Obviousness

Obviousness Analysis of US Patent 12305206B2 under 35 U.S.C. § 103

This analysis identifies combinations of prior art references that would render the claims of US Patent 12305206B2 obvious to a person having ordinary skill in the art (PHOSITA) as of the priority date of March 2, 2016. The motivation for combining these references stems from the clear desire to produce a more potent and efficient L-glufosinate product for herbicidal applications.

Prior Art References Considered

The following prior art references, explicitly mentioned in the patent text and confirmed to predate the priority date, are central to this obviousness analysis:

• Duke et al. (2010) Toxins 2:1943-1962: Discloses that current commercial glufosinate synthesis yields a racemic mixture of L- and D-glufosinate. [cite: Duke et al. 2010 Toxins 2:1943-1962]
• Ruhland et al. (2002) Environ. Biosafety Res. 1:29-37: Teaches that L-glufosinate is significantly more potent than D-glufosinate. [cite: Ruhland et al. (2002) Environ. Biosafety Res. 1:29-37]
• EP0030424 (1981): Indicates that PPO (2-oxo-4-(hydroxy(methyl)phosphinoyl)butyric acid) can contribute herbicidal activity. [cite: EP0030424]
• Hawkes et al. (2011) Plant Biotechnol J. 9(3):301-14: Describes the oxidative deamination of D-glufosinate to PPO using a mutant D-amino acid oxidase (DAAO) enzyme, specifically a mutant Rhodosporidium toruloides DAAO with F58K and M213S mutations. [cite: Hawkes et al. (2011) Plant Biotechnol J. 9(3):301-14] It also mentions oxygen electrode assays for DAAO activity. [cite: Hawkes et al. (2011) Plant Biotechnol J. 9(3):301-14]
• U.S. Pat. No. 7,939,709 (2011): Describes DAAO mutations, including at position 56 (e.g., T56M mutation is discussed in the current patent, and the '709 patent is cited in relation to position 56). [cite: U.S. Pat. No. 7,939,709]
• U.S. Pat. No. 8,227,228 (2012): Describes DAAO enzymes from Candida intermedia and notes that such enzymes can be modified for increased activity. [cite: U.S. Pat. No. 8,227,228]
• Berneman A et al. (2010) J Microbial Biochem Technol 2: 139-146: Describes colorimetric assays for determining D-amino acid oxidase activity. [cite: Berneman A, Alves-Ferreira M, Coatnoan N, Chamond N, Minoprio P (2010) Medium/High Throughput D-Amino Acid Oxidase Colorimetric Method for Determination of D-Amino Acids. Application for Amino Acid Racemases. J Microbial Biochem Technol 2: 139-146]
• Bartsch et al. (1990) Appl Environ Microbiol. 56(1):7-12: Teaches that Escherichia coli gabT transaminase (TA) can catalyze the amination of PPO to L-glufosinate. [cite: Bartsch et al. (1990) Appl Environ Microbiol. 56(1):7-12]
• Bhatia et al. (2004) Peptide Revolution: Genomics, Proteomics & Therapeutics, pp. 47-48: Discloses an evolved enzyme that catalyzes the desired transamination reaction at a higher rate using isopropylamine as an amine donor. [cite: Bhatia et al. (2004) Peptide Revolution: Genomics, Proteomics & Therapeutics, Proceedings of the Eighteenth American Peptide Symposium, Ed. Michael Chorev and Tomi K. Sawyer, Jul. 19-23, 2003, pp. 47-48]
• EP0249188 (1987) and U.S. Pat. No. 5,162,212 (1992): Both describe TA enzymes from various microorganisms (e.g., Streptomyces hygroscopicus, Streptomyces viridochromogenes, Candida albicans). [cite: EP0249188, U.S. Pat. No. 5,162,212]
• Schulz et al. (1990) Appl Environ Microbiol. 56(1):1-6: Outlines assays for selecting mutant TA enzymes with desired activity. [cite: Schulz et al., Appl Environ Microbiol. (1990) January 56(1):1-6]

Obviousness Analysis of Independent Claims

Independent Claim 1: Method for producing L-glufosinate

Claim language: A method comprising reacting D-glufosinate with a D-amino acid oxidase (DAAO) enzyme to form PPO (2-oxo-4-(hydroxy(methyl)phosphinoyl)butyric acid), wherein the DAAO enzyme has an activity of at least 3 µmol/min*mg, and wherein the D-glufosinate is originally present in a racemic mixture of D- and L-glufosinate; followed by aminating the PPO to L-glufosinate by a transaminase (TA) enzyme, using an amine group from one or more amine donors, wherein at least 70% of the D-glufosinate is eliminated and/or the yield of L-glufosinate is at least 85% of the input racemic glufosinate or at least 70% to 85% of the D-glufosinate is converted to L-glufosinate.

Combination of Prior Art References: Hawkes et al. (2011) and Bartsch et al. (1990), motivated by Ruhland et al. (2002) and Duke et al. (2010).

Reasoning for Obviousness:

1. Known Problem and Motivation: A PHOSITA would be well aware that commercial glufosinate is a racemic mixture [cite: Duke et al. (2010) Toxins 2:1943-1962] and that L-glufosinate is significantly more potent than its D-enantiomer [cite: Ruhland et al. (2002) Environ. Biosafety Res. 1:29-37]. This creates a strong motivation to develop a method to enrich L-glufosinate from racemic glufosinate.
2. Known Enzymatic Steps:
   • Oxidative deamination of D-glufosinate to PPO: Hawkes et al. (2011) explicitly teaches this first step, demonstrating the conversion of D-glufosinate to PPO using a mutant DAAO from Rhodosporidium toruloides. [cite: Hawkes et al. (2011) Plant Biotechnol J. 9(3):301-14]
   • Amination of PPO to L-glufosinate: Bartsch et al. (1990) describes the second step, showing that Escherichia coli gabT transaminase can aminate PPO to L-glufosinate. [cite: Bartsch et al. (1990) Appl Environ Microbiol. 56(1):7-12]
3. Motivation to Combine: The enzymatic deracemization of a compound by converting one enantiomer into an achiral intermediate and then converting that intermediate into the desired enantiomer is a well-established strategy in biocatalysis. Given the knowledge of the individual enzymatic steps (D-glufosinate to PPO and PPO to L-glufosinate) and the clear advantage of L-glufosinate, a PHOSITA would be motivated to combine these known biotransformations to achieve the desired L-glufosinate enrichment. The patent itself highlights that "By combining these two reactions, the proportion of L-glufosinate can be substantially increased in a racemic glufosinate mixture." [cite: US12305206B2 Description]
4. Enzyme Activity and Optimization:
   • DAAO activity of at least 3 µmol/min*mg: While this is a specific activity threshold, the prior art (e.g., U.S. Pat. No. 8,227,228 and Hawkes et al. (2011)) already taught the existence of DAAO enzymes capable of acting on D-glufosinate and the general approach of modifying enzymes to increase their activity. [cite: U.S. Pat. No. 8,227,228, Hawkes et al. (2011) Plant Biotechnol J. 9(3):301-14] The patent itself lists known DAAO sources and suggests mutagenesis to increase activity. [cite: US12305206B2 Summary, Description] Measuring enzyme activity was also a known practice (e.g., Berneman et al., 2010). [cite: Berneman A, Alves-Ferreira M, Coatnoan N, Chamond N, Minoprio P (2010) Medium/High Throughput D-Amino Acid Oxidase Colorimetric Method for Determination of D-Amino Acids. Application for Amino Acid Racemases. J Microbial Biochem Technol 2: 139-146] Achieving an optimal activity would be considered routine experimentation and optimization for a PHOSITA.
   • Amine donors: Bhatia et al. (2004) already demonstrated using isopropylamine as an amine donor for improved transamination rates. [cite: Bhatia et al. (2004) Peptide Revolution: Genomics, Proteomics & Therapeutics, Proceedings of the Eighteenth American Peptide Symposium, Ed. Michael Chorev and Tomi K. Sawyer, Jul. 19-23, 2003, pp. 47-48] The selection of other suitable amine donors would be within the skill of an ordinary artisan.
   • Yield/conversion criteria: These are results-oriented limitations. Once the two-step enzymatic process is deemed obvious, optimizing reaction conditions (enzyme loading, substrate concentration, pH, temperature, reaction time, etc.) to achieve desired conversion efficiencies and yields would be a matter of routine experimentation for a PHOSITA. The patent itself demonstrates achieving high L-glufosinate concentrations (e.g., Example 5 and FIG. 2, showing efficient deracemization).

Independent Claim 13: Composition for L-glufosinate

Claim language: A composition comprising a mixture of D-glufosinate, PPO, and L-glufosinate, wherein L-glufosinate is present in the composition in an amount of at least 80% by weight of the sum of L-glufosinate, PPO, and D-glufosinate.

Combination of Prior Art References: Hawkes et al. (2011), Bartsch et al. (1990), Ruhland et al. (2002), and EP0030424.

Reasoning for Obviousness:

1. Result of Obvious Method: The composition claimed in Claim 13 is the direct and expected result of performing the method outlined in Claim 1. If the method of producing enriched L-glufosinate is obvious, then the resulting composition would also be obvious.
2. Motivation for L-glufosinate Enrichment: As established, Ruhland et al. (2002) provides a clear motivation to produce compositions where L-glufosinate is predominant due to its higher potency. [cite: Ruhland et al. (2002) Environ. Biosafety Res. 1:29-37]
3. Presence of D-glufosinate and PPO: Since the starting material is racemic D/L-glufosinate [cite: Duke et al. (2010) Toxins 2:1943-1962], some residual D-glufosinate is expected if the conversion is not 100%. PPO is an intermediate in the described process. EP0030424 discloses that PPO itself has herbicidal activity, providing a motivation to allow some PPO to remain in the final composition, rather than incurring additional purification costs. [cite: EP0030424] A PHOSITA would anticipate and accept a mixture containing these components.
4. Concentration of L-glufosinate: Achieving "at least 80% L-glufosinate" would be an expected outcome of optimizing the known enzymatic deracemization process to maximize the more potent enantiomer, a routine goal for a PHOSITA.

Independent Claim 14: Formulation

Claim language: A formulation comprising L-glufosinate ammonium in an amount from 10-30% by weight of the formulation; and water as the balance of the formulation; and one or more additional components selected from the group consisting of sodium alkyl ether sulfate in an amount from 10-40% by weight of the formulation; 1-methoxy-2-propanol in an amount from 0.5-2% by weight of the formulation; dipropylene glycol in an amount from 4-18% by weight of the formulation; and alkyl polysaccharide in an amount from 4-20% by weight of the formulation.

Combination of Prior Art References: General knowledge in agricultural chemistry, including prior glufosinate formulations.

Reasoning for Obviousness:

1. Known Active Ingredient: L-glufosinate (or glufosinate in general) is a known herbicide, and its ammonium salt is a common form for herbicidal formulations.
2. Routine Formulation Components: The additional components listed (sodium alkyl ether sulfate as a surfactant, 1-methoxy-2-propanol and dipropylene glycol as solvents, and alkyl polysaccharide as a humectant/adjuvant) are all well-known and commonly used excipients in agricultural herbicide formulations. The patent itself broadly defines "adjuvant components" as "diluents, extenders, carriers, surfactants, organic solvents, humectants, or conditioning agents". [cite: US12305206B2 Description]
3. Routine Optimization of Ranges: A PHOSITA in the field of agricultural chemistry would routinely select such common formulation components and optimize their concentrations within typical ranges (as claimed) to achieve desired characteristics like solubility, stability, sprayability, and efficacy for a known active ingredient. The specific percentages claimed fall within the ranges routinely explored for such formulations.

Independent Claim 19: Method for selectively controlling weeds in an area containing glufosinate-resistant crops

Claim language: A method for selectively controlling weeds in an area containing a crop of planted seeds or crops that are resistant to glufosinate, comprising applying an effective amount of a composition comprising L-glufosinate at an enantiomeric excess of greater than 90% over D-glufosinate to the area.

Combination of Prior Art References: General knowledge in agriculture, Ruhland et al. (2002).

Reasoning for Obviousness:

1. Known Practice: The use of non-selective herbicides like glufosinate in conjunction with glufosinate-resistant crops for selective weed control is a well-established practice in agriculture, widely known before the priority date. The patent acknowledges this by stating "a crop of planted seeds or crops that may optionally be resistant to glufosinate". [cite: US12305206B2 Summary, Description]
2. Motivation for L-glufosinate: Ruhland et al. (2002) clearly teaches that L-glufosinate is more potent than D-glufosinate. [cite: Ruhland et al. (2002) Environ. Biosafety Res. 1:29-37] This provides a strong motivation for a PHOSITA to use compositions enriched in L-glufosinate (such as those with >90% enantiomeric excess, derived from the obvious production method of Claim 1) to achieve superior herbicidal effects, potentially at lower application rates.
3. "Effective Amount": Determining an "effective amount" is a routine experimental optimization for any herbicide.

Independent Claim 20: Method for controlling weeds, controlling weeds in non-field areas, defoliating plants or crops, and/or desiccating crops before harvest

Claim language: A method for selectively controlling weeds in a field, controlling weeds in non-field areas, defoliating plants or crops, and/or desiccating crops before harvest, comprising applying an effective amount of a composition comprising L-glufosinate at an enantiomeric excess of greater than 90% over D-glufosinate and more than 0.01% but less than 15% PPO to the field.

Combination of Prior Art References: General knowledge of glufosinate applications, Ruhland et al. (2002), and EP0030424.

Reasoning for Obviousness:

1. Known Herbicide Applications: The various applications (weed control, defoliation, desiccation) are standard uses for glufosinate-based herbicides and were known prior to the priority date.
2. Motivation for L-glufosinate: As discussed for Claim 19, the higher potency of L-glufosinate [cite: Ruhland et al. (2002) Environ. Biosafety Res. 1:29-37] motivates its use, including compositions with high enantiomeric excess.
3. Inclusion of PPO: EP0030424 teaches that PPO itself has herbicidal activity. [cite: EP0030424] Therefore, a PHOSITA would be motivated to include PPO, or would readily accept its presence within the claimed range (more than 0.01% but less than 15%), especially if it is a byproduct of an efficient enzymatic synthesis (Claim 1) and contributes to the overall herbicidal effect. The patent text itself describes compositions where "the amount of PPO is more than 1% but less than 20%, less than 15%, less than 10%, or less than 5%". [cite: US12305206B2 Description]
4. "Effective Amount": Again, determining an "effective amount" is routine optimization.

In conclusion, the core method of producing L-glufosinate via DAAO and TA enzymes, along with the resulting compositions and methods of use, would have been obvious to a PHOSITA given the cumulative teachings of the identified prior art. The motivation to combine these teachings is evident in the desire to produce a more potent L-glufosinate herbicide from readily available racemic starting material.