to the present invention comprises identifying said impurity, identifying at least one impurity signal that is representative of the impurity concentration in the sample, detecting said impurity signal in said amidite sample, detennining from the impurity signal the impurity concentration, comparing the quantity of impurity with a predetermined impurity threshold, rejecting the amidite sample if the quantity of impurity exceeds the predetermined impurity threshold, or accepting the amidite sample as an amidite starting material if the quantity ofthe identified impurity does not exceed the impurity threshold.
  • the impurity may be further classified as either a critical impurity or a non-critical impurity.
  • a critical impurity is an impurity that reacts with a nascent oligonucleotide chain during synthesis, and which permits further chain extension upon reaction. Certain critical impurities react with the 5' -OH ofthe nascent oligonucleotide chain, and themselves are capable of reacting with an amidite, thereby giving rise to oligonucleotides having inco ⁇ orated therein the impurity as a constituent part.
  • critical impurities react with the nascent oligonucleotide chain at positions other than the 5' -OH, and are inco ⁇ orated into the oligonucleotide as adducts. Some critical impurities give rise to branchmers. Other critical impurities give rise to adducts. Exemplary critical impurities will be illustrated in more detail below.
  • Non-critical impurities are impurities that are either inert with respect to the nascent oligonucleotide chain or non-inert.
  • Inert non-critical impurities are those impurities that do not react with the nascent oligonucleotide.
  • Non-inert non-critical impurities are those impurities that react with the oligonucleotide chain but result in the termination of the oligonucleotide sequence, and thus are similar in chemical behavior to capping.
  • a non-inert non-critical impurity may be referred to as a capping impurity.
  • Impurities may arise out of one or more of the process steps used to make the amidite sample, or may result from degradation of amidite.
  • amidites having the following fonnulae:
  • the impurity signal may be a signal that is representative of the concentration of the impurity in an amidite sample.
  • Suitable signals may include an abso ⁇ tion signal, a fluorescence signal, a phosphorescence signal, a mass spectrometry signal, etc.
  • An abso ⁇ tion signal includes ultraviolet light abso ⁇ tion signal, a nuclear magnetic resonance signal, a visible light abso ⁇ tion signal, etc.
  • Such a signal may be gathered by, for example, an HPLC instrument with an abso ⁇ tion detector, e.g. an ultraviolet detector.
  • a mass spectrometry signal may include an HPLC-mass spectrometry signal.
  • the impurity signal may vary linearly, log- linearly, log-log, or otherwise with respect to the impurity concentration.
  • an authentic impurity may be prepared and subjected to HPLC, whereby there is produced an impurity signal, the impurity signal comprising an HPLC retention time and a detector signal strength that varies in a consistent relationship to the amount of authentic impurity applied to the HPLC column.
  • an impurity signal may be multidimensional, comprising a first signal component representing the identity of the impurity (in the case of HPLC, retention time; in the case of mass- spectrometry, m/z) and a second signal component representative of impurity concentration (in the case of HPLC, UV absorbance; in the case of mass-spectrometry, electrode signal strength).
  • the signal component that is representative of impurity identity may be thought of as a qualitative component, while the signal component that is representative of the impurity's concentration in the sample may be thought of as a quantitative component.
  • an amidite sample may be subjected to the same testing method to determine whether the impurity is present in the sample (a qualitative determination) and if so, at what concentration (quantitative dete ⁇ nination). For example in HPLC, an amidite sample is applied to a column and the retention times and areas of at least two peaks (one representing the amidite and one representing the impurity) are recorded. The retention times are used to identify the signals relating to the amidite and the impurity, while the relative areas of the elution peaks of the amidite and impurity are used to determine the concentration of impurity relative to the amidite sample.
  • impurity concentration expressed as % impurity, is:
  • % impurity 100 % x (A; / (A ; + A A )), where A; is the area under the impurity's elution peak and A A is the area under the amidite's elution peak.
  • concentration of the impurity in the amidite sample is then compared to a predetermined impurity limit.
  • An amidite sample having an impurity concentration greater than the impurity limit is classified as rejected.
  • a rejected amidite sample may be destroyed, recycled, further purified, or otherwise disposed of, but not used in oligonucleotide synthesis.
  • An amidite sample in which no impurity's concentration exceeds its predetermined impurity limit is then classified as amidite starting material, which may be used as a starting material for making oligonucleotide.
  • n impurities are known to potentially be present in an amidite sample
  • a signal may be identified for each of the n impurities.
  • the concentration for impurity X is then calculated as:
  • %X l0Q% x Ax l(A A + ⁇ A ⁇ ) ,
  • a x is the area ofthe peak corresponding to impurity X, where is an integer from 1 to n
  • a A is the area ofthe peak corresponding to the amidite
  • each A is the area ofthe z ' th impurity peak, wherein i is an integer from 1 to n.
  • a predetermined impurity limit is selected for each impurity. If any impurity concentration exceeds its predetermined impurity threshold, the amidite sample is rejected. If each impurity concentration is less than or equal to its impurity threshold, the amidite sample is classified as an amidite starting material, which may be used in oligonucleotide synthesis. ,
  • the tenn “amidite sample” refers to a composition of matter, which contains a phosphoramidite, preferably of formula I, and optionally a detectable amount of at least one impurity.
  • An amidite sample may further be classified as an “amidite starting material” or as “rejected,” as described in more detail herein.
  • an amidite starting material refers to a composition of matter, which contains a phosphoramidite, preferably of formula I and optionally a detectable amount of at least one impurity, and which has been classified as “amidite starting material” according to a method of the present invention. In many cases, more than one impurity will be present in the "amidite starting material.”
  • an amidite starting material is an amidite sample that has been subjected to testing as described herein, and has been determined to contain no more than the impurity threshold of each impurity tested for.
  • the term "rejected amidite sample” or simply “rejected amidite,” refers to an amidite sample that has been subjected to testing by the inventive method described herein, and has been found to contain at least one impurity at a concentration greater than its impurity threshold. It should be noted that if there are n identified impurities potentially in an amidite sample, there will potentially be n signals corresponding to the n impurities. There also may be a signal relating to the amidite itself.
  • the tenn "signal” refers to a voltage, UV absorbance, current, or other value, whether analog or digital, that represents some characteristic of an impurity or amidite.
  • a signal may comprise more than one component, e.g. a qualitative component and a quantitative component.
  • an HPLC chromatogram comprises a time component (retention time), useful for identifying the signal relating to an impurity or amidite, and an absorbance component (UV absorbance), useful for determining the amount of impurity or amidite in the sample.
  • impurity includes any compound other than amidite or solvent, if any, whether actually present in the amidite sample or not, and whether identified or not.
  • a potential impurity is an impurity that has been hypothesized to be present in an amidite sample, whether actually present or not.
  • An unidentified impurity is an impurity whose structure is not known, but which is detected in the amidite sample. In some cases, a group of impurities may be collected into an "unidentified impurity" classification.
  • a known impurity is an impurity whose structure is known. In accordance with the present invention, a known impurity in an amidite sample can be identified and its quantity measured by obtaining its characteristic signal from both a reference (authentic compound) and the amidite sample.
  • Known impurities may be further subdivided into critical impurities and non-critical impurities, as described herein.
  • Detection of an impurity in an amidite sample may be conducted using a suitable analytical method, such as HPLC, Mass-Spectrometry, GC-Mass Spec, etc.
  • a suitable analytical method such as HPLC, Mass-Spectrometry, GC-Mass Spec, etc.
  • HPLC high-LC
  • Mass-Spectrometry mass-Spectrometry
  • GC-Mass Spec a method capable of unambiguously identifying and quantifying an impurity in an amidite sample will be suitable for the disclosed methodology.
  • oligonucleotide has the meaning of an oligomer having m subunits embraced within the brackets [ ] of the formula:
  • oligonucleotide to be made is depicted in a single stranded conformation, it is common for oligonucleotides to be used in a double stranded conformation.
  • siRNA antisense method
  • two strands of RNA or RNA-like oligonucleotide are prepared and annealed together, often with a two-nucleotide overlap at the ends.
  • the present invention contemplates manufacture of both single- and double-stranded oligonucleotides.
  • the nucleobases Bx may be the same or different, and include naturally occurring nucleobases adenine (A), guanine (G), ymine (T), uracil (U) and cytosine (C), as well as modified nucleobases.
  • Modified nucleobases include heterocyclic moieties that are structurally related to the naturally-occurring nucleobases, but which have been chemically modified to impart some property to the modified nucleobase that is not possessed by naturally-occurring nucleobases.
  • nucleobase is intended to by synonymous with “nucleic acid base or mimetic thereof.”
  • a nucleobase is any substructure that contains one or more atoms or groups of atoms capable of hydrogen bonding to abase of an oligonucleotide.
  • unmodified or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5- propynyl (-C ⁇ C-CH 3 ) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6- azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and
  • nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(lH-pyrimido[5,4-b][l,4]benzoxazin- 2(3H)-one), phenothiazine cytidine (lH-pyrimido[5,4-b][l,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g.
  • nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.
  • nucleobases include those disclosed in United States Patent No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J.I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S.T. and Lebleu, B. , ed., CRC Press, 1993.
  • nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention.
  • These include 5-substituted pyrimidines, 6- azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5- propynyluracil and 5-propynylcytosine.
  • 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, Y.S., Crooke, S.T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2'-0- methoxyethyl sugar modifications.
  • Additional modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide and the 5' position of 5' terminal nucleotide.
  • one additional modification of the ligand conjugated oligonucleotides of the present invention involves chemically linking to the oligonucleotide one or more additional non-ligand moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide.
  • Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci.
  • cholic acid Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053
  • a thioether e.g., hexyl-S-tritylthiol
  • a thiocholesterol (Oberhauser et al., Nucl.
  • oligomeric compounds e.g. oligonucleotides
  • polycyclic heterocyclic compounds in place of one or more heterocyclic base moieties.
  • fricyclic heterocyclic compounds have been previously reported. These compounds are routinely used in antisense applications to increase the binding properties of the modified strand to a target strand. The most studied modifications are targeted to guanosines hence they have been termed G-clamps or cytidine analogs. Many of these polycyclic heterocyclic compounds have the general formula:
  • the gain in helical stability does not compromise the specificity of the oligonucleotides.
  • the T m data indicate an even greater discrimination between the perfect match and mismatched sequences compared to dC5 me .
  • the tethered amino group serves as an additional hydrogen bond donor to interact with the Hoogsteen face, namely the 06, of a complementary guanine thereby forming 4 hydrogen bonds. This means that the increased affinity of G-clamp is mediated by the combination of extended base stacking and additional specific hydrogen bonding.
  • Rio a is O, S orN-CH 3 ;
  • Rn a is A(Z) xl , wherein A is a spacer and Z independently is a label bonding group bonding group optionally bonded to a detectable label, but Rn a is not amine, protected amine, nitro or cyano;
  • XI is 1, 2 or 3; and
  • R ⁇ 4 is N0 2 or both R M and R ⁇ 2 are independently -CH 3 .
  • the synthesis of these compounds is dicslosed in United States Patent Serial Number 5,434,257, which issued on July 18, 1995, United States Patent Serial Number 5,502,177, which issued on March 26, 1996, and United States Patent Serial Number 5,646, 269, which issued on July 8, 1997, the contents of which are commonly assigned with this application and are inco ⁇ orated herein in their entirety.
  • a and b are independently 0 or 1 with the total of a and b being 0 or 1;
  • A is N, C or CH;
  • Z is taken together with A to form an aryl or heteroaryl ring structure comprising 5 or 6 ring atoms wherein the heteroaryl ring comprises a single O ring heteroatom, a single N ring heteroatom, a single S ring heteroatom, a single O and a single N ring heteroatom separated by a carbon atom, a single S and a single N ring heteroatom separated by a C atom, 2 N ring heteroatoms separated by a carbon atom, or 3 N ring heteroatoms at least 2 of which are separated by a carbon atom, and wherein the
  • R 20 is , independently, H, C ⁇ . 6 alkyl, C 2 . 6 alkyl, C 2 . s alkenyl, C 2 .
  • R 20 is taken together with an adjacent R 20 to complete a ring containing 5 or 6 ring atoms, and tautomers, solvates and salts thereof;
  • R 21 is, independently, H or a protecting group;
  • R 3 is a protecting group or H; and tautomers, solvates and salts thereof.
  • each R 16 is, independently, selected from hydrogen and various substituent groups.
  • a ⁇ is O or S;
  • a 7 is CH 2 , N-CH 3 , 0 or S;
  • each Q 2 is, independently, H or Pg;
  • a 10 is H, Pg, substituted or unsubstituted C,-C 10 alkyl, acetyl, benzyl, -(CH 2 ) p3 NH 2 , -(CH
  • each dashed line ( — ) indicates a point of attachment to an adjacent phosphorus atom, represents the sugar portion of a general nucleoside or nucleotide as embraced by the present invention.
  • Suitable 2 '-substituents corresponding to R' 2 include: OH, F, O-alkyl (e.g. O-methyl), S- alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl; O-alkynyl, S-alkynyl, N-alkynyl; O-alkyl-O- alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C t to C ⁇ 0 alkyl or C 2 to Cio alkenyl or alkynyl, respectively.
  • O-alkyl e.g. O-methyl
  • Particularly preferred are 0[(CH 2 ) g O] h CH 3 , 0(CH 2 ) g OCH 3 , 0(CH 2 ) g NH 2 , 0(CH 2 ) g CH 3 , 0(CH 2 ) g ONH 2 , and 0(CH 2 ) g ON[(CH 2 ) g CH 3 ] 2 , where g and h are from 1 to about 10.
  • oligonucleotides comprise one of the following at the 2' position: Ci to Cio lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, CI, Br, CN, CF 3 , OCF 3 , SOCH 3 , S0 2 CH 3 , ON0 2 , N0 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, an ⁇ ioalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties.
  • a preferred 2'-modification includes 2'-methoxyethoxy (2'-O-CH 2 CH 2 0CH 3 , also known as 2'-0-(2-methoxyethyl) or 2'-MOE) (Martin et al, Helv. Chim. Acta, 1995, 78, 486- 504).
  • a further preferred modification includes 2'-dfmethylaminooxyethoxy, i.e., a 0(CH 2 ) 2 ON(CH 3 ) 2 group, also known as 2'-DMAOE, as described in examples hereinbelow, and 2'-dime ylaminoethoxyethoxy (also known in the art as 2'-0-dimethyl-amino-ethoxy-ethyl or 2'- DMAEOE), i.e., 2'-0-CH 2 -0-CH 2 -N(CH 3 ) 2 , also described in examples hereinbelow.
  • Each R s , R t , R u and R v is, independently, hydrogen, C(0)R w , substituted or unsubstituted Ci-Cio alkyl, substituted or unsubstituted C 2 -C ⁇ 0 alkenyl, substituted or unsubstituted C 2 -C ⁇ 0 alkynyl, alkylsulfonyl, arylsulfonyl, a chemical functional group or a conjugate group, wherein the substituent groups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl; or optionally, R u and R v , together form a phthalimido moiety with the nitrogen atom to which they are attached; each R w is, independently, substituted or unsubstituted -
  • R j is alkyl or haloalkyl having 1 to about 10 carbon atoms, alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 to about 10 carbon atoms, aryl having 6 to about 14 carbon atoms, N(R k )(R m ) OR k , halo, SR k or CN;
  • m a is 1 to about 10; each mb is, independently, 0 or 1;
  • mc is 0 or an integer from 1 to 10;
  • md is an integer from 1 to 10; me is from 0, 1 or 2; and provided that when mc is 0, md is greater than 1.
  • Particularly useful sugar substituent groups include 0[(CH 2 ) g O] h CH 3 , 0(CH 2 ) g OCH 3 , 0(CH 2 ) g NH 2> 0(CH 2 ) g CH 3 , 0(CH 2 ) g ONH 2 , and 0(CH 2 ) g ON[(CH 2 ) g CH 3 )] 2, where g and h are from 1 to about 10.
  • Some particularly useful oligomeric compounds of the invention contain at least one nucleoside having one of the following substituent groups: to Cio lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, CI, Br, CN, CF 3, OCF 3> SOCH 3; S0 2 CH 3 ⁇ ON0 2 ⁇ N0 2> N 3 , NH 2> heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligomeric compound, or a group for improving the pharmacodynamic properties of an oligomeric compound, and other substituents having similar properties.
  • a preferred modification includes 2'-methoxyethoxy [2'-0- CH 2 CH 2 OCH 3 , also known as 2'-0-(2-methoxyethyl) or 2'-MOE] (Martin et al., Helv. Chim. Acta, 1995, 78, 486), i.e., an alkoxyalkoxy group.
  • a further preferred modification is 2'- dimethylaminooxyethoxy, i.e., a 0(CH 2 ) 2 ON(CH 3 ) 2 group, also known as 2'-DMAOE.
  • 2 '-modifications include 2'-methoxy (2'-0-CH 3 ), 2'- a inopropoxy (2'-OCH 2 CH 2 CH 2 NH 2 ) and 2'-fluoro (2'-F). Similar modifications may also be made at other positions on nucleosides and oligomers, particularly the 3' position ofthe sugar on the 3' terminal nucleoside or at a 3'-position of a nucleoside that has a linkage from the 2'- position such as a 2'-5' linked oligomer and at the 5' position of a 5' terminal nucleoside.
  • Oligomers may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
  • Representative United States patents that teach the preparation of such modified sugars structures include, but are not limited to, U.S. Patents 4,981,957; 5,118,800; 5 ?
  • Representative acetamido substituent groups are disclosed in United States Patent 6,147,200 which is hereby inco ⁇ orated by reference in its entirety.
  • Representative dimethylaminoethyloxyethyl substituent groups are disclosed in International Patent Application PCT/US99/17895, entitled “2'-0-Dimethylaminoethyloxyethyl-Modified Oligonucleotides", filed August 6, 1999, hereby inco ⁇ orated by reference in its entirety.
  • the phosphate group can be linked to either the 2', 3' or 5' hydroxyl moiety ofthe sugar.
  • the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound.
  • the respective ends of this linear polymeric structure can be joined to form a circular structure by hybridization or by formation of a covalent bond, however, open linear structures are generally preferred.
  • the phosphate groups are commonly referred to as forming the internucleoside linkages ofthe oligonucleotide.
  • the normal internucleoside linkage of RNA and DNA is a 3' to 5' phosphodiester linkage.
  • RNAse H messenger RNA
  • RNAse H mechanism can be effectively used to modulate expression of target peptides or proteins.
  • an oligonucleotide inco ⁇ orating a stretch of DNA and a stretch of RNA or 2'-modified RNA can be used to effectively modulate gene expression.
  • the oligonucleotide comprises a stretch of DNA flanked by two stretches of 2 '-modified RNA.
  • Preferred 2 '-modifications include 2'-MOE as described herein.
  • the ribosyl sugar moiety has also been extensively studied to evaluate the effect its modification has on the properties of oligonucleotides relative to unmodified oligonucleotides.
  • the 2'-position ofthe sugar moiety is one ofthe most studied sites for modification.
  • Certain 2'- substituent groups have been shown to increase the lipohpilicity and enhance properties such as binding affinity to target RNA, chemical stability and nuclease resistance of oligonucleotides. Many of the modifications at the 2 '-position that show enhanced binding affinity also force the sugar ring into the C 3 -endo conformation.
  • RNA:RNA duplexes are more stable, or have higher melting temperatures (Tm) than DNA:DNA duplexes (Sanger et al, Principles of Nucleic Acid Structure, 1984, Springer-Verlag; New York, NY.; Lesnik et al, Biochemistry, 1995, 34, 10807-10815; Conte et al, Nucleic Acids Res., 1997, 25, 2627-2634).
  • RNA has been attributed to several structural features, most notably the improved base stacking interactions that result from an A-form geometry (Searle et al, Nucleic Acids Res., 1993, 21, 2051-2056).
  • the presence of the 2' hydroxyl in RNA biases the sugar toward a C3' endo pucker, i.e., also designated as Northern pucker, which causes the duplex to favor the A-form geometry.
  • deoxy nucleic acids prefer a C2' endo sugar pucker, i.e., also known as Southern pucker, which is thought to impart a less stable B-form geometry (Sanger, W. (1984) Principles of Nucleic Acid Structure, Springer-Verlag, New York, NY).
  • the 2' hydroxyl groups of RNA can form a network of water mediated hydrogen bonds that help stabilize the RNA duplex (Egli et al, Biochemistry, 1996, 35, 8489-8494).
  • DNA:RNA hybrid duplexes are usually less stable uian pure RNA:RNA duplexes, and depending on their sequence may be either more or less stable than DNA:DNA duplexes (Searle et al, Nucleic Acids Res., 1993, 21, 2051-2056).
  • the structure of a hybrid duplex is intermediate between A- and B-form geometries, which may result in poor stacking interactions (Lane et al, Eur. J. Biochem., 1993, 215, 297-306; Fedoroff et al, J. Mol. Biol, 1993, 233, 509-523; Gonzalez et al, Biochemistry, 1995, 34, 4969-4982; Horton et al, J. Mol.
  • the stability of a DNA:RNA hybrid is central to antisense therapies as the mechanism requires the binding of a modified DNA strand to a mRNA strand.
  • the antisense DNA should have a very high binding affinity with the mRNA. Otherwise the desired interaction between the DNA and target mRNA strand will occur infrequently, thereby decreasing the efficacy ofthe antisense oligonucleotide.
  • RNA:RNA duplexes are more stable, or have higher melting temperatures (Tm) than DNA:DNA duplexes (Sanger et al, Principles of Nucleic Acid Structure, 1984, Springer-Verlag; New York, NY.; Lesnik et al, Biochemistry, 1995, 34, 10807-10815; Conte et al, Nucleic Acids Res., 1997, 25, 2627-2634).
  • RNA has been attributed to several structural features, most notably the improved base stacking interactions that result from an A-form geometry (Searle et al, Nucleic Acids Res., 1993, 21, 2051-2056).
  • deoxy nucleic acids prefer a C2' endo sugar pucker, i.e., also known as Southern pucker, which is thought to impart a less stable B-form geometry (Sanger, W. (1984) Principles of Nucleic Acid Structure, Springer-Verlag, New York, NY).
  • DNA:RNA hybrid duplexes are usually less stable than pure RNA:RNA duplexes and, depending on their sequence, may be eiflier more or less stable than DNA:DNA duplexes (Searle et al, Nucleic Acids Res., 1993, 21, 2051-2056).
  • the structure of a hybrid duplex is intermediate between A- and B-form geometries, which may result in poor stacking interactions (Lane et al, Eur. J.
  • oligonucleotides also have been shown to be antisense inhibitors of gene expression with promising features for in vivo use (Martin, P., Helv. Chim. Acta, 1995, 78, 486-504; Altmann et al, Chimia, 1996, 50, 168-176; Altmann et al, Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al, Nucleosides Nucleotides, 1997, 16, 917-926). Relative to DNA, they display improved RNA affinity and higher nuclease resistance.
  • MOE substituted oligonucleotides have shown outstanding promise as antisense agents in several disease states.
  • One such MOE substituted oligonucleotide is presently being investigated in clinical trials for the treatment of CMV retinitis.
  • LNAs oligonucleotides wherein the 2' and 4' positions are connected by a bridge
  • CD Circular dichroism
  • spectra show that duplexes involving fully modified LNA (esp. LNA:RNA) structurally resemble an A-form RNA:RNA duplex.
  • Nuclear magnetic resonance (NMR) examination of an LNA:DNA duplex confirmed the 3'-endo conformation of an LNA monomer. Recognition of double-stranded DNA has also been demonstrated suggesting strand invasion by LNA. Studies of mismatched sequences show that LNAs obey the Watson-Crick base pairing rules with generally improved selectivity compared to the corresponding unmodified reference strands.
  • LNAs in which the 2'-hydroxyl group is linked to the 4' carbon atom of the sugar ring thereby forming a 2'-C,4'-C-oxymethylene linkage thereby forming a bicyclic sugar moiety.
  • the linkage may be a methelyne (-CH 2 -) n group bridging the 2' oxygen atom and the 4' carbon atom wherein n is 1 or 2 (Singh et al, Chem. Commun., 1998, 4, 455-456).
  • Other preferred bridge groups include the 2'-deoxy-2'-CH 2 OCH 2 -4' bridge.
  • Exemplary non-phosphate/phosphorothioate diester linkages contemplated within the skill of the art include: phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3 '-alkylene phosphonates, 5 '-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphorainidates, thionophosphoramidates, thionoalkylphos- phonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates.
  • Additional linkages include: thiodiester (-O-C(O)-S-), thionocarbamate (-0-C(0)(NJ)-S-), siloxane (-0- Si(J) 2 -0-), carbamate (-O-C(O)-NH- and -NH-C(O)-O-), sulfamate (-0-S(0)(0)-N- and -N- S(0)(0)-N-, mo ⁇ holino sulfamide (-0-S(0)(N(mo ⁇ holino)-), sulfonamide (-0-S0 2 -NH-), sulfide (-CH 2 -S-CH 2 -), sulfonate (-0-S0 2 -CH 2 -), N,N'-dimethylhydrazine (-CH 2 -N(CH 3 )- N(CH 3 )-), thioformacetal (-S-CH 2 -0-), formacetal (-0-CH 2 -0-),
  • J denotes a substituent group which is commonly hydrogen or an alkyl group or a more complicated group that varies from one type of linkage to another.
  • linking groups as described above that involve the modification or substitution of the -0-P-O- atoms of a naturally occurring linkage
  • linking groups that include modification of the 5'-methylene group as well as one or more of the -0-P-O- atoms.
  • Oligonucleotides are generally prepared, as described above, on a support medium, e.g. a solid support medium.
  • a first synthon e.g. a monomer, such as a nucleoside
  • the oligonucleotide is then synthesized by sequentially coupling monomers to the support-bound synthon. This iterative elongation eventually results in a final oligomeric compound or other polymer such as a polypeptide.
  • Suitable support media can be soluble or insoluble, or may possess variable solubility in different solvents to allow the growing support bound polymer to be either in or out of solution as desired.
  • support media is intended to include all forms of support known to the art skilled for the synthesis of oligomeric compounds and related compounds such as peptides.
  • Some representative support media that are amenable to the methods of the present invention include but are not limited to the following: controlled pore glass (CPG); oxalyl-controlled pore glass (see, e.g., Alul, et al., Nucleic Acids Research 1991, 19, 1527); silica-containing particles, such as porous glass beads and silica gel such as that formed by the reaction of trichloro-[3-(4- chloromethyl)phenyl]propylsilane and porous glass beads (see Parr and Grohmann, Angew. Chem. Internal Ed.
  • CPG controlled pore glass
  • oxalyl-controlled pore glass see, e.g., Alul, et al., Nucleic Acids Research 1991, 19, 1527
  • silica-containing particles such as porous glass beads and silica
  • Further support media amenable to the present invention include without limitation PEPS support a polyethylene (PE) film with pendant long-chain polystyrene (PS) grafts (molecular weight on the order of 10 ⁇ , (see Berg, et al, J. Am. Chem. Soc, 1989, 111, 8024 and International Patent Application WO 90/02749),).
  • the loading capacity of the film is as high as that of a beaded matrix with the additional flexibility to accomodate multiple syntheses simultaneously.
  • the PEPS film may be fashioned in the form of discrete, labeled sheets, each serving as an individual compartment.
  • the sheets are kept together in a single reaction vessel to permit concurrent preparation of a multitude of peptides at a rate close to that of a single peptide by conventional methods.
  • experiments with other geometries of the PEPS polymer such as, for example, non-woven felt, knitted net, sticks or microwellplates have not indicated any limitations of the synthetic efficacy.
  • Further support media amenable to the present invention include without limitation particles based upon copolymers of dimethylacrylamide cross-linked with N,N'- bisacryloylethylenediamine, including a known amount of N-tertbutoxycarbonyl-beta-alanyl-JV- acryloylhexametliylenediamine.
  • spacer molecules are typically added via the beta alanyl group, followed thereafter by the amino acid residue subunits.
  • the beta alanyl-containing monomer can be replaced with an acryloyl safcosine monomer during polymerization to form resin beads.
  • the polymerization is followed by reaction of the beads with emylenediamine to form resin particles that contain primary amines as the covalently linked functionality.
  • the polyacrylamide-based supports are relatively more hydrophilic than are the polystyrene-based supports and are usually used with polar aprotic solvents including dimethylformamide, dimethylacetamide, N-methylpyrrolidone and the like (see Atherton, et al, J. Am. Chem. Soc, 1975, 97, 6584, Bioorg. Chem. 1979, 8, 351, and J. C. S. Perkin 1538 (1981)).
  • Further support media amenable to the present invention include without limitation a composite of a resin and another material that is also substantially inert to the organic synthesis reaction conditions employed.
  • a composite see Scott, et al, J. Chrom. Sci., 1971, 9, 577) utilizes glass particles coated with a hydrophobic, cross-linked styrene polymer containing reactive chloromethyl groups, and is supplied by Northgate Laboratories, Inc., of Hamden, Conn., USA.
  • Another exemplary composite contains a core of fluorinated ethylene polymer onto which has been grafted polystyrene (see Kent and Merrifield, Israel J. Chem. 1978, 17, 243 and van Rietschoten in Peptides 1974, Y.
  • Contiguous solid supports other than PEPS such as cotton sheets (Lebl and Eichler, Peptide Res. 1989, 2, 232) and hydroxypropylacrylate-coated polypropylene membranes (Daniels, et al, Tetrahedron Lett. 1989, 4345).
  • a "tea bag” containing traditionally-used polymer beads.
  • Support bound oligonucleotide synthesis relies on sequential addition of nucleotides to one end of a growing chain.
  • a first nucleoside (having protecting groups on any exocyclic amine functionalities present) is attached to an appropriate glass bead support and activated phosphite compounds (typically nucleotide phosphoramidites, also bearing appropriate protecting groups) are added stepwise to elongate the growing oligonucleotide.
  • activated phosphite compounds typically nucleotide phosphoramidites, also bearing appropriate protecting groups
  • Additional methods for solid-phase synthesis may be found in Caruthers U.S. Patents Nos. 4,415,732; 4,458,066; 4,500,707; 4,668,777; 4,973,679; and 5,132,418; and Koster U.S. Patents Nos. 4,725,677 and Re. 34,069.
  • the phosphorus protecting group (pg) is an alkoxy or alkylthio group or O or S having a /S-eliminable group of the formula -CH 2 CH 2 -G W , wherein G w is an electron- withdrawing group.
  • Suitable examples of pg that are amenable to use in connection with the present invention include those set forth in the Caruthers U.S. Patents Nos. 4,415,732; 4,458,066; 4,500,707; 4,668,777; 4,973,679; and 5,132,418; and K ⁇ ster U.S. Patents Nos. 4,725,677 and Re. 34,069.
  • the alkyl or cyanoethyl withdrawing groups are preferred, as commercially available phosphoramidites generally inco ⁇ orate either the methyl or cyanoethyl phosphorus protecting group.
  • the method for removal of pg depends upon the specific pg to be removed.
  • an alkyl group is generally removed by nucleophilic attack on the ⁇ -carbon of the alkyl group.
  • Such PGs are described in the Caruthers et al. patents, as cited herein.
  • oxidation of P(III) to P(V) can be carried out by a variety of reagents.
  • the P(V) species can exist as phosphate triesters, phosphorothioate diesters, or phosphorodithioate diesters.
  • Each type of P(V) linkage has uses and advantages, as described herein.
  • the term "oxidizing agent" should be understood broadly as being any reagent capable of transforming a P(III) species (e.g. a phosphite) into a P(V) species.
  • oxidizing agent includes “sulfurizing agent,” which is also considered to have the same meaning as “thiation reagent.” Oxidation, unless otherwise modified, indicates introduction of oxygen or sulfur, with a concomitant increase in P oxidation state from III to V. Where it is important to indicate that an oxidizing agent introduces an oxygen into a P(III) species to make a P(V) species, the oxidizing agent will be referred to herein is “an oxygen-introducing oxidizing reagent.”
  • Oxidizing reagents for making phosphate diester linkages i.e. oxygen-introducing oxidizing reagents
  • phosphoramidite protocol e.g. Caruthers et al. and K ⁇ ster et al., as cited herein.
  • sulfurization reagents which have been used to synthesize oligonucleotides containing phosphorothioate bonds include elemental sulfur, dibenzoyltetrasulfide, 3-H-l,2-benzidithiol-3-one 1,1-dioxide (also known as Beaucage reagent), tetraethylthiuram disulfide (TETD), and bis(0,0-diisopropoxy phosphinothioyl) disulfide (known as Stec reagent).
  • Oxidizing reagents for making phosphorothioate diester linkages include phenylacetyldisulf ⁇ de (PADS), as described by Cole et al.
  • the phosphorothioate diester and phosphate diester linkages may alternate between sugar subunits. In other embodiments of the present invention, phosphorothioate linkages alone may be employed.
  • the thiation reagent may be a dithiuram disulfides. See US 5,166,387 for disclosure of some suitable dithiuram disulfides. It has been su ⁇ risingly found that one dithiuram disulfide may be used together with a standard capping reagent, so that capping and oxidation may be conducted in the same step. This is in contrast to standard oxidative reagents, such as Beaucage reagent, which require that capping and oxidation take place in separate steps, generally including a column wash between steps.
  • the 5'-protecting group bg or T' is a protecting group that is orthogonal to the protecting groups used to protect the nucleobases, and is also orthogonal, where appropriate to 2'-0- protecting groups, as well as to the 3 '-linker to the solid support.
  • the 5 '-protecting group is acid labile.
  • the 5'-protecting group is selected from an optionally substituted trityl group and an optionally substituted pixyl group.
  • the pixyl group is substituted with one or more substituents selected from alkyl, alkoxy, halo, alkenyl and alkynyl groups.
  • the trityl groups are substituted with from about 1 to about 3 alkoxy groups, specifically about 1 to about 3 methoxy groups. In particular embodiments of the invention, the trityl groups are substituted with 1 or 2 methoxy groups at the 4- and (if applicable) 4'- positions.
  • a particularly acceptable trityl group is 4,4'-dunethoxytrityl (DMT or DMTr).
  • the term "reagent push” has the meaning of a volume of solvent that is substantially free of any active compound (i.e. reagent, activator, byproduct, or other substance other than solvent), which volume of solvent is introduced to the column for the pu ⁇ ose, and with the effect, of pushing a reagent solution onto and tlirough the column ahead of a subsequent reagent solution.
  • a reagent push need not be an entire column volume, although in some cases it may include one or more column volumes.
  • a reagent push comprises at least the minimum volume necessary to substantially clear reagent, by-products and/or activator from a cross-section ofthe column immediately ahead of the front formed by the reagent solution used for the immediately subsequent synthetic step.
  • volume of solvent required for a "reagent push" will vary depending upon the solvent, the solubility in the solvent of the reagents, activators, by-products, etc., that are on the column, the amounts of reagents, activators, by-products, etc. that are to be cleared from the column, etc. It is considered within the skill of the artisan to select an appropriate volume for each reagent push, especially with an eye toward the Examples, below.
  • column wash may imply that at least one column volume is permitted to pass through the column before the subsequent reagent i , solution is applied to the column.
  • a column volume (CV) of the column wash is specified, this indicates that a volume of solvent equivalent to the interior volume of the unpacked column is used for the column wash.
  • a wash solvent is a solvent containing substantially no active compound that is applied to a column between synthetic steps.
  • a “wash step” is a step in which a wash solvent is applied to the column. Both "reagent push” and “column wash” are included within this definition of "wash step”.
  • a wash solvent may be a pure chemical compound or a mixture of chemical compounds, the solvent being capable of dissolving an active compound.
  • a wash solvent used in one of the wash steps may comprise some percentage of acetonitrile, not to exceed 50% v/v.
  • the sequence of capping and oxidation steps may be reversed, if desired. That is, capping may precede or follow oxidation. Also, with selection of a suitable thiation reagent, the oxidation and capping steps may be combined into a single step. For example, it has been su ⁇ risingly found that capping with acetic anhydride may be conducted in the presence of N,N'- dimethyldithiuram disulfide.
  • Suitable solvents are identified in the Caruthers et al. and K ⁇ ster et al. patents, cited herein.
  • the Cole et al. patent describes acetonitrile as a solvent for phenylacetyldisulfide.
  • Other suitable solvents include toluene, xanthenes, dichloromethane, etc.
  • Reagents for cleaving an oligonucleotide from a support are set forth, for example, in the Caruthers et al. and K ⁇ ster et al. patents, as cited herein. It is considered good practice to cleave oligonucleotide containing thymidine (T) nucleotides in the presence of an alkylated amine, such as triethylamine, when the phosphorus protecting group is 0-CH 2 CH 2 CN, because this is now known to avoid the creation if cyano-ethylated thymidine nucleotides (CNET). Avoidance of CNET adducts is described in general in US Patent No. 6,465,628, which is inco ⁇ orated herein by reference, and especially the Examples in columns 20-30, which are specifically inco ⁇ orated by reference.
  • CNET cyano-ethylated thymidine nucleotides
  • the oligonucleotide may be worked up by standard procedures known in the art, for example by size exclusion chromatography, high performance liquid chromatography (e.g. reverse-phase HPLC), differential precipitation, etc.
  • the oligonucleotide is cleaved from a solid support while the 5' -OH protecting group is still on the ultimate nucleoside.
  • This so-called DMT-on (or trityl-on) oligonucleotide is then subjected to chromatography, after which the DMT group is removed by treatment in an organic acid, after which the oligonucleotide is de-salted and further purified to form a final product.
  • the 5 '-hydroxyl protecting groups may be any groups that are selectively removed under suitable conditions.
  • the 4,4'-dimethoxytriphenylmethyl (DMT) group is a favored group for protecting at the 5'-position, because it is readily cleaved under acidic conditions (e.g. in the presence of dichlroacetic acid (DCA), trichloroacetic acid (TCA), or acetic acid.
  • DCA dichlroacetic acid
  • TCA trichloroacetic acid
  • acetic acid e.g. about 3 to about 10 percent DCA (v/v) in a suitable solvent.
  • DCA e.g. about 3 to about 10 percent DCA (v/v) in a suitable solvent.
  • Removal of oligonucleotide after cleavage from the support is generally performed with acetic acid.
  • oligonucleotides can be prepared as chimeras with other oligomeric moieties.
  • the term "oligomeric compound” refers to a polymeric structure capable of hybridizing a region of a nucleic acid molecule, and an "oligomeric moiety" a portion of such an oligomeric compound.
  • Oligomeric compounds include oligonucleotides, oligonucleosides, oligonucleotide analogs, modified oligonucleotides and oligonucleotide mimetics. Oligomeric compounds can be linear or circular, and may include branching.
  • an oligomeric compound comprises a backbone of linked monomeric subunits where each linked monomeric subunit is directly or indirectly attached to a heterocyclic base moiety.
  • the linkages joining the monomeric subunits, the monomeric subunits and the heterocyclic base moieties can be variable in structure giving rise to a plurality of motifs for the resulting oligomeric compounds including hemimers, gapmers and chimeras.
  • a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base moiety.
  • oligonucleoside refers to nucleosides that are joined by internucleoside linkages that do not have phosphorus atoms. Internucleoside linkages of this type include short chain alkyl, cycloalkyl, mixed heteroatom alkyl, mixed heteroatom cycloalkyl, one or more short chain heteroatomic and one or more short chain heterocyclic.
  • internucleoside linkages include but are not limited to siloxane, sulfide, sulfoxide, sulfone, acetyl, formacetyl, thioformacetyl, methylene formacetyl, thioformacetyl, alkeneyl, sulfamate; methyleneirnino, methylenehydrazino, sulfonate, sulfonamide, amide and others having mixed N, O, S and CH 2 component parts.
  • WO 91/08213 WO 90/15065; WO 91/15500; WO 92/20822; WO 92/20823; WO 91/15500; WO 89/12060; EP 216860; PCT/US 92/04294; PCT/US 90/03138; PCT/US 91/06855; PCT/US 92/03385; PCT/US 91/03680; U.S. Application Nos.
  • Phosphoramidites used in the synthesis of oligonucleotides are available from a variety of commercial sources (included are: Glen Research, Sterling, Virginia; Amersham Pharmacia Biotech Inc., Piscataway, New Jersey; Cruachem Inc., Aston, Pennsylvania; Chemgenes Co ⁇ oration, Waltham, Massachusetts; Proligo LLC, Boulder, Colorado; PE Biosystems, Foster City California; Beckman Coulter Inc., Fullerton, California). These commercial sources sell high purity phosphoramidites generally having a purity of better than 98%. Those not offering an across the board purity for all amidites sold will in most cases include an assay with each lot purchased giving at least the purity of the particular phosphoramidite purchased.
  • Phosphoramidites are prepared for the most part for automated DNA synthesis and as such are prepared for immediate use for synthesizing desired sequences of oligonucleotides.
  • Phosphoramidites may be prepared by methods disclosed by e.g. Caruthers et al. (US 4,415,732; 4,458,066; 4,500,707; 4,668,777; 4,973,679; and 5,132,418) and K ⁇ ster et al. (US RE 34,069).
  • Double stranded oligonucleotides such as double-stranded RNA, may be manufactured according to methods according to the present invention, as described herein.
  • RNA synthesis it is necessary to protect the 2' -OH of the amidite reagent with a suitable removable protecting groups.
  • Suitable protecting groups for 2' -OH are described in US Patent Nos. 6,008,400, 6,111,086 and 5,889,136.
  • a particularly suitable 2'-protecting group for RNA synthesis is the ACE protecting group as described in US 6,111,086. In some embodiments, it is considered advantageous to use a different 5 '-protecting group for amidites used in RNA synthesis.
  • Suitable 5 '-protecting groups are set forth in US 6,008,400.
  • a particularly suitable 5'- protecting group is the trimethylsilyloxy (TMSO) group as taught in US 6,008,400. See especially example 1, columns 10-13.
  • TMSO trimethylsilyloxy
  • the separate strands of the double stranded RNA may be separately synthesized and then annealed to form the double stranded (duplex) oligonucleotide.
  • Exemplary preferred antisense compounds include DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 5 '-terminus of one of the illustrative preferred antisense compounds (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately upstream of the 5 '-terminus of the antisense compound which is specifically hybridizable to the target nucleic acid and continuing until the DNA or RNA contains about 8 to about 80 nucleobases).
  • preferred antisense compounds are represented by DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 3 '-terminus of one of the illustrative preferred antisense compounds (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately downstream of the 3 '-terminus of the antisense compound which is specifically hybridizable to the target nucleic acid and continuing until the DNA or RNA contains about 8 to about 80 nucleobases).
  • One having skill in the art once armed with the empirically-derived preferred antisense compounds illustrated herein will be able, without undue experimentation, to identify further preferred antisense compounds.
  • Antisense and other compounds of the invention which hybridize to the target and inhibit expression of the target, are identified through experimentation, and representative sequences of these compounds are herein identified as preferred embodiments of the invention. While specific sequences of the antisense compounds are set forth herein, one of skill in the art will recognize that these serve to illustrate and describe particular embodiments within the scope of the present invention. Additional preferred antisense compounds may be identified by one having ordinary skill.
  • oligonucleotides containing modified backbones or non-natural internucleoside linkages include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.
  • modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.
  • Antisense technology involves directing oligonucleotides, or analogs thereof, to a specific, target messenger RNA (mRNA) sequence.
  • mRNA messenger RNA
  • the interaction of exogenous "antisense” molecules and endogenous mRNA modulates transcription by a variety of pathways. Such pathways include transcription arrest, RNAse H recruitment, and RNAi (e.g. siRNA).
  • Antisense technology permits modulation of specific protein activity in a relatively predictable manner.
  • non-critical means that the impurity in question is either inert or reactive under the test reaction conditions having no direct impact upon the quality ofthe oligonucleotide product.
  • critical means that the impurity in question is reactive under the test reaction conditions and does have a direct impact upon the quality of the oligonucleotide product.
  • Non-phosphorous containing impurities (Classes I - III) were subjected to the following test reaction conditions and the resulting products were analyzed by LC/MS: To a solution of phosphoramidite (100 ⁇ L, 0.2 M in acetonitrile) containing 10 mol-% of the corresponding impurity (classes I to in) add li ⁇ -tetrazole in acetonitrile (0.45 M, 150 ⁇ L). After 5 min add water (50 ⁇ L) to hydrolyze unreacted phosphoramidite. For LC/MS sample preparation, an aliquot of this solution (20 ⁇ L) was removed and to which was added a solution (980 ⁇ L) of internal standard (triphenylphosphine oxide).
  • Non-phosphorous containing impurities (Classes I - III) were subjected to the following test reaction Impurity Classes I to III comprise structures without phosphorus groups. Therefore, li7-tetrazole activated coupling of those impurities to free hydroxyl groups (e.g. free hydroxyl groups of the growing chain of support-bound oligonucleotide) was not considered to be a reaction likely to occur. It seemed more likely however, that upon activation of the phosphoramidite with 1/Y-tetrazole, impurities with free hydroxyl groups (classes I and II) might react with the phosphoramidite to form phosphite triester species (A, B, Scheme 2).
  • test reactions delineated herein perfo ⁇ ned on 2'-deoxy amidites may be repeated with 2'-0-substituted amidites, e.g. 2'-0-methyl, 2'-0-methoxyeu ⁇ yl (MOE) or -O- aminopropyl amidites, as well as 2'-deoxy-2'-ara-fluoro amidites.
  • the test reactions delineated herein may be conducted with 2'-0- methoxyethyl amidites.
  • the test reactions delineated herein may be perfo ⁇ ned with 2'-0-methyl amidites.
  • Class IV reduces the actual concentration of phosphoramidite, if not corrected for impurity profile. 31 P NMR analysis (Table 4) of Class IV impurities does not provide evidence of reactivity under the coupling conditions. Therefore, no impact on oligonucleotide quality is expected. Bis(cyanoethyl)P(N'Pr 2 ) contamination in the phosphitylation reagent could lead to formation of Class IV impurity. Classification: non-critical.
  • Class V reduces the actual concentration of phosphoramidite, if not corrected for impurity profile.
  • Class V impurities have two resonances near 150 ppm. (Table 5) Upon treatment with lH-tetrazole and ethanol the amidite resonances disappear and a new set of resonances appears at ca 140 ppm. The chemical shift ofthe newly formed product is consistent with the formation of a phosphite triester, confirming the reactivity ofthe Class V impurities with free hydroxyl groups.
  • Class V impurities compete with phosphoramidites for free 5 '-hydroxy groups of the solid- support bound oligonucleotide.
  • the reaction product is a 5' to 5' linked (instead of 5' to 3') phosphite triester which will be sulfurized to form a phosphorothioate triester.
  • This intermediate will be deprotected in the next deblock step and extended to form a 3 ' to 3 ' linkage.
  • the molecular mass of the modified oligonucleotide will be identical to the target oligonucleotide.
  • Class V impurities arise from carry-over of a side-product of the tritylation process (Class II) to the phosphitylation process. Classification: critical.
  • Class VI Base-protected 5'-0-DMT-3'-Q-cvanoethyl- H-phosphonate (compounds of formula ffll Table 6. 31 P NMR data of Class VI impurities with and without activator/ethanol.
  • Class VI reduces the actual concentration of phosphoramidite, if not corrected for impurity profile. 31 P NMR analysis (Table 6) of Class VI impurities does not provide evidence of reactivity under the coupling conditions. Therefore, no impact on oligonucleotide quality is expected.
  • Class VI impurities are hydrolysis products of phosphoramidites which may form during work-up of the phosphitylation reaction. Classification: non-critical
  • Class V ⁇ Base-protected 5'-0-DMT-3'-0-H-phosphonoamidate (compounds of formula (s)) Table 7. 31 P NMR data of Class VII impurities with and without activator/ethanol.
  • Class VTI reduces the actual concentration of phosphoramidite, if not corrected for impurity profile. 31 P NMR analysis (Table 7) of Class VTI impurities does not provide evidence of reactivity under the coupling conditions. Therefore, no impact on oligonucleotide quality is expected.
  • Class VTI impurities are elimination products of phosphoramidites which may form during work-up ofthe phosphitylation reaction. Classification: non-critical
  • Class ⁇ i reduces the actual concentration of phosphoramidite, if not corrected for impurity profile. 31 P NMR analysis (Table 9) of Class VHI impurities does not provide evidence of reactivity under the coupling conditions. Therefore, no impact on oligonucleotide quality is expected.
  • Class Vi ⁇ impurities are oxidation products of phosphoramidites which may form during work-up ofthe phosphitylation reaction. Classification: non-critical
  • Class IX Base-protected 5'-0-DMT-3'-O-cvanoethyl phosphonoamidate (compounds of formula ( ⁇ ) Table 9. 31 P NMR data of Class IX impurities with and without activator/ethanol.
  • Class IX reduces the actual concentration of phosphoramidite, if not corrected for impurity profile. 31 P NMR analysis (Table 9) of Class IX impurities does not provide evidence of reactivity under the coupling conditions. Therefore, no impact on oligonucleotide quality is expected. Class IX impurities are degradation products of phosphoramidites which may form during work-up of the phosphitylation reaction. Classification: non-critical
  • Class X impurities have resonances near 140 ppm and near 149 ppm. (Table 10) Upon treatment with lH-tetrazole and ethanol the amidite resonances near 149 ppm disappear and resonances near 140 ppm become more intense. The chemical shift of the newly formed product is consistent with the formation of a phosphite triester, confirming the reactivity of the Class X impurities.
  • Class X impurities compete with phosphoramidites for free 5 '-hydroxy groups.
  • the reaction product are phosphite triester with an extended linkage which will be sulfurized to form a phosphorothioate triester.
  • Class XI impurities have resonances near 149 ppm. (Table 11) Upon treatment with lH-tetrazole and ethanol the amidite resonances near 149 ppm disappear and resonances near 140 ppm appear. The chemical shift of the newly formed product is consistent with the formation of a phosphite triester, confirming the reactivity of the Class XI impurities.
  • Class XI impurities compete with phosphoramidites for free 5 '-hydroxy groups similarly to Class X impurities. Class XI impurities have two potential sites for reaction leading to different reaction products. The intermediates will be sulfurized and extended in the next coupling step.
  • Class XI impurities may be traced back to ethylene glycol contamination in the 2-cyanoethanol that is used in the production of phosphitylation reagent. Derivatization of ethylene glycol would lead to a phosphitylating compound contaminating the standard phosphitylating reagent. Phosphitylation with this contaminated reagent would then lead to formation of Class XI impurities.
  • Class X ⁇ Base-protected bis(5'-Q-DMT nucleoside) amidite (compounds of formula (l ⁇ l Table 12. 31 P NMR data of Class X ⁇ impurities with and without activator/ethanol.
  • Class XH impurities have one resonance between 146 to 149 ppm. (Table 12) Upon treatment with 1H- tetrazole and ethanol the amidite resonance disappears and a new set of resonances appears at ca 140 ppm. The chemical shift of the newly formed product is consistent with the formation of a phosphite triester, confirming the reactivity of the Class XII impurities.
  • Class XH impurities compete with phosphoramidites for free 5 '-hydroxy groups.
  • the reaction product is a phosphite triester with two 5'-0-DMT protected nucleosides.
  • Class X ⁇ i Base-protected bis(5' -DMT nucleoside) cvanoethyl phosphite (compounds of formula (m) Table 13. 31 P NMR data of Class XTH impurities with and without activator/ethanol.
  • Class XIII reduces the actual concentration of phosphoramidite, if not corrected for impurity profile. 31 P NMR analysis (Table 13) of Class XIH impurities does not provide evidence' of reactivity under the coupling conditions. Therefore, no impact on oligonucleotide quality is expected. Class XHI could possibly form during the phosphitylation of the protected nucleosides if the reaction product (standard phosphoramidite) is activated a second time under the reaction conditions and reacts with a second protected nucleoside. Classification: non-critical Class XTV. Base-protected 5'-DMT-3'-0-fO-cvanoethvl-N-methvl-N-isoproPvl phosphor amidite
  • Class XIV impurities have two resonances near 148 ppm. (Table 14) Upon treatment with lH-tetrazole and ethanol the amidite resonances disappear and a new set of resonances appears at ca 140 ppm. The chemical shift of the newly formed product is consistent with the formation of a phosphite triester, confirming the reactivity ofthe Class XTV impurities. During solid-phase synthesis Class XIV impurities compete with phosphoramidites for free 5 '-hydroxy groups. The oligonucleotide reaction product is identical to the phosphite triester which is formed from the standard amidite.
  • the oligonucleotide that is formed by Class XTV impurities is identical with the target oligonucleotide.
  • the modified amino group is washed off the column.
  • Class XIV impurities are reactive but have no impact on oligonucleotide quality.
  • the origin of Class XTV impurities may be due to methyl isopropyl amine contamination in the diisopropylamine that is used in the production of phosphitylation reagent. Phosphitylation with this contaminated reagent would then lead to formation of Class XIV impurities.
  • Class XV impurities reduces the actual concentration of phosphoramidite, if not corrected for impurity profile.
  • Class XV impurities have two resonances near 149.5 ppm. (Table 15) Upon treatment with lH-tetrazole and ethanol the amidite resonances disappear and a new set of resonances appears at ca 140 ppm. The chemical shift ofthe newly formed product is consistent with the formation of a phosphite triester, confirming the reactivity of the Class XV impurities with free hydroxyl groups.
  • Class XV impurities compete with phosphoramidites for free 5'- hydroxy groups of the solid-support bound oligonucleotide.
  • the molecular mass of the modified oligonucleotide will be identical to the target oligonucleotide and cannot be distinguished based on its molecular weight by mass specfroscopy.
  • Class XV impurities may arise from carry-over of a side-product ( ⁇ -anomer) formed during chemical synthesis of nucleosides.
  • Class XVT reduces the actual concentration of phosphoramidite, if not corrected for impurity profile.
  • Class XVI impurities have two resonances near 150 ppm. (Table 16) Upon treatment with IH-tefrazole and ethanol the amidite resonances disappear and a new set of resonances appears at ca 141 ppm. The chemical shift ofthe newly formed product is consistent with the formation of a phosphite triester, confirming the reactivity of the Class XVT impurities with free hydroxyl groups.
  • Class XVI impurities compete with phosphoramidites for free 5 '-hydroxy groups of the solid-support bound oligonucleotide.
  • Class XVH reduces the actual concentration of phosphoramidite, if not corrected for impurity profile.
  • Class XV ⁇ impurity has two resonances near 149 ppm. (Table 17) Upon treatment with lH-tetrazole and ethanol the amidite resonances disappear and a new set of resonances appears at ca 140 ppm. The chemical shift ofthe newly formed product is consistent with the formation of a phosphite triester, confirming the reactivity of the Class XV ⁇ impurities with free hydroxyl groups.
  • Class XVH impurities compete with T phosphoramidite for free 5 '-hydroxy groups of the solid-support bound oligonucleotide. The molecular mass of the modified oligonucleotide will be 53 amu larger than the target oligonucleotide (for one incorpoaration).
  • Class XVT ⁇ impurities have multiple resonances near 149 ppm due to the presence of 4 possible stereoisomers with two phosphoramidites each. (Table 20) Upon treatment with IH-tefrazole and ethanol the amidite resonances near 149 ppm disappear and resonances near 140.5 ppm appear. The chemical shift ofthe newly formed product is consistent with the formation of phosphite triester groups, confirming the reactivity of the Class XVT ⁇ impurities. During solid-phase synthesis Class XVBD impurities compete with phosphoramidites for free 5 '-hydroxy groups similarly to Class X impurities. Class XV ⁇ I impurities have two potential sites for reaction leading to different reaction products.
  • the intermediates will be sulfurized and extended in the next coupling step.
  • the origin of Class XVI ⁇ impurities may be fraced back to ethylene glycol contamination in the 2-cyanoethanol that is used in the production of phosphitylation reagent. Derivatization of ethylene glycol would lead to a phosphitylating compound contaminating the standard phosphitylating reagent. Phosphitylation with this contaminated reagent would then lead to formation of Class XV ⁇ l impurities.
  • an impurity threshold may be set in the range of about 1 to about 10,000 ppm, e.g. in the range of abut 1 to about 1000 ppm, and in some cases in the range of about 1 to about 100 ppm.
  • Each impurity threshold may be different, depending upon the degree to which it affects the integrity of the final oligonucleotide product (which is determined by experimentation).
  • the impurity threshold for a critical impurity may be set in the range of about 1 to about 1000 ppm, and in particular from about 1 to about 100 ppm.
  • a critical impurity threshold of 1, 10, 20, 30, 50, 80 or 100 ppm may be set.
  • a non-critical impurity threshold may be set in the range of about 1 to about 10,000 ppm, e.g. in the range of about 1 to about 1000 ppm, of about 1 to about 100 ppm, etc.
  • Some specific non-critical impurity thresholds may include 10, 50, 100, 200, 300, 500, 800 or 1000 ppm.
  • ppm may be replaced by another equivalent measure of concentration, e.g. wt %. The conversion of wt% to ppm and vice versa is well known to the artisan.

Abstract

Cette invention concerne la synthèse d'oligonucléotides. Cette invention concerne en particulier des procédés de production de phosphoramidites de haute qualité et d'oligonucléotides dérivés de ceux-ci. Dans certains modes de réalisation, ce procédé consiste à identifier et à mesurer un ou plusieurs signaux correspondant à une ou plusieurs impuretés critiques ou non critiques dans un échantillon d'amidite et à accepter ou à rejeter l'échantillon d'amidite selon que la quantité d'impureté critique ou non critique est inférieure ou égale à un seuil prédéterminé.
PCT/US2004/018704 2003-06-13 2004-06-14 Procede de production d'amidites pures et d'oligonucleotides WO2004113553A2 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US47839803P 2003-06-13 2003-06-13
US60/478,398 2003-06-13
US48870703P 2003-07-18 2003-07-18
US60/488,707 2003-07-18

Publications (3)

Publication Number Publication Date
WO2004113553A2 true WO2004113553A2 (fr) 2004-12-29
WO2004113553A8 WO2004113553A8 (fr) 2005-06-23
WO2004113553A3 WO2004113553A3 (fr) 2006-03-16

Family

ID=34681275

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2004/018704 WO2004113553A2 (fr) 2003-06-13 2004-06-14 Procede de production d'amidites pures et d'oligonucleotides

Country Status (1)

Country Link
WO (1) WO2004113553A2 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8084589B2 (en) 2007-08-31 2011-12-27 University Of Massachusetts Phosphoramidite nucleoside analogs
CN112409421A (zh) * 2020-12-01 2021-02-26 上海兆维科技发展有限公司 一种3’-磷酸酯核苷的制备方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
'DNA Phosphoramidites, Controlled Pore Glass and Columns' PROLIGO CATALOG. 2002, XP008047824 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8084589B2 (en) 2007-08-31 2011-12-27 University Of Massachusetts Phosphoramidite nucleoside analogs
CN112409421A (zh) * 2020-12-01 2021-02-26 上海兆维科技发展有限公司 一种3’-磷酸酯核苷的制备方法

Also Published As

Publication number Publication date
WO2004113553A8 (fr) 2005-06-23
WO2004113553A3 (fr) 2006-03-16

Similar Documents

Publication Publication Date Title
US7427675B2 (en) Compounds and methods for the characterization of oligonucleotides
US7759480B2 (en) Chloral-free DCA in oligonucleotide synthesis
WO2005108411A2 (fr) Amidites et methodes de synthese d'arn
US7276599B2 (en) Oligonucleotide synthesis with alternative solvents
EP1789460B1 (fr) Billes polymeres pour synthese d'oligomeres
EP1692139B1 (fr) Derives de 5, 6-dihydroxy-isoindole en tant que lieurs pour la synthese d'oligomeres en phase solide
WO2005077966A1 (fr) Groupes substitues de protection de pixyle destines a une synthese d'oligonucleotides
US7030230B2 (en) Process of purifying phosphoramidites
US7002006B2 (en) Protection of nucleosides
WO2004113553A2 (fr) Procede de production d'amidites pures et d'oligonucleotides
WO2009130328A1 (fr) Procédé de fabrication d’oligonucléotides
WO2021233406A1 (fr) Compositions comprenant de l'acide dichloroacétique, leurs procédés de préparation et leurs utilisations
WO2004009612A1 (fr) Suppression de la protection du phosphore dans la synthese des oligonucleotides
EP4313984A1 (fr) Réactifs lieurs universels pour synthèse de l'adn

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
CFP Corrected version of a pamphlet front page
CR1 Correction of entry in section i

Free format text: IN PCT GAZETTE 53/2004 UNDER (30) ADD "60/488,707, 18 JULY 2003 (18.07.2003), US"

122 Ep: pct application non-entry in european phase