DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
This office action is a response to applicant’s communication submitted June 21, 2024, wherein claims 3-14 were preliminarily amended, and claims 15-20 were added.
Claims 1-20 are pending in this application.
Priority
This application is a 371 of PCT/US2022/082239 filed 12/22/2022 and claims foreign priority to EP 21216879.3 filed 12/22/2021. Acknowledgment is made of applicant’s claim for foreign priority under 35 U.S.C. 119 (a)-(d). The certified copy has been received.
Specification
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code (pg. 8, paras. 0030-0031, pg. 10, para. 0041 pg. 16, para. 0068). Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01.
The disclosure is objected to because of the following informalities:
On page 14 in the phrase “CNET = cyanoethlyl” should read “CNET = cyanoethyl”.
On page 17, the bottom table recites the phrase “[weight 1%]”, it is believed this should read “[weight %]”
Appropriate correction is required.
Claim Objections
Claims 1-20 are objected to because of the following informalities:
In claims 1 and 4, “Nucleobase” should not be capitalized.
In claim 1, the claim should start with “A process” instead of “Process” to make it clear this is an independent claim.
In claims 2-20, the claim should start with “The process of claim” instead of “Process of claim” to make it clear these are dependent claims.
In claim 8,
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should not be in parenthesis. Appropriate correction is required.
In claims 14 and 20, the phrase “level cyanoethyl” should read “level of cyanoethyl”.
Appropriate correction is required.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 1-5, 9-11, 14, 17, and 20 are rejected under 35 U.S.C. 103 as being unpatentable over Wang (WO 2018/045317, IDS filed June 21, 2024) in view of Bhan (WO 0046231, IDS filed June 21, 2024) as evidenced by Diethylamine (Wikipedia, 2026, cited on PTO-892) and Acetonitrile (Wikipedia, 2026, cited on PTO-892).
Regarding claims 1-5, 9-11, 14, 17, and 20: Wang teaches a method of synthesizing an oligonucleotide containing 4'-Oxymethylphosphonate at 5'-terminus using dimethyl phosphonate ester Phosphoramidites represented by the following structure
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(pg. 60, example 6, para. 00369, 00371). Wang teaches oligonucleotide synthesis was carried out on a solid support in the 3' to 5 'direction using a commercial oligo synthesizer (pg. 60, para. 00370). The phosphonate groups of Phosphoramidite 3 and Phosphoramidite 4 each contain two methyl protected oxygen atoms. Depending on the deprotection step used, however, either one or both of the methyl groups are removed, resulting in a 5'-terminal nucleotide with one methyl protected oxygen atom in the phosphonate group as in the following structure, where R is F and OMe (pg. 61, para. 00372-00373). Wang teaches a monomethyl protected 4'-oxymethylphosphonate oligonucleotide can be prepared using concentrated ammonia (pg. 61, para. 00373). Wang teaches In the 4'-phosphate analog-containing oligonucleotides and nucleosides described above, B represents a natural nucleobase, a modified nucleobase or a universal nucleobase (pg. 42, para. 00289). Suitable natural nucleobases include purine and pyrimidine bases, e.g. adenine (A), thymine (T), cytosine (C), guanine (G), or uracil (U) (pg. 42, para. 00289).
Wang does not teach wherein the deprotection is carried out in the presence of a nucleophilic organic base in the presence of an organic solvent (i.e. Wang teaches concentrated ammonia).
Bhan teaches a method of purifying an oligonucleotide that comprises providing an oligonucleotide attached to a substrate, wherein the oligonucleotide contains phosphate protecting groups; contacting the oligonucleotide with a reagent, e.g., an organic amine, that cleaves the phosphate protecting groups from the oligonucleotide without detaching the oligonucleotide from the substrate; isolating the oligonucleotide attached to the substrate from the cleaved phosphate protecting groups; and cleaving the oligonucleotide from the substrate (abstract). Bhan teaches the method provides crude oligonucleotide mixtures that are easier to purify and from which the desired full-length oligonucleotide product can be isolated in higher yields (abstract). Bhan teaches the phosphate protecting group is a group capable of undergoing beta-, such as 2-cyanoethyl phosphate (pg. 4, para. 6). The reagent cleaves the phosphate protecting group from the oligonucleotide by beta-elimination. Preferably, the reagent comprises an amine with a formulaR-N-R1R2 wherein R, R1 and R2 are independently hydrogen, hydroxy, alkyl, allyl, aryl, cycloalkyl, alkenyl, alkoxy, allyloxy, aryloxy, and may include from one to twenty carbon atoms. (pg. 4, para. 6). Bhan teaches typically the 2-cyanoethyl protecting group is removed with concentrated ammonium hydroxide to produce acrylonitrile (pg. 5, para. 2). Bhan teaches the long exposure of ammonium hydroxide solution with oligonucleotide leads to acrylonitrile reacting with nucleobases resulting in observed n+1 and n+2 oligonucleotide modifications (pg. 5, para. 2). Bhan demonstrates that acrylonitrile is added to thymidine following ammonium hydroxide cleavage (pgs. 8-9, bridging para.). Bhan identified this impurity to be the N3 cyanoethyl adduct of thymidine (pg. 9, para. 2). The method of Bhan reduces exposure of acrylonitrile with oligonucleotides during deprotection, minimizing modification of nucleobases while rapidly and selectively removing phosphate protecting groups while the oligonucleotide is still tethered to the substrate (pg. 6, paras. 1-2). Bhan teaches a preferred method comprises using a solution of about 20% v/v diethylamine in anhydrous acetonitrile (pg. 8, para. 1). Acetonitrile discloses acetonitrile has a density of 0.786 g/cm3 (i.e. g/mL) (pg. 2, col. 2, bottom of page). Diethylamine discloses diethylamine has a density of 0.707 g/mL (pg. 2, col. 2, middle of page). Thus a 20% v/v diethylamine in acetonitrile equates to approximately 18% diethylamine by weight (100mL acetonitrile x (0.786g/ml)=78.6g, 20 mL diethylamine x (0.707g/mL)=14.14g, (14.14g/78.6g)x100=17.9% dimethylamine by weight). Bhan teaches the method reduced the n+ impurity (i.e. cyanoethyl impurity) to less than 0.05 % with corresponding increase in the yield of the full length oligomer by 3-7% (pg. 11, para. 1). According to the instant specification, diethylamine (i.e. DEA) has a nucleophilicity of 15.10 (pgs. 15-16, para. 0068).
Taken together it would have been prima facie obvious to modify the method of Wang such that the diethylamine in acetonitrile deprotection of Bhan is utilized to remove 2-cyanoethyl groups from the phosphate backbone. A person of ordinary skill in the art would have had the motivation to do so with a reasonable expectation of success in order to remove 2-cyanoethyl groups from the backbone while minimizing cyanoethyl adduct formation in order to improve yield and make purification easier. Although Bhan does not explicitly disclose that diethylamine would remove alkyl groups on a terminal phosphate, according to the instant specification, diethylamine in acetonitrile results in this transformation (pg. 17, para. 0069, example 2c). Thus application of diethylamine in acetonitrile procedure of Bhan to remove the cyanoethyl groups of Wang necessarily would result in the simultaneous removal of an alkyl group on the terminal phosphate group, absent evidence to the contrary.
Claim 6-7 and 15-16 are rejected under 35 U.S.C. 103 as being unpatentable over Wang (WO 2018/045317, IDS filed June 21, 2024), Bhan (WO 0046231, IDS filed June 21, 2024), Diethylamine (Wikipedia, 2026, cited on PTO-892) and Acetonitrile (Wikipedia, 2026, cited on PTO-892) as applied to claims 1-5, 9-11, 14, 17, and 20 above in view of ATDBio (Nucleic Acids, Solid-phase-oligonucleotide-synthesis, Web Archive, Online December 9, 2021, cited on PTO-892).
Regarding claims 6-7 and 15-16: As discussed above Wang and Bhan render obvious the method of claim 1 wherein the nucleophilic organic base is diethylamine. As discussed above, Bhan teaches the reagent amine can be an amine with a formula R-N-R1R2 wherein R, R1 and R2 are independently hydrogen, hydroxy, alkyl, allyl, aryl, cycloalkyl, alkenyl, alkoxy, allyloxy, aryloxy, and may include from one to twenty carbon atoms.
They do not explicitly teach wherein the nucleophilic organic base is triethylamine or N,N-dimethylethylamine, or with a pka of the protonated organic base of between 8 and 10.5 or a nucleophilicity between 16 and 25.
However, ATDBio teaches under the strong basic conditions used in oligonucleotide deprotection, 2-cyanoethyl adducts can form with the heterocyclic bases, particularly thymine (pg. 12, last para.). If these cyanoethyl adducts are a problem, the resin cleavage and phosphoramidite backbone deprotection steps can be reversed. If the support-bound oligonucleotide is treated with a solution of a weak base in an organic solvent (e.g. 10% diethylamine in acetonitrile, or 1:1triethylamine/acetonitrile), the cyanoethyl protecting groups are removed from the phosphate backbone, but the oligo remains bound to the support (pg. 14, para. 1).
Taken together, it would have been prima facie obvious to further modify the method of Wang and Bhan such that diethylamine is replaced with triethylamine as taught by ATDBio. A person of ordinary skill in the art would have had the motivation to do so with a reasonable expectation of success as both reagents are known in the art to remove 2-cyanoethyl groups in a manner to prevent 2-cyanoethyl adducts from forming. Wherein both diethylamine and triethylamine are known in the art for this purpose it is prima facie obvious to substitute equivalents known for the same purpose (See MPEP 2144.06 (II)).
Additionally, given the teaching of diethylamine as a preferred embodiment and alkyl amines generally being encompassed by the claimed formula of Bhan, recognition of triethylamine being used to remove 2-cyanoethyl protecting groups as taught by ATDBio, a person of ordinary skill in the art would recognize alkylamines such as triethylamine and N,N-dimethylethylamine being encompassed by the formula. Compounds which are homologs (compounds differing regularly by the successive addition of the same chemical group, e.g., by -CH2- groups) are generally of sufficiently close structural similarity that there is a presumed expectation that such compounds possess similar properties (See MPEP 2144.09 (II)).
Thus, in view of Wang, Bhan and ATDBio it would it would have been prima facie obvious to further modify the method such that diethylamine is replaced with N,N-dimethylethylamine. A person of ordinary skill in the art would have had the motivation to do so with a reasonable expectation of success given that alkylamines diethylamine and triethylamine are known in the art for deprotecting 2-cyanoethyl groups and homologs (compounds differing regularly by the successive addition of the same chemical group, e.g., by -CH2- groups) are generally of sufficiently close structural similarity that there is a presumed expectation that such compounds possess similar properties (See MPEP 2144.09 (II)).
According to the instant specification N,N-dimethylamine (DMEA) has a pka of 9.99, and triethylamine (TEA) has a pKa of 10.65 and nucleophilicity of 17.10 (pg. 16, top of page).
Claims 6, 12, 15-16, and 18 are rejected under 35 U.S.C. 103 as being unpatentable over Wang (WO 2018/045317, IDS filed June 21, 2024), Bhan (WO 0046231, IDS filed June 21, 2024), Diethylamine (Wikipedia, 2026, cited on PTO-892) and Acetonitrile (Wikipedia, 2026, cited on PTO-892) as applied to claims 1-5, 9-11, 14, 17, and 20 above in view of Wyrzykiewicz (US 2011/0087014, cited on PTO-892).
Regarding claims 12 and 18: As discussed above, Wang and Bhan render obvious the method of claim 1. Wang teaches oligonucleotide synthesis was carried out on a solid support in the 3' to 5' direction using a commercial oligo synthesizer (pg. 60, para. 00370). Bhan teaches the reagent is passed through a reaction column at a flow rate of about 1ml/min to selectively remove phosphate protecting groups (pg. 8, para. 2).
They do not teach wherein the nucleophilic organic base is applied in a range of 1.5 CV to 30.0 CV and the flow rate is selected in the range of 0.1 CV/min to 0.5 CV/min.
However, Wyrzykiewicz teaches a process for manufacturing an oligonucleotide which comprises removing β-eliminating phosphorus-protecting groups, in particular β-cyanoethyl protective groups from a protected oligonucleotide, wherein said removing comprises contacting the protected oligonucleotide with an amine solution in a solvent which preferably does not consist of pyridine, wherein the conjugate acid of the amine has preferably a pKa of greater than 11.5, and wherein the concentration of the amine in the solution is less than 0.5 mole/liters (abstract). Wyrzykiewicz teaches the amine may be contacted with the protected oligonucleotide by passing the amine solution through the said device. Preferably, the process is automated using a commercially available automated synthesizer, programmed to deliver the amine in a solvent through one of the delivery lines of the synthesizer (pg. 3, para. 0030). Wyrzykiewicz teaches the amine solution may be passed through the column in an amount of at least 1 often at least 2 column volumes, most preferably in an amount of at least 5 column volumes (pg. 2, para. 0031). Wyrzykiewicz teaches the amine base can alternatively be diethylamine, triethylamine or piperidine or amines in which the conjugate acid has pKa from 8 to 11.5 (pgs. 4-5, para. 0057). In an example, Wyrzykiewicz teaches an amine wash using DBU in acetonitrile for at least 200 min, using 10-60 column volumes (10CV/200min to 60CV/200min = 0.05CV/min to 0.3 CV/min, pg. 5, para. 0063). Wyrzykiewicz also teaches a decyanoethylation treatment on 1 mmol scale which used 2 CV with 6 minutes contact time (2CV/6min=0.33CV/min, pg. 5, para. 0069, pg. 6, table 2). Wyrzykiewicz teaches that various contact times and various column volumes are optimizable variables to allow for efficient and selective deprotection with low concentration of base in order to avoid formation of undesired CNET adducts (pg. 6, para. 0070).
Taken together, it would have been prima facie obvious to modify the method of Wang and Bhan such that the amine solution is applied on 2 column volumes at rate of 033CV/min passed through the synthesizer to remove cyanoethyl groups as taught by Wyrzykiewicz
Additionally, where column volume and column contact time are known result-effective variables in the art of cyanoethyl deprotection, it is not inventive to discover the optimum or workable ranges by routine experimentation (See MPEP 2144.05 (II)).
Regarding claims 6, 15-16: As discussed above, Wang and Bhan render obvious the method of claim 1.
They do not explicitly teach wherein the nucleophilic organic base is triethylamine or N,N-dimethylethylamine, or with a pka of the protonated organic base of between 8 and 10.5 or a nucleophilicity between 16 and 25.
However, as discussed above, Wyrzykiewicz teaches the amine base alternatively can be diethylamine, triethylamine or piperidine or amines in which the conjugate acid has pKa from 8 to 11.5 (pgs. 4-5, para. 0057). According to the instant specification triethylamine (TEA) has a pKa of 10.65 and nucleophilicity of 17.10 (pg. 16, top of page).
Taken together, it would have been prima facie obvious to further modify the method of Wang and Bhan such that diethylamine is replaced with triethylamine or an amine with a conjugate acid having a pKA from 8 to 11.5 as taught by Wyrzykiewicz. A person of ordinary skill in the art would have had the motivation to do so with a reasonable expectation of success as both reagents are known in the art to remove 2-cyanoethyl groups in a manner to prevent 2-cyanoethyl adducts from forming. Wherein both diethylamine , triethylamine, and amines with a conjugate acid having a pKA from 8 to 11.5 are known in the art for this purpose it is prima facie obvious to substitute equivalents known for the same purpose (See MPEP 2144.06 (II)). In the case where the claimed ranges "overlap or lie inside ranges disclosed by the prior art" a prima facie case of obviousness exists (i.e. 8 to 11.5 overlaps with between 8 and 10.5, See MPEP 2144.05 (I)).
Claim 8 is rejected under 35 U.S.C. 103 as being unpatentable over Wang (WO 2018/045317, IDS filed June 21, 2024), Bhan (WO 0046231, IDS filed June 21, 2024), Diethylamine (Wikipedia, 2026, cited on PTO-892) and Acetonitrile (Wikipedia, 2026, cited on PTO-892) as applied to claims 1-5, 9-11, 14, 17, and 20 above in view of Sinha (WO 03405969, cited on PTO-892).
Regarding claim 8: As discussed above, Wang and Bhan render obvious the method of claim 1.
They do not teach wherein the nucleophilic organic base is DABCO.
However, Sinha teaches methods of preparing H-phosphonate diesters and oligonucleotides (abstract). Sinha teaches cyanoethyl groups on phosphate backbones can be removed via a strongly basic amine such as DABCO, 1,5-diazabicylo[4.3.0]non-5-ene (DBN), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) or triethylamine (pg. 10, lines 5-9).
Taken together, it would have been prima facie obvious to a person of ordinary skill in the art to modify the method of Wang and Bhan such that diethylamine is replaced with DABCO as suggested by Sinha. A person of ordinary skill would have had the motivation to do so with a reasonable expectation of success given that DABCO is a known alternative reagent for removing cyanoethyl groups from phosphate backbones. Wherein DABCO is recognized as a compound capable of removing 2-cyanoethyl groups, it is prima facie obvious to substitute equivalents for the same purpose (See MPEP 2144.06 (II)).
Claims 8, 13, and 19 are rejected under 35 U.S.C. 103 as being unpatentable over Wang (WO 2018/045317, IDS filed June 21, 2024), Bhan (WO 0046231, IDS filed June 21, 2024), Diethylamine (Wikipedia, 2026, cited on PTO-892), Acetonitrile (Wikipedia, 2026, cited on PTO-892), and Wyrzykiewicz (US 2011/0087014, cited on PTO-892) as applied to claims 1-6, 9-12, 14-18, and 20 above in view of Sinha (WO 03405969, cited on PTO-892).
Regarding claim 8: As discussed above, Wang and Bhan render obvious the method of claim 1.
They do not teach wherein the nucleophilic organic base is DABCO.
However, Sinha teaches methods of preparing H-phosphonate diesters and oligonucleotides (abstract). Sinha teaches cyanoethyl groups on phosphate backbones can be removed via a strongly basic amine such as DABCO, 1,5-diazabicylo[4.3.0]non-5-ene (DBN), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) or triethylamine (pg. 10, lines 5-9).
Taken together, it would have been prima facie obvious to a person of ordinary skill in the art to modify the method of Wang and Bhan such that diethylamine is replaced with DABCO as suggested by Sinha. A person of ordinary skill would have had the motivation to do so with a reasonable expectation of success given that DABCO is a known alternative reagent for removing cyanoethyl groups from phosphate backbones. Wherein DABCO is recognized as a compound capable of removing 2-cyanoethyl groups, it is prima facie obvious to substitute equivalents for the same purpose (See MPEP 2144.06 (II)).
Regarding claims 13 and 19: As discussed above, Wang, Bhan, and Wyrzykiewicz render obvious the methods of claim 5, 12, 15-16 and 18. Wyrzykiewicz teaches an amine wash for at least 200 min, using 10-60 column volumes (10CV/200min to 60CV/200min = 0.05CV/min to 0.3 CV/min, pg. 5, para. 0063). Additionally, Wang, Bhan, Wyrzykiewicz, and Sinha render obvious a method of using DABCO. The methods do not recognize that less than 0.5% N+ alkyl impurities is achieved. However, according to the instant specification, treatment with 20% DABCO in acetonitrile, results in the sum of N+methyl impurities of 0.3 % (pg. 17, para. 0069). The instant specification demonstrates that column volumes and delivery times can vary significantly and still provide this result (pg. 17, para. 0069, pg. 18, para. 0070, see 2j, 2k). Wherein it would have been obvious to substitute DABCO for diethylamine into the method of as taught by Sinha, and optimize column application/column contact time as taught by Wyrzykiewicz, the result of less than 0.5 % N+ alkyl impurities naturally flows as a result of practicing the method suggested by the prior art, absent evidence to the contrary.
Conclusion
No claims are allowed in this action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to SAMUEL L GALSTER whose telephone number is (571)270-0933. The examiner can normally be reached Monday - Friday 8:00 AM - 5:00 PM.
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/SAMUEL L GALSTER/Examiner, Art Unit 1693
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