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 .
Priority
This application is a 371 of PCT/EP2021/085636 filed 12/14/2021. Applicant’s claim for the benefit of a prior-filed application FR2013142 filed 12/14/2020, under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged. A copy of the priority documents has been received in this National Stage application from the International Bureau (PCT Rule 17.2(a)) - a copy of the certified copy of FR2013142 was provided on the filing date of the national stage application (6/14/2023, 22 pages long).
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 6/14/2023 is acknowledged. The submission is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner. The signed IDS form is attached with the instant office action.
Specification
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code. 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. A hyperlink appears on page 8, line 26.
Appropriate correction is required.
Claim Interpretation
The term “raspberry ketone” is being interpreted under the broadest reasonable interpretation (B.R.I.) in light of the specification, as referring to the compound 4-(4-hydroxyphenyl)-2-butanone, which is also known, inter alia, as frambinone, oxyphenalon, 4-(p-hydroxyphenyl)-2-butanone (PubChem entry CID 21648, cited on PTO-892).
Additionally, p-hydroxybenzalacetone, formed in step (b), is also known in the art as p-hydroxybenzylidene acetone, 4-hydroxycinnamoylmethane, p-(E)-4-(4-hydroxyphenyl)but-3-en-2-one, and/or 4-(p-Hydroxyphenyl)-3-buten-2-one (PubChem CID 796857, cited on PTO-892).
The terms ene-reductase and enone-reductase, recited in claim 6, has been interpreted under the B.R.I. as referring to reductases with activity towards alkenes and/or α,β-unsaturated compounds, including enzymes of the classes E.C. 1.6.99.1 (KEGG ENZYME: 1.6.99.1; genome.jp/dbget-bin/www_bget?ec:1.6.99.1) or E.C. 1.3.1.31 (KEGG ENZYME: 1.3.1.31; genome.jp/dbget-bin/www_bget?enzyme+1.3.1.31).
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 3 and 9-13 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 3 recites the limitation "the bioconversion reaction" in line 2. There is insufficient antecedent basis for this limitation in the claim. Claim 1, from which claim 3 depends, recites two bioconversion reactions. Step (a) recites the bioconversion of p-coumaric acid to p-hydroxybenzaldehyde and step (c) recites the conversion of p-hydroxybenzalacetone into raspberry ketone by bioconversion or biocatalysis. Thus, it is impossible to determine if “the bioconversion reaction” referred to in claim 3 is the reaction of step (a) or step (c).
Claims 11-13 depend directly or indirectly from the indefinite claim 3 and are, therefore, also rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, for the reasons set forth above.
Claims 9 and 10 each recite “used without further purification”. The term “further purification” is a relative term which renders the claims indefinite. The term “further” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. In the instant case, no purification is described before the recitation of “further purification” in claims 9 and 10. There is no manner to determine what further is in reference to and therefore the amount of purification permitted or not permitted for these compounds cannot be determined. The resulting claim scope is indefinite. According to the specification (see pg. 5, lines 6-14, and pg. 6, lines 21-26), the Applicant appears to have meant that the intermediate products (p-hydroxybenzaldehyde or p-hydroxybenzalacetone) is not removed from the biomass or the reaction mixture before the next step of the process, or in other words: no purification is performed. However, limitations from the specification are not imported into the claims, and the claims as presented remain indefinite. Correction is required. The following suggestions are provided as merely possible means to address this indefiniteness.
Claim 9 may be amended to include that “step (b) is performed in the presence of the biomass from step (a) without purification” (see spec., pg. 5). Claim 10 may be amended to recite that “the p-hydroxybenzalacetone obtained on conclusion of step (b) is directly subjected to step (c) without purification” (as in pg. 6 of the spec).
Claim Rejections - 35 USC § 112(a) - Enablement
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claim 13 is rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the enablement requirement. The claim contains subject matter which was not described in the specification in such a way as to enable one skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention.
The specification lacks complete deposit information for the microorganisms deposited under the identifiers of ATCC39116, DSMZ 9991, DSMZ 9992, CCTCC 2015329 and/or IMI 390106, as recited in claim 13. No addresses or names of the depositories have been provided. Further, it is unclear from the disclosure alone if these are established publicly-available strains or if these are novel strains deposited by the inventors.
Because it is not clear that the properties of each of these strains are known and publicly available or can be reproducibly isolated from nature without undue experimentation and because the best mode disclosed by the specification requires the use of one or more of these specific strains, a suitable deposit for patent purposes is required. Applicant or applicant's representative may provide assurance of compliance with the requirements of 35 U.S.C § 112, first paragraph, in the following manner.
SUGGESTION FOR DEPOSIT OF BIOLOGICAL MATERIAL
A declaration by applicant, assignee, or applicant's agent identifying a deposit of biological material and averring the following may be sufficient to overcome an objection and rejection based on a lack of availability of biological material:
1. Identifies declarant.
2. States that a deposit of the material has been made in a depository affording permanence of the deposit and ready accessibility thereto by the public if a patent is granted. The depository is to be identified by name and address.
3. States that the deposited material has been accorded a specific (recited) accession number.
4. States that all restriction on the availability to the public of the material so deposited will be irrevocably removed upon the granting of a patent.
5. States that the material has been deposited under conditions that access to the material will be available during the pendency of the patent application to one determined by the Commissioner to be entitled thereto under 37 CFR 1.14 and 35 U.S.C § 122.
6. States that the deposited material will be maintained with all the care necessary to keep it viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposited microorganism, and in any case, for a period of at least thirty (30) years after the date of deposit for the enforceable life of the patent, whichever period is longer.
7. That he/she declares further that all statements made therein of his/her own knowledge are true and that all statements made on information and belief are believed to be true, and further that these statements were made with knowledge that willful false statements and the like so made are punishable by fine or imprisonment, or both, under section 1001 of Title 18 of the United States Code and that such willful false statements may jeopardize the validity of the instant patent application or any patent issuing thereon.
Alternatively, it may be averred that deposited material has been accepted for deposit under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the purpose of Patent Procedure (e.g. see 961 OG 21, 1977) and that all restrictions on the availability to the public of the material so deposited will be irrevocably removed upon the granting of a patent.
It is noted that the microorganisms required to practice the claimed invention are said to be available from one more repositories (ATCC, DSMZ, CCTCC, or IMI). The deposit must be referred to in the body of the specification and be identified by deposit (accession) number, name and address of the depository and the complete taxonomic description. It appears that these microorganisms should remain available to the public beyond the effective life of the patent. Any information to the contrary which comes to an applicant's attention during the prosecution of this application, must be entered in the record or otherwise be brought to the attention of the Office by the applicant.
If an Applicant has adequately established that a biological material is known and readily available, the Office will accept that showing. In those instances, however, the Applicant takes the risk that the material may cease to be known and readily available. Such a defect cannot be cured by reissue after the grant of a patent. (MPEP 2404.01)
Applicant's attention is directed to In re Lundak, 773 F.2d. 1216, 227 USPQ 90 (CAFC 1985) and 37 CFR §1.801-1.809 for further information concerning deposit practice.
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-3, 7-10, and 16 are rejected under 35 U.S.C. 103 as being unpatentable over Fronza et al. ("Stereochemistry of the double bond saturation in the formation in baker's yeast of 4-(4-hydroxyphenyl)-2-butanone (raspberry ketone)." Tetrahedron 52.11 (1996): 4041-4052., on IDS filed 6/14/2023), in view of Alvarado et al. (“Evidence of a new biotransformation pathway of p-coumaric acid into p-hydroxybenzaldehyde in Pycnoporus cinnabarinus.” Applied microbiology and biotechnology vol. 57,5-6 (2001): 725-30. doi:10.1007/s002530100761, on IDS filed 6/14/2023).
Fronza et al. describes the baker’s yeast (i.e. Saccharomyces spp.) mediated reduction of 4-(4-hydroxyphenyl)-but-3-en-2-one (identical to p-Hydroxybenzalacetone or 4-Hydroxybenzalacetone) to 4-(4-hydroxyphenyl)-2-butanone, also known as raspberry ketone (Abstract, and pg. 4042: Chemicals 3 and 1, respectively). Fronza teaches that 'natural' products are preferred by consumers, and, therefore, it is desirable to obtain substantial quantities of these valuable materials- such as the flavoring raspberry ketone- either by extraction from botanical sources or by biotransformation of abundant natural precursors (pg 4042, first paragraph).
Fronza teaches that p-hydroxybenzalacetone is a natural precursor of raspberry ketone, and is available by condensation of the sodium salt of 4-hydroxybenzaldehyde (e.g. p-hydroxybenzaldehyde) with acetone, two materials which are “readily available in the natural modification” (pg. 4042, second paragraph). Fronza teaches that the subsequent bioconversion of p-hydroxybenzalacetone into raspberry ketone using fermenting baker's yeast is a viable entry to 'natural' raspberry ketone and states that “moist commercial baker's yeast effectively reduces the unsaturated ketone 3 to raspberry ketone 1” (pg. 4042, last paragraph, first line). In Figure 1, Fronza demonstrates that raspberry ketone (triangles) is produced during a fermentation of baker’s yeast with glucose and EtOH via the reduction of p-hydroxybenzalacetone (squares).
Therefore, Fronza teaches a process for the bioproduction of raspberry ketone in which p-hydroxybenzaldehyde is condensed with acetone to produce p-hydroxybenzalacetone, which is converted to raspberry ketone by bioconversion (e.g. in the presence of fermenting baker’s yeast or a yeast extract) in the presence of the cofactor NADPH (see Abstract and Scheme 1).
However, Fronza does not explicitly teach a step (a) in which p-coumaric acid undergoes bioconversion to prepare p-hydroxybenzaldehyde.
Alvarado et al. teaches the biotransformation of p-coumaric acid into p-hydroxybenzaldehyde by a fungus Pycnoporus cinnabarinus (Title, Abstract). Alvarado teaches that microbiological means to produce p-hydroxybenzaldehyde is desirable in the favoring industry, as p-hydroxybenzaldehyde is important in producing natural vanilla flavors (pg. 725, right hand col, third paragraph). Alvarado also teaches that due to the abundance of the natural aromatic products ferulic and p-coumaric acids (which are hydroxycinnamic acids found in plants), there is strong interest in utilizing them as substrates in biotechnological processes for the production of flavoring compounds to be labelled as “natural” (pg. 725, under Introduction).
Specifically, Alvarado teaches growth conditions under which p-coumaric acid is bioconverted into p-hydroxybenzaldehyde by the strain Pycnoporus cinnabarinus MUCL 39533 (Fig. 2B, the black circle representing p-hydroxybenzaldehyde).
Therefore, prior to the effective filing date of the instant application, to one of ordinary skill in the art it would have been prima facie obvious to combine the teachings of Fronza and Alvarado to arrive at a method in which p-coumaric acid is bioconverted by a microbiological process into the intermediate p-hydroxybenzaldehyde, which is then condensed with acetone to yield p-hydroxybenzalacetone which in turn is converted to raspberry ketone by bioconversion (e.g. in the presence of baker’s yeast or a yeast enzyme extract), as taught in Fronza for a natural process of making raspberry ketone.
Fronza describes both the condensation of p-hydroxybenzaldehyde with acetone to produce p-hydroxybenzalacetone and the subsequent reduction using an extract of baker’s yeast enzymes (i.e. a biomaterial from Saccharomyces) to produce the desirable flavoring product raspberry ketone. One would have been motivated by the teachings of Fronza to seek a naturally occurring source of p-hydroxybenzaldehyde because Fronza teaches that 'natural' products are preferred by consumers, and it is desirable to obtain starting materials by extraction from botanical sources and/or using biotransformation of abundant natural precursors. Therefore, one would have sought natural means known to the art to obtain p-hydroxybenzaldehyde, including the method of Alvarado comprising the production of p-hydroxybenzaldehyde from plant-derived p-coumaric acid using the fungal strain Pycnoporus cinnabarinus MUCL 39533.
See MPEP § 2143.1.(A) regarding the combining of prior art elements according to known methods to yield predictable results. In this case, each of the claimed steps in the conversion of p-coumaric acid to raspberry ketone were known individually to the art, and in combination, each element is performing the same function as it does separately. The predictable result is a multi-step method to produce raspberry ketone using natural materials and steps including bioconversion of the natural products.
Regarding claims 2 and 3, the process taught in Alvarado which is analogous to step (a) is a microbiological process involving a fermentation of the selected fungi Pycnoporus cinnabarinus MUCL 39533 in the presence of a suitable microorganism.
Regarding claim 7, Fronza explicitly teaches that reduction of p-hydroxybenzalacetone to raspberry ketone is performed in the presence of the cofactor NADPH.
Regarding claims 8 and 16, Fronza does not explicitly state that the baker’s yeast is a Saccharomyces species, but the identity of baker’s yeast as a strain of a Saccharomyces species is well-known in the art, and the term baker’s yeast is often used interchangeably with Saccharomyces (and with Saccharomyces cerevisiae). It would have been prima facie obvious to one of ordinary skill in the art to use Saccharomyces sp., in particular Saccharomyces cerevisiae, to perform the method of Fronza for reducing p-hydroxybenzalacetone to raspberry ketone.
Claims 9 and 10 have been best interpreted under the B.R.I. to mean that the products of steps (a) and (b) are used without removal from the reaction mixtures of the proceeding steps. Fronza does not teach any purification after the condensation of p-hydroxybenzaldehyde with acetone to produce p-hydroxybenzalacetone, in fact Fronza states merely that the two precursor materials which are “readily available in the natural modification” (pg. 4042, second paragraph). There is no teaching or suggestion of purification of the p-hydroxybenzaldehyde or the p-hydroxybenzalacetone in Fronza. In regards to performing the steps utilizing the products p-hydroxybenzaldehyde and p-hydroxybenzalacetone, one skilled in the art of microbial fermentation and bioproduction would be intrinsically motivated to optimize each step of the production process. The purification level of the products, or the selection of a “one-pot” fermentation and synthesis, would have been an obvious point of optimization for these bioproduction steps. Further, purification of the intermediates may be time-consuming or inefficient in terms of the amount of recovered product. Coculturing of different microorganisms and/or enzymes is commonly performed in the art of fermentation. Indeed, the nutrients provided by yeast extract were found important for high-quality cultures of the mycelium fungus used in Alvarado (see page 726, left hand col, under “Medium and culture conditions). One would predict that the baker’s yeast can be used in the presence of residual fungus from step (a).
Thus, one having ordinary skill in the art would have predictably arrived at the methods of the instant claims 9 and 10, in view of the cited art and general skill level in the art. It is further noted that all of the method steps claimed herein are comprising steps, and there are no limitations regarding the amounts of product produced or the efficiency of the steps. Thus, there would have been at least a reasonable expectation that at least some amount of the product of (a) will be utilized in step (b) and that at least some amount of the product of (b) will be utilized in (c), even when the steps are performed without any purification.
From the teachings of the cited references, it is apparent that there would have been a reasonable expectation of success in combining the methods therein to arrive at the claimed invention because both Fronza and Alvarado are directed to the production of natural flavor chemicals, and both teach either the production or use of p-hydroxybenzaldehyde.
Therefore, the combination of these methods to yield a route to convert p-coumaric acid to raspberry ketone would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date, as evidenced by the cited references, especially in the absence of evidence to the contrary.
Claims 1-3, 6-10, and 16 are rejected under 35 U.S.C. 103 as being unpatentable over Fronza et al. (Tetrahedron 52.11 (1996): 4041-4052) and Alvarado et al. (Applied microbiology and biotechnology vol. 57,5-6 (2001): 725-30), as applied to claims 1-3, 7-10, and 16 above, and further in view of Romagnolo et al. ("Identification of fungal ene-reductase activity by means of a functional screening." Fungal Biology 119.6 (2015): 487-493) (to include the rejections of claim 6).
The combination of Fronza and Alvarado makes obvious a method for producing natural raspberry ketone, comprising the steps (a), (b), and (c), as defined in claim 1, for all of the reasons described above. Fronza teaches that fermenting baker’s yeast (i.e. Saccharomyces cerevisiae) or an enzymatic extract thereof can be used for the reduction of the double bond in p-hydroxybenzalacetone to produce 4-(4-hydroxyphenyl)-2-butanone (raspberry ketone).
Claim 6 recites that step (c) is performed in the presence of an ene-reductase or an enone-reductase (interpreted according to the B.R.I., as indicated above).
The combined teachings of Fronza and Alvarado do not explicitly disclose nor teach that an ene-reductase or an enone-reductase is present.
However, Romagnolo, a research article directed to the characterization of fungal ene-reductases (Abstract), teaches that the reduction of C=C double bonds conjugated with different electron-withdrawing groups (EWG)- such as ketone, nitro, and aldehyde- is catalyzed by Ene-Reductases of the class E.C. 1.6.99.1, termed ERs (pg. 487, right col.). Romagnolo teaches that “Most of the known ERs are flavin-dependent oxidoreductases belonging to the Old Yellow Enzyme family, which require NAD(P)H as cofactor” and that these are present in S. cerevisiae. (see paragraph and sentence spanning pgs. 487-488). Further, Romagnolo teaches that “Since the purification of fungal ERs is not a common practice, a whole-cell system is still the method of choice for biocatalysis experiments” (page 488, left col, last paragraph). Romagnolo also teaches that among the 28 novel fungi tested 96.4% of these also had sufficient reducing activity towards a ketone EWG, that of cyclohexene (referred to as CE therein, Fig. 1), and Romagnolo teaches this is analogous to the reduction of C=C double bonds of ketonic substrates in benzalacetone derivatives, known in the art, which is the reaction of step (c) (pg. 489: CE biotransformation).
Therefore, in view of the knowledge in the art, before the instantly effective filing date, it would have been prima facie obvious to one of ordinary skill that when performing the method for making raspberry ketone according to the combination of Fronza and Alvarado, due to the inherent properties of Saccharomyces cerevisiae and/or an enzymatic extract thereof, that the reduction of p-hydroxybenzalacetone is performed in the presence of at least one ene-reductase and/or enone-reductase, which are found in S. cerevisiae (baker’s yeast) according to the teachings of Romagnolo.
One having ordinary skill in the art would understand that inherently, the treatment of the double C=C in p-hydroxybenzalacetone to obtain reduced 4-(4-hydroxyphenyl)-2-butanone (raspberry ketone) by using S. cerevisiae or a similar yeast extract, as made obvious by the teachings of Fronza and Alvarado, would result in the presence of ene-reductases and/or enone-reductases, because these enzymes and the critical activity they possessed is inherently found in the yeast cells or extract thereof, as taught in Romagnolo.
Further, even if the yeast used was not completely identical to the composition of the instant claim, Romagnolo teaches the existence and application of many different ene-reductases (these obtained from different fungal species) which can be selected from to catalyze the reduction of the C=C double bonds in substrates with ketones. Romagnolo cites references including the reduction of benzalacetone derivatives, akin to those instantly claimed. Therefore, in the alternative interpretation of the claim requiring an additional enzyme, the inclusion of one or more ene-reductases would also have been obvious to one of ordinary skill.
It is apparent from the teachings of Romagnolo and Fronza that there would have been a reasonable expectation of success in performing the claimed method, because both references are directed to the reduction of carbon-carbon double bonds in α’β-unsaturated compounds using biocatalysts, include whole cells or cell extracts, and Romagnolo teaches the biotransformation of such compounds using ene-reductases is known in the art.
Thus, the instant invention would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date, as evidenced by the cited references, especially in the absence of evidence to the contrary.
Claims 1-5, 7-10, and 14-16 are rejected under 35 U.S.C. 103 as being unpatentable over Fronza et al. (Tetrahedron 52.11 (1996): 4041-4052) and Alvarado et al. (Applied microbiology and biotechnology vol. 57,5-6 (2001): 725-30), as applied to claims 1-3, 7-10, and 16 above, and further in view of Sharma et al. ("First bovine serum albumin‐promoted synthesis of enones, cinnamic acids and coumarins in ionic liquid: An insight into the role of protein impurities in porcine pancreas lipase for olefinic bond formation." Advanced Synthesis & Catalysis 353.6 (2011): 871-878., on IDS filed 6/14/2023) and Benedetti et al. (“Aldolase activity of serum albumins.” Org Biomol Chem. 2011 Jun 21;9(12):4417-20. doi: 10.1039/c0ob01219j) (to include the rejections of claims 4-5 and 14-15).
The combination of Fronza and Alvarado makes obvious a method for producing natural raspberry ketone, comprising the steps (a), (b), and (c), as defined in claim 1, for all of the reasons described above. Fronza teaches that step (b), comprising a condensation of p-hydroxybenzaldehyde with acetone is known to the art (pg. 4042, second paragraph).
However, the combined teachings of Fronza and Alvarado do not explicitly teach that the condensation is performed in the presence of a biocatalyst, as recited in claim 4, or in the presence of an amino acid, as in claim 5, nor that the biocatalyst is an albumin including bovine serum albumin (BSA), as in claims 14 and 15 respectively.
Sharma et al. pertains to the use of bovine serum albumin (BSA) and an ionic liquid for the synthesis of (E)-a,b-unsaturated compounds (i.e. enones, cinnamic Acids and coumarins) including a one-pot cascade synthesis of cinnamic acids and coumarins via aldol, Knoevenagel and Knoevenagel–Doebner condensations (Abstract, Title). Sharma teaches that the use of bovine serum albumin resulted in 89% yield when condensing 4-methoxybenzaldehyde (1a) with acetone and using 1-butyl-3-methylimidazolium bromide ([bmim]Br) as a representative ionic liquid (Table 1, entry 24, and pgs. 874-873). Sharma also teaches that “five different fractions from the crude enzyme were prepared and tested for the aldol condensation. Interestingly, fractions F4 and F5 containing peptides and residual amino acids of molecular weight less than 10 kDa provided best results while the “PPL”-containing fractions (F1 and F2) gave significantly lower yields; suggesting the role of protein catalysis in the above reaction (Figure 1).” (pg. 873, left col). Sharma also teaches that the use of amino acids as a promoter in ionic liquids has also been established in the art (pg. 873, right col, last sentence). Sharma teaches that BSA can efficiently catalyze the condensation of various aldols and acetone producing an olefinic (C=C) bond (see Table 2). Specifically, regarding the formation of raspberry ketones and its precursors, Sharma teaches that “preparative scale reactions (1 g batch) of some substituted benzaldehydes were effectively accomplished ... two of the isolated condensates 4b and 5b (Table 2) were subjected to chemoselective hydrogenation, providing raspberry ketone and zingerone, respectively, in good yields (Scheme 2)” (page 875, right hand col).
Benedetti et al. teaches that bovine and human serum albumins catalyze the aldol reaction of aromatic aldehydes and acetone, with saturation kinetics and moderate and opposite enantioselectivity (First paragraph, Abstract). Benedetti teaches that a low pKa lysine, Lys-222 for BSA, in the binding site of the albumin is involved in the covalent binding of substrates and is responsible for albumin’s ability to behave as an enzyme-like catalyst (pg. 4417, left col.) Benedetti teaches that BSA catalyzed reactions exhibited multiple turnover and allowed full conversion of 1a to aldol 2a, and Benedetti teaches that partial dehydration of 2a to the a,b-unsaturated ketone was observed in over 80% conversion (pg. 4417, right col, and Table 1).
Therefore, to one of ordinary skill in the art, before the instant effective filing date, it would have been prima facie obvious to modify the method made obvious by the combination of Fronza and Alvarado, to provide a biocatalyst in step (b) to mediate the aldol condensation of acetone with p-hydroxybenzaldehyde, wherein the biocatalyst includes the albumin BSA, as taught in Sharma and Benedetti.
One would have been motivated by the teachings of Benedetti and Sharma to provide the biocatalyst BSA because both of these references teach the improved efficacy of the aldol condensation reaction between acetone and a substituted aldehyde when provided with BSA. Although Sharma is predominately concerned with the reaction using BSA with an specific ionic solution, the teachings of Benedetti suggest that no such solution is required for the albumin catalyzed reaction (Supplementary Material (ESI) section 1, included with this action: “Both the albumin-catalyzed and the background reactions were run at 37.0 °C, in 10% acetone (1.36 M) in 100 mM phosphate buffer pH 7.5, 200 mM NaCl...”). Thus, one of ordinary skill in the art would have been motivated by the knowledge of the aldol condensation reactions catalyzed by BSA according to Benedetti in view of the teachings of Sharma in which a similar BSA-catalyzed condensation is used to prepare a precursor to raspberry ketone to arrive at the instantly claimed method wherein a biocatalyst, including the albumin protein BSA, is provided for step (b).
Regarding claim 5, the claim as interpreted under the B.R.I. recites that an amino acid is present. Because BSA is a protein that is comprised of amino acids which include- for merely an example- the catalytic basic side chain Lys-222 as taught in Benedetti, the inclusion of BSA in the reaction as a catalyst would appear to naturally fulfill this limitation. If the limitation is meant to be further limited to purified or unbonded amino acids, then such a condition is suggested by the literature discussed in Sharma which teaches that “the use of... amino acids as a promoter in ionic liquids has also been established” (pg. 873, right col, last sentence).
There would have existed a reasonable expectation of success in modifying the methods of Fronza and Alvarado to include using BSA as a biocatalyst for the step of condensation with acetone because Sharma and Benedetti each teach the catalytic properties of BSA for this reaction between an aromatic aldehyde and acetone, and Sharma demonstrates that such a technique can be successfully used to produce a raspberry ketone precursor.
Therefore, the invention as a whole would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date, as evidenced by the references, especially in the absence of evidence to the contrary.
Claims 1-3, 7-13, and 16 are rejected under 35 U.S.C. 103 as being unpatentable over Fronza et al. (Tetrahedron 52.11 (1996): 4041-4052) and Alvarado et al. (Applied microbiology and biotechnology vol. 57,5-6 (2001): 725-30), as applied to claims 1-3, 7-10, and 16 above, and further in view of Sutherland et al. (“Metabolism of cinnamic, p-coumaric, and ferulic acids by Streptomyces setonii.” Canadian journal of microbiology vol. 29,10 (1983): 1253-7. doi:10.1139/m83-195, on IDS filed 6/14/2023) with supporting evidence from “Amycolatopsis tucumanensis Albarracín et al. ATCC 39116” (Product Information for the deposited strain ATCC 39116, copyright 2023, accessed on 1/5/2026 from https://www.atcc.org/products/39116) (to include the rejection of claims 11-13).
The combination of Fronza and Alvarado makes obvious a method for producing natural raspberry ketone, comprising the steps (a), (b), and (c), as defined in claim 1, for all of the reasons described above. Regarding the microorganism used for the bioproduction step (a), wherein p-coumaric acid is converted to p-hydroxybenzaldehyde, Alvarado teaches using a fungal strain, specifically Pycnoporus cinnabarinus MUCL 39533.
The combination of Fronza and Alvarado does not teach using a microorganism comprising one or more belonging to the order Actinomycetes, the family Streptomycetacae, Pseudonocardiacae, or any combination thereof, as in claim 11, nor one of the specific species or strains thereof recited in claims 12 and 13.
Sutherland teaches that the actinomycetes bacteria Streptomyces setonii strain 75Vi2 (“=ATCC 39116”, according to pg. 1253, right col, line 5 under Introduction) catabolizes p-coumaric acid via the production of p-hydroxybenzaldehyde as an intermediate before the formation of p-hydroxybenzoic acid and protocatechuic acid (see Abstract and Fig. 1). Sutherland teaches that when this strain is cultured with p-coumaric acid (i.e. p-coumarate), p-hydroxybenzaldehyde production is detected (see Table 1, line 7, and pg. 1254, right col).
Further, analysis of the deposit information for ATCC 39116 (see the cited product information sheet for ATCC Amycolatopsis tucumanensis Albarracín et al. 39116, provided herein as an evidentiary reference) demonstrates that Amycolatopsis sp. strain ATCC 39116 is a deposited strain of Streptomyces setonii 75iv2, of the classification Pseudonocardiaceae, and thus comprises the same strain characterized in Sutherland.
Therefore, to one of ordinary skill in the art, before the effective filing date of the instant invention, it would have been prima facie obvious to modify the method taught by the combination of Fronza and Alvarado, by substituting Amycolatopsis sp. strain ATCC 39116 (of the order Actinomycetes, and formerly known as Streptomyces setonii) for the Pycnoporus cinnabarinus MUCL 39533 taught in Alvarado for the predictable equivalent production of p-hydroxybenzaldehyde from p-coumaric acid.
MPEP § 2143.I.B. discusses the case of obviousness for the simple substitution of one known element for another to obtain predictable results and cites In re Fout, 675 F.2d 297, 213 USPQ 532 (CCPA 1982) in which “The court found that "[b]ecause both Pagliaro and Waterman teach a method for separating caffeine from oil, it would have been prima facie obvious to substitute one method for the other. Express suggestion to substitute one equivalent for another need not be present to render such substitution obvious." Id. at 301, 213 USPQ at 536”.
In the instant case, the bioconversion activity of transforming p-coumaric acid to p-hydroxybenzaldehyde is known in both the fungal species used in Alvarado, and in the Amycolatopsis sp. strain ATCC 39116, e.g. Streptomyces setonii 75iv2, taught in Sutherland. Thus, the substitution of Amycolatopsis sp. for the Pycnoporus cinnabarinus MUCL 39533 taught in Alvarado would have been obvious with the predictable result of performing the same transformation of p-coumaric acid to p-hydroxybenzaldehyde. The instant claims are comprising claims, and there are no express limitations regarding the production of secondary chemicals or limiting the desired results, i.e. the efficiency, of the process. Regardless, one of ordinary skill in the art having the knowledge of Sutherland that the catabolism of p-coumaric acid by this organism produces, inter alia, p-hydroxybenzaldehyde, would have been adequately motivated to optimize the process to improve the yield of the intermediate p-hydroxybenzaldehyde, known to be useful for making raspberry ketone, as taught in Fronza. The selection of the p-hydroxybenzaldehyde-producing organism from among those known in the art to have this activity would have been a matter of judicious selection, absent evidence to the contrary.
From the teachings of the cited references, it is apparent that there would have been a reasonable expectation of success in combining the teachings therein to arrive at the claimed invention because Fronza pertains to the use of p-hydroxybenzaldehyde, which is demonstrated to be produced from p-coumaric acid via several organisms including the fungus of Alvarado or the Amycolatopsis sp. strain ATCC 39116, e.g. Streptomyces setonii 75iv2, of Sutherland.
Therefore, the invention of claims 11-13, as a whole, would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date, as evidenced by the cited references, especially in the absence of evidence to the contrary.
Citation of Pertinent Art
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure.
Beekwilder et al. (GB2416769 to Danisco, on IDS filed 6/14/2023) is drawn to a host cell comprising a benzalacetone synthase (BAS) polypeptide sequence and a 4-coumarate:CoA ligase (4CL) sequence useful in the production of benzalacetone and/or raspberry ketone from precursors including p-coumaric acid (Abstract). Beekwilder uses genetically modified E. coli to perform the conversion of p-coumaric acid into raspberry ketone through p-coumaryl CoA and benzalacetone intermediates.
Schloesser et al. (WO2018/210432 A1 to Wacker Chemi AG, on IDS filed 6/14/2023) pertains to the fermentative production of raspberry ketone using a genetically modified microorganism strain comprising and expressing at least one TAL gene, a 4-CL gene, a BAS gene and a tyrA gene (Abstract, claim 1).
Feron et al. (“Microbial production of 4-hydroxybenzylidene acetone, the direct precursor of raspberry ketone.” Letters in applied microbiology vol. 45,1 (2007): 29-35, on IDS filed 6/14/2023) pertains to the biological production of 4-hydroxybenzylidene acetone (4-(4-hydroxyphenyl)-but-3-ene-2-one) via an enzymatic aldol reaction of acetone and 4-hydoxybenzaldehyde using bacteria possessing 2-deoxyribose-5-phosphate aldolase (DERA) activity (Abstract).
Conclusion
No claims are allowable.
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/A.T.M./Examiner, Art Unit 1655
/ANAND U DESAI/Supervisory Patent Examiner, Art Unit 1655