EP4695412A1

EP4695412A1 - Phosphonoalanine oligopeptides and methods of making and use thereof

Info

Publication number: EP4695412A1
Application number: EP24789532.9A
Authority: EP
Inventors: Kou-san JU; Yeying ZHANG; Jerry Cui
Original assignee: Ohio State Innovation Foundation
Current assignee: Ohio State Innovation Foundation
Priority date: 2023-04-13
Filing date: 2024-04-12
Publication date: 2026-02-18
Also published as: WO2024216030A1

Abstract

Disclosed herein are compositions and methods of making and use thereof. For example, described herein are compounds defined by Formula I (I). Also disclosed herein are compounds comprising a head group and a tail group, the head group being bound to the tail group using one or more enzymes derived from Bacillus, wherein the head group comprises a phosphonic acid, a phosphinic acid, or a derivative thereof, and the tail group comprises one or more (canonical or non-canonical) amino acids. In some examples, the compound is a Bacillus isolate or a derivative or salt thereof. Also disclosed herein are compositions comprising any of the compounds disclosed herein. Also disclosed herein are methods of making and methods of use any of the compounds or compositions disclosed herein.

Description

PHOSPHONOALANINE OLIGOPEPTIDES

AND METHODS OF MAKING AND USE THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. Provisional Application No. 63/459,008, filed April 13, 2023, which is hereby incorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant/ contract no. GM137135 awarded by the National Institutes of Health. The government has certain rights in the invention.

REFERENCE TO SEQUENCE LISTING

The sequence listing submitted on April 12, 2024, as an .XML file entitled “103361- 487WO1_ST26” created on April 12, 2024, and having a file size of 40,158 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).

BACKGROUND

Some phosphonate natural products, including isolates from naturally-occurring microorganisms, have been shown to have inhibitory activities. The inhibitory activities underly their development as antibiotics and pesticides. Most bio-active phosphonate natural products have been isolated from Actinobacteria. Many plant, animal, and insect pathologies have poor or no modalities of control and new compositions are needed. The compositions and methods discussed herein address these and other needs.

SUMMARY

In accordance with the purposes of the disclosed compositions and methods as embodied and broadly described herein, the disclosed subject matter relates to compositions and methods of making and use thereof.

For example, described herein are compounds defined by Formula I:

I wherein R¹ is hydrogen, halide, substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C1-C20 alkoxy, substituted or unsubstituted C3-C20 aryl (e.g., substituted or unsubstituted phenyl), substituted or unsubstituted C4-C21 alkylaryl, NR^xR^y, or OR⁹; R² is hydrogen, halide, substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C1-C20 alkoxy, substituted or unsubstituted C3-C20 aryl (e.g., substituted or unsubstituted phenyl), substituted or unsubstituted C4-C21 alkylaryl, NR^xR^y, or OR¹⁰; R³ is hydrogen, hydroxyl, halide, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C1-C10 alkoxy, substituted or unsubstituted C3-C10 aryl (e.g., substituted or unsubstituted phenyl), substituted or unsubstituted C4-C11 alkylaryl, or -C(O)OR⁸; R⁴ is hydrogen, hydroxyl, halide, substituted or unsubstituted Ci- C10 alkyl, substituted or unsubstituted C1-C10 acyl, substituted or unsubstituted C1-C10 amide, NR^xR^y, or one or more amino acids (e.g., one or more canonical or non-canonical amino acids); and R⁸, R⁹, and R¹⁰ are each independently hydrogen, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C3-C10 aryl (e.g., substituted or unsubstituted phenyl), or substituted or unsubstituted C4-C11 alkylaryl; and R^x and R^y are independently selected from hydrogen, or substituted or unsubstituted C1-C5 alkyl, or substituted or unsubstituted C1-C5 acyl; or a derivative or salt thereof; with the proviso that the compound is not phosphonoalamide A, B, C, D, E, or F.

Also disclosed herein are compounds comprising a head group and a tail group, the head group being bound to the tail group using one or more enzymes derived from Bacillus, wherein the head group comprises a phosphonic acid, a phosphinic acid, or a derivative thereof, and the tail group comprises one or more (canonical or non-canonical) amino acids, with the proviso that the compound is not phosphonoalamide A, B, C, D, E, or F.

In some examples, the compound is a Bacillus isolate or a derivative or salt thereof. Also disclosed herein are compositions comprising any of the compounds disclosed herein.

Also disclosed herein are nucleic acids encoding any of the compounds or compositions disclosed herein, vectors encoding said nucleic acids, cells comprising said vector, and cells comprising any of the compounds or compositions disclosed herein.

Also disclosed herein are methods of making any of the compounds or compositions disclosed herein.

Also disclosed herein are methods of making a compound comprising a head group and a tail group, the head group being bound to the tail group, wherein the head group comprises a phosphonic acid, a phosphinic acid, or a derivative thereof, and the tail group comprises one or more (canonical or non-canonical) amino acids, using one or more enzymes derived from Bacillus, with the proviso that the compound is not phosphonoalamide A, B, C, D, E, or F.

Also disclosed herein are methods of use of any of the compounds, compositions, nucleic acids, vectors, or cells disclosed herein.

Also disclosed herein are methods of reducing the activity of bacteria, the methods comprising exposing the bacteria to an effective amount of any of the compounds, compositions, nucleic acids, vectors, or cells disclosed herein.

Also disclosed herein are methods of reducing bacterial population, the methods comprising exposing the bacteria to an effective amount of any of the compounds, compositions, nucleic acids, vectors, or cells disclosed herein.

Also disclosed herein are methods of killing bacteria, the methods comprising exposing the bacteria to an effective amount of any of the compounds, compositions, nucleic acids, vectors, or cells disclosed herein.

Also disclosed herein are methods for treating, preventing, inhibiting, and/or ameliorating a disease or disorder in a plant or a subject in need thereof, the methods comprising administering to the plant or subject a therapeutically effective amount of any of the compounds, compositions, nucleic acids, vectors, or cells disclosed herein.

Also disclosed herein are methods for treating, preventing, inhibiting, and/or ameliorating a microbial infection in a plant or a subject, comprising administering to the plant or subject an effective amount of any of the compounds, compositions, nucleic acids, vectors, or cells disclosed herein.

Additional advantages of the disclosed compositions and methods will be set forth in part in the description which follows, and in part will be obvious from the description. The advantages of the disclosed compositions and methods will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosed systems and methods, as claimed.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects of the disclosure, and together with the description, serve to explain the principles of the disclosure.

Figure 1. The structures of L-phosphonoalanine, phosphonoalamide E, and phosphonoalamide F. Figure 2. Synteny between B. amyloliquefaciens DSM 7, B. velezensis NRRL B-41580, B. swezeyi NRRL B-41282, B. cabrialesii TE3, and B. subtilis 168. Genes conserved between B. velezensis and B. swezeyi but not the other Bacillus strains demonstrate the boundaries of the biosynthetic gene cluster.

Figure 3A. ³¹P NMR spectra of the pDG1730 and pKSJ657 integrant extracts, showing phosphonic/phosphinic acid production with pKSJ657.

Figure 3B. ¹H-³¹P HMBC of the pKSJ657 extract, showing the correlated proton resonances.

Figure 3C. LC-HRMS showing EICs for [M+H]⁺ m/z corresponding to phosphonoalanine (170.02128), phosphonoalamide E (241.0584), and phosphonoalamide F (312.0955).

Figure 4. ³¹P NMR spectra of in vitro assays assessing the ability of each canonical amino acid to serve as an amino donor for the conversion of phosphonopyruvate to phosphonoalanine by PnfD in a coupled reaction with PnfC: Alanine (Ala) (i), Arginine (Arg) (ii), Asparagine (Asn) (iii), Aspartic Acid (Asp) (iv), Cysteine (Cys) (v), Glutamine (Gin) (vi), Glutamic acid (Glu) (vii), Glycine (Gly) (viii), Histidine (His) (ix), Isoleucine (He) (x), Leucine (Leu) (xi), Lysine (Lys) (xii), Methionine (Met) (xiii), Phenylalanine (Phe) (xiv), Proline (Pro) (xv), Serine (Ser) (xvi), Threonine (Thr) (xvii), Tryptophan (Trp) (xviii), Tyrosine (Tyr) (xix), and Valine (Vai) (xx). Phosphoenolpyruvate is highlighted in grey while phosphonoalanine is highlighted in yellow.

Figure 5A. Chromophore analysis of PnfD (20 uM) via UV-Vis scans. Native protein demonstrated an absorbance maximum at 439 nm. To examine pyridoxal-5-phosphate occupancy of the isolated protein, 20 uM of pyridoxal-5-phosphate was added, demonstrating a free absorbance peak of 410 nm. By comparing the absorbance at 439 nm, the pyridoxal-5- phosphate occupancy of native enzyme was estimated to be 40.3%.

Figure 5B. Chromophore analysis of PnfD (20 uM) via UV-Vis scans. Native protein demonstrated an absorbance maximum at 439 nm. The internal Schiff base was reduced by addition of 1 mM NaBH4, shifting the absorbance maximum to 344 nm.

Figure 5C. Chromophore analysis of PnfD (20 uM) via UV-Vis scans. Native protein demonstrated an absorbance maximum at 439 nm. The enzyme was also incubated with 5 mM of L-cycloserine to form the pyridoxal-5-phosphate adduct, which shifted the absorbance maximum to 381 nm.

Figure 6 A. ³¹P NMR spectra of PnfA in vitro assays: i. Complete reaction containing PnfA, L-alanine, L-phosphonoalanine, ATP, and Mg²⁺; ii. Reaction lacking L-phosphonoalanine; iii. Reaction lacking PnfA; iv. Reaction lacking ATP; v. Reaction lacking L-alanine; and vi. Reaction lacking Mg²⁺. The characteristic shift of phosphonoalanine is highlighted in yellow, while the product shift is highlighted in green.

Figure 6B. ³¹P NMR spectra of PnfA in vitro assays investigating substrate stereoselectivity: i. L-alanine and L-phosphonoalanine; ii. D-alanine and L-phosphonoalanine; and iii. L-alanine and DL-phosphonoalanine. The characteristic shift of phosphonoalanine is highlighted in yellow, while the product shift is highlighted in green.

Figure 7 A. ³¹P NMR spectra of PnfA in vitro assays containing phosphonoalanine and each of the 20 canonical amino acids: Ala (i), Arg (ii), Asn (iii), Asp (iv), Cys (v), Gin (vi), Glu (vii), Gly (viii), His (ix), He (x), Leu (xi), Lys (xii), Met (xiii), Phe (xiv), Pro (xv), Ser (xvi), Thr (xvii), Trp (xviii), Tyr (xix), and Vai (xx). Phosphonoalanine is highlighted in yellow while any products are highlighted in green.

Figure 7B. [M+H]⁺ Extracted ion chromatograms for each potential dipeptide product (vertical axis = intensity; horizontal axis = retention time (min)): m/z 241.0584 (i, Ala-PnAla), 326.1224 (ii, Arg-PnAla), 284.0642 (iii, Asn-PnAla), 285.0482 (iv, Asp-PnAla), 273.0305 (v, Cys-PnAla), 298.0799 (vi, Gln-PnAla), 299.0639 (vii, Glu-PnAla), 227.0428 (viii, Gly-PnAla), 293.0646 (ix, His-PnAla), 283.1054 (x, Ile-PnAla), 283.1054 (xi, Leu-PnAla), 298.1163 (xii, Lys-PnAla), 301.0618 (xiii, Met-PnAla), 317.0897 (xiv, Phe-PnAla), 267.0741 (xv, Pro-PnAla), 257.0533 (xvi, Ser-PnAla), 240.0952 (xvii, Thr-PnAla), 356.1006 (xviii, Trp-PnAla), 333.0846 (xix, Tyr-PnAla), and 269.0897 (xx, Val-PnAla).

Figure 8. [M+H]⁺ Extracted ion chromatograms for each potential dipeptide product from PnfA in vitro assays containing alanine and various aminophosphonates (vertical axis = intensity; horizontal axis = retention time (min)): m/z 241.0584 (Ala-PnAla, i), 269.0897 (Ala-L- AP4, ii), 283.1054 (Ala-L-AP5, iii), 197.0686 (Ala-Ala(P), iv), 225.0999 (Ala-Val(P), v), 211.0842 (Ala-3ApPn, vi), 245.0686 (Ala-4APhePn, vii), 197.0686 (Ala-2AEP, viii), 183.0529 (Ala-AmPn, ix), and 223.0842 (Ala-PT, x).

Figure 9. The three products produced by PnfA, with differences in chemical structure from the canonical product shown in red.

Figure 10. ³¹P NMR spectra of PnfB in vitro assays containing Ala-PnAla (phosphonoalamide E) and each of the 20 canonical amino acids: Ala (i), Arg (ii), Asn (iii), Asp (iv), Cys (v), Gin (vi), Glu (vii), Gly (viii), His (ix), He (x), Leu (xi), Lys (xii), Met (xiii), Phe (xiv), Pro (xv), Ser (xvi), Thr (xvii), Trp (xviii), Tyr (xix), and Vai (xx). Ala-PnAla is highlighted in yellow while any products are highlighted in green.

Figure 11. The biosynthetic pathway for phosphonoalamide E and F.

Figure 12. Biosynthetic pathway for phosphonoalamides E and F, antimicrobial phosphonopeptides with a conserved C-terminal L-phosphonoalanine (PnAla) residue.

Figure 13. Synteny analysis of the B. velezensis pepM gene neighborhood. pnfABCDT were conserved between B. velezensis and B. swezeyi, which encode pepM (pnfC), but not between B. velezensis and closely related strains lacking pepM. Upstream genes were excluded based on their conservation within organisms lacking the ability to produce Pn. Downstream genes were excluded by the same basis but also by their identification as a o factor, and members of the j’c/i and pur (purine biosynthesis) operons. Orfs are numbered as space allows, with detailed annotation in Table 5.

Figure 14A-Figure 14C. Heterologous expression of pnfABCD. A) ³¹P NMR spectra of the empty vector (pDG1730) and pKSJ652 integrant extracts. B) ¹H-³¹P HMBC of the pKSJ652 integrant extract, demonstrating correlated proton resonances. C) LC-HRMS showing EICs for [M+H]⁺ ions of PnAla (m/z 170.0213), phosphonoalamide E (m/z 241.0584), and phosphonoalamide F (m/z 312.0955).

Figure 15A-Figure 15C. PnAla production by PnfC and PnfD. A) Biosynthetic scheme for L-PnAla formation. B) ³¹P NMR spectrum of PnfCD reactions resulting in conversion of PEP (grey) to PnAla (green). C) ³¹P NMR spectrum of PnfD reverse reaction resulting in conversion of PnAla to PnPy (yellow).

Figure 16A-Figure 16C. Biosynthesis of phosphonoalamide F. A) Potential biosynthetic routes to phosphonoalamide F. The convergent pathway (grey) was excluded based on the biochemical activity of the ATP-Grasp ligases. B) Reactions with Ala and PnAla, highlighting the formation of Ala-PnAla (cyan) by PnfA. C) Reactions with Ala and Ala-PnAla, highlighting the formation of Ala- Ala-PnAla (blue) by PnfB.

Figure 17. Comparison of the Bacillus and Streptomyces pepM gene neighborhoods. Fifteen gene windows centered around pepM show that the only genes conserved between the putative Bacillus and Streptomyces phosphonoalamide biosynthetic gene clusters encode PepM, a transaminase, two ATP -grasp ligases, and a MFS transporter.

Figure 18. SDS-PAGE of purified Pnf proteins. 3 ug of each protein was loaded and bands of expected size were observed. 1 : PnfA (49.7 kDa), 2: PnfB (47.7 kDa), 3: PnfC (49.0 kDa), 4: PnfD (45.3 kDa).

Figure 19A. UV-Vis spectroscopy of recombinant PnfD. The native protein demonstrated an absorbance maximum at 439 nm. To examine PLP occupancy, stoichiometric PLP was added to ensure full occupancy of the binding site. By subsequently comparing the absorbance at 439 nm before (native occupancy) and after addition of PLP (full occupancy), the PLP occupancy of the recombinant protein as purified was estimated to be 40%. Figure 19B. UV-Vis spectroscopy of recombinant PnfD. Addition of 1 mM NaB Lj resulted in reduction of the internal Schiff base, shifting the absorbance maximum to 344 nm.

Figure 19C. UV-Vis spectroscopy of recombinant PnfD. Incubation with 5 mM L- cycloserine (LCS) resulted in PLP adduct formation, shifting the absorbance maximum to 381 nm.

Figure 20. Acceptance of amino donors by PnfD. ³¹P NMR spectra for the conversion of PEP to PnAla by PnfC and PnfD. In each reaction, a different proteinogenic amino acid was used as a potential amino donor for PnfD: Ala (i), Arg (ii), Asn (iii), Asp (iv), Cys (v), Gin (vi), Glu (vii), Gly (viii), His (ix), He (x), Leu (xi), Lys (xii), Met (xiii), Phe (xiv), Pro (xv), Ser (xvi), Thr (xvii), Trp (xviii), Tyr (xix), and Vai (xx). PEP is highlighted in grey while PnAla is highlighted in green.

Figure 21. LC-HRMS of PnPy produced by the PnfD reverse reaction. An EIC of the [M- H]’ ion of phosphonopyruvate (m/z 166.9751)

Figure 22A-Figure 22D. Time courses of transamination reactions. A) The PnfD forward reaction with 0.1 mM PLP, 1 mM PnPy, and 10 mM Asp, with PnAla highlighted in green and PnPy in yellow. B) The conversion of PnPy to PnAla over time. C) The PnfD reverse reaction with 0.1 mM PLP, 1 mM PnAla, and 10 mM OAA, with PnAla highlighted in green and PnPy in yellow. D) The conversion of PnAla to PnPy over time.

Figure 23. Essentiality of PnfA reaction components. ³¹P NMR spectra of PnfA reactions, from top to bottom: the complete reaction, omission of PnAla, omission of PnfA, omission of ATP, omission of Ala, and omission of Mg²⁺. The characteristic shift of PnAla is highlighted in green while the shift of Ala-PnAla is highlighted in cyan.

Figure 24 A. Carboxylate specificity of PnfA. Reactions containing PnfA, PnAla, ATP, Mg²⁺, and each of the proteinogenic amino acids. ³¹P NMR spectra of assays with Ala (i), Arg (ii), Asn (iii), Asp (iv), Cys (v), Gin (vi), Glu (vii), Gly (viii), His (ix), He (x), Leu (xi), Lys (xii), Met (xiii), Phe (xiv), Pro (xv), Ser (xvi), Thr (xvii), Trp (xviii), Tyr (xix), and Vai (xx). PnAla is highlighted in green, Ala-PnAla in cyan, and Ser-PnAla in purple.

Figure 24B. Carboxylate specificity of PnfA. Reactions containing PnfA, PnAla, ATP, Mg²⁺, and each of the proteinogenic amino acids. LC-HRMS analysis of reactions. [M+H]⁺ Extracted ion chromatograms for each potential dipeptide product: m/z 241.0584 (i, Ala-PnAla), 326.1224 (ii, Arg-PnAla), 284.0642 (iii, Asn-PnAla), 285.0482 (iv, Asp-PnAla), 273.0305 (v, Cys-PnAla), 298.0799 (vi, Gln-PnAla), 299.0639 (vii, Glu-PnAla), 227.0428 (viii, Gly-PnAla), 293.0646 (ix, His-PnAla), 283.1054 (x, Ile-PnAla), 283.1054 (xi, Leu-PnAla), 298.1163 (xii, Lys-PnAla), 301.0618 (xiii, Met-PnAla), 317.0897 (xiv, Phe-PnAla), 267.0741 (xv, Pro-PnAla), 257.0533 (xvi, Ser-PnAla), 240.0952 (xvii, Thr-PnAla), 356.1006 (xviii, Trp-PnAla), 333.0846 (xix, Tyr-PnAla), and 269.0897 (xx, Val-PnAla).

Figure 25A-Figure 25C. LC-HRMS/MS of PnfA and PnfB reaction products. LC- HRMS/MS fragmentation of A) phosphonoalamide F (Ala-Ala-PnAla), B) phosphonoalamide E (Ala-PnAla), and C) Ser-PnAla. Structural assignment for each ion is found in Table 6.

Figure 26. ^JH NMR of Ala-PnAla. Water-suppressed ^JH NMR of Ala-PnAla in 90% H2O/10% D2O. Protons and corresponding peaks are labeled with the same letter.

Figure 27A. Carboxylate specificity of PnfB. Reactions containing PnfB, Ala-PnAla, ATP, Mg²⁺, and each of the proteinogenic amino acids were monitored by ³¹P NMR. ³¹P NMR spectra of assays with Ala (i), Arg (ii), Asn (iii), Asp (iv), Cys (v), Gin (vi), Glu (vii), Gly (viii), His (ix), He (x), Leu (xi), Lys (xii), Met (xiii), Phe (xiv), Pro (xv), Ser (xvi), Thr (xvii), Trp (xviii), Tyr (xix), and Vai (xx). Ala-PnAla is highlighted in cyan while Ala-Ala-PnAla is highlighted in dark blue.

Figure 27B. Carboxylate specificity of PnfB. Reactions containing PnfB, Ala-PnAla, ATP, Mg²⁺, and each of the proteinogenic amino acids were monitored by LC-HRMS. LC- HRMS analysis of reactions. [M+H]⁺ Extracted ion chromatograms for each potential tripeptide product: m/z 312.0955 (i, Ala-Ala-PnAla), 397.1595 (ii, Arg-Ala-PnAla), 355.1013 (iii, Asn- Ala-PnAla), 356.0854 (iv, Asp-Ala-PnAla), 344.0676 (v, Cys-Ala-PnAla), 369.1170 (vi, Gln- Ala-PnAla), 370.1010 (vii, Glu-Ala-PnAla), 298.0799 (viii, Gly-Ala-PnAla), 364.1017 (ix, His- Ala-PnAla), 354.1425 (x, Ile-Ala-PnAla), 354.1425 (xi, Leu-Ala-PnAla), 369.1534 (xii, Lys- Ala-PnAla), 372.0989 (xiii, Met-Ala-PnAla), 388.1268 (xiv, Phe- Ala-PnAla), 338.1112 (xv, Pro-Ala-PnAla), 328.0904 (xvi, Ser-Ala-PnAla), 342.1061 (xvii, Thr-Ala-PnAla), 427.1377 (xviii, Trp-Ala-PnAla), 404.1217 (xix, Tyr-Ala-PnAla), 340.1268 (xx, Val-Ala-PnAla).

Figure 28A-Figure 28B. PnfA and PnfB reactions with Ala- Ala. A) Reactions containing Ala- Ala, PnAla, ATP, Mg²⁺, and either PnfA or PnfB, were analyzed by ³¹P NMR. A single product observed in the PnfA reaction. PnAla is highlighted in green while product is highlighted in cyan. B) LC-HRMS revealed the product of the PnfA reaction to be Ala-PnAla m/z 241.0584) rather than Ala-Ala-PnAla m/z 312.0955). Ala-Ala m/z 161.0926, boxed) was present within the reaction mixture, but free Ala was also observed m/z 90.0555).

Figure 29A-Figure 29B. Ala- Ala degradation in reaction buffer. A) ¹ H NMR of Ala- Ala in 90% H₂O/10% D₂O. B) LC-HRMS analysis of Ala m/z 90.0550) and Ala-Ala m/z 161.0922) in reaction buffer over time.

Figure 30A-Figure 30B. Di- and tripeptide formation by PnfAB. A) ³¹P NMR spectrum of the reaction including PnfA, PnfB, Ala, PnAla, ATP, and Mg²⁺ demonstrating production of Ala-PnAla (cyan) and Ala-Ala-PnAla (dark blue) from PnAla (green). B) LC-HRMS confirmed the presence of PnAla (m/z 170.0218), Ala-PnAla (m/z 241.0584), and Ala-Ala-PnAla (m/z 312.0955).

Figure 31 A-Figure 3 ID. Ala-Ala is not formed by PnfAB, PnfA, or PnfB. A) LC-HRMS of an Ala- Ala standard (m/z 161.0922) shows that it is not formed in B) PnfAB reactions, C) PnfA reactions, or D) PnfB reactions containing enzyme, Ala, ATP, and Mg²⁺.

Figure 32A-Figure 32B. One-pot biosynthesis with PnfABCD. A) ³¹P NMR analysis of the reaction containing PnfABCD with PEP, PLP, Asp, Ala, PnAla, ATP, and Mg²⁺. PEP is highlighted in grey while product is highlighted in cyan. B) LC-HRMS revealed the major product of the one-pot reaction to be Ala-PnAla (m/z 241.0584), though PnAla (m/z 170.02184) and Ala-Ala-PnAla (m/z 312.0955) were also observed.

Figure 33A-Figure 33B. Nucleophile specificity of PnfA. Reactions containing PnfA, Ala, ATP, Mg²⁺, and the panel of aminophosphonates (A) were monitored by LC-HRMS (B). [M+H]⁺ EICs were obtained for each potential dipeptide m z. i) Ala-PnAla (241.0584), ii) Ala- 2AEP (197.0686), iii) Ala-1H2AEP (213.0640), iv) Ala-3ApPn (211.0842), v) Ala-PT (223.0842), vi) Ala-L-AP4 (269.0897), vii) Ala-L-AP5 (283.1054), viii) Ala-AmPn (183.0529), ix) Ala-Ala(P) (197.0686), x) Ala-Val(P) (225.0999), xi) Ala-4APhePn (245.0686)

Figure 34A-Figure 34B. LC-HRMS/MS of Ala-2AEP and Ala-1H2AEP. LC-HRMS/MS fragmentation of A) Ala-2AEP (m/z 197.0686) and B) Ala-1H2AEP (m/z 213.0640). Structural assignment for each ion is found in Table 7.

Figure 35. LC-HRMS/MS of Ala-AP4. LC-HRMS/MS fragmentation of Ala-AP4 (m/z 255.0740). Structural assignment for each ion is found in Table 10.

Figure 36A-Figure 36B. PnfA reactions with Ala, Ser, , PnAla, 2AEP, and 1H2AEP. A) Structural similarities between Ala and Ser with PnAla, 2AEP, and 1H2AEP. B) ³¹P NMR of PnfA reactions using Ala as carboxylate and PnAla, 2AEP, and 1H2AEP as nucleophiles as well as Ser as carboxylate and PnAla, 2AEP, and 1H2AEP as nucleophiles. PnAla is highlighted in green, Ala-PnAla in cyan, Ser-PnAla in purple, 2AEP in peach, Ala-2AEP in magenta, 1H2AEP in pink, and Ala-1H2AEP in orange.

Figure 37A-Figure 37D. PnfA reactions containing Ala and Asp, and Ser and Asp. LC- HRMS of PnfA reactions containing A) Ala and Asp (for Ala- Asp, m/z 205.0824) and B) Ser and Asp (for Ser-Asp, m/z 221.0774). LC-HRMS/MS fragmentation of C) Ala-Asp and D) Ser- Asp. Structural assignment for each ion is found in Table 8.

Figure 38 A-Figure 38C. Stereoselectivity of PnfA. ³¹P NMR spectra of the PnfA reaction with A) D-Ala and L-PnAla and B) L-Ala and DL -PnAla. C) Marfey’s analysis of DL- (top) and L- PnAla (middle) standards along with the PnfA + Ala + DL-PnAla reaction mixture (bottom).

Figure 39A-Figure 39B. NMR analysis of enzymatically prepared 1H2AEP. A) Water- suppressed ’H NMR spectrum and B) ¹H-³¹P HMBC analysis of enzymatically prepared 1H2AEP. The data are in agreement with literature values [S6],

DETAILED DESCRIPTION

The compositions, methods, and systems described herein may be understood more readily by reference to the following detailed description of specific aspects of the disclosed subject matter and the Examples included therein.

Before the present compositions, methods, and systems are disclosed and described, it is to be understood that the aspects described below are not limited to specific synthetic methods or specific reagents, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.

Also, throughout this specification, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which the disclosed matter pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.

General Definitions

In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings.

Throughout the description and claims of this specification, the word “comprise” and other forms of the word, such as “comprising” and “comprises,” means including but not limited to, and is not intended to exclude, for example, other additives, components, integers, or steps.

As used in the description and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a composition” includes mixtures of two or more such compositions, reference to “an agent” includes mixtures of two or more such agents, reference to “the component” includes mixtures of two or more such components, and the like.

“Optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. By “about” is meant within 5% of the value, e.g., within 4, 3, 2, or 1% of the value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

Values can be expressed herein as an “average” value. “Average” generally refers to the statistical mean value.

By “substantially” is meant within 5%, e.g., within 4%, 3%, 2%, or 1%.

“Exemplary” means “an example of’ and is not intended to convey an indication of a preferred or ideal embodiment. “Such as” is not used in a restrictive sense, but for explanatory purposes.

It is understood that throughout this specification the identifiers “first” and “second” are used solely to aid in distinguishing the various components and steps of the disclosed subject matter. The identifiers “first” and “second” are not intended to imply any particular order, amount, preference, or importance to the components or steps modified by these terms.

References in the specification and concluding claims to parts by weight of a particular element or component in a composition denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a compound containing 2 parts by weight of component X and 5 parts by weight component Y, X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.

A weight percent (wt. %) of a component, unless specifically stated to the contrary, is based on the total weight of the formulation or composition in which the component is included.

The term “or combinations thereof’ as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof’ is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CAB ABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

As used herein, Me refers to a methyl group; OMe refers to a methoxy group; and z-Pr refers to an isopropyl group. As used herein, agriculturally acceptable salts and esters refer to salts and esters that exhibit herbicidal activity, or that are or can be converted in plants, water, or soil to the referenced herbicide. Exemplary agriculturally acceptable esters are those that are or can be hydrolyzed, oxidized, metabolized, or otherwise converted, e.g., in plants, water, or soil, to the corresponding carboxylic acid which, depending on the pH, may be in the dissociated or undissociated form.

As used herein, by a “subject” is meant an individual. Thus, the “subject” can include domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.), birds, and insects. “Subject” can also include a mammal, such as a primate or a human. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.

As used herein, antimicrobials include, for example, antibacterials, antifungals, and antivirals. As used herein, “antimicrobial” refers to the ability to treat or control (e.g., reduce, prevent, treat, or eliminate) the growth of a microbe at any concentration. Similarly, the terms “antibacterial,” “antifungal,” and “antiviral” refer to the ability to treat or control the growth of bacteria, fungi, and viruses at any concentration, respectively.

The term “inhibit” refers to a decrease in an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This can also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.

As used herein, “reduce” or other forms of the word, such as “reducing” or “reduction,” refers to lowering of an event or characteristic (e.g., microbe population/infection). It is understood that the reduction is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. For example, “reducing microbial infection” means reducing the spread of a microbial infection relative to a standard or a control.

As used herein, “prevent” or other forms of the word, such as “preventing” or “prevention,” refers to stopping a particular event or characteristic, stabilizing or delaying the development or progression of a particular event or characteristic, or minimizing the chances that a particular event or characteristic will occur. “Prevent” does not require comparison to a control as it is typically more absolute than, for example, “reduce.” As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed. For example, the terms “prevent” or “suppress” can refer to a treatment that forestalls or slows the onset of a disease or condition or reduced the severity of the disease or condition. Thus, if a treatment can treat a disease in a subject having symptoms of the disease, it can also prevent or suppress that disease in a subject who has yet to suffer some or all of the symptoms.

As used herein, “treat” or other forms of the word, such as “treated” or “treatment,” refers to administration of a composition or performing a method in order to reduce, prevent, inhibit, or eliminate a particular characteristic or event (e.g., microbe growth or survival). The term “control” is used synonymously with the term “treat.”

The term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. By way of example, in the context of microbial infections, “treating,” “treat,” and “treatment” as used herein, refers to partially or completely inhibiting or reducing the microbial infections which the subject is suffering. In one embodiment, this term refers to an action that occurs while a patient is suffering from, or is diagnosed with, the microbial infections, which reduces the severity of the condition, or retards or slows the progression of the condition. Treatment need not result in a complete cure of the condition; partial inhibition or reduction of the microbial infections is encompassed by this term.

The term “therapeutically effective amount” refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.

The term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.

As used herein, “molecular weight” refers to number average molecular weight as measured by 'H NMR spectroscopy, unless indicated otherwise.

As used herein, the term “delivery” encompasses both local and systemic delivery. For example, delivery of mRNA encompasses situations in which an mRNA is delivered to a target tissue and the encoded protein or peptide is expressed and retained within the target tissue (also referred to as “local distribution” or “local delivery”), and situations in which an mRNA is delivered to a target tissue and the encoded protein or peptide is expressed and secreted into patient's circulation system (e.g., serum) and systematically distributed and taken up by other tissues (also referred to as “systemic distribution” or “systemic delivery).

As used herein, the term “encapsulation,” or grammatical equivalent, refers to the process of confining an individual nucleic acid molecule within a nanoparticle.

As used herein, “expression” of a mRNA refers to translation of an mRNA into a peptide (e.g., an antigen), polypeptide, or protein (e.g., an enzyme) and also can include, as indicated by context, the post-translational modification of the peptide, polypeptide or fully assembled protein (e.g., enzyme). In this application, the terms “expression” and “production,” and grammatical equivalent, are used inter-changeably.

As used herein, the term “messenger RNA (mRNA)” refers to a polynucleotide that encodes at least one peptide, polypeptide or protein. mRNA as used herein encompasses both modified and unmodified RNA. mRNA may contain one or more coding and non-coding regions. mRNA can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, mRNA can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, backbone modifications, etc. An mRNA sequence is presented in the 5' to 3' direction unless otherwise indicated. In some embodiments, an mRNA is or comprises natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl- cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaad enosine, 7-deazaguanosine, 8- oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, pseudouridine, and 5- methylcytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and/or modified phosphate groups (e.g., phosphorothioates and 5'-N- phosphoramidite linkages).

As used herein, the term “nucleic acid,” in its broadest sense, refers to any compound and/or substance that is or can be incorporated into a polynucleotide chain. In some embodiments, a nucleic acid is a compound and/or substance that is or can be incorporated into a polynucleotide chain via a phosphodiester linkage. In some embodiments, “nucleic acid” refers to individual nucleic acid residues (e.g., nucleotides and/or nucleosides). In some embodiments, “nucleic acid” refers to a polynucleotide chain comprising individual nucleic acid residues. In some embodiments, “nucleic acid” encompasses RNA as well as single and/or double-stranded DNA and/or cDNA. Furthermore, the terms “nucleic acid,” “DNA,” “RNA,” and/or similar terms include nucleic acid analogs, i.e., analogs having other than a phosphodiester backbone. In some examples, the term “nucleic acid” as used herein means natural and synthetic DNA, RNA, oligonucleotides, oligonucleosides, and derivatives thereof. For ease of discussion, such nucleic acids are at times collectively referred to herein as “constructs,” “plasmids,” or “vectors.”

The term “gene” as used in this specification refers to a segment of deoxyribonucleotides (DNA) possessing the information required for synthesis of a functional biological product such as a protein or ribonucleic acid (RNA).

The term “genetic engineering” is used to indicate various methods involved in gene manipulation including isolationjoining, introducing of gene(s) as well as methods to isolate select organisms containing the manipulated gene(s).

As specified herein, the term “DNA construct” refers to a sequence of deoxyribonucleotides including deoxyribonucleotides obtained from one or more sources.

The term “gene expression” refers to efficient transcription and translation of genetic information contained in concerned genes.

The term “recombinant” cells or population of cells refers to cells or population of cells into which an exogenous nucleic acid sequence is introduced using a delivery vehicle such as a plasmid.

Chemical Definitions

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The organic moieties mentioned when defining variable positions within the general formulae described herein (e.g., the term “halogen”) are collective terms for the individual substituents encompassed by the organic moiety. The prefix C_n-C_m preceding a group or moiety indicates, in each case, the possible number of carbon atoms in the group or moiety that follows.

The term “ion,” as used herein, refers to any molecule, portion of a molecule, cluster of molecules, molecular complex, moiety, or atom that contains a charge (positive, negative, or both at the same time within one molecule, cluster of molecules, molecular complex, or moiety (e.g., zwitterions)) or that can be made to contain a charge. Methods for producing a charge in a molecule, portion of a molecule, cluster of molecules, molecular complex, moiety, or atom are disclosed herein and can be accomplished by methods known in the art, e.g., protonation, deprotonation, oxidation, reduction, alkylation, acetylation, esterification, de-esterification, hydrolysis, etc.

The term “anion” is a type of ion and is included within the meaning of the term “ion.” An “anion” is any molecule, portion of a molecule (e.g., zwitterion), cluster of molecules, molecular complex, moiety, or atom that contains a net negative charge or that can be made to contain a net negative charge. The term “anion precursor” is used herein to specifically refer to a molecule that can be converted to an anion via a chemical reaction (e.g., deprotonation).

The term “cation” is a type of ion and is included within the meaning of the term “ion.” A “cation” is any molecule, portion of a molecule (e.g., zwitterion), cluster of molecules, molecular complex, moiety, or atom, that contains a net positive charge or that can be made to contain a net positive charge. The term “cation precursor” is used herein to specifically refer to a molecule that can be converted to a cation via a chemical reaction (e.g., protonation or alkylation).

As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described below. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, the heteroatoms, such as nitrogen, can have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms. This disclosure is not intended to be limited in any manner by the permissible substituents of organic compounds. Also, the terms “substitution” or “substituted with” include the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.

“Z¹,” “Z²,” “Z³,” and “Z⁴” are used herein as generic symbols to represent various specific substituents. These symbols can be any substituent, not limited to those disclosed herein, and when they are defined to be certain substituents in one instance, they can, in another instance, be defined as some other substituents.

The term “aliphatic” as used herein refers to a non-aromatic hydrocarbon group and includes branched and unbranched, alkyl, alkenyl, or alkynyl groups.

As used herein, the term “alkyl” refers to saturated, straight-chained or branched saturated hydrocarbon moieties. Unless otherwise specified, C1-C24 (e.g., C1-C22, C1-C20, Ci-Cis, C1-C16, C1-C14, C1-C12, C1-C10, Ci-Cs, Ci-Ce, or C1-C4) alkyl groups are intended. Examples of alkyl groups include methyl, ethyl, propyl, 1-methyl-ethyl, butyl, 1-methyl-propyl, 2-methyl- propyl, 1,1 -dimethyl -ethyl, pentyl, 1 -methyl -butyl, 2-methyl-butyl, 3 -methyl -butyl, 2,2- dimethyl-propyl, 1 -ethyl -propyl, hexyl, 1,1-dimethyl-propyl, 1,2-dimethyl-propyl, 1 -methylpentyl, 2-methyl-pentyl, 3-methyl-pentyl, 4-methyl-pentyl, 1,1 -dimethyl -butyl, 1,2-dimethyl- butyl, 1,3-dimethyl-butyl, 2,2-dimethyl-butyl, 2,3-dimethyl-butyl, 3,3-dimethyl-butyl, 1-ethyl- butyl, 2-ethyl -butyl, 1,1,2-trimethyl-propyl, 1,2,2-trimethyl-propyl, 1 -ethyl- 1-methyl-propyl, 1- ethyl-2-methyl-propyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like. Alkyl substituents may be unsubstituted or substituted with one or more chemical moieties. The alkyl group can be substituted with one or more groups including, but not limited to, hydroxyl, halogen, acyl, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, cyano, carboxylic acid, ester, ether, ketone, nitro, phosphonyl, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol, as described below, provided that the substituents are sterically compatible and the rules of chemical bonding and strain energy are satisfied.

Throughout the specification “alkyl” is generally used to refer to both unsubstituted alkyl groups and substituted alkyl groups; however, substituted alkyl groups are also specifically referred to herein by identifying the specific substituent(s) on the alkyl group. For example, the term “halogenated alkyl” specifically refers to an alkyl group that is substituted with one or more halides (halogens; e.g., fluorine, chlorine, bromine, or iodine). The term “alkoxyalkyl” specifically refers to an alkyl group that is substituted with one or more alkoxy groups, as described below. The term “alkylamino” specifically refers to an alkyl group that is substituted with one or more amino groups, as described below, and the like. When “alkyl” is used in one instance and a specific term such as “alkylalcohol” is used in another, it is not meant to imply that the term “alkyl” does not also refer to specific terms such as “alkylalcohol” and the like.

This practice is also used for other groups described herein. That is, while a term such as “cycloalkyl” refers to both unsubstituted and substituted cycloalkyl moieties, the substituted moieties can, in addition, be specifically identified herein; for example, a particular substituted cycloalkyl can be referred to as, e.g., an “alkylcycloalkyl.” Similarly, a substituted alkoxy can be specifically referred to as, e.g., a “halogenated alkoxy,” a particular substituted alkenyl can be, e.g., an “alkenylalcohol,” and the like. Again, the practice of using a general term, such as “cycloalkyl,” and a specific term, such as “alkylcycloalkyl,” is not meant to imply that the general term does not also include the specific term.

As used herein, the term “alkenyl” refers to unsaturated, straight-chained, or branched hydrocarbon moieties containing a double bond. Unless otherwise specified, C2-C24 (e.g., C2-C22, C2-C20, C2-C18, C2-C16, C2-C14, C2-C12, C2-C10, C₂-C₈, C₂-C₆, or C2-C4) alkenyl groups are intended. Alkenyl groups may contain more than one unsaturated bond. Examples include ethenyl, 1-propenyl, 2-propenyl, 1 -methylethenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-methyl-l- propenyl, 2-methyl- 1-propenyl, 1 -methyl -2-propenyl, 2-methyl -2-propenyl, 1 -pentenyl, 2- pentenyl, 3-pentenyl, 4-pentenyl, 1 -methyl- 1-butenyl, 2-methyl- 1-butenyl, 3-methyl-l-butenyl, 1 -methyl -2-butenyl, 2-methyl-2-butenyl, 3-methyl-2-butenyl, l-methyl-3-butenyl, 2-methyl-3- butenyl, 3 -methyl -3-butenyl, l,l-dimethyl-2-propenyl, 1,2-dimethyl- 1-propenyl, l,2-dimethyl-2- propenyl, 1 -ethyl- 1-propenyl, l-ethyl-2-propenyl, 1 -hexenyl, 2-hexenyl, 3 -hexenyl, 4-hexenyl, 5-hexenyl, 1 -methyl- 1 -pentenyl, 2-methyl-l-pentenyl, 3-methyl-l-pentenyl, 4-methyl-l- pentenyl, 1 -methyl -2-pentenyl, 2-methyl-2-pentenyl, 3-methyl-2-pentenyl, 4-methyl-2-pentenyl, l-methyl-3-pentenyl, 2-methyl-3-pentenyl, 3-methyl-3-pentenyl, 4-methyl-3-pentenyl, 1-methyl- 4-pentenyl, 2-methyl-4-pentenyl, 3-methyl-4-pentenyl, 4-methyl-4-pentenyl, 1,1 -dimethyl -2- butenyl, l,l-dimethyl-3-butenyl, 1,2-dimethyl- 1-butenyl, l,2-dimethyl-2-butenyl, 1,2-dimethyl- 3-butenyl, 1,3-dimethyl-l-butenyl, l,3-dimethyl-2-butenyl, l,3-dimethyl-3-butenyl, 2,2- dimethyl-3-butenyl, 2,3 -dimethyl- 1-butenyl, 2,3-dimethyl-2-butenyl, 2,3-dimethyl-3-butenyl, 3, 3 -dimethyl- 1-butenyl, 3,3-dimethyl-2-butenyl, 1 -ethyl- 1-butenyl, 1 -ethyl -2-butenyl, l-ethyl-3- butenyl, 2-ethyl-l-butenyl, 2-ethyl-2-butenyl, 2-ethyl-3-butenyl, l,l,2-trimethyl-2-propenyl, 1- ethyl-l-methyl-2-propenyl, l-ethyl-2-m ethyl- 1-propenyl, and l-ethyl-2-methyl-2-propenyl. The term “vinyl” refers to a group having the structure -CEUCH2; 1-propenyl refers to a group with the structure -CEUCH-CH3; and 2-propenyl refers to a group with the structure -CH2-CEUCH2. Asymmetric structures such as (Z¹Z²)C=C(Z³Z⁴) are intended to include both the E and Z isomers. This can be presumed in structural formulae herein wherein an asymmetric alkene is present, or it can be explicitly indicated by the bond symbol C=C. Alkenyl substituents may be unsubstituted or substituted with one or more chemical moieties. Examples of suitable substituents include, for example, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, acyl, aldehyde, amino, cyano, carboxylic acid, ester, ether, halide, hydroxyl, ketone, nitro, phosphonyl, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol, as described below, provided that the substituents are sterically compatible and the rules of chemical bonding and strain energy are satisfied.

As used herein, the term “alkynyl” represents straight-chained or branched hydrocarbon moieties containing a triple bond. Unless otherwise specified, C2-C24 (e.g., C2-C24, C2-C20, C2- Ci8, C2-C16, C2-C14, C2-C12, C2-C10, C2-C8, C2-C6, or C2-C4) alkynyl groups are intended. Alkynyl groups may contain more than one unsaturated bond. Examples include C2-Ce-alkynyl, such as ethynyl, 1-propynyl, 2-propynyl (or propargyl), 1-butynyl, 2-butynyl, 3-butynyl, 1- methyl-2-propynyl, 1 -pentynyl, 2-pentynyl, 3 -pentynyl, 4-pentynyl, 3 -methyl- 1-butynyl, 1- methyl-2-butynyl, 1 -methyl -3-butynyl, 2-methyl-3-butynyl, l,l-dimethyl-2-propynyl, l-ethyl-2- propynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, 3 -methyl- 1 -pentynyl, 4- methyl-1 -pentynyl, l-methyl-2-pentynyl, 4-methyl-2-pentynyl, l-methyl-3 -pentynyl, 2-methyl- 3-pentynyl, l-methyl-4-pentynyl, 2-methyl-4-pentynyl, 3-methyl-4-pentynyl, 1,1 -dimethyl -2- butynyl, l,l-dimethyl-3-butynyl, l,2-dimethyl-3-butynyl, 2,2-dimethyl-3-butynyl, 3,3-dimethyl- 1-butynyl, l-ethyl-2-butynyl, l-ethyl-3-butynyl, 2-ethyl-3-butynyl, and 1 -ethyl -1 -methyl -2- propynyl. Alkynyl substituents may be unsubstituted or substituted with one or more chemical moieties. Examples of suitable substituents include, for example, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, acyl, aldehyde, amino, cyano, carboxylic acid, ester, ether, halide, hydroxyl, ketone, nitro, phosphonyl, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol, as described below.

As used herein, the term “aryl,” as well as derivative terms such as aryloxy, refers to groups that include a monovalent aromatic carbocyclic group of from 3 to 50 carbon atoms. Aryl groups can include a single ring or multiple condensed rings. In some examples, aryl groups include Ce-Cio aryl groups. Examples of aryl groups include, but are not limited to, benzene, phenyl, biphenyl, naphthyl, tetrahydronaphthyl, phenylcyclopropyl, phenoxybenzene, and indanyl. The term “aryl” also includes “heteroaryl,” which is defined as a group that contains an aromatic group that has at least one heteroatom incorporated within the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorus. The term “non-heteroaryl,” which is also included in the term “aryl,” defines a group that contains an aromatic group that does not contain a heteroatom. The aryl substituents may be unsubstituted or substituted with one or more chemical moieties. Examples of suitable substituents include, for example, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, acyl, aldehyde, amino, cyano, carboxylic acid, ester, ether, halide, hydroxyl, ketone, nitro, phosphonyl, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol as described herein. The term “biaryl” is a specific type of aryl group and is included in the definition of aryl. Biaryl refers to two aryl groups that are bound together via a fused ring structure, as in naphthalene, or are attached via one or more carbon-carbon bonds, as in biphenyl.

The term “cycloalkyl” as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc. The term “heterocycloalkyl” is a cycloalkyl group as defined above where at least one of the carbon atoms of the ring is substituted with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkyl group and heterocycloalkyl group can be substituted or unsubstituted. The cycloalkyl group and heterocycloalkyl group can be substituted with one or more groups including, but not limited to, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, acyl, aldehyde, amino, cyano, carboxylic acid, ester, ether, halide, hydroxyl, ketone, nitro, phosphonyl, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol as described herein.

The term “cycloalkenyl” as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms and containing at least one double bound, z.e., C=C. Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, and the like. The term “heterocycloalkenyl” is a type of cycloalkenyl group as defined above and is included within the meaning of the term “cycloalkenyl,” where at least one of the carbon atoms of the ring is substituted with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkenyl group and heterocycloalkenyl group can be substituted or unsubstituted. The cycloalkenyl group and heterocycloalkenyl group can be substituted with one or more groups including, but not limited to, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, acyl, aldehyde, amino, cyano, carboxylic acid, ester, ether, halide, hydroxyl, ketone, nitro, phosphonyl, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol as described herein.

The term “cyclic group” is used herein to refer to either aryl groups, non-aryl groups (z.e., cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl groups), or both. Cyclic groups have one or more ring systems (e.g., monocyclic, bicyclic, tricyclic, polycyclic, etc.) that can be substituted or unsubstituted. A cyclic group can contain one or more aryl groups, one or more non-aryl groups, or one or more aryl groups and one or more non-aryl groups.

The term “acyl” as used herein is represented by the formula -C(O) ¹ where Z¹ can be a hydrogen, hydroxyl, alkoxy, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above. As used herein, the term “acyl” can be used interchangeably with “carbonyl.” Throughout this specification “C(O)” or “CO” is a shorthand notation for C=O.

The term “acetal” as used herein is represented by the formula (Z¹Z²)C(=OZ³)(=OZ⁴), where Z¹, Z², Z³, and Z⁴ can be, independently, a hydrogen, halogen, hydroxyl, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

The term “alkanol” as used herein is represented by the formula Z'OH, where Z¹ can be an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

As used herein, the term “alkoxy” as used herein is an alkyl group bound through a single, terminal ether linkage; that is, an “alkoxy” group can be defined as to a group of the formula Z where Z¹ is unsubstituted or substituted alkyl as defined above. Unless otherwise specified, alkoxy groups wherein Z¹ is a C1-C24 (e.g., C1-C22, C1-C20, Ci-Cis, C1-C16, C1-C14, Ci- C12, C1-C10, Ci-Cs, Ci-Ce, or C1-C4) alkyl group are intended. Examples include methoxy, ethoxy, propoxy, 1 -methyl-ethoxy, butoxy, 1 -methyl -propoxy, 2-methyl-propoxy, 1,1 -dimethyl- ethoxy, pentoxy, 1-methyl-butyloxy, 2-methyl-butoxy, 3-methyl-butoxy, 2,2-di-methyl-propoxy, 1 -ethyl -propoxy, hexoxy, 1,1-dimethyl-propoxy, 1,2-dimethyl-propoxy, 1-methyl-pentoxy, 2- methyl-pentoxy, 3-methyl-pentoxy, 4-methyl-penoxy, 1,1 -dimethyl -butoxy, 1,2-dimethyl- butoxy, 1,3-dimethyl-butoxy, 2,2-dimethyl-butoxy, 2,3-dimethyl-butoxy, 3, 3 -dimethyl -butoxy, 1 -ethyl -butoxy, 2-ethylbutoxy, 1,1,2-trimethyl-propoxy, 1,2,2-trimethyl-propoxy, 1 -ethyl- 1- methyl-propoxy, and 1 -ethyl -2-methyl-propoxy.

The term “aldehyde” as used herein is represented by the formula — C(O)H. Throughout this specification “C(O)” is a shorthand notation for C=O.

The term “amino” as used herein are represented by the formula — NZ^JZ²Z³, where Z¹, Z², and Z³ can each be substitution group as described herein, such as hydrogen, an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

The terms “amide” or “amido” as used herein are represented by the formula — C(O)NZ¹Z², where Z¹ and Z² can each be substitution group as described herein, such as hydrogen, an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

The term “anhydride” as used herein is represented by the formula Z¹C(O)OC(O)Z² where Z¹ and Z², independently, can be an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above. The term “cyclic anhydride” as used herein is represented by the formula: where Z¹ can be an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

The term “azide” as used herein is represented by the formula -N=N=N.

The term “carboxylic acid” as used herein is represented by the formula — C(O)OH.

A “carboxylate” or “carboxyl” group as used herein is represented by the formula — C(O)O’

The term “cyano” as used herein is represented by the formula — CN.

The term “ester” as used herein is represented by the formula — OC(O)Z¹ or — C(O)OZ¹, where Z¹ can be an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

The term “ether” as used herein is represented by the formula Z^XOZ², where Z¹ and Z² can be, independently, an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

The term “epoxy” or “epoxide” as used herein refers to a cyclic ether with a three atom ring and can represented by the formula:

_Z1 o z³

_Z2^<Z⁴ where Z¹, Z², Z³, and Z⁴ can be, independently, an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above

The term “ketone” as used herein is represented by the formula Z¹C(O)Z², where Z¹ and Z² can be, independently, an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

The term “halide” or “halogen” or “halo” as used herein refers to fluorine, chlorine, bromine, and iodine.

The term “hydroxyl” as used herein is represented by the formula — OH.

The term “nitro” as used herein is represented by the formula — NO2.

The term “phosphonyl” is used herein to refer to the phospho-oxo group represented by the formula — P(O)(OZ¹)2, where Z¹ can be hydrogen, an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above. The term “silyl” as used herein is represented by the formula — SiZ^JZ²Z³, where Z¹, Z², and Z³ can be, independently, hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

The term “sulfonyl” or “sulfone” is used herein to refer to the sulfo-oxo group represented by the formula — S(O)2Z^X, where Z¹ can be hydrogen, an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

The term “sulfide” as used herein comprises the formula — S — .

The term “thiol” as used herein is represented by the formula — SH.

“R¹,” “R²,” “R³,” “Rⁿ,” etc., where n is some integer, as used herein can, independently, possess one or more of the groups listed above. For example, if R¹ is a straight chain alkyl group, one of the hydrogen atoms of the alkyl group can optionally be substituted with a hydroxyl group, an alkoxy group, an amino group, an alkyl group, a halide, and the like. Depending upon the groups that are selected, a first group can be incorporated within a second group or, alternatively, the first group can be pendant (i.e., attached) to the second group. For example, with the phrase “an alkyl group comprising an amino group,” the amino group can be incorporated within the backbone of the alkyl group. Alternatively, the amino group can be attached to the backbone of the alkyl group. The nature of the group(s) that is (are) selected will determine if the first group is embedded or attached to the second group.

Unless stated to the contrary, a formula with chemical bonds shown only as solid lines and not as wedges or dashed lines contemplates each possible stereoisomer or mixture of stereoisomer (e.g., each enantiomer, each diastereomer, each meso compound, a racemic mixture, or scalemic mixture).

Compounds

Disclosed herein are compounds comprising phosphonic/phosphonic acids or derivatives thereof.

For example, disclosed herein are compounds defined by Formula I:

I wherein R¹ is hydrogen, halide, substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C1-C20 alkoxy, substituted or unsubstituted C3-C20 aryl (e.g., substituted or unsubstituted phenyl), substituted or unsubstituted C4-C21 alkylaryl, NR^xR^y, or OR⁹;

R² is hydrogen, halide, substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C1-C20 alkoxy, substituted or unsubstituted C3-C20 aryl (e.g., substituted or unsubstituted phenyl), substituted or unsubstituted C4-C21 alkylaryl, NR^xR^y, or OR¹⁰;

R³ is hydrogen, hydroxyl, halide, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C1-C10 alkoxy, substituted or unsubstituted C3-C10 aryl (e.g., substituted or unsubstituted phenyl), substituted or unsubstituted C4-C11 alkylaryl, or -C(O)OR⁸;

R⁴ is hydrogen, hydroxyl, halide, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C1-C10 acyl, substituted or unsubstituted C1-C10 amide, NR^xR^y, or one or more amino acids (e.g., one or more canonical or non-canonical amino acids); and

R⁸, R⁹, and R¹⁰ are each independently hydrogen, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C3-C10 aryl (e.g., substituted or unsubstituted phenyl), or substituted or unsubstituted C4-C11 alkylaryl; and

R^x and R^y are independently selected from hydrogen, or substituted or unsubstituted Ci- C5 alkyl, or substituted or unsubstituted C1-C5 acyl; or a derivative or salt thereof; with the proviso that the compound is not phosphonoalamide A, B, C, D, E, or F.

In some examples of Formula I, R⁴ is substituted or unsubstituted C1-C10 amide, or one or more amino acids (e.g., one or more canonical or non-canonical amino acids). In some examples of Formula I, R⁴ is one or more amino acids (e.g., one or more canonical or non-canonical amino acids). In some examples of Formula I, R⁴ is one or more canonical amino acids. In some examples of Formula I, R⁴ is one or more canonical amino acids selected from the group consisting of Alanine (Ala), Arginine (Arg), Asparagine (Asn), Aspartic Acid (Asp), Cysteine (Cys), Glutamine (Gin), Glutamic acid (Glu), Glycine (Gly), Histidine (His), Isoleucine (He), Leucine (Leu), Lysine (Lys), Methionine (Met), Phenylalanine (Phe), Proline (Pro), Serine (Ser), Threonine (Thr), Tryptophan (Trp), Tyrosine (Tyr), and Valine (Vai). In some examples of Formula I, R⁴ is one or more amino acids selected from the group consisting of alanine, serine, and combinations thereof.

In some examples of Formula I, R¹ is OR⁹ and/or R² is OR¹⁰. In some examples of Formula I, R¹ is OR⁹ and R² is OR¹⁰. In some examples of Formula I, R¹ is OR⁹ and/or R² is OR¹⁰, and R⁹ and/or R¹⁰ is hydrogen. In some examples of Formula I, R¹ is OR⁹, R² is OR¹⁰, R⁹ is hydrogen, and R¹⁰ is hydrogen.

In some examples of Formula I, R³ is hydrogen or -C(O)OR⁸. In some examples of Formula I, R³ is hydrogen. In some examples of Formula I, R³ -C(O)OR⁸. In some examples of Formula I, R³ -C(O)OR⁸ and R⁸ is hydrogen.

In some examples of Formula I, R⁴ is one or more amino acids (e.g., one or more canonical or non-canonical amino acids), R¹ is OR⁹, R² is OR¹⁰, R³ is hydrogen or -C(O)OR⁸, or a combination thereof. In some examples of Formula I, R⁴ is one or more amino acids (e.g., one or more canonical or non-canonical amino acids), R¹ is OR⁹, R² is OR¹⁰, and R³ is hydrogen or - C(O)OR⁸.

In some examples, the compound is defined by Formula II:

II wherein

R¹ is hydrogen, halide, substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C1-C20 alkoxy, substituted or unsubstituted C3-C20 aryl (e.g., substituted or unsubstituted phenyl), substituted or unsubstituted C4-C21 alkylaryl, NR^xR^y, or OR⁹;

R⁵ is hydrogen, hydroxyl, halide, substituted or unsubstituted C1-C5 alkyl, or substituted or unsubstituted C1-C5 alkoxy;

R⁶ is hydrogen, hydroxyl, halide, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C1-C10 acyl, substituted or unsubstituted C1-C10 amide, NR^xR^y, or one or more amino acids (e.g., one or more canonical or non-canonical amino acids); R⁷ is hydrogen, hydroxyl, halide, substituted or unsubstituted C1-C5 alkyl, or substituted or unsubstituted C1-C5 alkoxy;

R^x and R^y are independently selected from H, or substituted or unsubstituted C1-C5 alkyl, or substituted or unsubstituted C1-C5 acyl; or a derivative or salt thereof; with the proviso that the compound is not phosphonoalamide A, B, C, D, E, or F.

In some examples of Formula II, R¹ is OR⁹ and/or R² is OR¹⁰. In some examples of Formula II, R¹ is OR⁹ and R² is OR¹⁰. In some examples of Formula II, R¹ is OR⁹ and/or R² is OR¹⁰, and R⁹ and/or R¹⁰ is hydrogen. In some examples of Formula II, R¹ is OR⁹, R² is OR¹⁰, R⁹ is hydrogen, and R¹⁰ is hydrogen.

In some examples of Formula II, R³ is hydrogen or -C(O)OR⁸. In some examples of Formula II, R³ is hydrogen. In some examples of Formula II, R³ -C(O)OR⁸. In some examples of Formula II, R³ -C(O)OR⁸ and R⁸ is hydrogen.

In some examples of Formula II, R⁷ is substituted or unsubstituted C1-C5 alkyl. In some examples of Formula II, R⁷ is substituted or unsubstituted Ci alkyl. In some examples of Formula II, R⁷ is CH₃ or CH₂OH.

In some examples of Formula II, R⁵ and/or R⁶ is hydrogen. In some examples of Formula II, R⁵ and R⁶ are hydrogen.

In some examples of Formula II, R¹ is OR⁹, R² is OR¹⁰, R³ is hydrogen or -C(O)OR⁸, R⁷ is substituted or unsubstituted Ci alkyl, R⁵ is hydrogen, R⁶ is hydrogen, or a combination thereof. In some examples of Formula II, R¹ is OR⁹, R² is OR¹⁰, R³ is hydrogen or -C(O)OR⁸, R⁷ is substituted or unsubstituted Ci alkyl, R⁵ is hydrogen, and R⁶ is hydrogen.

In some examples, the compound is defined by Formula III: ill wherein

R⁶ is hydrogen, hydroxyl, halide, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C1-C10 acyl, substituted or unsubstituted C1-C10 amide, NR^xR^y, or one or more amino acids (e.g., one or more canonical or non-canonical amino acids);

R⁷ is hydrogen, hydroxyl, halide, substituted or unsubstituted C1-C5 alkyl, or substituted or unsubstituted C1-C5 alkoxy;

In some examples of Formula III, R⁹ and/or R¹⁰ is hydrogen. In some examples of Formula III, R⁹ and R¹⁰ are hydrogen.

In some examples of Formula III, R³ is hydrogen or -C(O)OR⁸. In some examples of Formula III, R³ is hydrogen. In some examples of Formula III, R³ -C(O)OR⁸. In some examples of Formula III, R³ -C(O)OR⁸ and R⁸ is hydrogen.

In some examples of Formula III, R⁷ is substituted or unsubstituted C1-C5 alkyl. In some examples of Formula III, R⁷ is substituted or unsubstituted Ci alkyl. In some examples of Formula III, R⁷ is CH₃ or CH₂OH.

In some examples of Formula III, R⁵ and/or R⁶ is hydrogen. In some examples of Formula III, R⁵ and R⁶ are hydrogen.

In some examples of Formula III, R⁹ is hydrogen, R¹⁰ is hydrogen, R³ is hydrogen or - C(O)OR⁸, R⁷ is substituted or unsubstituted Ci alkyl, R⁵ is hydrogen, R⁶ is hydrogen, or a combination thereof. In some examples of Formula III, R⁹ is hydrogen, R¹⁰ is hydrogen, R³ is hydrogen or -C(O)OR⁸, R⁷ is substituted or unsubstituted Ci alkyl, R⁵ is hydrogen, and R⁶ is hydrogen.

In some examples, the compound is defined by Formula IV: wherein

R⁹ and R¹⁰ are each independently hydrogen, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C3-C10 aryl (e.g., substituted or unsubstituted phenyl), or substituted or unsubstituted C4-C11 alkylaryl; and

R^x and R^y are independently selected from H, or substituted or unsubstituted C1-C5 alkyl, or substituted or unsubstituted C1-C5 acyl; or a derivative or salt thereof.

In some examples of Formula IV, R⁹ and/or R¹⁰ is hydrogen. In some examples of Formula IV, R⁹ and R¹⁰ are hydrogen.

In some examples of Formula IV, R⁷ is substituted or unsubstituted C1-C5 alkyl. In some examples of Formula IV, R⁷ is substituted or unsubstituted Ci alkyl. In some examples of Formula IV, R⁷ is CH₃ or CH₂OH.

In some examples of Formula IV, R⁵ and/or R⁶ is hydrogen. In some examples of Formula IV, R⁵ and R⁶ are hydrogen.

In some examples of Formula IV, R⁹ is hydrogen, R¹⁰ is hydrogen, R⁷ is substituted or unsubstituted Ci alkyl, R⁵ is hydrogen, R⁶ is hydrogen, or a combination thereof. In some examples of Formula IV, R⁹ is hydrogen, R¹⁰ is hydrogen, R⁷ is substituted or unsubstituted Ci alkyl, R⁵ is hydrogen, and R⁶ is hydrogen.

In some examples, the compound is defined by Formula IV-A:

IV-A wherein

R⁹ is hydrogen, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C3- C10 aryl (e.g., substituted or unsubstituted phenyl), or substituted or unsubstituted C4-C11 alkylaryl; and

In some examples of Formula IV-A, R⁹ is hydrogen.

In some examples of Formula IV-A, R⁷ is substituted or unsubstituted C1-C5 alkyl. In some examples of Formula IV-A, R⁷ is substituted or unsubstituted Ci alkyl. In some examples of Formula IV-A, R⁷ is CH₃ or CH₂OH.

In some examples of Formula IV-A, R⁵ and/or R⁶ is hydrogen. In some examples of Formula IV-A, R⁵ and R⁶ are hydrogen.

In some examples of Formula IV-A, R⁹ is hydrogen, R⁷ is substituted or unsubstituted Ci alkyl, R⁵ is hydrogen, R⁶ is hydrogen, or a combination thereof. In some examples of Formula IV-A, R⁹ is hydrogen, R⁷ is substituted or unsubstituted Ci alkyl, R⁵ is hydrogen, and R⁶ is hydrogen.

In some examples, the compound is defined by Formula IV-B:

IV-B wherein

R⁷ is hydrogen, hydroxyl, halide, substituted or unsubstituted C1-C5 alkyl, or substituted or unsubstituted C1-C5 alkoxy; and

In some examples of Formula IV-B, R⁷ is substituted or unsubstituted C1-C5 alkyl. In some examples of Formula IV-B, R⁷ is substituted or unsubstituted Ci alkyl. In some examples of Formula IV-B, R⁷ is CH₃ or CH₂OH.

In some examples of Formula IV-B, R⁵ and/or R⁶ is hydrogen. In some examples of Formula IV-B, R⁵ and R⁶ are hydrogen.

In some examples of Formula IV-B, R⁷ is substituted or unsubstituted Ci alkyl, R⁵ is hydrogen, R⁶ is hydrogen, or a combination thereof. In some examples of Formula IV-B, R⁷ is substituted or unsubstituted Ci alkyl, R⁵ is hydrogen, and R⁶ is hydrogen.

In some examples, the compound is defined by Formula IV-C: wherein

R⁶ is hydrogen, hydroxyl, halide, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C1-C10 acyl, substituted or unsubstituted C1-C10 amide, NR^xR^y, or one or more amino acids (e.g., one or more canonical or non-canonical amino acids); and

In some examples of Formula IV-C, R⁵ and/or R⁶ is hydrogen. In some examples of Formula IV-C, R⁵ and R⁶ are hydrogen.

In some examples, the compound is defined by Formula IV-D: or a derivative or salt thereof.

In some examples, the compound is defined by Formula IV-E:

IV-E or a derivative or salt thereof.

In some examples, the compound is defined by Formula V:

V wherein

In some examples of Formula V, R⁹ and/or R¹⁰ is hydrogen. In some examples of Formula V, R⁹ and R¹⁰ are hydrogen.

In some examples of Formula V, R⁸ is hydrogen.

In some examples of Formula V, R⁷ is substituted or unsubstituted C1-C5 alkyl. In some examples of Formula V, R⁷ is substituted or unsubstituted Ci alkyl. In some examples of Formula V, R⁷ is CH₃ or CH₂OH.

In some examples of Formula V, R⁵ and/or R⁶ is hydrogen. In some examples of Formula V, R⁵ and R⁶ are hydrogen.

In some examples of Formula V, R⁹ is hydrogen, R¹⁰ is hydrogen, R⁸ is hydrogen, R⁷ is substituted or unsubstituted Ci alkyl, R⁵ is hydrogen, R⁶ is hydrogen, or a combination thereof. In some examples of Formula V, R⁹ is hydrogen, R¹⁰ is hydrogen, R⁸ is hydrogen, R⁷ is substituted or unsubstituted Ci alkyl, R⁵ is hydrogen, and R⁶ is hydrogen.

In some examples, the compound is defined by Formula V-A: wherein

In some examples of Formula V-A, R⁹ and/or R¹⁰ is hydrogen. In some examples of Formula V-A, R⁹ and R¹⁰ are hydrogen.

In some examples of Formula V-A, R⁷ is substituted or unsubstituted C1-C5 alkyl. In some examples of Formula V-A, R⁷ is substituted or unsubstituted Ci alkyl. In some examples of Formula V-A, R⁷ is CH₃ or CH₂OH.

In some examples of Formula V-A, R⁵ and/or R⁶ is hydrogen. In some examples of Formula V-A, R⁵ and R⁶ are hydrogen.

In some examples of Formula V-A, R⁹ is hydrogen, R¹⁰ is hydrogen, R⁷ is substituted or unsubstituted Ci alkyl, R⁵ is hydrogen, R⁶ is hydrogen, or a combination thereof. In some examples of Formula V-A, R⁹ is hydrogen, R¹⁰ is hydrogen, R⁷ is substituted or unsubstituted Ci alkyl, R⁵ is hydrogen, and R⁶ is hydrogen.

In some examples, the compound is defined by Formula V-B:

V-B wherein

R⁵ is hydrogen, hydroxyl, halide, substituted or unsubstituted C1-C5 alkyl, or substituted or unsubstituted C1-C5 alkoxy; R⁶ is hydrogen, hydroxyl, halide, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C1-C10 acyl, substituted or unsubstituted C1-C10 amide, NR^xR^y, or one or more amino acids (e.g., one or more canonical or non-canonical amino acids);

In some examples of Formula V-B, R⁹ is hydrogen.

In some examples of Formula V-B, R⁷ is substituted or unsubstituted C1-C5 alkyl. In some examples of Formula V-B, R⁷ is substituted or unsubstituted Ci alkyl. In some examples of Formula V-B, R⁷ is CH₃ or CH₂OH.

In some examples of Formula V-B, R⁵ and/or R⁶ is hydrogen. In some examples of Formula V-B, R⁵ and R⁶ are hydrogen.

In some examples of Formula V-B, R⁹ is hydrogen, R⁷ is substituted or unsubstituted Ci alkyl, R⁵ is hydrogen, R⁶ is hydrogen, or a combination thereof. In some examples of Formula V-B, R⁹ is hydrogen, R⁷ is substituted or unsubstituted Ci alkyl, R⁵ is hydrogen, and R⁶ is hydrogen.

In some examples, the compound is defined by Formula V-C:

V-C wherein

In some examples of Formula V-C, R⁷ is substituted or unsubstituted C1-C5 alkyl. In some examples of Formula V-C, R⁷ is substituted or unsubstituted Ci alkyl. In some examples of Formula V-C, R⁷ is CH₃ or CH₂OH.

In some examples of Formula V-C, R⁵ and/or R⁶ is hydrogen. In some examples of Formula V-C, R⁵ and R⁶ are hydrogen.

In some examples of Formula V-C, R⁷ is substituted or unsubstituted Ci alkyl, R⁵ is hydrogen, R⁶ is hydrogen, or a combination thereof. In some examples of Formula V-C, R⁷ is substituted or unsubstituted Ci alkyl, R⁵ is hydrogen, and R⁶ is hydrogen.

In some examples, the compound is defined by Formula V-D:

V-D wherein

In some examples of Formula V-C, R⁵ and/or R⁶ is hydrogen. In some examples of Formula V-C, R⁵ and R⁶ are hydrogen. In some examples, the compound is defined by Formula V-E: or a derivative or salt thereof. In some examples, the compound is defined by Formula V-F:

V-F or a derivative or salt thereof.

In some examples, the compound is a salt. In some examples, the compound is a salt form of Formula I, Formula II, Formula III, Formula IV, Formula V, or a combination thereof with a counterion. In some examples, the compound is a salt form of Formula IV and/or Formula V with a counterion. In some examples, the compound is a salt form of Formula IV and/or Formula V with a counterion and the salt form of the compound is selected from the group consisting of

and combinations thereof.

In some examples, the counterion is a monovalent, divalent, or trivalent counterion. In some examples, the counterion is selected from the group consisting of sodium, potassium, calcium, lithium, magnesium, manganese, ammonium, iron, and combinations thereof.

In some examples, the compound is a potassium salt, sodium salt, calcium salt, iron salt, ammonium salt, or a combination thereof. In some examples, the compound comprises an agriculturally acceptable salt thereof and/or a pharmaceutically acceptable salt thereof.

Also disclosed herein are compounds comprising a head group and a tail group, the head group being bound to the tail group using one or more enzymes derived from Bacillus, wherein the head group comprises a phosphonic acid, a phosphinic acid, or a derivative thereof, and the tail group comprises one or more (canonical or non-canonical) amino acids, with the proviso that the compound is not phosphonoalamide A, B, C, D, E, or F. In some examples, the head group comprises phosphonoalanine (PnAla), 2-aminoethylphosphonic acid (2AEP), or a derivative thereof. In some examples, the tail group comprises alanine, serine, or a derivative thereof. In some examples, the head group comprises PnAla or a derivative thereof and tail group comprises serine or a derivative thereof. In some examples, the head group comprises 2AEP or a derivative thereof and tail group comprises alanine or a derivative thereof. In some examples, the compound comprises Ala-PnAla, Ser-PnAla, Ala-2AEP, or a derivative or salt thereof. In some examples, the head group is C-terminal. In some examples, the compound is a di-peptide or a tripeptide. In some examples, the compound is a salt. In some examples, the compound is a potassium salt, sodium salt, calcium salt, iron salt, ammonium salt, or a combination thereof. In some examples, the compound comprises an agriculturally acceptable salt thereof and/or a pharmaceutically acceptable salt thereof. In some examples, the compound is of Formula I.

In some examples, the compound is a Bacillus isolate or a derivative or salt thereof. In some examples, the compound is an isolate of B. velezensis, B. swezeyi, B. amyloliquefaciens, B. cabrialesii, B. sublilis. or a combination thereof. In some examples, the compound is an isolate of B. velezensis NRRL B-41850.

Compositions

Also disclosed herein are compositions comprising any of the compounds disclosed herein. In some examples, the compositions further comprise one or more agriculturally acceptable and/or pharmaceutically acceptable carriers. In some examples, the composition comprises a pharmaceutical composition, an agricultural composition, or a combination thereof.

In some examples, the composition comprises a pesticide. In some examples, the composition comprises an herbicide.

In some examples, the composition exhibits antimicrobial activity. In some examples, the composition results in at least 5 log reduction in a population of microbes.

In some examples, the composition further comprises a solvent, a carrier, an excipient, or a combination thereof. In some examples, the composition further comprises an agriculturally acceptable adjuvant or carrier.

In some examples, the composition is formulated for delivery to a plant or animal. In some examples, the composition is formulated for delivery to a plant. In some examples, the plant is a crop.

In some examples, the composition is formulated for delivery to an animal. In some examples, the animal is a companion animal, livestock, research animal, insect, or human.

Also disclosed herein are nucleic acids encoding any of the compounds or compositions disclosed herein. Also disclosed herein are vectors encoding said nucleic acids. Also disclosed herein are cells comprising said vectors. Also disclosed herein are cells comprising any of the compounds or compositions disclosed herein. In some examples, the cell comprises a Bacillus cell. In some examples, the cell comprises B. velezensis, B. swezeyi, B. amyloliquefaciens, B. cabrialesii, B. sublilis. or a combination thereof.. In some examples, the cell comprises B. velezensis NRRL B-41850.

Methods of Making

Also disclosed herein are methods of making any of the compounds disclosed herein.

For example, the methods can comprise a biosynthetic method. In some examples, the method can use one or more enzymes derived from Bacillus.

For example, also disclosed herein are methods of making a compound comprising a head group and a tail group, the head group being bound to the tail group, wherein the head group comprises a phosphonic acid, a phosphinic acid, or a derivative thereof, and the tail group comprises one or more (canonical or non-canonical) amino acids, using one or more enzymes derived from Bacillus, with the proviso that the compound is not phosphonoalamide A, B, C, D, E, or F.

In some examples, the head group comprises PnAla, 2AEP, or a derivative thereof. In some examples, the tail group comprises alanine, serine, or a derivative thereof. In some examples, the head group comprises PnAla or a derivative thereof and tail group comprises serine or a derivative thereof. In some examples, the head group comprises 2AEP or a derivative thereof and tail group comprises alanine or a derivative thereof.

In some examples, the compound is any of the compounds disclosed herein.

In some examples, the one or more enzymes are derived from B. velezensis, B. swezeyi, B. amyloliquefaciens, B. cabrialesii, B. subtilis, or a combination thereof. In some examples, the one or more enzymes are derived from B. velezensis NRRL B-41850.

In some examples, the one or more enzymes comprise one or more ATP-grasp enzymes.

In some examples, the one or more enzymes are encoded by a gene comprising at least 90% identity to pnfA,pnfB, or a combination thereof.

In some examples, the method proceeds via a linear pathway.

In some examples, the method comprises contacting a first nucleophile and a first carboxylate with a first enzyme, the first nucleophile comprising a phosphonic acid, a phosphinic acid, or a derivative thereof, and the first carboxylate comprising a first amino acid or a derivative thereof, to thereby form a first compound the first nucleophile bound to the first carboxylate (e.g. a carboxylate-nucleophile). In some examples, the first enzyme is encoded by a gene comprising at least 90% identity to pnfA. In some examples, the first enzyme comprises PnfA. In some examples, the first enzyme comprises recombinant PnfA. In some examples, the first carboxylate comprises alanine (e.g., L-alanine), serine, or a combination thereof. In some examples, the first nucleophile comprises PnAla, 2AEP, or a combination thereof.

In some examples, the method further comprises contacting the first compound and a second carboxylate with a second enzyme, the first compound being a nucleophile, the second carboxylate comprising a second amino acid or a derivative thereof, to thereby form a second compound comprising the first compound bound to the second carboxylate (e.g. a carboxylate- nucleophile). In some examples, the second enzyme is encoded by a gene comprising at least 90% identity to pnfB. In some examples, the second enzyme comprises PnfB. In some examples, the second enzyme comprises recombinant PnfB. In some examples, the second carboxylate comprises alanine (e.g., L-alanine).

In some examples, the method is further performed in the presence of one or more additional components. In some examples, the method is further performed in the presence of ATP.

Methods of Use

For example, also disclosed herein are methods of using any of the compounds, compositions, nucleic acids, vectors, or cells as an antimicrobial, an herbicide, a pesticide, or combination thereof, for example to control (e.g., treat, reduce, inhibit, and/or ameliorate) an undesirable population.

In some examples, the methods comprise using any of the compounds, compositions, nucleic acids, vectors, or cells as a pesticide.

In some examples, the methods comprise using any of the compounds, compositions, nucleic acids, vectors, or cells to control (e.g., treat, reduce, inhibit, and/or ameliorate) an undesirable population in plants. In some examples, the method comprises contacting the plants or the locus thereof with or applying to the soil or water any of the compounds, compositions, nucleic acids, vectors, or cells. In some examples, the methods further comprise applying an additional pesticide.

In some examples, the undesirable population is a herbicide resistant or tolerant population, a pesticide resistant or tolerant population, an antimicrobial resistant or tolerant population, or a combination thereof. In some examples, the undesirable population comprises bacteria. Also disclosed herein are methods of reducing the activity of bacteria, the methods comprising exposing the bacteria to an effective amount of any of the compounds, compositions, nucleic acids, vectors, or cells disclosed herein.

Also disclosed herein are methods of reducing bacterial population, the method comprising exposing the bacteria to an effective amount of any of the compounds, compositions, nucleic acids, vectors, or cells disclosed herein.

Also disclosed herein are methods of treating, preventing, and/or ameliorating a disease or a disorder in a plant or a subject in need thereof, the method comprising administering to the plant or subject a therapeutically effective amount of any of the compounds, compositions, nucleic acids, vectors, or cells disclosed herein.

In some examples, the disease or disorder comprises an infection, such as with an infectious microbe (e.g., bacteria, virus, fungi, protozoa, etc.). In some examples, the disease or disorder comprises a microbial infection.

In some examples, the plant is a crop.

In some examples, the subject is an animal. In some examples, the animal is a companion animal, livestock, research animal, insect, or human.

In some examples, the compound, composition, nucleic acid, or vector is delivered via cultured Bacillus. In some examples, the compound, composition, nucleic acid, or vector is delivered via cultured Bacillus velezensis. In some examples, the compound, composition, nucleic acid, or vector is delivered via cultured: B. velezensis, B. swezeyi, B. amyloliquefaciens, B. cabrialesii, B. sublilis, or a combination thereof. In some examples, the compound, composition, nucleic acid, or vector is delivered via cultured B. velezensis NRRL B-41850.

In some examples, the compounds, compositions, nucleic acids, vectors and/or cells can display broad-spectrum antibacterial activity, with strong inhibition against pathogenic microbes.

The methods of treatment of the disease or disorder described herein can further include treatment with one or more additional agents. The one or more additional agents and the compounds and compositions or pharmaceutically acceptable salts thereof as described herein can be administered in any order, including simultaneous administration, as well as temporally spaced order of up to several days apart. The methods can also include more than a single administration of the one or more additional agents and/or the compounds and compositions or pharmaceutically acceptable salts thereof as described herein. The administration of the one or more additional agents and the compounds and compositions or pharmaceutically acceptable salts thereof as described herein can be by the same or different routes. When treating with one or more additional agents, the compounds and compositions or pharmaceutically acceptable salts thereof as described herein can be combined into a pharmaceutical composition that includes the one or more additional agents.

It is understood, however, that the specific dose level for any particular subject will depend upon a variety of factors. Such factors include the age, body weight, general health, sex, and diet of the subject. Other factors include the time and route of administration, rate of excretion, drug combination, and the type and severity of the particular disease or disorder.

The methods, compounds, and compositions as described herein are useful for both prophylactic and therapeutic treatment. As used herein the term treating or treatment includes prevention; delay in onset; diminution, eradication, or delay in exacerbation of signs or symptoms after onset; and prevention of relapse. For prophylactic use, a therapeutically effective amount of the compounds and compositions or pharmaceutically acceptable salts thereof as described herein are administered to a subject prior to onset (e.g., before obvious signs of the disease or disorder), during early onset (e.g., upon initial signs and symptoms of the disease or disorder), or after an established development of the disease or disorder. Prophylactic administration can occur for several days to years prior to the manifestation of symptoms of a disease or disorder. Therapeutic treatment involves administering to a subject a therapeutically effective amount of the compounds and compositions or pharmaceutically acceptable salts thereof as described herein after the disease or disorder is diagnosed.

Pharmaceutical Compositions

Also disclosed herein are pharmaceutical compositions comprising any of the compounds or compositions disclosed herein.

In some examples, the pharmaceutical composition is administered to a subject. In some examples, the subject is an animal. In some examples, the animal is a companion animal, livestock, research animal, insect, or human.

In some examples, the disclosed compositions comprise the disclosed compounds (including pharmaceutically acceptable salt(s) thereof) as an active ingredient, a pharmaceutically acceptable carrier, and, optionally, other therapeutic ingredients or adjuvants. The instant compositions include those suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration, although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the active ingredient is being administered. The compositions can be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy.

Pharmaceutical Compositions, Formulations, Methods of Administration, and Kits

In vivo application of the disclosed compounds, and compositions containing them, can be accomplished by any suitable method and technique presently or prospectively known to those skilled in the art. For example, the disclosed compounds can be formulated in a physiologically- or pharmaceutically-acceptable form and administered by any suitable route known in the art including, for example, oral, nasal, rectal, topical, and parenteral routes of administration. As used herein, the term parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, and intrasternal administration, such as by injection. Administration of the disclosed compounds or compositions can be a single administration, or at continuous or distinct intervals as can be readily determined by a person skilled in the art.

The compounds disclosed herein, and compositions comprising them, can also be administered utilizing liposome technology, slow release capsules, implantable pumps, and biodegradable containers. These delivery methods can, advantageously, provide a uniform dosage over an extended period of time. The compounds can also be administered in their salt derivative forms or crystalline forms.

The compounds disclosed herein can be formulated according to known methods for preparing pharmaceutically acceptable compositions. Formulations are described in detail in a number of sources which are well known and readily available to those skilled in the art. For example, Remington ’s Pharmaceutical Science by E.W. Martin (1995) describes formulations that can be used in connection with the disclosed methods. In general, the compounds disclosed herein can be formulated such that an effective amount of the compound is combined with a suitable excipient in order to facilitate effective administration of the compound. The compositions used can also be in a variety of forms. These include, for example, solid, semisolid, and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspension, suppositories, injectable and infusible solutions, and sprays. The preferred form depends on the intended mode of administration and application. The compositions can also include conventional pharmaceutically-acceptable carriers and diluents which are known to those skilled in the art. Examples of carriers or diluents for use with the compounds include ethanol, dimethyl sulfoxide, glycerol, alumina, starch, saline, and equivalent carriers and diluents. To provide for the administration of such dosages for the desired application, compositions disclosed herein can comprise between about 0.1% and 100% by weight of the total of one or more of the subject compounds based on the weight of the total composition including carrier or diluent.

The pharmaceutical carrier employed can be, for example, a solid, liquid, or gas. Examples of solid carriers include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. Examples of liquid carriers are sugar syrup, peanut oil, olive oil, and water. Examples of gaseous carriers include carbon dioxide and nitrogen.

Formulations suitable for administration include, for example, aqueous sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions, which can include suspending agents and thickening agents. The formulations can be presented in unit-dose or multi -dose containers, for example sealed ampoules and vials, and can be stored in a freeze dried (lyophilized) condition requiring only the condition of the sterile liquid carrier, for example, water for injections, prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powder, granules, tablets, etc. It should be understood that in addition to the excipients particularly mentioned above, the compositions disclosed herein can include other agents conventional in the art having regard to the type of formulation in question.

Compounds disclosed herein, and compositions comprising them, can be delivered to a cell either through direct contact with the cell or via a carrier means. Carrier means for delivering compounds and compositions to cells are known in the art.

For the treatment of oncological disorders, the compounds or compositions disclosed herein can be administered to a patient in need of treatment in combination with other substances and/or therapies and/or with surgical treatment. These other substances or treatments can be given at the same as or at different times from the compounds or compositions disclosed herein.

In certain examples, compounds and compositions disclosed herein can be locally administered at one or more anatomical sites, such as sites of microbial infection, optionally in combination with a pharmaceutically acceptable carrier such as an inert diluent. Compounds and compositions disclosed herein can be systemically administered, such as intravenously or orally, optionally in combination with a pharmaceutically acceptable carrier such as an inert diluent, or an assimilable edible carrier for oral delivery. They can be enclosed in hard or soft shell gelatin capsules, can be compressed into tablets, or can be incorporated directly with the food of the patient’s diet. For oral therapeutic administration, the active compound can be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, aerosol sprays, and the like.

The tablets, troches, pills, capsules, and the like can also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; diluents such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring can be added. When the unit dosage form is a capsule, it can contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials can be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules can be coated with gelatin, wax, shellac, or sugar and the like. A syrup or elixir can contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound can be incorporated into sustained-release preparations and devices.

Compounds and compositions disclosed herein, including pharmaceutically acceptable salts thereof, can be administered intravenously, intramuscularly, or intraperitoneally by infusion or injection. Solutions of the active agent or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient, which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. The ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. Optionally, the prevention of the action of microorganisms can be brought about by various other antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the inclusion of agents that delay absorption, for example, aluminum monostearate and gelatin.

Pharmaceutical compositions disclosed herein suitable for injectable use include sterile aqueous solutions or dispersions. Furthermore, the compositions can be in the form of sterile powders for the extemporaneous preparation of such sterile injectable solutions or dispersions. In some examples, the final injectable form can be sterile and can be effectively fluid for easy syringability. In some examples, the pharmaceutical compositions can be stable under the conditions of manufacture and storage; thus, they can be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof.

Sterile injectable solutions are prepared by incorporating a compound and/or agent disclosed herein in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.

Pharmaceutical compositions disclosed herein can be in a form suitable for topical use such as, for example, an aerosol, cream, ointment, lotion, dusting powder, mouth washes, gargles, solution, tincture, and the like. In some examples, the compositions can be in a form suitable for use in transdermal devices. In some examples, it will be desirable to administer them topically to the skin as compositions, in combination with a dermatologically acceptable carrier, which can be a solid or a liquid. Compounds and agents and compositions disclosed herein can be applied topically to a subject’s skin. These formulations can be prepared, utilizing any of the compounds disclosed herein or pharmaceutically acceptable salts thereof, via conventional processing methods.

Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers, for example.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.

Pharmaceutical compositions disclosed herein can be in a form suitable for rectal administration wherein the carrier is a solid. In some examples, the mixture forms unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. The suppositories can be conveniently formed by first admixing the composition with the softened or melted carriers) followed by chilling and shaping in molds.

In addition to the aforementioned carrier ingredients, the pharmaceutical formulations described above can include, as appropriate, one or more additional carrier ingredients such as diluents, buffers, flavoring agents, binders, surface-active agents, thickeners, lubricants, preservatives (including anti-oxidants) and the like. Furthermore, other adjuvants can be included to render the formulation isotonic with the blood of the intended recipient. Compositions containing any of the compounds disclosed herein, and/or pharmaceutically acceptable salts thereof, can also be prepared in powder or liquid concentrate form.

Useful dosages of the compounds and agents and pharmaceutical compositions disclosed herein can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art.

The dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms or disorder are affected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days.

Also disclosed are kits that comprise a compound disclosed herein in one or more containers. The disclosed kits can optionally include pharmaceutically acceptable carriers and/or diluents. In one embodiment, a kit includes one or more other components, adjuncts, or adjuvants as described herein. In one embodiment, a kit includes instructions or packaging materials that describe how to administer a compound or composition of the kit. Containers of the kit can be of any suitable material, e.g., glass, plastic, metal, etc., and of any suitable size, shape, or configuration. In one embodiment, a compound and/or agent disclosed herein is provided in the kit as a solid, such as a tablet, pill, or powder form. In another embodiment, a compound and/or agent disclosed herein is provided in the kit as a liquid or solution. In one embodiment, the kit comprises an ampoule or syringe containing a compound and/or agent disclosed herein in liquid or solution form.

In some examples, the kit further comprises at least one agent, wherein the compound and the agent are co-formulated.

In some examples, the compound and the agent are co-packaged.

The kits can also comprise compounds and/or products co-packaged, co-formulated, and/or co-delivered with other components. For example, a drug manufacturer, a drug reseller, a physician, a compounding shop, or a pharmacist can provide a kit comprising a disclosed compound and/or product and another component for delivery to a patient.

It is contemplated that the disclosed kits can be used in connection with the disclosed methods of making, the disclosed methods of using, and/or the disclosed compositions.

Agricultural Compositions, Formulations, and Methods of Administration

Also disclosed herein are agricultural compositions comprising any of the compounds or compositions disclosed herein, and methods of use thereof.

For example, the compound or composition can be applied to vegetation or an area adjacent the vegetation or applied to soil or water to prevent the emergence or growth of vegetation in an amount sufficient to induce an effect, such as an antimicrobial effect. In some embodiments, compounds or compositions are used in an amount sufficient to induce an antimicrobial effect while still showing good crop compatibility.

The present disclosure also relates to formulations of the compositions and methods disclosed herein. In some embodiments, the formulation can be in the form of a single package formulation including any of the compounds disclosed herein. In some embodiments, the formulation can be in the form of a single package formulation including any of the compounds disclosed herein and further including at least one additive. In some embodiments, the formulation can be in the form of a two-package formulation, wherein one package contains any of the compounds disclosed herein and while the other package contains at least one additive. In some embodiments of the two-package formulation, the formulation including any of the compounds disclosed herein and the formulation including at least one additive are mixed before application and then applied simultaneously. In some embodiments, the mixing is performed as a tank mix (i.e., the formulations are mixed immediately before or upon dilution with water). In some embodiments, the formulation including (a) and the formulation including (b) are not mixed but are applied sequentially (in succession), for example, immediately or within 1 hour, within 2 hours, within 4 hours, within 8 hours, within 16 hours, within 24 hours, within 2 days, or within 3 days, of each other.

In some embodiments, the formulation of any of the compounds disclosed herein is present in suspended, emulsified, or dissolved form. Exemplary formulations include, but are not limited to, aqueous solutions, powders, suspensions, also highly-concentrated aqueous, oily or other suspensions or dispersions, aqueous emulsions, aqueous microemulsions, aqueous suspo- emulsions, oil dispersions, self-emulsifying formulations, pastes, dusts, and materials for spreading or granules.

In some embodiments, the compound or composition is an aqueous solution that can be diluted before use. In some embodiments, the compound or composition is provided as a high- strength formulation such as a concentrate. In some embodiments, the concentrate is stable and retains potency during storage and shipping. In some embodiments, the concentrate is a clear, homogeneous liquid that is stable at temperatures of 54 °C or greater. In some embodiments, the concentrate does not exhibit any precipitation of solids at temperatures of -10 °C or higher. In some embodiments, the concentrate does not exhibit separation, precipitation, or crystallization of any components at low temperatures. For example, the concentrate remains a clear solution at temperatures below 0 °C (e.g., below -5 °C, below -10 °C, below -15 °C). In some embodiments, the concentrate exhibits a viscosity of less than 50 centipoise (50 megapascals), even at temperatures as low as 5 °C.

The compositions and methods disclosed herein can also be mixed with or applied with an additive. In some embodiments, the additive can be diluted in water or can be concentrated. In some embodiments, the additive is added sequentially. In some embodiments, the additive is added simultaneously. In some embodiments, the additive is premixed with the compound.

In some embodiments, the additive is an additional pesticide. For example, the compositions described herein can be applied in conjunction with one or more additional pesticides. The composition can be formulated with the one or more additional pesticides, tank mixed with the one or more additional pesticides, or applied sequentially with the one or more additional pesticides.

In some embodiments, the additional pesticide or an agriculturally acceptable salt or ester thereof is provided in a premixed formulation with the compound.

In some embodiments, the additive includes an agriculturally acceptable adjuvant. Exemplary agriculturally acceptable adjuvants include, but are not limited to, antifreeze agents, antifoam agents, compatibilizing agents, sequestering agents, neutralizing agents and buffers, corrosion inhibitors, colorants, odorants, penetration aids, wetting agents, spreading agents, dispersing agents, thickening agents, freeze point depressants, antimicrobial agents, crop oil, herbicide safeners, adhesives (for instance, for use in seed formulations), surfactants, protective colloids, emulsifiers, tackifiers, and mixtures thereof.

Exemplary agriculturally acceptable adjuvants include, but are not limited to, crop oil concentrate (mineral oil (85%) +emulsifiers (15%)); nonylphenol ethoxylate; benzylcocoalkyldimethyl quaternary ammonium salt; blend of petroleum hydrocarbon, alkyl esters, organic acid, and anionic surfactant; C9-C11 alkylpolyglycoside; phosphate alcohol ethoxylate; natural primary alcohol (C12-C16) ethoxylate or less, di- ec-butylphenol EO-PO block copolymer; polysiloxane-methyl cap; nonylphenol ethoxy late+urea ammonium nitrate; emulsified methylated seed oil; tridecyl alcohol (synthetic) ethoxylate (8 EO); tallow amine ethoxylate (15 EO); and PEG(400) dioleate-99.

In some embodiments, the additive is a safener, which is an organic compound leading to better crop plant compatibility when applied with a pesticide. In some embodiments, the safener itself is herbicidally active. In some embodiments, the safener acts as an antidote or antagonist in the crop plants and can reduce or prevent damage to the crop plants.

Exemplary surfactants (e.g., wetting agents, tackifiers, dispersants, emulsifiers) include, but are not limited to, the alkali metal salts, alkaline earth metal salts and ammonium salts of aromatic sulfonic acids, for example lignosulfonic acids, phenolsulfonic acids, naphthalenesulfonic acids, and dibutylnaphthalenesulfonic acid, and of fatty acids, alkyl- and alkylarylsulfonates, alkyl sulfates, lauryl ether sulfates and fatty alcohol sulfates, and salts of sulfated hexa-, hepta- and octadecanols, and also of fatty alcohol glycol ethers, condensates of sulfonated naphthalene and its derivatives with formaldehyde, condensates of naphthalene or of the naphthalene sulfonic acids with phenol and formaldehyde, polyoxyethylene octylphenol ether, ethoxylated isooctyl-, octyl- or nonylphenol, alkylphenyl or tributylphenyl polyglycol ether, alkyl aryl polyether alcohols, isotridecyl alcohol, fatty alcohol/ethylene oxide condensates, ethoxylated castor oil, polyoxyethylene alkyl ethers or polyoxypropylene alkyl ethers, lauryl alcohol polyglycol ether acetate, sorbitol esters, lignosulfite waste liquors and proteins, denatured proteins, polysaccharides (e.g., methylcellulose), hydrophobically modified starches, polyvinyl alcohol, polycarboxylates, polyalkoxylates, polyvinyl amine, polyethyleneimine, polyvinylpyrrolidone and copolymers thereof.

Exemplary thickeners include, but are not limited to, polysaccharides, such as xanthan gum, and organic and inorganic sheet minerals, and mixtures thereof.

Exemplary antifoam agents include, but are not limited to, silicone emulsions, long-chain alcohols, fatty acids, salts of fatty acids, organofluorine compounds, and mixtures thereof.

Exemplary antimicrobial agents include, but are not limited to, bactericides based on dichlorophen and benzyl alcohol hemiformal, and isothiazolinone derivatives, such as alkylisothiazolinones and benzisothiazolinones, and mixtures thereof.

Exemplary antifreeze agents include, but are not limited to ethylene glycol, propylene glycol, urea, glycerol, and mixtures thereof.

Exemplary colorants include, but are not limited to, the dyes known under the names Rhodamine B, pigment blue 15:4, pigment blue 15:3, pigment blue 15:2, pigment blue 15: 1, pigment blue 80, pigment yellow 1, pigment yellow 13, pigment red 112, pigment red 48:2, pigment red 48: 1, pigment red 57: 1, pigment red 53: 1, pigment orange 43, pigment orange 34, pigment orange 5, pigment green 36, pigment green 7, pigment white 6, pigment brown 25, basic violet 10, basic violet 49, acid red 51, acid red 52, acid red 14, acid blue 9, acid yellow 23, basic red 10, basic red 108, and mixtures thereof.

Exemplary adhesives include, but are not limited to, polyvinylpyrrolidone, polyvinyl acetate, polyvinyl alcohol, tylose, and mixtures thereof.

In some embodiments, the additive includes a carrier. In some embodiments, the additive includes a liquid or solid carrier. In some embodiments, the additive includes an organic or inorganic carrier. Exemplary liquid carriers include, but are not limited to, petroleum fractions or hydrocarbons such as mineral oil, aromatic solvents, paraffinic oils, and the like or less, vegetable oils such as soybean oil, rapeseed oil, olive oil, castor oil, sunflower seed oil, coconut oil, com oil, cottonseed oil, linseed oil, palm oil, peanut oil, safflower oil, sesame oil, tung oil and the like or less, esters of the above vegetable oils or less, esters of monoalcohols or dihydric, trihydric, or other lower polyalcohols (4-6 hydroxy containing), such as 2-ethyl hexyl stearate, n- butyl oleate, isopropyl myristate, propylene glycol dioleate, di-octyl succinate, di-butyl adipate, di-octyl phthalate and the like or less, esters of mono, di and polycarboxylic acids and the like, toluene, xylene, petroleum naphtha, crop oil, acetone, methyl ethyl ketone, cyclohexanone, trichloroethylene, perchloroethylene, ethyl acetate, amyl acetate, butyl acetate, propylene glycol monomethyl ether and diethylene glycol monomethyl ether, methyl alcohol, ethyl alcohol, isopropyl alcohol, amyl alcohol, ethylene glycol, propylene glycol, glycerine, /f-methyl-2- pyrrolidinone, /f-di methyl alkylamides, dimethyl sulfoxide, liquid fertilizers and the like, and water as well as mixtures thereof. Exemplary solid carriers include, but are not limited to, silicas, silica gels, silicates, talc, kaolin, limestone, lime, chalk, bole, loess, clay, dolomite, diatomaceous earth, calcium sulfate, magnesium sulfate, magnesium oxide, ground synthetic materials, pyrophyllite clay, attapulgus clay, kieselguhr, calcium carbonate, bentonite clay, Fuller's earth, cottonseed hulls, wheat flour, soybean flour, pumice, wood flour, walnut shell flour, lignin, ammonium sulfate, ammonium phosphate, ammonium nitrate, ureas, cereal meal, tree bark meal, wood meal and nutshell meal, cellulose powders, and mixtures thereof.

In some embodiments, emulsions, pastes or oil dispersions can be prepared by homogenizing the compound in water by means of wetting agent, tackifier, dispersant or emulsifier. In some embodiments, concentrates suitable for dilution with water are prepared, comprising the compound, a wetting agent, a tackifier, and a dispersant or emulsifier.

In some embodiments, powders or materials for spreading and dusts can be prepared by mixing or concomitant grinding of the compound and optionally a safener with a solid carrier.

In some embodiments, granules (e.g., coated granules, impregnated granules and homogeneous granules) can be prepared by binding the compound to solid carriers.

The compositions disclosed herein can be applied in any known technique for applying pesticides. Exemplary application techniques include, but are not limited to, spraying, atomizing, dusting, spreading, or direct application into water (in-water). The method of application can vary depending on the intended purpose. In some embodiments, the method of application can be chosen to ensure the finest possible distribution of the compositions disclosed herein.

If desired, the compositions can be applied as an in-water application.

When the compositions are used in crops, the compositions can be applied after seeding and before or after the emergence of the crop plants. In some embodiments, when the compositions are used in crops, the compositions can be applied before seeding of the crop plants.

In some embodiments, the compositions disclosed herein are applied to vegetation or an area adjacent the vegetation or applied to soil or water by spraying (e.g., foliar spraying). In some embodiments, the spraying techniques use, for example, water as carrier and spray liquor rates of from 10 liters per hectare (L/ha) to 2000 L/ha (e.g., from 50 L/ha to 1000 L/ha, or from 100 to 500 L/ha). In some embodiments, the compositions disclosed herein are applied by the low-volume or the ultra-low- volume method, wherein the application is in the form of micro granules. In some embodiments, wherein the compositions disclosed herein are less well tolerated by certain crop plants, the compositions can be applied with the aid of the spray apparatus in such a way that they come into little contact, if any, with the leaves of the sensitive crop plants while reaching the undesirable population or the bare soil (e.g., post-directed or layby). In some embodiments, the compositions disclosed herein can be applied as dry formulations (e.g., granules, WDGs, etc.) into water.

The compositions and methods disclosed herein can also be used in plants that are resistant to, for instance, pesticides, pathogens, and/or insects. In some embodiments, the compositions and methods disclosed herein can be used in plants that are resistant to one or more pesticides because of genetic engineering or breeding.

In some embodiments, the compositions described herein and other complementary pesticides are applied at the same time, either as a combination formulation or as a tank mix, or as sequential applications.

The compositions and methods may be used in controlling undesirable populations in crops possessing agronomic stress tolerance (including but not limited to drought, cold, heat, salt, water, nutrient, fertility, pH), pest tolerance (including but not limited to insects, fungi and pathogens) and crop improvement traits (including but not limited to yield; protein, carbohydrate, or oil content; protein, carbohydrate, or oil composition; plant stature and plant architecture).

The herbicidal compositions described herein can be used to control herbicide resistant or tolerant populations. The methods employing the compositions described herein may also be employed to control herbicide resistant or tolerant populations. Exemplary resistant or tolerant populations include, but are not limited to, biotypes with resistance or tolerance to multiple herbicides, biotypes with resistance or tolerance to multiple chemical classes, biotypes with resistance or tolerance to multiple herbicide modes-of-action, and biotypes with multiple resistance or tolerance mechanisms (e.g., target site resistance or metabolic resistance).

The present compositions may be formulated and delivered to host plants by methods known in the art, including soil drench via soil drench formulations, seed inoculation via seed inoculation formulations, and plant inoculation via plant inoculation formulations. Seed inoculation formulations can include a carrier such as peat slurry or a film coat consisting of alginate polymers, to protect the compositions from environmental stresses such as desiccation and temperature perturbations. Soil drench or in-furrow composition delivery to plants may be performed by applying the compositions and/or composition formulations in soil before or after planting. Soil drench has several advantages over seed inoculation: 1) prevents the compositions or composition formulations from being inhibited by the chemicals coated on seeds (e.g., fungicides and pesticides) and 2) delivers compositions or composition formulations at higher density without being constrained by seed size. A higher composition or composition formulation concentration is usually required for soil inoculation. Foliar spray and root dipping are also suitable for composition or composition formulation delivery of plants. Plants may be treated at the seedling stage to increase persistence in the plant. In addition, seedling priming, direct seed coating, alginate seed coating, and 12-h coating are within the scope of the present disclosure.

The compositions in the present invention may be formulated and administered to insect hives as a liquid suspension, powder, or solid substrates, such as lipid-based patties.

Liquid formulations may optionally comprise water, sugar syrup and/or other carbohydrate, vitamins, stabilizers, and any other nutrients supportive of bee health. Dry formulations may optionally comprise powdered sugar or other carbohydrate, vitamins, stabilizers, and any other nutrients supportive of bee health. Patty formulations may comprise sugar and/or other carbohydrate, vegetable and/or animal fat, vitamins, stabilizers, and any other nutrients supportive of bee health.

The compositions may be administered as a treatment and/or prophylactically. The compositions may also be administered as a protocol that includes vaccination, phage therapy, the use of lactic acid-producing bacteria.

The formulations optionally include additional foulbrood treatments, such as tylosin tartrate (produced by Elanco, e.g., tylosin A, B, C, and D), and/or Terramycin® (produced by Pfizer, e.g. TM25®, TM50®, TM100®), including Terra-Pro®, and/or the active ingredient of Terramycin®, oxytetracycline HCL. For example, the compounds and compositions disclosed herein can be formulated and/or used in conjunction with the known foulbrood treatments. Therefore, the methods include treatment with one or more of the present compositions and can optionally include additional treatments from previously-known modalities.

A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

The examples below are intended to further illustrate certain aspects of the systems and methods described herein, and are not intended to limit the scope of the claims.

EXAMPLES

The following examples are set forth below to illustrate the methods and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention which are apparent to one skilled in the art.

Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric. There are numerous variations and combinations of measurement conditions, e.g., component concentrations, temperatures, pressures and other measurement ranges and conditions that can be used to optimize the described process.

Example 1 - Biosynthesis of l-phosphonoalanine oligopeptides proceeds in a linear pathway in Bacillus

Introduction. Phosphonic and phosphinic acid compounds (Pn), as a class, are defined by their carbon-phosphorus bonds. This moiety enables chemical mimicry of phosphate esters and carboxylates scattered throughout metabolism, allowing for potent inhibition of essential processes. The high rate of bioactivity demonstrated by phosphonic and phosphinic acid natural products (NPs) has rendered them a rich source of commercialized compounds, including the antibiotic fosfomycin (Monurol), the antiviral phosphonoformate (Foscamet), and the herbicide phosphinothricin (glufosinate). Fosfomycin inhibits LTDP-A-acetylglucosamine- enolpyruvyltransferase by mimicking phosphoenolpyruvate (PEP), disabling peptidoglycan biosynthesis [1], Phosphonoformate inhibits viral DNA polymerases by mimicking pyrophosphate, preventing replication [2], Lastly, phosphinothricin inhibits glutamine synthetase by mimicking glutamate, disrupting nitrogen homeostasis [3],

The urgent need for new therapeutics has led to resurgent interest in natural products. In spite of their historical success, phosphonic and phosphinic acid natural products remain understudied compared to other classes of natural products. Roughly 7% of all microorganisms contain the genetic potential for phosphonic/phosphinic acid biosynthesis, with >85% of biosynthetic gene clusters (BGCs) encoding unknown products [4,5], In the past decade alone, genome mining and targeted isolation has resulted in the discovery of over 20 new phosphonic/phosphinic acid natural products, with new chemotypes, previously unknown headgroups, and a wide array of bioactivities [5-9],

The phosphonoalamides are one new chemotype of phosphonic/phosphinic acid natural products, made up of phosphonopeptides with a common L-phosphonoalanine (PnAla) head group. Both free phosphonoalanine and phosphonoalamide A demonstrated antibacterial activity, and the discovery that phosphonopyruvate is transaminated to phosphonoalanine established a new branch of phosphonic/phosphinic acid biosynthesis [6],

Here, the understanding of phosphonic/phosphinic acid metabolism is advanced by elucidating the biosynthesis of phosphonoalamides E and F, recently discovered antimicrobial phosphonopeptides isolated from Bacillus velezensis NRRL B-41580 [10], Like phosphonoalamides A-D, these compounds contain a phosphonoalanine headgroup, but in E & F, it is the C-terminal, rather than N-terminal, residue.

Results

Syntenic determination of the biosynthetic gene cluster. During a search for phosphonoalanine-encoding biosynthetic gene clusters within actinobacteria, a biosynthetic gene cluster attributed to Mycobacteroides abscessus subsp. massiliense strain aerosol_aerosol_3 was identified. It was established that this assembly was heavily contaminated and that the contig containing pepM — which initiates all characterized routes of phosphonic/phosphinic acid biosynthesis by converting phosphoenolpyruvate into phosphonopyruvate (PnPy) — belonged to a member of the Bacillus subitilis species complex. Production screening of phosphonic/phosphinic acid from several strains encoding this biosynthetic gene cluster ultimately led to the isolation of the phosphonoalanine-containing peptides phosphonoalamide E and F from B. velezensis NRRL B-41580 (Figure 1) [10],

To establish the boundaries for this biosynthetic gene cluster, a fifteen-gene window centered on the PepM found within B-41580 (henceforth referred to as PnfC, Table 1) was first analyzed before being compared with genomic neighborhoods encoding PepMs with >80% shared identity PnfC, as biosynthetic gene clusters meeting this threshold should produce similar compounds. Therefore, the genes conserved within these related neighborhoods are predicted to encode the enzymes required for biosynthesis of phosphonoalamide F. However, all genomes containing this biosynthetic gene cluster displayed extensive conservation, extending tens of kb upstream and downstream of pnfC, as demonstrated by the synteny between B. velezensis and B. swezeyi (Figure 2).

To delineate the boundaries of this biosynthetic gene cluster, a reciprocal search was performed. As genomes lacking pepM would lack the capability for phosphonic/phosphinic acid biosynthesis, genes conserved between B-41580 and closely related strains, such as B. amyloliquefaciens, B. cabrialesii, and B. subtilis — such as a c-factor and purine biosynthesis genes — could be excluded, resulting in a putative assignment of pnfABCDT as the biosynthetic genes (Figure 2).

Table 1: Gene annotations for //. velezensis NRRL B-41580.

Heterologous expression of the biosynthetic gene cluster. Given the similarity between this biosynthetic gene cluster and that of the Streptomyces phosphonoalamides [6], it was hypothesized that pnfA-D would be important for biosynthesis of phosphonoalamide F while pnfT would serve as its transporter. It was hypothesized that PnfC (PepM) would convert phosphoenolpyruvate to phosphonopyruvate, while PnfD would transaminate phosphonopyruvate to phosphonoalanine, and the ATP-grasp ligases encoded by pnfA and pnfB would ligate alanine to phosphonoalanine to produce phosphonoalamides E and F.

To determine if these genes were sufficient for phosphonoalamide F biosynthesis, the putative biosynthetic gene cluster pnfABCDT) was cloned and assembled into pDG1730 via Gibson assembly. This plasmid (pKSJ657), along with the empty vector, was transformed and integrated into 7>. subtilis 168, and the resulting strains were fermented for 7 days in TSB. The resulting spent media extracts were analyzed by ³¹P NMR for the presence of phosphonic/phosphinic acid species, demonstrating the accumulation of phosphonoalamide F in the biosynthetic gene cluster integrant, which was corroborated by ¹H-³¹P HMBC and LC- HRMS analysis (Figure 3A-Figure 3C).

Phosphonoalanine production by PnfC and PnfD. The conversion of phosphoenolpyruvate to phosphonopyruvate is highly unfavorable, such that subsequent reactions, including the transamination of phosphonopyruvate to phosphonoalanine, are required to drive the conversion forward. Given the annotation of PnfD as an aspartate aminotransferase (AST), it was proposed that this transamination would parallel that of a-ketoglutarate to glutamate by AST. Recombinant forms of PnfC and PnfD were purified from Escherichia coli and incubated with phosphoenolpyruvate, Mg²⁺, pyridoxal-5-phosphate (PLP), and each of the 20 canonical amino acids prior to analysis by ³¹P NMR. A new phosphonic/phosphinic acid species (5p 16.7) was observed in reactions containing asparagine (Asn), aspartic acid (Asp), cysteine (Cys), and glutamic acid (Glu), which was confirmed to be phosphonoalanine by LC- HRMS (Figure 4). An apparent preference was observed for aspartic acid (Asp) as the amino donor, which was not unusual given the similarity of PnfD to aspartate aminotransferase.

Spectroscopic analysis of transaminase (TA) (e.g., aminotransferase). When recombinant PnfD was purified, it demonstrated a yellow color, suggesting the presence of a bound chromophore. Given its annotation as a pyridoxal-5-phosphate-dependent aminotransferase, this was suspected to be an internal aldimine with pyridoxal-5-phosphate, and the UV-Vis spectra revealed absorbance at 439 nm. This absorbance was shifted upon reduction by sodium borohydride as well as adduct formation by L-cycloserine. By comparing the absorbance at 439 nm upon addition of stoichiometric pyridoxal-5-phosphate, the occupancy of native recombinant PnfD was estimated to be 40.3% (Figure 5A-Figure 5C).

Formation of Phosphonoalamide E. The original isolation of phosphonoalamides E and F suggested that ligation of alanine to phosphonoalanine occurs in a linear manner, first forming phosphonoalamide E (H-Ala-PnAla-OH) and then forming phosphonoalamide F (H-Ala-Ala- PnAla-OH) [10], As ATP-grasp enzymes (pnfA, pnfB) are known to form peptide bonds and present within the biosynthetic gene cluster, these proteins were predicted to be responsible for these ligations.

Recombinant PnfA was purified from E. coli and biochemical reactions containing PnfA with phosphonoalanine, ATP, alanine, and Mg²⁺ resulted in the formation of a new ³¹P NMR species (5p 17.1) and an MS signal of m/z 241.0584 [M+H]⁺. After scaling up the reaction, this compound was purified and characterized by ^JH NMR and LC-HRMS to be phosphonoalamide E. Exclusion of any reaction component prevented the formation of phosphonoalamide E (Figure 6A).

Specificity of PnfA. The general reaction for ATP -grasp amino acid ligases involves a carboxylate substrate and a nucleophilic substrate. The carboxylate first reacts with ATP to form a reactive acylphosphate intermediate, which is attacked by the nucleophile to yield a tetrahedral intermediate which decomposes into the final product and inorganic phosphate.

As ATP -grasp ligases demonstrate a wide degree of selectivity for their substrates, the specificity of PnfA was examined, starting with its canonical substrates. To test stereoselectivity of the carboxylate substrate, PnfA biochemical assays were set up with D-alanine and L- phosphonoalanine, with no reaction observed. To test stereoselectivity for the nucleophilic substrate, PnfA biochemical assays were performed with L-alanine DL-phosphonoalanine, where exactly half of the initial DL-phosphonoalanine was converted to dipeptide (Figure 6A-Figure 6B).

Each of the other 20 canonical amino acids was tested for acceptance by PnfA as a carboxylate, using phosphonoalanine as a nucleophile, with only the serine (Ser) product (Ser- PnAla) observed by ³¹P NMR and LC-HRMS (Figure 7A-Figure 7B).

Likewise, an array of aminophosphonates (L-AP4, L-AP5, Ala(P), Val(P), 3- aminopropylphosphonic acid [3ApPn], 4-aminophenylphosphonic acid [4APhePn], 2- aminoethylphosphonic acid [2AEP], aminomethyl phosphonic acid [AmPn], and phosphinothricin [PT]) were tested as potential nucleophiles using L-alanine as the carboxylate substrate, with only the 2AEP product (Ala-2AEP) observed by ³¹P NMR and LC-HRMS (Figure 8).

Given the chemical similarity between alanine and serine as well as phosphonoalanine and 2AEP, it appears that PnfA demonstrates a significant degree of substrate-selectivity (Figure 9).

Formation of Phosphonoalamide F. Having established the function for all other genes within the minimal biosynthetic gene cluster for phosphonoalamide F, recombinant PnfB was purified from E. coli and incubated with L-alanine, purified phosphonoalamide E from enzymatic synthesis, ATP, and Mg²⁺, resulting in observation of a new ³¹P NMR species (5p 17.3) matching phosphonoalamide F (Figure 10, panel i).

Specificity of PnfB. To determine the stereoselectivity of PnfB, biochemical assays were performed with D-alanine and phosphonoalamide E, yielding no observed product. Subsequently, the carboxylate specificity of PnfB was assayed with phosphonoalamide E and each of the 20 canonical amino acids, from which phosphonoalamide F was the only observed product, demonstrating that PnfB is highly selective for L-alanine as a carboxylate (Figure 10).

Conclusion. Through a combination of comparative genomics, heterologous expression, and in vitro reconstitution, the complete biosynthetic pathway for phosphonoalamide F was established (Figure 11). First, phosphoenolpyruvate is converted phosphonopyruvate by PepM (PnfC), driven by the transamination of phosphonopyruvate to phosphonoalanine by PnfD, the second reported example of a pyridoxal-5-phosphate-dependent phosphonopyruvate transaminase. The canonical ATP -grasp ligases PnfA and PnfB then act, respectively, to ligate alanine to phosphonoalanine to form phosphonoalamide E and alanine to phosphonoalamide E to form phosphonoalamide F.

Unlike phosphonoalamides A-D, which share phosphonoalanine as an N-terminal residue, phosphonoalamides E and F have C-terminal phosphonoalanine residues. In the context of ATP -grasp amide bond formation, this indicates that PnfA utilizes the phosphonic/phosphinic acid headgroup as a nucleophile, while the Streptomyces PnaB instead uses phosphonoalanine as a carboxylate. As some degree of substrate flexibility with regards to the PnfA reaction has been shown, this highlights the possibility for biocatalytic production of phosphonopeptide analogues, combining multiple warheads into the same compound. The biosynthetic pathway for phosphonoalamides E and F indicate how the same phosphonic/phosphinic acid moieties can be utilized in different fashions, resulting in different spectrums of activity.

Materials and Methods

Chemicals. General chemical reagents were purchased from Sigma-Aldrich, Fischer Scientific, VWR, or Santa Cruz Biotechnologies.

Strains, Media, and General Culture Conditions. Strains and plasmids used in this study are listed in Table 2 and Table 3. Escherichia coli and Bacillus subitilis strains were routinely grown on LB broth or agar at 37°C, while the following antibiotics were included for plasmid maintenance and selection as appropriate: 50 pg/mL kanamycin (Km), 100 pg/mL ampicillin (Amp), 15 pg/mL chloramphenicol (Clm), 100 pg/mL spectinomycin (Spc). Formulations are given per liter. All components were dissolved in deionized water. To pour plates, 16 g of agar was added per liter of media.

LB: 10g tryptone, 5g yeast extract, 5g NaCl

TSB: 17g soytone, 3g glucose, 2.5g NaCl, 5g K2HPO4, adjust pH to 7.3 prior to autoclaving SOB: 20g tryptone, 5g yeast extract, lOmL of 250mM KC1, adjust pH to 7.0 and just before use add lOmL of IM MgCh and IM MgSO4

SOC: Add 20mL of filter-sterilized IM glucose into re-constituted SOC

MCM: lOOmL of IM potassium phosphate pH 7, 0.88g trisodium citrate dihydrate, 0.36g MgSO4, ImL of 22mg/mL ferric ammonium citrate, 1g casein hydrolysate, 2g potassium glutamate, 11 ImL of IM glucose, and 50mg tryptophan

Table 2: Strains used in this study Table 3: Plasmids used in this study

AmpR: ampicillin resistant; KmR: kanamycin resistant; SpcR: spectinomycin resistant;

ClmR: chloramphenicol resistant

Molecular Biology. DNA manipulations were performed according to manufacturer protocols. Genomic DNA was isolated using DNeasy UltraClean Microbial Kits from Qiagen (Germantown, MD). Restriction endonucleases were from New England Biolabs (Beverly, MA). Plasmids were purified using Zymopure miniprep kits from Zymo Research (Irvine, CA). PCR reactions to generate DNA fragments for cloning were performed with Phusion polymerase (New England Biolabs), while PCR reactions for construct verification used OneTaq polymerase (New England Biolabs). Gibson assembly was performed at 50% scale per reaction using HiFi DNA assembly mix from New England Biolabs. Oligonucleotide primers were purchased from Life Technologies (Carlsbad, CA) and listed in Table 4. Sanger sequencing was performed by the Ohio State University Comprehensive Cancer Center Genomics Shared Resource. Nanopore sequencing was performed by Plasmidsaurus (Eugene, OR).

Table 4: Primers used in this study

For primers used in Gibson assembly, lowercase indicates the oligonucleotides homologous to the gene being amplified while uppercase indicates oligonucleotides homologous to the vector

Identification and annotation of the valinophos biosynthetic gene cluster. The draft genome of B. velezensis NRRL B-41580 (GCF_001461825.1) was downloaded from NCBI and analyzed locally. BLAST [11] and Pfam [12] were used to analyze encoded genes and proteins while Clinker [13] was used for syntenic comparison.

Heterologous expression. Fragments containing each of the putative biosynthetic gene cluster genes were PCR amplified from B. velezensis NRRL B-41580 genomic DNA using the Bvel BGC primers (Table 4). Linear pDG1730 was obtained from PCR amplification of

Hindlll-digested pDG1730 using primers pDG1730_EcoRI_F and pDG1730_BamHI_R (Table 4). The fragments were then assembled into linear pDG1730 by Gibson assembly prior to transformation into chemically competent E. coli DH5a X-pir. After recovery for 1 hour at 37°C, cells were plated onto LB-Amp and grown overnight at 37°C. Colonies were sequentially passaged and plasmids were isolated from overnight cultures grown in 4mL of LB-Amp. Diagnostic restriction digestion analysis and Nanopore sequencing of resultant plasmids were performed to validate constructs. Validated plasmid pKSJ657 was transformed into E. coli BL21(DE3) and miniprepped out to yield plasmid which was transformed into B. subtilis 168, which was carried out by adding Ipg of plasmid to ImL of culture which had grown 90 minutes past To in MCM. After recovery for 1 hour at 37°C, cells were pelleted by centrifugation at 10,000x g for 1 minute, decanted, and resuspended in 200uL of MCM before being plated onto LB-Spc and grown overnight at 37°C. Colonies were passaged, and PCR was used to confirm presence of pnfA-T.

Production Screening. Strains were cultivated overnight in 20 x 150 mm test tubes containing 5 mL of TSB in a drum roller at 30°C and used to inoculate 125 mL UltraYield flasks containing 25 mL of TSB, which were incubated in an orbital shaker (200 rpm) at 30°C for 7 days. Cultures were centrifuged for 15 minutes at 3000 rpm to obtain supernatant, which was lyophilized and reconstituted in 1 mL of DI water before precipitation with 90% methanol. The soluble fraction was collected by centrifugation as above and the methanol was removed on a rotary evaporator before lyophilizing. The dried sample was reconstituted in 1 mL di H2O and analyzed by NMR spectroscopy and mass spectrometry as described below.

NMR Spectroscopy. NMR spectroscopy was performed at the OSU Campus Chemical Instrument Center on a Bruker Avance Neo 400 MHz spectrometer (400 MHz for 'H and 162 MHz for ³¹P) equipped with a 5 mm Prodigy Cry oprobe. Proton chemical shifts are reported in 5 values relative to an external standard of 0.1% tetramethylsilane in D2O while phosphorus chemical shifts are reported in 5 values relative to an external standard of 85% phosphoric acid. ¹H-³¹P gHMBC (gradient Heteronuclear Multiple-Bond Correlation) spectra were collected after optimization of long-range proton-phosphorus coupling at 18 Hz. Spectra were processed in MestReNova 14 software.

Mass Spectrometry. Weak-anion exchange was routinely used as a preparative step to enrich phosphonic/phosphinic acid compounds from extracts and improve signal to noise. Chelex-100 resin (Bio-Rad) was charged with 0.15 M FeCh, washed to remove free iron, and adjusted to pH 3 with acetic acid. For each sample, 100 pL was diluted in 1 mL di H2O and acidified to pH 3 with acetic acid. Chelex-Fe (200 pL of 50% resin) was added to each sample and gently mixed at room temperature for 30 min. The unbound fraction was removed, and the resin was washed with 1 mL di H2O for 10 min before decanting again, phosphonic/phosphinic acid were eluted twice with 1 mL 500 mM NH4HCO3 (pH 9) and then 1 mL of di H2O. These three fractions were combined, neutralized with acetic acid, and lyophilized. Dried material was re-dissolved in 100 pL di H2O for LC-MS analysis.

Mass spectrometry analyses were performed on a Thermo Q-Exactive Orbitrap with a Vanquish-H UHPLC system. The data were acquired under high resolution mode (RP = 70,000) with an AGC target of 1E6 and a maximum IT of 200 ms. Source parameters included a sheath gas flow rate of 58 units, an aux gas flow rate of 16 units, and a sweep gas flow rate of 3 units, a spray voltage of 2.50 kV, a capillary temperature of 281°C, a S-lens RF level of 50.0, and an aux gas heater temperature of 463 °C.

For LC-MS positive mode analyses, 10 pL of weak anion exchanged sample was mixed with 5 pL of di H2O and 85 pL MeCN with 0.1% formic acid. A 5pL sample was injected onto a Waters XB ridge Amide (2.1 x 150 mm) HPLC column. The buffer used for UHPLC was H2O with 0.1% formic acid (solvent A) and MeCN with 0.1% formic acid (solvent B). The flow rate was set at 0.35 mL/min. The elution gradient started at 85% solvent B for 2 minutes followed by a linear gradient to 40% solvent B over 4 min, a maintenance at 40% solvent B over 3 minutes, a return to 85% solvent B over 6 seconds, and re-equilibrated for 5.4 min before the next injection. The mass window was set to m/z 70-450.

Expression and purification of His6-PnfA. The pnfA gene was PCR amplified from the genomic DNA of B. velezensis B-41580 using primers pET28B-NHis-PnfA-F and pET28B- NHis-PnfA-R (Table 4). The resulting 1.3 kb fragment was gel purified. Linear pET28B for Gibson assembly was obtained by PCR using primers pET28B-Xho!-F and pET28B-NdeI-R. The 5.5 kb PCR product was gel purified. The gel purified PCR product of pnfA was then cloned into the Ndel and Xhol restriction sites of pET28B by Gibson assembly to yield pKSJ601, which encodes for Hise-PnfA. pKSJ601 was transformed into A. coli Rosetta (DE3) pLysRARE. The strain was grown in 1 L of LB-Km-Clm at 37°C and 220 rpm to ODeoo of 0.4 and cold-shocked for 10 min. Hise- PnfA production was induced by the addition of 1 mM IPTG and the culture was returned to 18°C, 220 rpm for 16 hours. The culture was harvested by centrifugation at 5000 rpm for 10 minutes and the cell pellets were frozen at -80°C. Cell pellets were then re-suspended in 20mL lysis buffer (50 mM HEPES pH 7.5, 250 mM NaCl, 10% glycerol, 30 mM imidazole) containing 10 mg lysozyme, 1 mg RNase A, and 100U Dnase. The suspension was gently mixed at 4°C for 20 minutes before being lysed by sonication and centrifuged at 11,000 rpm for 40 minutes at 4°C. Clarified cell lysate was combined with 5 mL HisPur Ni-NTA affinity resin (Thermo) in a column and gently mixed for 30 min at 4°C. The resin was washed with 50mL of wash buffer A (50 mM HEPES pH 7.5, 250 mM NaCl, 30 mM imidazole, 10% glycerol) followed by 25 mL of wash buffer B (50 mM HEPES pH 7.5, 250 mM NaCl, 50 mM imidazole, 10% glycerol) and elution with 20 mL of elution buffer C (50 mM HEPES pH 7.5, 250 mM NaCl, 100 mM imidazole, 10% glycerol) and 20 mL of elution buffer D (50 mM HEPES pH 7.5, 250 mM NaCl, 250 mM imidazole, 10% glycerol). Fractions were analyzed by Bradford and SDS-PAGE, and those containing the target protein were concentrated to 2.5 mL via 10 kDa Amicon Ultra-15 Millipore centrifugal filters and desalted by PD-10 columns using storage buffer (50mM HEPES pH 7.5, 250 mM NaCl, 10% glycerol).

Expression and purification of His6-PnfB. The pnfB gene was PCR amplified from the genomic DNA of B. velezensis B-41580 using primers pET28B-NHis-PnfB-F and pET28B- NHis-PnfB-R (Table 4). The resulting 1.2 kb fragment was gel purified and cloned into the Ndel and Xhol restriction sites of linearized pET28B as described above to yield pKSJ602, which encodes for Hise-PnfB. Overproduction and purification procedures for Hise-PnfB were the same as for Hise-PnfA.

Expression and purification of His nfC. The pnfC gene was PCR amplified from the genomic DNA of B. velezensis B-41580 using primers pET28B-NHis-PnfC-F and pET28B- NHis-PnfC-R (Table 4). The resulting 0.9 kb fragment was gel purified and cloned into the Ndel and Xhol restriction sites of linearized pET28B as described above to yield pKSJ600, which encodes for Hise-PnfC. Overproduction and purification procedures for Hise-PnfC were the same as for Hise-PnfA.

Expression and purification of His6-SUMO-PnfD. The pnfD gene was PCR amplified from the genomic DNA of B. velezensis B-41580 using primers 2ST-Gibson-PnfD-F and 2ST- Gibson-PnfD-R (Table 4). The resulting 1.2kb fragment was gel purified and cloned into the LIC sites of linearized 2-ST by Gibson assembly to yield pKSJ603, which encodes for Hise-SUMO- PnfD. Overproduction and purification procedures for Hise-SUMO-PnfD were the same as for Hise-PnfA.

Biochemical assay of PnfC and PnfD. Typical reaction mixtures (500 pL) contained 10 pM Hise-PnfC, 10 pM Hise-SUMO-PnfD, 1 mM phosphoenolpyruvate, 2 mM MgCL, 100 pM pyridoxal -5 -phosphate, and 3 mM of L-amino acid in 50 mM HEPES 250 mM NaCl buffer pH 7.5 Reactions were incubated at 30°C for 18 hr followed by heat inactivation at 65°C for 15 min. Reaction supernatants were analyzed by NMR and LC-MS as described above.

Biochemical assay of PnfA. Typical reaction mixtures (500 uL) contained 10 pM Hise- PnfA, 2 mM MgCh, 5 mM ATP, 3 mM carboxylate substrate, and 1 mM nucleophilic substrate in 50 mM Tris 100 mM KC1 pH 9. All canonical amino acids were tested as carboxylate substrates with phosphonoalanine as a nucleophile, while an array of aminophosphonates (L- AP4, L-AP5, L-Ala(P), L-Val(P), 3-aminopropylphosphonic acid, 4-aminophenylphosphonic acid, 2-aminoethylphosphonic acid, aminomethyl phosphonic acid, and phosphinothricin) were tested with L-alanine as a carboxylate. Reactions were incubated, inactivated, and analyzed as described above.

Enzymatic preparation of alanine-phosphonoalanine (Ala-PnAla). A 11.45 mL reaction containing 20 pM PnfA, 2 mM MgCL, 10 mM ATP, 10 mM L-alanine, and 2.6 mM L- phosphonoalanine in 50 mM Tris 100 mM KC1 pH 9 was incubated at 30°C for 24 hours. The reaction was heat inactivated by incubating at 65°C for 15 min and a 75% MeOH precipitation was used to remove protein. The sample was centrifuged at 3000 rpm for 15 min and the supernatant was evaporated in a rotary evaporator to remove methanol before lyophilizing to dryness.

The sample was rehydrated in 1 mL di H2O and strong cation exchange (SCX) was performed using a 5 mL bed volume of Dowex 50WX8 (Bio-Rad) in a 2.5 x 10 cm EconoColumn (Bio-Rad). The sample was eluted with 10 volumes of di H2O and 5 volumes of 1% NH4OH. All fractions were neutralized and analyzed by NMR, and the fractions containing alanine-phosphonoalanine but not phosphonoalanine were combined and lyophilized, yielding 5.7 mg of material. This was dissolved in 63% MeCN with 10 mM NH4HCO3 for HPLC purification. 500 pL was injected onto a XB ridge Amide 10 x 250 mm column. The solvent system was H2O with 10 mM NH4HCO3 (solvent A) and 90% MeCN with 10 mM NH4HCO3 (solvent B). The flow rate was set at 4 mL/min with a fraction size of 4 mL. The elution gradient started at 70% solvent B for 5 minutes followed by a linear gradient to 40% solvent B over 30 min, maintenance at 40% solvent B for 5 min followed by a return to 70% solvent B over 5 min and maintenance at 70% solvent B for 20 min to re-equilibrate the column. The fractions with UV-Vis signals were concentrated by rotary evaporation to 2 mL and analyzed by NMR. alanine-phosphonoalanine eluted between 65-67% (8 to 10 minutes). Yield: 2.3 mg.

Biochemical assay of PnfB. Typical reaction mixtures (250 uL) contained 10 pM Hise- PnfB, 2 mM MgCh, 5 mM ATP, 3 mM carboxylate substrate, and 1 mM alanine- phosphonoalanine (phosphonoalamide E) in 50 mM Tris 100 mM KC1 pH 9. All canonical amino acids were tested as carboxylate substrates with phosphonoalamide E as a nucleophile. Reactions were incubated, inactivated, and analyzed as described above.

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6 Kayrouz CM et al. Genome Mining Reveals the Phosphonoalamide Natural Products and a New Route in Phosphonic Acid Biosynthesis. ACS Chem Biol 15, 1921-1929, doi: 10.1021/acschembio.0c00256 (2020).

7 Polidore ALA et al. Phosphonate Natural Product Made by Pantoea ananatis is Necessary and Sufficient for the Hallmark Lesions of Onion Center Rot. mBio 12, doi: 10.1128/mBio.03402-20 (2021).

8 Zhang Y et al. Valinophos Reveals a New Route in Microbial Phosphonate Biosynthesis That Is Broadly Conserved in Nature. J Am Chem Soc 144, 9938-9948, doi: 10.1021/jacs.2c02854 (2022).

9 Zhang Y et al. Biosynthesis of Argolaphos Illuminates the Unusual Biochemical Origins of Aminomethylphosphonate and N(epsilon)-Hydroxyarginine Containing Natural Products. J Am Chem Soc 144, 9634-9644, doi: 10.1021/jacs.2c00627 (2022).

10 Wilson JC et al. Discovery of Anti-Phytopathogenic Phosphonopeptide Natural Products from Bacillus velezensis by Genome Mining. [Manuscript submitted for publication] (2023).

11 Clark K et al. GenBank. Nucleic Acids Res 44, D67-72, doi: 10.1093/nar/gkvl276 (2016).

12 Mistry J et al. Pfam: The protein families database in 2021. Nucleic Acids Res 49, D412-D419, doi: 10.1093/nar/gkaa913 (2021).

13 Gilchrist CL et al. Clinker & clustermap .js: Automatic generation of gene cluster comparison figures. Bioinformatics, doi: 10.1093/bioinformatics/btab007 (2021). Example 2

Described herein are genetic and enzymatic methods to produce and obtain phosphonopyruvate, phosphonoalanine and phosphonoalanine-peptide compounds using engineered strains and recombinant protein catalysts developed from Bacillus microorganisms. These compounds are valuable chemical synthons, neuroactive agents, research reagents, and have potential application as antimicrobial agents against plant and animal pathogens of human and agricultural concern. Potent inhibition of bacterial pathogens was previously demonstrated using the phosphonoalanine containing di- and tripeptides, specifically against the causative agents of soft rots and pink seed disease. These compounds also inhibit bacterial pathogens attributed to the devasting collapse of honeybees worldwide. Herein methods are reported for utilizing the purified enzymes as effective biocatalysts for these compounds in both individual and combinatorial reactions. Their utility in phosphonopeptide synthesis shown by rapidly producing all previously described phosphonoalamide compounds (six) and generating new peptides not previously observed in Nature.

Example 3 - Biosynthesis of Bacillus Phosphonoalamides Reveals Highly Specific Amino Acid Ligation

ABSTRACT. Phosphonate natural products have a history of commercial success across numerous industries due to their potent inhibition of metabolic processes. Over the past decade, genome mining approaches have successfully led to the discovery of numerous bioactive phosphonates. However, continued success is dependent upon a greater understanding of phosphonate metabolism, which will enable the prioritization and prediction of biosynthetic gene clusters for targeted isolation. Here, the complete biosynthetic pathway is reported for phosphonoalamides E and F, antimicrobial phosphonopeptides with a conserved C-terminal L- phosphonoalanine (PnAla) residue (Figure 12). These peptides, produced by Bacillus, are the direct result of PnAla biosynthesis and serial ligation by two ATP -grasp ligases. A critical step of this pathway was the reversible transamination of phosphonopyruvate to PnAla by a dedicated transaminase with preference for the forward reaction. The dipeptide ligase PnfA was shown to ligate alanine to PnAla to afford phosphonoalamide E, which was subsequently ligated to alanine by PnfB to form phosphonoalamide F. Specificity profiling of both ligases found each to be highly specific, unusual for ATP-grasp ligases in phosphonopeptide biosynthesis, though the limited acceptance of non-canonical substrates by PnfA allowed for in vitro formation of products incorporating alternative pharmacophores. These findings further establish the transaminative branch of phosphonate metabolism, unveil insights into the specificity of ATP- grasp ligation, and highlight the biocatalytic potential of biosynthetic enzymes. INTRODUCTION. Phosphonate and phosphinate (Pn) compounds are a class of natural products (NPs) characterized by direct carbon-phosphorus bonds. This moiety, an isostere of phosphate esters and carboxylic acids, is responsible for the bioactivity of phosphonate and phosphinate natural products, the majority of which are potent metabolic inhibitors [Al], Notable examples include the antibacterial fosfomycin, the herbicide phosphinothricin, and the antimalarial fosmidomycin. Fosfomycin (Monurol) is a clinically approved antibiotic which covalently inhibits MurA by mimicking phosphoenolpyruvate, preventing peptidoglycan biosynthesis [A2], Fosmidomycin has shown promise in clinical trials against malaria, where it inhibits 1 -deoxy -D-xylulose 5-phosphate reductoisom erase to block the non-mevalonate pathway of isoprenoid biosynthesis [A3], Outside of medicine, phosphinothricin (glufosinate) and its peptide derivatives, which irreversibly inhibit glutamine synthetase, are the active ingredients of multiple herbicide lines produced by BASF [A4],

Phosphonate and phosphinate natural products have found commercial success in multiple industries, inspiring new genomics-driven techniques for their discovery [A5], In the last decade alone, genome mining and targeted isolation have yielded multiple new phosphonate and phosphinate scaffolds, with new chemotypes, previously unknown headgroups, and a wide array of bioactivities [A6-A10], However, genomic data suggests that an even greater number of phosphonate and phosphinate natural products await discovery, as nearly 7% of all bacteria are predicted to harbor biosynthetic gene clusters (BGCs) for the compounds, very few of which encode for known products [Al l], To tap into this wealth of compounds, a greater understanding of phosphonate and phosphinate biosynthesis is required to accurately predict and classify the pathways, products, and potential biological activities of these biosynthetic gene clusters.

All known phosphonate and phosphinate biosynthesis begins with the conversion of phosphoenolpyruvate (PEP) to phosphonpyruvate (PnPy) by phosphoenolpyruvate mutase (PepM). By itself, this reaction is highly unfavorable, requiring a coupled enzymatic reaction to thermodynamically drive C-P bond formation [Al 2] . Previously, this principle was leveraged to search for phosphonate and phosphinate biosynthetic gene clusters lacking a known coupling enzyme. This resulted in the isolation of L-phosphonoalanine (PnAla) and four tripeptides with an TV-terminal PnAla (phosphonoalamides A-D) from Streptomyces sp. NRRL B-2790 [A7], A subsequent search for additional phosphonoalamide biosynthetic gene clusters led to the discovery of C-terminal PnAla peptides (Ala-PnAla and Ala-Ala-PnAla, phosphonoalamides E and F) produced by Bacillus velezensis NRRL B-41580 [Al 3].

While those studies have revealed PnAla and its peptide derivatives to have broadspectrum antibacterial activity [A7, A13], the role which PnAla plays in Nature is still unknown, six decades after its first isolation from biological material [A14], The bioactivity of phosphonoalamides E and F, along with their isolation from B. velezensis, a noted biocontrol agent, further supports a potential role in microbial chemical ecology. Strains of this species have demonstrated efficacy against numerous bacterial and fungal phytopathogens, and are used in commercial products such as Rhizo Vital®, Botrybel, Serenade®, Kodiak™, and Taegro® [Al 5], B. velezensis dedicates a substantial portion of its genome to secondary metabolite production. The B-41580 genome contains the biosynthetic gene clusters for 11 known antimicrobials compounds in addition to the phosphonoalamide cluster [Al 5], suggesting that PnAla is yet another warhead within the B. velezensis arsenal.

In this study, the genetic and biochemical logic is established for phosphonoalamide biosynthesis in Bacillus. The biosynthetic gene cluster was defined using heterologous expression and biochemically demonstrated phosphonoalamide F to be the product of four enzymatic reactions. PnfD was shown to catalyze the interconversion of phosphonopyruvate (PnPy) and PnAla, confirming it as an example of the transaminative early branch of phosphonate and phosphinate biosynthesis. Tripeptide formation was revealed to be a linear process catalyzed by two distinct ATP -grasp L-amino acid ligases, both of which displayed strict substrate specificity.

RESULTS

Delineation of the phosphonoalamide biosynthetic gene cluster. During a previous search for PnAla-encoding biosynthetic gene clusters within Actinobacteria, a biosynthetic gene cluster attributed to Mycobacteroides abcessus subsp. massiliense strain aerosol_aerosol_3 was identified. It was established that this genome was heavily contaminated and that the contig containing pepM belonged to a member of the Bacillus subtilis species complex. Fermentation of several strains encoding this biosynthetic gene cluster ultimately resulted in the isolation of two PnAla-containing peptides, phosphonoalamide E (Ala-PnAla) and phosphonoalamide F (Ala- Ala-PnAla) from B. velezensis NRRL B-41580 [A 13],

To establish the boundaries of this biosynthetic gene cluster, a fifteen-gene window (Table 5) centered on the pepM within the B-41580 genome (henceforth designated pnfC) was first analyzed. This genomic neighborhood was compared to those of other Bacillus spp. encoding a PepM with >80% shared identity to PnfC, as biosynthetic gene clusters meeting this similarity threshold generally produce similar phosphonate and phosphinate natural products [A6], However, all genomes (90, Table 6) containing this biosynthetic gene cluster belonged to members of the B. subtilis group and displayed extensive conservation. As strains lacking pepM would be incapable of phosphonate and phosphinate biosynthesis, it would be highly unlikely that genes conserved between pepM⁺ and pepM~ strains would be involved in phosphonoalamide biosynthesis. Thus, the reciprocal search of neighboring genes was performed to delineate the cluster (Figure 13). Although orfsl-5 could not be confidently assigned to known functions, their conservation within closely related strains lacking pepM (B. subtilis 168 and B. amyloliquefascians DSM7) led to their exclusion from the putative gene cluster. Genes downstream of pnfT were also excluded because their annotations as c factor (prflT), yebDEG (prfl5-17), and members of the pur operon (orfs 18-29)) suggest functions unrelated to phosphonate and phosphinate metabolism. This resulted in a putative assignment of pnfABCDT as the biosynthetic genes. The B-41580 pnfABCDT were individually compared to those of all other pepM⁺ Bacillus strains. The minimum percent identity observed was 81%, while the median percent identity across all comparisons was 99-100%.

Table 5. Annotation of the B-41580 pepM neighborhood

Table 6. List of genomes used for bioinformatic analysis

This cluster assignment was further supported by similarities between the Bacillus and Streptomyces pepM gene neighborhoods: both encode a PepM, aminotransferase, two ATP-grasp ligases, and a transporter, and result in the biosynthesis of PnAla-containing tripeptides [A7, A13], Thus, it was reasoned that pnfA-D would be essential for the production of phosphonoalamide F, while pnjT may serve as a transporter. It was proposed that PnfC would convert PEP to PnPy, PnfD would transaminate PnPy to PnAla, and the ATP-grasp ligases PnfA and PnfB would catalyze peptide bond formation to produce phosphonoalamides E and F.

To test this hypothesis, pnfABCD from B-41580 was cloned into pDG1730 for expression within a heterologous host. The resulting plasmid (pKSJ652) was transformed and integrated into genome of B. subtilis 168, and the process was repeated with pDG1730 to provide a negative control. Metabolites produced from the resulting strain, B. subtilis KSJ2140 (168 a/77j7v:pKSJ652) were analyzed by ³¹P NMR and compared to the empty vector control strain, B. subtilis KSJ2139 (168 a/iqVUpDG I 730). Significant accumulation of a Pn was observed within B. subtilis KSJ2140 extracts (Figure 14A) and but absent from the control strain. These were identified as phosphonoalamide F by ³¹P NMR (5p 17.8), ¹H-³¹P HMBC (correlated 5H 4.03, 1.87, and 1.69), and LC-HRMS analyses (m/z 312.0955), which were all consistent with literature values (Figure 14A-Figure 14C) [A13], These results are congruent with synteny analyses and clearly demonstrate pnfABCD as the only genes required for phosphonoalamide F biosynthesis.

PnAla production by PnfC and PnfD. After establishing the minimal biosynthetic gene cluster, attention was focused on the biosynthesis of PnAla. Previous heterologous expression experiments demonstrated that introduction of genes encoding PepM and a PLP-dependent aminotransferase from Streptomyces sp. NRRL S-515 were sufficient for PnAla production [A7], As PnfD was similarly annotated as a PLP-dependent aspartate aminotransferase (AAT), it was proposed that it would catalyze transamination of PnPy using aspartate as an amino donor (Figure 15 A).

To test this hypothesis, recombinant PnfC and PnfD from E. coli were overexpressed and purified for in vitro assays (Figure 18). Interestingly, purified recombinant PnfD was yellow in color, suggesting the presence of a bound chromophore. This was suspected to be an internal aldimine with PLP, and UV-Vis spectroscopy did reveal an absorbance maxima at 439 nm, consistent with other PLP-dependent aminotransferases (Figure 19A) [A16, A17], A series of spectrophotometric experiments were performed to understand the nature of the protein. Upon treatment of PnfD with sodium borohydride, a decrease in absorbance at 439 nm with a concomitant absorption increase at 344 nm was observed (Figure 19B). These characteristics were consistent of reduction of the aldimine linkage between PLP and a lysine residue [Al 6], Incubation of PnfC with L-cycloserine a shift of the absorbance maxima to 381 nm (Figure 19C). This was consistent with formation of a stable adduct between L-cycloserine and PLP, resulting from the formation a transient oxime intermediate [A17], Having established PLP as the chromophore, PnfC was incubated with a stoichiometric amount of PLP to allow for full occupancy. By comparing the absorbance at 439 nm with and without exogenous PLP, the occupancy of as purified recombinant PnfD was estimated to be 40% (Figure 19 A).

The activity of PnfD as a PepM-coupling enzyme was demonstrated through in-vitro reconstitution. Purified PnfD was incubated with PnfC, PEP, L-Asp (amino donor), and PLP. Over 24 hours, this resulted in consumption of PEP (-1.0 ppm) and production of a new species (16.7 ppm, 27% yield) in ³¹P NMR spectra (Figure 15B). To determine if other amino donors would be more favorable, L-Asp was substituted with each of the other proteinogenic amino acids (Figure 20). While L-G1U (19%), L-Asn (17%), and L-Cys (11%) were all accepted, the yield of PnAla remained highest with L-Asp (Figure 15B).

Having established PnAla biosynthesis with PnfD, its ability to catalyze the reverse reaction was examined, using PnAla as an amino donor to convert OAA to L-Asp. Incubating PnfD with PnAla, OAA, and PLP resulted in consumption of PnAla (16.6 ppm) and formation of a new species (9.9 ppm) in ³¹P NMR (Figure 15C). LC-HRMS confirmed this new species as PnPy (Figure 21). Intriguingly, the yield of PnPy was low, suggesting that the equilibrium constant of the PnfD reaction may favor PnAla. To further examine the effects of substrate concentration on this directionality, both forward reactions containing 1 mM PnPy and a large excess (10 mM) of amino donor (Asp) and reverse reactions containing of 1 mM PnAla with a large excess (10 mM) of keto-acid acceptor (OAA) were monitored using ³¹P NMR over the course of 2 hours (Figure 22A-Figure 22D). In both the forward and reverse PnfD reactions, conversion occurred rapidly, reaching apparent equilibrium within 10 minutes. As the majority of enzymatic transamination reactions are freely reversible, with equilibrium constants close to one [Al 8], both reactions were expected to proceed towards completion. However, the yield of PnAla in the forward reaction (96%) was far higher than the yield of PnPy in the reverse reaction (60%). This suggests that PnfD has an inherent preference for the transamination of PnPy over the transamination of PnAla, allowing it to drive PnAla biosynthesis.

These combined results demonstrate PnfD as a reversible PLP-dependent transaminase that functions as PepM-coupling enzyme by converting PnPy to PnAla. While an excess of amino donor could be used to drive the forward reaction to near completion, an equivalent excess of keto-acid acceptor was unable to drive the reverse reaction past 60% conversion, suggesting that reaction characteristics inherent to PnfD control the directionality of biosynthesis.

Phosphonoalamide biosynthesis in Bacillus is a linear pathway. Having established the synthesis of PnAla from PEP by PnfC and PnfD, it was sought to reconstitute the reactions leading to peptide formation. The remaining genes in the biosynthetic gene cluster, pnfA and pnfB, encoded putative ATP-grasp ligases, are a family of enzymes implicated formation of other phosphonopeptides including valinophos, rhizocticin, and plumbemycin pathways [A9, A19-A21], In canonical peptide bond formation by ATP-grasp ligases, one amino acid is first activated as a carboxylate, forming an acylphosphate intermediate upon ATP hydrolysis. The amine of the second amino acid is then primed for nucleophilic attack, forming an amide bond. The C-terminal position of PnAla in the Bacillus compounds suggested it would be a nucleophile in the biosynthesis of phosphonoalamide E and F. Based on this logic, there would be two possible routes to phosphonoalamide F (Figure 16 A). In the convergent pathway, one ligase would produce Ala-Ala, and the second would ligate PnAla to the alanyl dipeptide to form phosphonoalamide F. However, this would not provide a clear explanation for the isolation of phosphonoalamide E (Ala-PnAla). Alternatively, ligation of Ala to PnAla may occur in a linear manner, first forming phosphonoalamide E and then forming phosphonoalamide F, with one ligase catalyzing each reaction.

To test this hypothesis, recombinant PnfA and PnfB were expressed and purified from E. coli. As it was unclear which enzyme would catalyze the first ligation, Ala, PnAla, Mg²⁺ and ATP were incubated with either PnfA or PnfB, and the reactions were monitored by ³¹P NMR (Figure 16B). In the reaction with PnfA, PnAla (16.6 ppm) was completely consumed, with appearance of a new species (17.0 ppm). No product was observed when any component was omitted (Figure 23). LC-HRMS identified this product as phosphonoalamide E (Ala-PnAla, Figure 24Ai), which was confirmed by fragmentation analysis (Figure 25B, Table 7). PnAla remained untransformed in the reaction with PnfB.

Table 7. LC-HRMS/MS fragment assignment for PnAla-containing peptides

The PnfA reaction was scaled-up and Ala-PnAla was purified for use in tripeptide ligation assays. Ala-PnAla was isolated from the reaction mixture by strong cation exchange chromatography and HILIC HPLC. The purified dipeptide was >95% pure as determined by 'H NMR (Figure 26). Reactions containing Ala, Ala-PnAla, Mg²⁺, and ATP with either PnfA or PnfB were prepared and monitored by ³¹P NMR (Figure 16C). A new species (17.3 ppm) was observed in the PnfB reaction, which was identified as phosphonoalamide F by LC-HRMS (Figure 27Ai) and confirmed by fragmentation analysis (Figure 25A, Table 7). Ala-PnAla remained untransformed in the reaction with PnfA. These results demonstrated the phosphonoalamide biosynthesis proceeds through a linear pathway in Bacillus.

To further exclude the possibility the peptides may result by convergent biosynthesis from secondary activity of the ATP-Grasp ligases, additional control reactions were prepared using the recombinant enzymes. First, Ala-Ala, PnAla, ATP, and Mg²⁺ were incubated with either PnfA or PnfB. Surprisingly, a new species (17.1 ppm) was observed in the ³¹P NMR spectra of the PnfA reaction (Figure 28A). The NMR shift of the product was consistent with Ala-PnAla instead of Ala-Ala-PnAla (17.4 ppm). Indeed, the LC-HRMS revealed the signal as the Ala-PnAla dipeptide. (Figure 28B). As free Ala was not added to the reaction, Ala contamination in the stock of Ala-Ala was suspected. However, this possibility was eliminated based on the absence of signals corresponding to free Ala in the ¹ H NMR spectrum (Figure 29A). As the ligation reactions took place at alkaline pH, it was hypothesized that Ala- Ala may be undergoing base-mediated hydrolysis to generate free L-Ala. Indeed, when Ala-Ala was incubated in reaction buffer and analyzed by LC-HRMS at sequential timepoints, free Ala steadily increased, revealing the source of Ala for Ala-PnAla formation (Figure 29B).

To further exclude the involvement of Ala-Ala in phosphonoalamide F biosynthesis, PnfA was incubated with PnfB, Ala, PnAla, ATP, and Mg²⁺. Analysis of the reaction by ³¹P NMR and LC-HRMS demonstrated the formation of Ala-PnAla and Ala-Ala-PnAla (Figure 30A-Figure 30B), but not Ala-Ala (Figure 3 IB). Ala-Ala was also absent from separate reactions containing Ala, ATP, Mg²⁺ with only PnfA (Figure 31C) or PnfB (Figure 3 ID). These data demonstrate the Ala-Ala dipeptide is not a product of the pathway.

Inspired by the success of the PnfAB reaction, the entire pathway was reconstituted in a one-pot reaction containing PnfA, PnfB, PnfC, PnfD, PEP, PLP, Asp, Ala, PnAla, ATP, and Mg²⁺. Conversion of PEP was low (8.3%), with only one product signal visible by ³¹P NMR (Figure 32A). Nonetheless, LC-HRMS revealed the formation of PnAla, Ala-PnAla, and Ala- Ala-PnAla, with the dipeptide as the major product (Figure 32B). In vivo biosynthesis heavily favored tripeptide production, as demonstrated by isolation from the native producer [A22] and heterologous expression, suggesting that the in vitro reaction conditions require optimization. While the independent PnfA (Figure 24Ai) and PnfB (Figure 27Aii) reactions result in nearcomplete turnover of Pn substrates, the PnfAB reaction (Figure 30A) produces equivalent amounts of dipeptide and tripeptide, further highlighting the complexity behind coordinating biosynthesis.

PnfA and PnfB are highly specific amino acid ligases. In contrast to the diverse tripeptides isolated from Streptomyces [A7], the only phosphonopeptides isolated from B-41580 were Ala-Ala-PnAla and its immediate precursor Ala-PnAla [A22], This suggested PnfA and PnfB may display strict substrate specificity. To test this hypothesis, PnfA was first incubated with PnAla and each of the proteinogenic amino acids (Figure 24A-Figure 24B). Monitoring the reactions by ³¹P NMR and LC-HRMS identified Ser-PnAla to be the only other dipeptide produced by PnfA, the structure of which was established by fragmentation analysis (Figure 25C, Table 7). Overall turnover of PnAla was much lower with Ser (8%) than with Ala (100%), indicating that replacement of a methyl group hydrogen with a hydroxyl moiety reduced carboxylate acceptance. The methyl side chain was required for catalysis, as Gly (noralanine), was not ligated to PnAla by PnfA. The second ATP-Grasp ligase in the pathway, PnfB, also exhibited high substrate specificity for Ala. Reactions containing PnfB, Ala-PnAla, and each of proteinogenic amino acid yielded no products as detected by ³¹P NMR and LC-HRMS other than Ala-Ala-PnAla (Figure 27A-Figure 27B).

It was determined whether PnfA could be used to synthesize other phosphonodipeptides using reactions containing Ala other aminophosphonate substrates. These included 2- aminoethylphosphonate (2AEP), 1 -hydroxy -2 AEP (1H2AEP), 3-aminopropylphosphonate (3- APPn), phosphinothricin (PT), L-2-amino-4-phosphonobutyrate (L-AP4), L-2-amino-5- phosphonopentanoate (L-AP5), aminomethylphosphonate (AmPn), (A)-l- aminoethylphosphonate (L-Ala(P)), (lA)-l-amino-2-methylpropylphosphonate (L-Val(P)), and 4-aminophenylphosphonate (4-APhePn) (Figure 33 A). These substrates were chosen to provide structural insights into nucleophile selectivity by PnfA, as they differed from PnAla with respect to sidechain length (L-AP4 and L-AP5), presence of a carboxyl group (2AEP), use of a phosphinate moiety (PT), or a combination of the above and other modifications. Reactions with Ala and these alternative nucleophiles were monitored by LC-HRMS (Figure 33B). All signals corresponding to products were further characterized by fragmentation analysis to unambiguously establish their chemical identities. These revealed PnfA to synthesize additional phosphonate dipeptides including Ala-2AEP (9.4% yield), Ala-1H2AEP (6.4%), and Ala-AP4 (trace amounts; Figure 34A-Figure 34B, Figure 35, Table 8, Table 9).

Table 8. LC-HRMS/MS fragment assignment for Ala-2AEP and Ala-1H2AEP

Table 9. LC-HRMS/MS fragment assignment for Ala-AP4

The activity profile of PnfA with the tested substrates allowed for rudimentary rules for the nucleophile specificity to be proposed. Using PnAla as the canonical substrate, acceptance of 2AEP revealed that a carboxyl group was not required, as 2AEP is equivalent to decarboxylated PnAla. Acceptance of L-AP4, but not L-AP5, demonstrated that the amino acid side chain could be extended by one carbon, but not two. However, these moi eties could not be combined, as 3- APPn, which exhibits side chain extension by one carbon and loss of the carboxylate moiety, was not accepted by PnfA. Likewise, PT, the phosphinate analogue of L-AP4, failed to ligate with PnAla. While PnfA accepted 1H2AEP as a substrate, which is decarboxylated and substituted at the P carbon, other compounds with multiple structural differences from PnAla (AmPn, Ala(P), Val(P), and 4-APhePn) were not ligated. Although PnfA formed dipeptides between Ala and PnAla, 2AEP, and 1H2AEP. PnAla was the only substrate among the three substrates to be successfully ligated to Ser (7.2% yield) (Figure 36B).

As all the accepted nucleophilic substrates contained a Pn group, it was wondered if this moiety was strictly required by PnfA. Therefore, Asp, the proteinogenic isostere of PnAla, was incubated with Ala or Ser. LC-HRMS revealed product formation in both reactions (Figure 37A) and fragment analysis identified the products as Ala-Asp and Ser-Asp (Figure 37B, Table 10). Nonetheless, Pns remained the preferred substrate, with significantly greater amounts of unligated Ala and Ser remaining in reactions containing Asp versus PnAla.

Table 10. LC-HRMS/MS fragment assignment for Ala-Asp and Ser-Asp

Finally, it was determined whether the specificity of PnfA would include substrate stereochemistry. Dipeptide products were absent in the reactions containing D-Ala and L-PnAla (Figure 38 A). However, when PnfA was supplied with L-Ala and DL-PnAla, roughly half of the DL-PnAla was converted to dipeptide (Figure 38B). The stoichiometry of this reaction immediately suggested that PnfA had consumed one enantiomer of PnAla while ignoring the other. Indeed, Marfey’s analysis of the reaction mixture revealed the unreacted PnAla to be overwhelmingly in D configuration, with little, if any, L-PnAla remaining (Figure 38C). Altogether, these data demonstrate that PnfA is an entantioselective dipeptide ligase with strict specificity for both carboxylate and nucleophilic substrates.

DISCUSSION. The complete pathway for the phosphonoalamide biosynthesis was elucidated using a combination of comparative genomics, heterologous expression, and biochemical reconstitution experiments. Of the contiguous genes conserved between Bacillus strains, only 4 encoded enzymes were essential for biosynthesis. The pathway begins with the interconversion of PEP to PnPy by PepM (PnfC), which is coupled to the transamination of PnPy to PnAla by PnfD. Ligation of PnAla to Ala is catalyzed by PnfA, forming Ala-PnAla (phosphonoalamide E). Subsequent ligation of Ala-PnAla to Ala by PnfB results in production of Ala- Ala-PnAla (phosphonoalamide F, Figure 16A).

Similar to other aminotransferases, PnfD exhibited relaxed substrate specificity, accepting Asp, Glu, Asn, and Cys as amino donors for the transamination of PnPy into PnAla. While utilization of Asn and Cys as amino donors is unusual for aspartate aminotransferases [A23], this ability may simply be overlooked, as recent studies have discovered other bacterial aspartate aminotransferases capable of cysteine- and asparagine-oxo-acid transamination [A24, A25], Transamination of PnPy to PnAla was a reversible reaction, but the limited degree to which substrate concentration affected the reverse reaction was unexpected. While a large excess of amino donor was able to drive the forward reaction to near completion (96% yield of PnAla), an equivalent excess of keto-acid acceptor was unable to drive the reverse reaction as far (60% yield of PnPy. Taken together, this suggests that PnfD has an inherent preference for the transamination of PnPy over the transamination of PnAla, allowing this reversible enzyme to dictate the directionality of Pn biosynthesis.

PnfA and PnfB provide further examples of ATP-grasp amino acid ligases involved in phosphonopeptide biosynthesis. Both ligases were highly specific, such that PnfB did not accept any alternate substrates provided, and PnfA only tolerated small differences in carboxylate and nucleophile structure. The high selectivity of these enzymes provides an opportunity to understand structural differences between di- and tri-peptide ligases, as they activate the same carboxylate substrate (Ala), are produced by the same organism, and act within the same biosynthetic pathway. Additionally, the specificity of PnfA for L-PnAla, even in the presence of stoichiometric D-PnAla, offers a method for separation of the two enantiomers. The commercial cost of pure D-PnAla is roughly twelvefold higher than that of L-PnAla and nearly eightyfold that of DL -PnAla, highlighting the difficulty of obtaining specific isomers.

The activation of PnAla as a nucleophile by PnfA, rather than as a carboxylate, results the in the alternate connectivity of the Bacillus phosphonoalamides (C-terminal PnAla) as compared to the Streptomyces phosphonoalamides (TV-terminal PnAla). This is the only example in natural product biosynthesis where the same pharmacophore is differentially incorporated (carboxylate vs. nucleophile) by ATP-grasp ligases. This is unusual for phosphonopeptides, as other groups with conserved Pn pharmacophores retain the same connectivity. The rhizocticins and plumbemycins contain the threonine synthase inhibitor (Z)-L-2-amino-5-phosphono-3- pentenoate (APPA) as a C-terminal residue [A21, A26-A28], while bialaphos, trialaphos, and phosalacine contain the glutamine synthetase inhibitor phosphinothricin at their TV-terminus [A4],

Also unique to this pathway is the production of a single end-product (phosphonoalamide F) rather than a mixture of tripeptides. In this respect, the Bacillus phosphonoalamides are more similar to phosphonopeptides produced by non-ribosomal peptide synthetases (phosphinothricin (PT) tripeptide, phosalacine) [A4, A29] or tRNA-dependent GCN5-related N-acetyltransf erases (argolaphos, dehydrophos, fosfazinomycin) [A10, A30-A32], which result in specific, invariant natural products.

Outside of the Bacillus phosphonoalamides, ATP-grasp ligases appear to underly a strategy for producing multiple phosphonopeptides from a single pathway. The rhizocticin, plumbemycin, valinophos, and Streptomyces phosphonoalamide pathways all use ATP-grasp ligases to produce multiple compounds with the same Pn warhead [A7, A9, A19, A20, A26], Moreover, the composition of proteinogenic amino acids within these antimicrobial phosphonopeptides has been shown to influence their selectivity [A27, A33, A34], Most strikingly, the rhizocticins and plumbemycins both contain the threonine synthase inhibitor APPA as a C-terminal residue. However, the rhizocticins are antifungals while the plumbemycins display antibacterial activity [A26-A28], Phosphonoalamide A (PnAla-Ala-Val, from Streptomyces) and phosphonoalamide F (Ala-Ala-PnAla, from Bacillus) also exhibit different spectrums of antimicrobial activity [A7, A13], but it remains to be seen whether this is due to amino acid composition or position of the PnAla moiety.

The convergent alanyl derivatization of Pn moi eties observed in bialaphos, trialaphos, and the Bacillus phosphonoalamides, which are produced by taxonomically distant organisms and use different mechanisms for amino acid ligation, suggests that alanylation may be advantageous. Indeed, bialaphos is a more potent antimicrobial than phosphinothricin [A29], and each Ala residue ligated to PnAla results in lower MICs [A7, A13], Ala appears to be a preferred constituent of antimicrobial phosphonopeptides, as roughly half of these natural products contain at least one Ala residue: phosphonoalamides A (PnAla-Ala-Val), C (PnAla-Ala-Ile), E (Ala- PnAla), and F (Ala-Ala-PnAla) [A7, A13], plumbemycins A (Ala-Asp-APPA) and B (Ala-Asn- APPA) [A26], phosalacine (PT-Ala-Leu) [A4], bialaphos/phosphinothricin tripeptide (PT-Ala- Ala) [A35], and trialaphos (PT- Ala- Ala- Ala) [A36],

B. velezensis also utilizes alanyl derivatization in the production of the antimicrobial dipeptide bacilysin. Coincidentally, bacilysin is also assembled by an ATP-grasp ligase and composed of an TV-terminal Ala and a C-terminal L-anticapsin pharmacophore [A37], Likewise, bacilysin is a much more potent antibacterial than free anticapsin [A38], Thus, it stands to reason that alanyl incorporation may afford the broadest uptake of these peptides, obviating the need for peptide diversification. Alternatively, these single products may be ignored by the oligopeptide transporters of producing strains, offering a means of self-resistance. Further investigation is required to address these hypotheses, yielding insights which can be applied to the rational design of antimicrobial peptides.

The biosynthesis of phosphonoalamides E and F in Bacillus further establishes the transaminative branch of Pn natural product metabolism. Peptide biosynthesis reactions revealed the strict specificity of ligases within this pathway, while demonstrating the importance of different functional groups for substrate acceptance. The alternative incorporation of PnAla (C- vs N-) within this group of phosphonopeptides suggest further exploration of this biosynthetic branch will yield additional variations on this theme, all of which can be leveraged to decipher the molecular mechanisms underlying their bioactivity. MATERIALS and METHODS

Chemicals. General chemical reagents were purchased from Sigma-Aldrich, Fisher

Scientific, VWR, or Santa Cruz Biotechnology.

Strains, Media, General Culture Conditions. The strains and plasmids used in this study are listed in Table 11 and Table 12. Escherichia coli and Bacillus subtilis strains were routinely grown on LB broth or agar at 37 °C. Bacillus strains were fermented at 30 °C in TSB. The following additives and antibiotics were included for plasmid maintenance and selection as appropriate: 50 pg mL'¹ kanamycin (Km), 100 pg mL'¹ ampicillin (Amp), 15 pg mL'¹ chloramphenicol (Clm), 100 pg mL'¹ spectinomycin (Spc). All components were dissolved in deionized water (di H2O). For plates, 16 g agar was added per liter of media. All media formulations are given per liter.

LB: 10 g tryptone, 5 g yeast extract, 5 g NaCl

TSB: 17 g soytone, 3 g glucose, 2.5 g NaCl, 5 g K2HPO4, adjust pH to 7.3 prior to autoclaving

SOB: 20 g tryptone, 5 g yeast extract, 10 mL of 250 mM KC1, adjust pH to 7.0 and just before use add 10 mL of 1 M MgCh and 1 M MgSO4

SOC: Add 20 mL of filter-sterilized 1 M glucose into re-constituted SOC

MCM: 100 mL of 1 M potassium phosphate pH 7, 0.88 g trisodium citrate dihydrate, 0.36 g MgSO4, 1 mL of 22 mg/mL ferric ammonium citrate, 1 g casein hydrolysate, 2 g potassium glutamate, 111 mL of 1 M glucose, and 50 mg tryptophan

Table 11. List of strains used in this study

Table 12. List of plasmids used in this study

Amp^R: ampicillin resistant; Km^R: kanamycin resistant; Spc^R: spectinomycin resistant;

Clm^R: chloramphenicol resistant

Molecular Biology. DNA manipulations were performed according to manufacturer protocols. Genomic DNA was isolated using DNeasy UltraClean Microbial Kits from Qiagen (Germantown, MD). Restriction endonucleases were from New England Biolabs (Beverly, MA). Plasmids were purified using Zymopure miniprep kits from Zymo Research (Irvine, CA). PCR reactions to generate DNA fragments for cloning were performed with Phusion polymerase (New England Biolabs), while PCR reactions for construct verification used OneTaq polymerase (New England Biolabs). Gibson assembly was performed at 50% scale per reaction using HiFi DNA assembly mix from New England Biolabs. Oligonucleotide primers were purchased from Life Technologies (Carlsbad, CA) and listed in Table 13. Sanger sequencing was performed by the Ohio State University Comprehensive Cancer Center Genomics Shared Resource. Nanopore sequencing was performed by Plasmidsaurus (Eugene, OR).

Table 13. List of primers used in this study

For primers used in Gibson assembly, lowercase indicates the oligonucleotides homologous to the gene being amplified while uppercase indicates oligonucleotides homologous to the vector.

Identification and annotation of the phosphonoalamide biosynthetic gene cluster.

The draft genome of B. velezensis NRRL B-41580 (GCF OO 1461825.1) was downloaded from NCBI and analyzed locally. BLAST [SI] and Pfam [S2] were used to analyze encoded genes and proteins while Clinker [S3] was used for syntenic comparisons. The biosynthetic gene cluster was deposited to NCBI under the accession PP372566.

Heterologous expression. Fragments containing each of the putative biosynthetic gene cluster genes were PCR amplified from B. velezensis NRRL B-41580 genomic DNA using the Bvel BGC primers (Table 13). Linear pDG1730 was obtained from PCR amplification of Hindlll-digested pDG1730 using primers pDG1730_EcoRI_F and pDG1730_BamHI_R (Table 13). The fragments were then assembled into linear pDG1730 by Gibson assembly prior to transformation into chemically competent A. coli DH5a k-pir. After recovery for 1 hour at 37°C, cells were plated onto LB-Amp and grown overnight at 37°C. Colonies were sequentially passaged and plasmids were isolated from overnight cultures grown in 4 mL of LB-Amp. Diagnostic restriction digestion analysis and Nanopore sequencing of resultant plasmids were performed to validate constructs. Validated plasmid pKSJ657 was transformed into E. coli BL21(DE3), miniprepped, and then transformed into A subtilis 168 by the following procedure. One pg of plasmid was added to 1 mL of culture which had grown 90 minutes past To in MCM. After recovery for 1 hour at 37°C, cells were pelleted by centrifugation at 10,000x g for 1 minute, decanted, and resuspended in 200 uL of MCM before being plated onto LB-Spc and grown overnight at 37°C. Colonies were passaged, and diagnostic PCR reactions was used to confirm integration of pnfA-D.

Production screening. Strains were cultivated overnight in 20 x 150 mm test tubes containing 5 mL of TSB in a drum roller at 30°C. These were used to inoculate 125 mL UltraYield flasks containing 25 mL of TSB, which were incubated in an orbital shaker (200 rpm) at 30°C for 7 days. Cultures were centrifuged for 15 minutes at 3000 rpm to obtain supernatant, which was then lyophilized and reconstituted in 1 mL of DI water before precipitation with 90% methanol. The soluble fraction was collected by centrifugation as above. Methanol was removed by rotary evaporation before lyophilization. The dried sample was reconstituted in 1 mL di H2O and analyzed by NMR spectroscopy and mass spectrometry as described below.

Mass Spectrometry. Weak-anion exchange was routinely used as a preparative step to enrich phosphonic acids from extracts and improve signal to noise. This was performed using Chelex-100-Fe resin as previously described [S4] .

Mass spectrometry analyses were performed on a Thermo Q-Exactive Orbitrap with a Vanquish-H UHPLC system. Data were acquired under high resolution mode (RP = 70,000) with an AGC target of 1E6 and a maximum IT of 200 ms. Source parameters included a sheath gas flow rate of 58 units, an aux gas flow rate of 16 units, and a sweep gas flow rate of 3 units, a spray voltage of 2.50 kV, a capillary temperature of 281°C, a S-lens RF level of 50.0, and an aux gas heater temperature of 463 °C.

For LC-MS positive mode analyses, 10 pL of weak anion exchanged sample was mixed with 5 pL of di H2O and 85 pL MeCN with 0.1% formic acid. A 5 pL sample was injected onto a Waters XB ridge Amide (2.1 x 150 mm) HPLC column. The buffer used for UHPLC was H2O with 0.1% formic acid (solvent A) and MeCN with 0.1% formic acid (solvent B). The flow rate was set at 0.35 mL/min. The elution gradient started at 85% solvent B for 2 minutes followed by a linear gradient to 40% solvent B over 4 min, maintenance at 40% solvent B over 3 minutes, a return to 85% solvent B over 6 seconds, and re-equilibration for 5.4 min before the next injection. The mass window was set to m/z 70-450 for dipeptide analysis and m/z 100-600 for tripeptide analysis. For MS/MS, the same settings, gradient, and column were used. Target ion(s) were added to the inclusion list with a starting collision energy of 10 eV for dipeptides and 15 eV for tripeptides, adjusted in 5 eV increments in subsequent runs if needed. Published analyses of amino acid fragmentation were referenced in structural assignment of fragment ions [S5] .

Expression and purification of Hise-PnfA. The pnfA gene was PCR amplified from the genomic DNA of B. velezensis B-41580 using primers pET28B-NHis-PnfA-F and pET28B- NHis-PnfA-R (Table 13). The resulting 1.3 kb fragment was gel purified. Linear pET28B for Gibson assembly was obtained by PCR using primers pET28B-XhoI-F and pET28B-NdeI-R (Table 13). The 5.5 kb PCR product was gel purified. The gel purified PCR product of pnfA was then cloned into the Ndel and Xhol restriction sites of pET28B by Gibson assembly to yield pKSJ601, which encodes for Hise-PnfA. pKSJ601 was transformed into E. coli Rosetta (DE3) pLysRARE. The strain was grown in 1 L of LB-Km-Clm at 37°C and 220 rpm to OD600 of 0.4 and cold-shocked for 10 min. Hise- PnfA production was induced by the addition of 1 mM IPTG and the culture was returned to 18°C, 220 rpm for 16 hours. The culture was harvested by centrifugation at 5000 rpm for 10 minutes and the cell pellets were frozen at -80°C. Cell pellets were then re-suspended in 20 mL lysis buffer (50 mM HEPES pH 7.5, 250 mM NaCl, 10% glycerol, 30 mM imidazole) containing 10 mg lysozyme, 1 mg RNase A, and 100U Dnase. The suspension was gently mixed at 4°C for 20 minutes, lysed by sonication, and centrifuged at 11,000 rpm for 40 minutes at 4°C. Clarified cell lysate was combined with 5 mL HisPur Ni-NTA affinity resin (Thermo) in a column and gently mixed for 30 min at 4°C. The resin was washed with 50 mL of wash buffer A (50 mM HEPES pH 7.5, 250 mM NaCl, 30 mM imidazole, 10% glycerol) followed by 25 mL of wash buffer B (50 mM HEPES pH 7.5, 250 mM NaCl, 50 mM imidazole, 10% glycerol) and elution with 20 mL of elution buffer C (50 mM HEPES pH 7.5, 250 mM NaCl, 100 mM imidazole, 10% glycerol) and 20 mL of elution buffer D (50 mM HEPES pH 7.5, 250 mM NaCl, 250 mM imidazole, 10% glycerol). Fractions were analyzed by Bradford and SDS-PAGE, and those containing the target protein were concentrated to 2.5 mL via 10 kDa Amicon Ultra-15 Millipore centrifugal filters. Purified proteins were then desalted by PD-10 columns using storage buffer (50 mM HEPES pH 7.5, 250 mM NaCl, 10% glycerol).

Expression and purification of Hise-PnfB. The pnfB gene was PCR amplified from the genomic DNA of B. velezensis B-41580 using primers pET28B-NHis-PnfB-F and pET28B- NHis-PnfB-R (Table 13). The resulting 1.2 kb fragment was gel purified and cloned into the Ndel and Xhol restriction sites of linearized pET28B as described above to yield pKSJ602, which encodes for His6-PnfB. Overproduction and purification procedures for Hise-PnfB were the same as for Hise-PnfA.

Expression and purification of Hise-PnfC. The pnfC gene was PCR amplified from the genomic DNA of B. velezensis B-41580 using primers pET28B-NHis-PnfC-F and pET28B- NHis-PnfC-R (Table 13). The resulting 0.9 kb fragment was gel purified and cloned into the Ndel and Xhol restriction sites of linearized pET28B as described above to yield pKSJ600, which encodes for Hise-PnfC. Overproduction and purification procedures for Hise-PnfC were the same as for Hise-PnfA. Expression and purification of His6-SUMO-PnfD. The pnfD gene was PCR amplified from the genomic DNA of B. velezensis B-41580 using primers 2ST-Gibson-PnfD-F and 2ST- Gibson-PnfD-R (Table 13). The resulting 1.2 kb fragment was gel purified and cloned into the LIC sites of linearized 2-ST by Gibson assembly to yield pKSJ603, which encodes for Hise- SUMO-PnfD. Overproduction and purification procedures for Hise-SUMO-PnfD were the same as for Hise-PnfA.

Biochemical assays of Hise-PnfC with Hise-PnfD. Typical reaction mixtures (500 pL) contained 10 pM His₆-PnfC, 10 pM Hise-SUMO-PnfD, 1 mM PEP, 2 mM MgCh, 100 pM PLP, and 3 mM of L-amino acid in 50 mM HEPES 250 mM NaCl buffer pH 7.5 Reactions were incubated at 30°C for 24 hr followed by heat inactivation at 65°C for 15 min. Reaction supernatants were analyzed by NMR and LC-MS as described above.

Biochemical assays of Hise-Pnf . Typical reaction mixtures (500 uL) contained 10 pM Hise-PnfA, 2 mM MgCh, 5 mM ATP, 3 mM carboxylate substrate, and 1 mM nucleophilic substrate in 50 mM Tris 100 mM KC1 pH 9. All canonical amino acids were tested as carboxylate substrates with PnAla as a nucleophile, while an array of aminophosphonates (L- AP4, L-AP5, L-Ala(P), L-Val(P), 3-aminopropylphosphonic acid, 4-aminophenylphosphonic acid, 2-aminoethylphosphonic acid, aminomethyl phosphonic acid, and phosphinothricin) were tested with L-Ala as a carboxylate. Reactions were incubated, inactivated, and analyzed as described above.

Enzymatic preparation of Ala-PnAla. A 11.45 mL reaction containing 20 pM Hise- PnfA, 2 mM MgCh, 10 mM ATP, 10 mM L-Ala, and 2.6 mM L-PnAla in 50 mM Tris 100 mM KC1 pH 9 was incubated at 30°C for 24 hours. The reaction was heat inactivated by incubating at 65°C for 15 min and a 75% MeOH precipitation was used to remove protein. The sample was centrifuged at 3000 rpm for 15 min and the supernatant was evaporated in a rotary evaporator to remove methanol before lyophilizing to dryness.

The sample was rehydrated in 1 mL di H2O and acidified to pH 3. Strong cation exchange (SCX) was performed using a 5 mL bed volume of Dowex 50WX8 (Bio-Rad) in a 2.5 x 10 cm Econo-Column (Bio-Rad). The sample was eluted with 10 volumes of di H2O and 5 volumes of 1% NH4OH. All fractions were neutralized and analyzed by NMR, and the fractions containing Ala-PnAla but not PnAla were combined and lyophilized, yielding 5.7 mg of material.

This was dissolved in 63% MeCN with 10 mM NH4HCO3 for HPLC purification. 500 pL was injected onto a XB ridge Amide 10 x 250 mm column. The solvent system was H2O with 10 mM NH4HCO3 (solvent A) and 90% MeCN with 10 mM NH4HCO3 (solvent B). The flow rate was set at 4 mL/min with a fraction size of 4 mL. The elution gradient started at 70% solvent B for 5 minutes followed by a linear gradient to 40% solvent B over 30 min, maintenance at 40% solvent B for 5 min followed by a return to 70% solvent B over 5 min and maintenance at 70% solvent B for 20 min to re-equilibrate the column. The fractions with UV-Vis signals were concentrated by rotary evaporation to 2 mL and analyzed by NMR. Ala-PnAla eluted between 65-67% (8 to 10 minutes). Yield: 2.3 mg.

Biochemical assays of Hise-PnfB. Typical reaction mixtures (250 uL) contained 10 pM Hise-PnfB, 2mM MgCL, 5 mM ATP, 3 mM carboxylate substrate, and 1 mM Ala-PnAla (phosphonoalamide E) in 50 mM Tris 100 mM KC1 pH 9. All canonical amino acids were tested as carboxylate substrates with phosphonoalamide E as a nucleophile. Reactions were incubated, inactivated, and analyzed as described above.

Biochemical assays of Hise-PnfA and Hise-PnfB. Typical reaction mixtures (500 uL) contained 10 pM Hise-PnfA, 10 pM Hise-PnfB, 2 mM MgCh, 10 mM ATP, 10 mM Ala, and 1 mM PnAla substrate in 50 mM Tris 100 mM KC1 pH 9. Reactions were incubated, inactivated, and analyzed as described above.

Biochemical assays of Hise-Pnf with Hise-PnfB, Hise-PnfC, and Hise-PnfD. Typical reaction mixtures (500 uL) contained 10 pM of each enzyme, 2 mM MgCL, 0.1 mM PLP, 1 mM PEP, 10 mM Asp, 5 mM Ala, and 10 mM ATP in 50 mM Tris 100 mM KC1 pH 9. Reactions were incubated, inactivated, and analyzed as described above.

Expression and purification of Hise-HpxV. The hpxV gene was PCR amplified from the genomic DNA of Streptomyces regensis WC-3744 using primers pET28B-NHis-WC744- HpxV-F and pET28B-NHis-WC3774-HpxV-R (Table 13). The resulting 1.0 kb fragment was gel purified and cloned into the Ndel and Xhol restriction sites of linearized pET28B as described above to yield pKSJ515, which encodes for Hise-HpxV. Overproduction and purification procedures for Hise-HpxV were the same as for Hise-PnfA.

Enzymatic preparation of 1H2AEP. To produce (5)-l-hydroxy-2- aminoethylphosphonate (1H2AEP), a 31.98 mL reaction containing 30 pM Hise-HpxV, 30 mM a-ketoglutarate, 0.2 mM ferrous ammonium sulfate, and 4 mM L-ascorbic acid in pH 7.4 phosphate-buffered saline was incubated at 30°C for 24 hours. The reaction was heat-inactivated by incubating at 65°C for 30 minutes, and centrifuged at 15,000 rpm for 2 minutes to remove precipitated protein. This was followed with a 90% MeOH precipitation to remove any residual protein. The sample was centrifuged at 3000 rpm for 15 minutes and the supernatant was evaporated in a rotary evaporator to remove methanol before lyophilizing to dryness.

The resulting powder was reconstituted into 25 mL of di H2O and loaded onto a l m bed of Sephadex LH-20 resin (GE Healthcare) in a 1.5 x 120 cm Econo-Column (Bio-Rad). The sample was eluted with di H2O in 7 mL fractions. Fractions were lyophilized to dryness, reconstituted in 1 mL di H2O and analyzed by ³¹P NMR. All fractions containing Pn species were pooled, concentrated by rotary evaporation, and lyophilized to dryness.

The sample was rehydrated in 1 mL di H2O and acidified to pH 3. Strong cation exchange (SCX) was performed using a 5 mL bed volume of Dowex 50WX8 (Bio-Rad) in a 2.5 x 10 cm Econo-Column (Bio-Rad). The sample was eluted with 10 volumes of di H2O and 5 volumes of 1% NH4OH. All fractions were neutralized and analyzed by NMR, and two separate pools were created, one including a mix of 2AEP and 1H2AEP, and another with the fractions containing 1H2AEP but not 2AEP. The 1H2AEP pool was lyophilized to dryness, reconstituted in 1 mL di H2O, acidified to pH 3, and the above SCX process was repeated. Fractions were neutralized and analyzed by NMR. All fractions containing pure 1H2AEP, as demonstrated by 'H NMR, were combined and lyophilized, yielding 8.3 mg. This material was analyzed by 'H, ³¹P, and ¹H-³¹P HMBC NMR (Figure 39A-Figure 39B), which agreed with literature values [S6] .

Marfey’s analysis. Following the incubation of PnfA with Ala, DL-PnAla, ATP, and Mg²⁺, the reaction mixture, as well as 5 mM standards of DL-PnAla and L-PnAla, were derivatized by incubating 25 pL aliquots with 10 pL of 1 M NaHCCL and 50 pL of 1% w/v FDAA in acetone at 40°C for 1 hour. The derivatization reaction was allowed to cool to room temperature before being quenched with 10 pL of 1 M HC1. This quenched reaction was then diluted tenfold to prepare samples for LC-MS analysis.

Mass spectrometry was performed as above but with the following changes. The mass window was set to m/z 200-1000. 5 pL of each samples was injected onto a 2 x 100 mm Synergi Fusion-RP column. The solvent system was H2O with 0.1% formic acid (solvent A) and MeCN with 0.1% formic acid (solvent B). The flow rate was set at 0.2 mL/min and the gradient started at 0% solvent B for 2 minutes followed by a linear gradient to 100% solvent B over 30 minutes, maintenance at 100% solvent B for 2 minutes, followed by a return to 0% solvent B over 0.1 minute and maintenance at 0% solvent B for 3.9 minutes to re-equilibrate the column.

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EXEMPLARY ASPECTS

In view of the described compositions, devices, systems, and methods, herein below are described certain more particularly described aspects of the inventions. The particularly recited aspects should not, however, be interpreted to have any limiting effect on any different claims containing different or more general teachings described herein or that the “particular” aspects are somehow limited in some way other than the inherent meanings of the language and formulas literally used therein.

Example 1 : A compound defined by Formula I: wherein

Example 2: The compound of any examples herein, particularly example 1, wherein R⁴ is substituted or unsubstituted C1-C10 amide, or one or more amino acids (e.g., one or more canonical or non-canonical amino acids).

Example 3 : The compound of any examples herein, particularly example 1 or example 2, wherein R⁴ is one or more amino acids (e.g., one or more canonical or non-canonical amino acids).

Example 4: The compound of any examples herein, particularly examples 1-3, wherein R⁴ is one or more canonical amino acids.

Example 5: The compound of any examples herein, particularly examples 1-4, wherein R⁴ is one or more amino acids selected from the group consisting of alanine, serine, and combinations thereof.

Example 6: The compound of any examples herein, particularly examples 1-5, wherein the compound is of Formula IE wherein

Example 7: The compound of any examples herein, particularly examples 1-6, wherein R¹ is OR⁹ and/or R² is OR¹⁰.

Example 8: The compound of any examples herein, particularly examples 1-7, wherein the compound is of Formula III: wherein

Example 9: The compound of any examples herein, particularly examples 1-8, wherein R³ is hydrogen or -C(O)OR⁸.

Example 10: The compound of any examples herein, particularly examples 1-9, wherein the compound is of Formula IV: wherein

Example 11 : The compound of any examples herein, particularly examples 1-10, wherein R⁹ and/or R¹⁰ is hydrogen.

Example 12: The compound of any examples herein, particularly examples 1-11, wherein the compound is of Formula IV-A: wherein

Example 13: The compound of any examples herein, particularly examples 1-12, wherein the compound is of Formula IV-B: wherein

Example 14: The compound of any examples herein, particularly examples 1-13, wherein R⁷ is substituted or unsubstituted C1-C5 alkyl.

Example 15: The compound of any examples herein, particularly examples 1-14, wherein R⁷ is substituted or unsubstituted Ci alkyl.

Example 16: The compound of any examples herein, particularly examples 1-15, wherein R⁷ is CH3 or CH2OH.

Example 17: The compound of any examples herein, particularly examples 1-16, wherein the compound is of Formula IV-C: wherein

Example 18: The compound of any examples herein, particularly examples 1-17, wherein R⁵ and/or R⁶ is hydrogen.

Example 19: The compound of any examples herein, particularly examples 1-18, wherein the compound is of Formula IV-D: or a derivative or salt thereof.

Example 20: The compound of any examples herein, particularly examples 1-19, wherein the compound is of Formula IV-E or a derivative or salt thereof.

Example 21 : The compound of any examples herein, particularly examples 1-9, wherein the compound is of Formula V: wherein

Example 22: The compound of any examples herein, particularly example 21, wherein R⁸ is hydrogen.

Example 23: The compound of any examples herein, particularly example 21 or example 22, wherein the compound is of Formula V-A: wherein

Example 24: The compound of any examples herein, particularly examples 21-23, wherein R⁹ and/or R¹⁰ is hydrogen.

Example 25: The compound of any examples herein, particularly examples 21-24, wherein the compound is of Formula V-B: wherein

Example 26: The compound of any examples herein, particularly examples 21-25, wherein the compound is of Formula V-C: wherein

R⁷ is hydrogen, hydroxyl, halide, substituted or unsubstituted C1-C5 alkyl, or substituted or unsubstituted C1-C5 alkoxy; and R^x and R^y are independently selected from H, or substituted or unsubstituted C1-C5 alkyl, or substituted or unsubstituted C1-C5 acyl; or a derivative or salt thereof; with the proviso that the compound is not phosphonoalamide A, B, C, D, E, or F.

Example 27: The compound of any examples herein, particularly examples 21-26, wherein R⁷ is substituted or unsubstituted C1-C5 alkyl.

Example 28: The compound of any examples herein, particularly examples 21-27, wherein R⁷ is substituted or unsubstituted Ci alkyl.

Example 29: The compound of any examples herein, particularly examples 21-28, wherein R⁷ is CEE or CH2OH.

Example 30: The compound of any examples herein, particularly examples 21-29, wherein the compound is of Formula V-D: wherein

Example 31 : The compound of any examples herein, particularly examples 21-30, wherein R⁵ and/or R⁶ is hydrogen.

Example 32: The compound of any examples herein, particularly examples 21-31, wherein the compound is of Formula V-E: or a derivative or salt thereof.

Example 33: The compound of any examples herein, particularly examples 21-32, wherein the compound is of Formula V-F: or a derivative or salt thereof.

Example 34: The compound of any examples herein, particularly examples 1-33, wherein the compound is a salt.

Example 35: The compound of any examples herein, particularly examples 1-34, wherein the compound is a salt form of Formula I, Formula II, Formula III, Formula IV, Formula V, or a combination thereof with a counterion.

Example 36: The compound of any examples herein, particularly examples 1-35, wherein the compound is a salt form of Formula IV and/or Formula V with a counterion.

Example 37: The compound of any examples herein, particularly examples 1-36, wherein the compound is a salt form of Formula IV and/or Formula V with a counterion and the salt form of the compound is selected from the group consisting of: combinations thereof.

Example 38: The compound of any examples herein, particularly examples 35-37, wherein the counterion is a monovalent, divalent, or trivalent counterion.

Example 39: The compound of any examples herein, particularly examples 35-38, wherein the counterion is selected from the group consisting of sodium, potassium, calcium, lithium, magnesium, manganese, ammonium, iron, and combinations thereof.

Example 40: The compound of any examples herein, particularly examples 1-39, wherein the compound is a potassium salt, sodium salt, calcium salt, iron salt, ammonium salt, or a combination thereof.

Example 41 : The compound of any examples herein, particularly examples 1-40, wherein the compound comprises an agriculturally acceptable salt thereof and/or a pharmaceutically acceptable salt thereof.

Example 42: A compound comprising a head group and a tail group, the head group being bound to the tail group using one or more enzymes derived from Bacillus, wherein the head group comprises a phosphonic acid, a phosphinic acid, or a derivative thereof, and the tail group comprises one or more (canonical or non-canonical) amino acids, with the proviso that the compound is not phosphonoalamide A, B, C, D, E, or F.

Example 43 : The compound of any examples herein, particularly example 42, wherein the head group comprises phosphonoalanine (PnAla), 2-aminoethylphosphonic acid (2AEP), or a derivative thereof.

Example 44: The compound of any examples herein, particularly example 42 or example 43, wherein the tail group comprises alanine, serine, or a derivative thereof.

Example 45: The compound of any examples herein, particularly examples 42-44, wherein the head group comprises PnAla or a derivative thereof and tail group comprises serine or a derivative thereof.

Example 46: The compound of any examples herein, particularly examples 42-45, wherein the head group comprises 2AEP or a derivative thereof and tail group comprises alanine or a derivative thereof.

Example 47: The compound of any examples herein, particularly examples 42-46, wherein the compound comprises Ala-PnAla, Ser-PnAla, Ala-2AEP, or a derivative or salt thereof.

Example 48: The compound of any examples herein, particularly examples 42-47, wherein the head group is C-terminal.

Example 49: The compound of any examples herein, particularly examples 42-48, wherein the compound is a di-peptide or a tri-peptide.

Example 50: The compound of any examples herein, particularly examples 42-49, wherein the compound is a salt.

Example 51 : The compound of any examples herein, particularly examples 42-50, wherein the compound is a potassium salt, sodium salt, calcium salt, iron salt, ammonium salt, or a combination thereof.

Example 52: The compound of any examples herein, particularly examples 42-51, wherein the compound comprises an agriculturally acceptable salt thereof and/or a pharmaceutically acceptable salt thereof.

Example 53: The compound of any examples herein, particularly examples 42-52, wherein the compound is of any examples herein, particularly examples 1-41.

Example 54: The compound of any examples herein, particularly examples 1-53, wherein the compound is a Bacillus isolate or a derivative or salt thereof.

Example 55: The compound of any examples herein, particularly examples 1-54, wherein the compound is an isolate of B. velezensis, B. swezeyi, B. amyloliquefaciens, B. cabrialesii, B. sublilis. or a combination thereof.

Example 56: The compound of any examples herein, particularly examples 1-55, wherein the compound is an isolate of B. velezensis NRRL B-41850.

Example 57: A composition comprising the compound of any examples herein, particularly examples 1-56.

Example 58: The composition of any examples herein, particularly example 57, wherein the composition further comprises one or more agriculturally acceptable and/or pharmaceutically acceptable carriers.

Example 59: The composition of any examples herein, particularly example 57 or example 58, wherein the composition comprises a pharmaceutical composition, an agricultural composition, or a combination thereof.

Example 60: The composition of any examples herein, particularly examples 57-59, wherein the composition comprises a pesticide.

Example 61 : The composition of any examples herein, particularly examples 57-60, wherein the composition comprises an herbicide.

Example 62: The composition of any examples herein, particularly examples 57-61, wherein the composition exhibits antimicrobial activity.

Example 63: The composition of any examples herein, particularly examples 57-62, wherein the composition results in at least 5 log reduction of a population of microbes.

Example 64: The composition of any examples herein, particularly examples 57-63, further comprising a solvent, a carrier, an excipient, or a combination thereof.

Example 65: The composition of any examples herein, particularly examples 57-64, further comprising an agriculturally acceptable adjuvant or carrier.

Example 66: The composition of any examples herein, particularly examples 57-65, wherein the composition is formulated for delivery to a plant or animal.

Example 67: The composition of any examples herein, particularly examples 57-66, wherein the composition is formulated for delivery to a plant.

Example 68: The composition of any examples herein, particularly example 67, wherein the plant comprises a crop.

Example 69: The composition of any examples herein, particularly examples 57-66, wherein the composition is formulated for delivery to an animal.

Example 70: The composition of any examples herein, particularly example 69, wherein the animal is a companion animal, livestock, research animal, insect, or human.

Example 71 : A nucleic acid encoding the compound or composition of any examples herein, particularly examples 1-70.

Example 72: A vector encoding the nucleic acid of any examples herein, particularly example 71.

Example 73: A cell comprising the vector of any examples herein, particularly example 72.

Example 74: A cell comprising the compound or composition of any examples herein, particularly examples 1-70.

Example 75: The cell of any examples herein, particularly example 73 or example 74, wherein the cell comprises a Bacillus cell.

Example 76: The cell of any examples herein, particularly examples 73-75, wherein the cell comprises B. velezensis, B. swezeyi, B. amyloliquefaciens, B. cabrialesii, B. subtilis, or a combination thereof.

I l l Example 77: The cell of any examples herein, particularly examples 73-76, wherein the cell comprises B. velezensis NRRL B-41850.

Example 78: A method of making the compound of any examples herein, particularly examples 1-56.

Example 79: The method of any examples herein, particularly example 78, wherein the method is a biosynthetic method.

Example 80: The method of any examples herein, particularly example 78 or example 79, wherein the method uses one or more enzymes derived from Bacillus.

Example 81 : A method of making a compound comprising a head group and a tail group, the head group being bound to the tail group, wherein the head group comprises a phosphonic acid, a phosphinic acid, or a derivative thereof, and the tail group comprises one or more (canonical or non-canonical) amino acids, using one or more enzymes derived from Bacillus, with the proviso that the compound is not phosphonoalamide A, B, C, D, E, or F.

Example 82: The method of any examples herein, particularly example 81, wherein the head group comprises PnAla, 2AEP, or a derivative thereof.

Example 83: The method of any examples herein, particularly example 81 or example 82, wherein the tail group comprises alanine, serine, or a derivative thereof.

Example 84: The method of any examples herein, particularly examples 81-83, wherein the head group comprises PnAla or a derivative thereof and tail group comprises serine or a derivative thereof.

Example 85: The method of any examples herein, particularly examples 81-84, wherein the head group comprises 2AEP or a derivative thereof and tail group comprises alanine or a derivative thereof.

Example 86: The method of any examples herein, particularly examples 81-85, wherein the compound is the compound of any examples herein, particularly examples 1-56.

Example 87: The method of any examples herein, particularly examples 80-86, wherein the one or more enzymes are derived from B. velezensis, B. swezeyi, B. amyloliquefaciens, B. cabrialesii, B. subtilis, or a combination thereof.

Example 88: The method of any examples herein, particularly examples 80-87, wherein the one or more enzymes are derived from B. velezensis NRRL B-41850.

Example 89: The method of any examples herein, particularly examples 80-88, wherein the one or more enzymes comprise one or more ATP -grasp enzymes.

Example 90: The method of any examples herein, particularly examples 80-89, wherein the one or more enzymes are encoded by a gene comprising at least 90% identity to pnfA,pnfB, or a combination thereof.

Example 91 : The method of any examples herein, particularly examples 80-90, wherein the method proceeds via a linear pathway.

Example 92: The method of any examples herein, particularly examples 78-91, wherein the method comprises contacting a first nucleophile and a first carboxylate with a first enzyme, the first nucleophile comprising a phosphonic acid, a phosphinic acid, or a derivative thereof, and the first carboxylate comprising a first amino acid or a derivative thereof, to thereby form a first compound the first nucleophile bound to the first carboxylate (e.g. a carboxylate- nucleophile).

Example 93 : The method of any examples herein, particularly example 92, wherein the first enzyme is encoded by a gene comprising at least 90% identity to pnfA.

Example 94: The method of any examples herein, particularly example 92 or example 93, wherein the first enzyme comprises PnfA.

Example 95: The method of any examples herein, particularly examples 92-94, wherein the first enzyme comprises recombinant PnfA

Example 96: The method of any examples herein, particularly examples 92-95, wherein the first carboxylate comprises alanine (e.g., L-alanine), serine, or a combination thereof.

Example 97: The method of any examples herein, particularly examples 92-96, wherein the first nucleophile comprises PnAla, 2AEP, or a combination thereof.

Example 98: The method of any examples herein, particularly examples 92-97, wherein the method further comprises contacting the first compound and a second carboxylate with a second enzyme, the first compound being a nucleophile, the second carboxylate comprising a second amino acid or a derivative thereof, to thereby form a second compound comprising the first compound bound to the second carboxylate (e.g. a carboxylate-nucleophile).

Example 99: The method of any examples herein, particularly example 98, wherein the second enzyme is encoded by a gene comprising at least 90% identity to pnfB.

Example 100: The method of any examples herein, particularly example 98 or example 99, wherein the second enzyme comprises PnfB.

Example 101 : The method of any examples herein, particularly examples 98-100, wherein the second enzyme comprises recombinant PnfB.

Example 102: The method of any examples herein, particularly examples 98-101, wherein the second carboxylate comprises alanine (e.g., L-alanine).

Example 103: The method of any examples herein, particularly examples 89-102, wherein the method further performed in the presence of ATP. Example 104: A method of use of the compound, composition, nucleic acid, vector, or cell of any examples herein, particularly examples 1-77.

Example 105: The method of any examples herein, particularly example 104, wherein the method comprises using the compound, composition, nucleic acid, vector, or cell as an antimicrobial, herbicide, pesticide, or combination thereof to control an undesirable population.

Example 106: The method of any examples herein, particularly example 105, wherein the method comprises using the compound, composition, nucleic acid, vector, or cell as a pesticide.

Example 107: The method of any examples herein, particularly example 106, wherein the method comprises using the compound, composition, nucleic acid, vector, or cell to control an undesirable population in plants.

Example 108: The method of any examples herein, particularly example 107, wherein the method comprises contacting the plants or the locus thereof with or applying to the soil or water the compound, composition, nucleic acid, vector, or cell.

Example 109: The method of any examples herein, particularly examples 106-108, further comprising applying an additional pesticide.

Example 110: The method of any examples herein, particularly examples 105-109, wherein the undesirable population is a herbicide resistant or tolerant population, a pesticide resistant or tolerant population, an antimicrobial resistant or tolerant population, or a combination thereof.

Example 111 : The method of any examples herein, particularly examples 105-110, wherein the undesirable population comprises bacteria.

Example 112: A method of reducing the activity of bacteria, the method comprising exposing the bacteria to an effective amount of the compound, composition, nucleic acid, vector, or cell of any examples herein, particularly examples 1-77.

Example 113: A method of reducing bacterial population, the method comprising exposing the bacteria to an effective amount of the compound, composition, nucleic acid, vector, or cell of any examples herein, particularly examples 1-77.

Example 114: A method of killing bacteria, the method comprising exposing the bacteria to an effective amount of the compound, composition, nucleic acid, vector, or cell of any examples herein, particularly examples 1-77.

Example 115: A method for treating, preventing, inhibiting, and/or ameliorating a disease or disorder in a plant or a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the compound, composition, nucleic acid, vector, or cell of any examples herein, particularly examples 1-77.

Example 116: The method of any examples herein, particularly example 115, wherein the disease or disorder comprises an infection, such as a microbial infection.

Example 117: A method for treating, preventing, inhibiting, and/or ameliorating a microbial infection in a plant or a subject, comprising administering to the plant or subject an effective amount of the compound, composition, nucleic acid, vector, or cell of any examples herein, particularly examples 1-77.

Example 118: The method of any examples herein, particularly examples 115-117, wherein the plant is a crop.

Example 119: The method of any examples herein, particularly examples 115-117, wherein the subject is an animal.

Example 120: The method of any examples herein, particularly example 119, wherein the animal is a companion animal, livestock, research animal, insect, or human.

Example 121 : The method of any examples herein, particularly examples 111-120, wherein the compound, composition, nucleic acid, or vector is delivered via cultured Bacillus.

Example 122: The method of any examples herein, particularly examples 111-121, wherein the compound, composition, nucleic acid, or vector is delivered via cultured Bacillus velezensis.

Example 123: The method of any examples herein, particularly examples 111-122, wherein the compound, composition, nucleic acid, or vector is delivered via cultured: B. velezensis, B. swezeyi, B. amyloliquefaciens, B. cabrialesii, B. sublilis, or a combination thereof.

Example 124: The method of any examples herein, particularly examples 111-123, wherein the compound, composition, nucleic acid, or vector is delivered via cultured B. velezensis NRRL B-41850.

Other advantages which are obvious and which are inherent to the invention will be evident to one skilled in the art. It will be understood that certain features and sub-combinations are of utility and may be employed without reference to other features and sub-combinations. This is contemplated by and is within the scope of the claims. Since many possible embodiments may be made of the invention without departing from the scope thereof, it is to be understood that all matter herein set forth or shown in the accompanying drawings is to be interpreted as illustrative and not in a limiting sense.

The compositions and methods of the appended claims are not limited in scope by the specific compositions methods described herein, which are intended as illustrations of a few aspects of the claims and any compositions and methods that are functionally equivalent are intended to fall within the scope of the claims. Various modifications of the compositions and methods in addition to those shown and described herein are intended to fall within the scope of the appended claims. Further, while only certain representative method steps disclosed herein are specifically described, other combinations of the method steps also are intended to fall within the scope of the appended claims, even if not specifically recited. Thus, a combination of steps, elements, components, or constituents may be explicitly mentioned herein or less, however, other combinations of steps, elements, components, and constituents are included, even though not explicitly stated.

Claims

CLAIMS What is claimed:

1. A compound defined by Formula I: wherein

2. The compound of claim 1, wherein R⁴ is substituted or unsubstituted C1-C10 amide, or one or more amino acids (e.g., one or more canonical or non-canonical amino acids).

3. The compound of claim 1 or claim 2, wherein R⁴ is one or more amino acids (e.g., one or more canonical or non-canonical amino acids).

4. The compound of any one of claims 1-3, wherein R⁴ is one or more canonical amino acids.

5. The compound of any one of claims 1-4, wherein R⁴ is one or more amino acids selected from the group consisting of alanine, serine, and combinations thereof.

6. The compound of any one of claims 1-5, wherein the compound is of Formula II: wherein

7. The compound of any one of claims 1-6, wherein R¹ is OR⁹ and/or R² is OR¹⁰.

8. The compound of any one of claims 1-7, wherein the compound is of Formula III: wherein

9. The compound of any one of claims 1-8, wherein R³ is hydrogen or -C(O)OR⁸.

10. The compound of any one of claims 1-9, wherein the compound is of Formula IV: wherein

11. The compound of any one of claims 1-10, wherein R⁹ and/or R¹⁰ is hydrogen.

12. The compound of any one of claims 1-11, wherein the compound is of Formula IV-A: wherein

13. The compound of any one of claims 1-12, wherein the compound is of Formula IV-B: wherein

14. The compound of any one of claims 1-13, wherein R⁷ is substituted or unsubstituted Ci- C5 alkyl.

15. The compound of any one of claims 1-14, wherein R⁷ is substituted or unsubstituted Ci alkyl.

16. The compound of any one of claims 1-15, wherein R⁷ is CH3 or CH2OH.

17. The compound of any one of claims 1-16, wherein the compound is of Formula IV-C: wherein

18. The compound of any one of claims 1-17, wherein R⁵ and/or R⁶ is hydrogen.

19. The compound of any one of claims 1-18, wherein the compound is of Formula IV-D: or a derivative or salt thereof.

20. The compound of any one of claims 1-19, wherein the compound is of Formula IV-E or a derivative or salt thereof.

21. The compound of any one of claims 1-9, wherein the compound is of Formula V: wherein

22. The compound of claim 21, wherein R⁸ is hydrogen.

23. The compound of claim 21 or claim 22, wherein the compound is of Formula V-A: wherein

24. The compound of any one of claims 21-23, wherein R⁹ and/or R¹⁰ is hydrogen.

25. The compound of any one of claims 21-24, wherein the compound is of Formula V-B: wherein

26. The compound of any one of claims 21-25, wherein the compound is of Formula V-C: wherein

27. The compound of any one of claims 21-26, wherein R⁷ is substituted or unsubstituted Ci- C5 alkyl.

28. The compound of any one of claims 21-27, wherein R⁷ is substituted or unsubstituted Ci alkyl.

29. The compound of any one of claims 21-28, wherein R⁷ is CH3 or CH2OH.

30. The compound of any one of claims 21-29, wherein the compound is of Formula V-D: wherein

31. The compound of any one of claims 21-30, wherein R⁵ and/or R⁶ is hydrogen.

32. The compound of any one of claims 21-31, wherein the compound is of Formula V-E: or a derivative or salt thereof.

33. The compound of any one of claims 21-32, wherein the compound is of Formula V-F: or a derivative or salt thereof.

34. The compound of any one of claims 1-33, wherein the compound is a salt.

35. The compound of any one of claims 1-34, wherein the compound is a salt form of Formula I, Formula II, Formula III, Formula IV, Formula V, or a combination thereof with a counterion.

36. The compound of any one of claims 1-35, wherein the compound is a salt form of Formula IV and/or Formula V with a counterion.

37. The compound of any one of claims 1-36, wherein the compound is a salt form of Formula IV and/or Formula V with a counterion and the salt form of the compound is selected from the group consisting of: and combinations thereof.

38. The compound of any one of claims 35-37, wherein the counterion is a monovalent, divalent, or tri valent counterion.

39. The compound of any one of claims 35-38, wherein the counterion is selected from the group consisting of sodium, potassium, calcium, lithium, magnesium, manganese, ammonium, iron, and combinations thereof.

40. The compound of any one of claims 1-39, wherein the compound is a potassium salt, sodium salt, calcium salt, iron salt, ammonium salt, or a combination thereof.

41. The compound of any one of claims 1-40, wherein the compound comprises an agriculturally acceptable salt thereof and/or a pharmaceutically acceptable salt thereof.

42. A compound comprising a head group and a tail group, the head group being bound to the tail group using one or more enzymes derived from Bacillus, wherein the head group comprises a phosphonic acid, a phosphinic acid, or a derivative thereof, and the tail group comprises one or more (canonical or non-canonical) amino acids, with the proviso that the compound is not phosphonoalamide A, B, C, D, E, or F.

43. The compound of claim 42, wherein the head group comprises phosphonoalanine (PnAla), 2-aminoethylphosphonic acid (2AEP), or a derivative thereof.

44. The compound of claim 42 or claim 43, wherein the tail group comprises alanine, serine, or a derivative thereof.

45. The compound of any one of claims 42-44, wherein the head group comprises PnAla or a derivative thereof and tail group comprises serine or a derivative thereof.

46. The compound of any one of claims 42-45, wherein the head group comprises 2AEP or a derivative thereof and tail group comprises alanine or a derivative thereof.

47. The compound of any one of claims 42-46, wherein the compound comprises Ala-PnAla, Ser-PnAla, Ala-2AEP, or a derivative or salt thereof.

48. The compound of any one of claims 42-47, wherein the head group is C-terminal.

49. The compound of any one of claims 42-48, wherein the compound is a di-peptide or a tripeptide.

50. The compound of any one of claims 42-49, wherein the compound is a salt.

51. The compound of any one of claims 42-50, wherein the compound is a potassium salt, sodium salt, calcium salt, iron salt, ammonium salt, or a combination thereof.

52. The compound of any one of claims 42-51, wherein the compound comprises an agriculturally acceptable salt thereof and/or a pharmaceutically acceptable salt thereof.

53. The compound of any one of claims 42-52, wherein the compound is of any one of claims 1-41.

54. The compound of any one of claims 1-53, wherein the compound is a Bacillus isolate or a derivative or salt thereof.

55. The compound of any one of claims 1-54, wherein the compound is an isolate of B. velezensis, B. swezeyi, B. amyloliquefaciens, B. cabrialesii, B. sublilis. or a combination thereof.

56. The compound of any one of claims 1-55, wherein the compound is an isolate of B. velezensis NRRL B-41850.

57. A composition comprising the compound of any one of claims 1-56.

58. The composition of claim 57, wherein the composition further comprises one or more agriculturally acceptable and/or pharmaceutically acceptable carriers.

59. The composition of claim 57 or claim 58, wherein the composition comprises a pharmaceutical composition, an agricultural composition, or a combination thereof.

60. The composition of any one of claims 57-59, wherein the composition comprises a pesticide.

61. The composition of any one of claims 57-60, wherein the composition comprises an herbicide.

62. The composition of any one of claims 57-61, wherein the composition exhibits antimicrobial activity.

63. The composition of any one of claims 57-62, wherein the composition results in at least 5 log reduction of a population of microbes.

64. The composition of any one of claims 57-63, further comprising a solvent, a carrier, an excipient, or a combination thereof.

65. The composition of any one of claims 57-64, further comprising an agriculturally acceptable adjuvant or carrier.

66. The composition of any one of claims 57-65, wherein the composition is formulated for delivery to a plant or animal.

67. The composition of any one of claims 57-66, wherein the composition is formulated for delivery to a plant.

68. The composition of claim 67, wherein the plant comprises a crop.

69. The composition of any one of claims 57-66, wherein the composition is formulated for delivery to an animal.

70. The composition of claim 69, wherein the animal is a companion animal, livestock, research animal, insect, or human.

71. A nucleic acid encoding the compound or composition of any one of claims 1-70.

72. A vector encoding the nucleic acid of claim 71.

73. A cell comprising the vector of claim 72.

74. A cell comprising the compound or composition of any one of claims 1-70.

75. The cell of claim 73 or claim 74, wherein the cell comprises a Bacillus cell.

76. The cell of any one of claims 73-75, wherein the cell comprises B. velezensis, B. swezeyi, B. amyloliquefaciens, B. cabrialesii, B. sublilis. or a combination thereof.

77. The cell of any one of claims 73-76, wherein the cell comprises B. velezensis NRRL B- 41850.

78. A method of making the compound of any one of claims 1-56.

79. The method of claim 78, wherein the method is a biosynthetic method.

80. The method of claim 78 or claim 79, wherein the method uses one or more enzymes derived from Bacillus.

81. A method of making a compound comprising a head group and a tail group, the head group being bound to the tail group, wherein the head group comprises a phosphonic acid, a phosphinic acid, or a derivative thereof, and the tail group comprises one or more (canonical or non-canonical) amino acids, using one or more enzymes derived from Bacillus, with the proviso that the compound is not phosphonoalamide A, B, C, D, E, or F.

82. The method of claim 81, wherein the head group comprises PnAla, 2AEP, or a derivative thereof.

83. The method of claim 81 or claim 82, wherein the tail group comprises alanine, serine, or a derivative thereof.

84. The method of any one of claims 81-83, wherein the head group comprises PnAla or a derivative thereof and tail group comprises serine or a derivative thereof.

85. The method of any one of claims 81-84, wherein the head group comprises 2AEP or a derivative thereof and tail group comprises alanine or a derivative thereof.

86. The method of any one of claims 81-85, wherein the compound is the compound of any one of claims 1-56.

87. The method of any one of claims 80-86, wherein the one or more enzymes are derived from B. velezensis, B. swezeyi, B. amyloliquefaciens, B. cabrialesii, B. subtilis, or a combination thereof.

88. The method of any one of claims 80-87, wherein the one or more enzymes are derived from A velezensis NRRL B-41850.

89. The method of any one of claims 80-88, wherein the one or more enzymes comprise one or more ATP -grasp enzymes.

90. The method of any one of claims 80-89, wherein the one or more enzymes are encoded by a gene comprising at least 90% identity to pnfA,pnfB, or a combination thereof.

91. The method of any one of claims 80-90, wherein the method proceeds via a linear pathway.

92. The method of any one of claims 78-91, wherein the method comprises contacting a first nucleophile and a first carboxylate with a first enzyme, the first nucleophile comprising a phosphonic acid, a phosphinic acid, or a derivative thereof, and the first carboxylate comprising a first amino acid or a derivative thereof, to thereby form a first compound the first nucleophile bound to the first carboxylate (e.g. a carboxylate-nucleophile).

93. The method of claim 92, wherein the first enzyme is encoded by a gene comprising at least 90% identity to pnfA.

94. The method of claim 92 or claim 93, wherein the first enzyme comprises PnfA.

95. The method of any one of claims 92-94, wherein the first enzyme comprises recombinant PnfA.

96. The method of any one of claims 92-95, wherein the first carboxylate comprises alanine (e.g., L-alanine), serine, or a combination thereof.

97. The method of any one of claims 92-96, wherein the first nucleophile comprises PnAla, 2AEP, or a combination thereof.

98. The method of any one of claims 92-97, wherein the method further comprises contacting the first compound and a second carboxylate with a second enzyme, the first compound being a nucleophile, the second carboxylate comprising a second amino acid or a derivative thereof, to thereby form a second compound comprising the first compound bound to the second carboxylate (e.g. a carboxylate-nucleophile).

99. The method of claim 98, wherein the second enzyme is encoded by a gene comprising at least 90% identity to pnfB.

100. The method of claim 98 or claim 99, wherein the second enzyme comprises PnfB.

101. The method of any one of claims 98-100, wherein the second enzyme comprises recombinant PnfB.

102. The method of any one of claims 98-101, wherein the second carboxylate comprises alanine (e.g., L-alanine).

103. The method of any one of claims 89-102, wherein the method further performed in the presence of ATP.

104. A method of use of the compound, composition, nucleic acid, vector, or cell of any one of claims 1-77.

105. The method of claim 104, wherein the method comprises using the compound, composition, nucleic acid, vector, or cell as an antimicrobial, herbicide, pesticide, or combination thereof to control an undesirable population.

106. The method of claim 105, wherein the method comprises using the compound, composition, nucleic acid, vector, or cell as a pesticide.

107. The method of claim 106, wherein the method comprises using the compound, composition, nucleic acid, vector, or cell to control an undesirable population in plants.

108. The method of claim 107, wherein the method comprises contacting the plants or the locus thereof with or applying to the soil or water the compound, composition, nucleic acid, vector, or cell.

109. The method of any one of claims 106-108, further comprising applying an additional pesticide.

110. The method of any one of claims 105-109, wherein the undesirable population is a herbicide resistant or tolerant population, a pesticide resistant or tolerant population, an antimicrobial resistant or tolerant population, or a combination thereof.

111. The method of any one of claims 105-110, wherein the undesirable population comprises bacteria.

112. A method of reducing the activity of bacteria, the method comprising exposing the bacteria to an effective amount of the compound, composition, nucleic acid, vector, or cell of any one of claims 1-77.

113. A method of reducing bacterial population, the method comprising exposing the bacteria to an effective amount of the compound, composition, nucleic acid, vector, or cell of any one of claims 1-77.

114. A method of killing bacteria, the method comprising exposing the bacteria to an effective amount of the compound, composition, nucleic acid, vector, or cell of any one of claims 1-77.

115. A method for treating, preventing, inhibiting, and/or ameliorating a disease or disorder in a plant or a subject in need thereof, the method comprising administering to the plant or subject a therapeutically effective amount of the compound, composition, nucleic acid, vector, or cell of any one of claims 1-77.

116. The method of claim 115, wherein the disease or disorder comprises an infection, such as a microbial infection.

117. A method for treating, preventing, inhibiting, and/or ameliorating a microbial infection in a plant or a subject, comprising administering to the plant or subject an effective amount of the compound, composition, nucleic acid, vector, or cell of any one of claims 1-77.

118. The method of any one of claims 115-117, wherein the plant is a crop.

119. The method of any one of claims 115-117, wherein the subject is an animal.

120. The method of claim 119, wherein the animal is a companion animal, livestock, research animal, insect, or human.

121. The method of any one of claims 111-120, wherein the compound, composition, nucleic acid, or vector is delivered via cultured Bacillus.

122. The method of any one of claims 111-121, wherein the compound, composition, nucleic acid, or vector is delivered via cultured Bacillus velezensis.

123. The method of any one of claims 111-122, wherein the compound, composition, nucleic acid, or vector is delivered via cultured: B. velezensis, B. swezeyi, B. amyloliquefaciens, B. cabrialesii, B. sublilis, or a combination thereof.

124. The method of any one of claims 111-123, wherein the compound, composition, nucleic acid, or vector is delivered via cultured B. velezensis NRRL B-41850.