CN116529254A - Phosphonate products and methods - Google Patents
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- CN116529254A CN116529254A CN202180074732.3A CN202180074732A CN116529254A CN 116529254 A CN116529254 A CN 116529254A CN 202180074732 A CN202180074732 A CN 202180074732A CN 116529254 A CN116529254 A CN 116529254A
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Abstract
The inventors herein demonstrate that pantoea ananatis (p.ananatis) produces at least three phosphonates, two of which are purified and structurally characterized. The first, named Pan Dalin (pantaphos), was demonstrated as 2- (hydroxy (phosphono) methyl) maleate; the second, one possible biosynthetic precursor, proved to be 2- (phosphonomethyl) maleate. Purified pantaphos are both necessary and sufficient for the marker lesions of onion heart rot. In addition, when mustard seedlings are tested, the phytotoxic activity of pantaphos is comparable to that of the widely used herbicides glyphosate and phosphinothricin. Panthahos is also active against a variety of human cell lines.
Description
RELATED APPLICATIONS
The present application claims priority from U.S. provisional patent application 63/181,745, filed 29 on 4 months 2021, and U.S. provisional patent application 63/075,138, filed 5 months 2020, and the preceding applications are hereby incorporated by reference.
Government funding
The present invention was funded by the U.S. government support under the grant R01 GM127659 provided by National Institutes of Health. The united states government has certain rights in this invention.
Background
Pantoea (Pantoea) species have been identified as plant pathogens since 1928. These gram-negative enterobacteria were initially classified as members of the Erwinia genus, but were subsequently transferred to the Pantoea genus according to the DNA hybridization experiment. Although many Pantoea species are benign or beneficial plant symbiotes, pantoea pineapple (p. Ananatis) strains are always associated with harmful crops and forest insect pests. Since 1983, the host of P.ananatis has been known to have increased to 8 plants, including rice, maize, onion, melon and pineapple, among 11 countries. Once a plant is infected, these bacteria cause internal rot, death and wilt, resulting in serious economic losses. In addition to the major infection in the field, severe post-harvest losses of onion heart rot have also been reported. In addition, such plant pathogens can also infect humans and insects, which act as a vehicle for plant infection. Thus, there is an urgent need to understand the pathogenesis of p.
Although p.ananatis infection has an impact on economy and food safety, the mechanism of plant pathogenicity has recently been studied. Comparative genomic analysis revealed significant differences between the p.ananatis strains, possibly accounting for their ability to colonize and thrive in so many different hosts. Different p.ananatis genomes encode pathogenicity determinants including quorum sensing systems, type VI secretion systems, motor factors, cell wall degrading enzymes and thiosulfate resistant alleles. Recently, by comparing the genomic sequences of two pathogenic and two non-pathogenic P.ananatis strains, a new pathogenic determinant of onion heart rot was revealed (Mol Plant Microbe Interact 2018,31: 1291).
The method identified a genomic island named "HiVir" which was subsequently shown to be present in 14 pathogenic strains and absent in 16 non-pathogenic strains via PCR-based screening. The HiVir locus encodes 11 gene operators (hereinafter hvr), which are suggested to encode the biosynthetic pathway of unknown phosphonate natural products based on the presence of the putative pepM gene. This gene encodes a Phosphonate Enolpyruvate (PEP) phosphonoase that catalyzes the first step of all characterized phosphonate biosynthetic pathways and is widely used as a genetic marker for the ability to produce phosphonate metabolites. Deletion of pepM in P.ananatis OC5a resulted in a severe attenuation of pathogenicity in Allium cepa (onion), demonstrating the required role of the hvr operon in onion heart rot. Based on this finding, asselin et al believe that the small molecule phosphonate is involved in plant disease caused by P.ananatis.
Phosphonates (phosphinates), defined by the presence of chemically stable carbon-phosphorus bonds, are a class of underdeveloped bioactive molecules, which find important applications in both medicine and agriculture. The biological activity of these molecules stems from their structural similarity to phosphates (phosphates) and carboxylic acids (carboxylic acids), which enable them to bind enzymes acting on similar substrates, thus inhibiting the enzymatic activity. One prominent example is the artificial herbicide glyphosate, which was first synthesized by chemists in the 50 s of the 20 th century. The phytotoxicity of glyphosate is due to its inhibition of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, a key enzyme in aromatic amino acid biosynthesis in plants. Notably, the enzyme inhibition of individual phosphonates is very specific and is generally limited to enzymes acting on chemically homologous substrates. Thus, phosphonates may be toxic to one group of organisms and harmless to another group of organisms. Thus, depending on the organism in which the enzyme of interest is found, phosphonates may be used as specific antibacterial, antifungal, antiparasitic and herbicidal compounds. Given the ubiquity of phosphate esters and carboxylic acids in metabolism, phosphonate inhibitors have a wide range of potential biological targets. Indeed, as demonstrated by the known biomass of biologically active phosphonate production, the deadly vulnerability of this metabolism is often exploited in nature. Examples include phosphinothricin tripeptide (phosphinothricin tripeptide) and fosfomycin (fosmidomycin) produced by members of the genus streptomyces, which have potent herbicidal and antibacterial activity due to inhibition of the essential enzymes glutamate synthase and deoxyxylulose-5-phosphate reductase, respectively. Naturally also exploits the fact that the C-P bond is highly stable and resistant to chemical and enzymatic degradation. Thus, many organisms replace labile biomolecules such as phospholipids and phosphate modified extracellular polysaccharides with similar phosphates.
Given their useful biological properties, it is not surprising that biosynthesis of phosphonate compounds is common in microorganisms. Based on the presence of pepM in the sequencing genome and metagenome, about 5% of bacteria have phosphonate biosynthetic capacity. The biosynthetic gene cluster including pepM is known to direct biosynthesis of phospholipids, phosphoglycans, and various small molecule secondary metabolites. As with the streptomycin-derived natural products described above, many small molecule phosphates are biologically active. Despite considerable progress in understanding the biological activity and biosynthesis of small molecule phosphonates, only a portion of the observed gene cluster encoding pepM was characterized. Thus, the degree of chemical diversity of phosphonates in nature has not been determined.
Consistent with the view that phosphonate biosynthesis is common in nature, it is also observed that about 30% of the sequenced bacterial genome contains genes for phosphonate catabolism, which allows them to be used as a source of phosphorus, carbon or nitrogen. Genes encoding carbon-phosphorus (C-P) lyase systems are particularly common in bacteria, which catalyze multi-step phosphonate degradation pathways with broad substrate specificity. Other examples include enzymatic phosphonates specific for aminoethylphosphonate, and the recently characterized oxidative pathways using hydroxymethylphosphonate.
One of the challenges facing agriculture is the emergence of resistance to synthetic herbicides, and the lack of new, effective natural product herbicides. It is estimated that only 7% of conventional pest control agents (including pesticides, fungicides and herbicides) are natural products or natural products. However, as far as herbicidal compounds are concerned, since 1997 only one class of natural product derived herbicides has been registered, and only 8% of herbicidal compounds are natural product derived compared to 30% of bactericides and pesticides. Derivatives of this natural product produced by this strain can be used as alternative organic treatment strategies for herbicide-resistant crops.
There is a problem in that the increased resistance to commercial herbicides poses a threat to agriculture. Thus, there is a need to develop new herbicides to combat this resistance.
Disclosure of Invention
Pantoea ananatis (Pantoea ananatis) is an important plant pathogen, and this problem is compounded by the lack of effective treatments to prevent its spread for many important crops. We determined that Pan Dalin (pantaphos) is a key virulence factor for onion heart rot, suggesting that a variety of approaches may be employed to address this important plant disease. Furthermore, the general phytotoxicity of this molecule suggests that it may be developed as an effective herbicide against the dramatic increase in herbicide-resistant weeds.
Accordingly, the present disclosure provides a composition comprising a compound of formula I:
or a salt thereof; wherein:
represents a single bond or a double bond;
represents a double bond or a single bond, wherein->And->Not both double bonds;
g is X A CHOR 5 ,O,C(＝O),C(＝CH 2 ),CHP(＝O)(R 6 ) 2 Or CX (CX) B 2 ；
X A Is a defect or O;
each X is B Each independently is H or halogen;
R 1 and R is 2 Each independently is OR A Or an amino acid;
R 3 is-C (=O) R 7 Or triazole or tetrazole;
R 4 is-C (=O) R 8 Or triazole or tetrazole;
R 5 is H, - (C) 1 -C 6 ) Alkyl, - (C) 3 -C 6 ) Cycloalkyl, aryl or heteroaryl;
each R 6 Each independently is OR B Or an amino acid;
R 7 and R is 8 Each independently is OR C Or an amino acid; and
R A ，R B and R is C Each independently is H, - (C) 1 -C 6 ) Alkyl, - (C) 3 -C 6 ) Cycloalkyl, aryl or heteroaryl; and
a non-aqueous fluid, an additive, or a combination thereof.
The present disclosure also provides a method of inhibiting weed growth or development comprising contacting the weed and/or soil in which the weed can develop, and a herbicidally effective amount of a composition or compound of the present disclosure, wherein weed growth or development is inhibited.
In addition, the present disclosure provides a method of inhibiting the growth of cancer cells to treat cancer in a subject in need of cancer treatment. In addition, the present disclosure provides methods of forming or preparing 2- (hydroxy (phosphono) methyl) maleic acid and 2-phosphono-methyl maleic acid.
The technology described herein provides novel compounds of formula I and formula II, synthetic intermediates for compounds of formula I and formula II, and methods of preparing compounds of formula I and formula II. The technology also provides compounds of formula I and formula II which are useful as intermediates in the synthesis of other useful compounds.
The technology provides for the use of compounds of formula I and formula II for the manufacture of a medicament for the treatment of cancer in a mammal (e.g., a human).
The technology provides for the use of the compositions described herein in medical treatment or as herbicides. The medical treatment may be treatment of cancer, such as brain, breast, lung, pancreas, prostate or colon cancer. The invention also provides the use of a composition as described herein for the manufacture of a medicament for the treatment of a disease in a mammal (e.g. human cancer). The medicament may include a pharmaceutically acceptable diluent, excipient or carrier.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description, serve to explain the principles of the invention. In some instances, embodiments of the invention may be better understood by reference to the drawings in combination with the detailed description provided herein. The description and drawings may highlight certain specific embodiments or certain aspects of the invention. However, those skilled in the art will appreciate that embodiments or aspects of these parts may also be used in conjunction with other embodiments or aspects of the invention.
FIG. 1. Phenotype of onion heart rot of P.ananatis strain used in the study. Surface sterilized onion bulbs were inoculated as indicated in each picture, then cultured at 30 ℃ for 14 days, and then sectioned to show a central rot phenotype. Panels a-C show sterile water control and inoculation of wild type strains. Panels D-J show vaccination with p.ananatis LMG 5342 mutant derivatives.
FIG. 2 construction of P.ananatis LMG 5342 derivatives with IPTG-induced hvr expression. Plasmid pAP01, which carries the hvrA gene under the control of the IPTG-induced Ptac promoter, was transferred to P.ananatis MMG1998 by conjugation from an E.coli donor. Since pAP01 was unable to autonomously replicate in P.ananatis, a kanamycin-resistant (conferred by the aph gene) conjugation post-cursor was obtained only by chromosomal integration of the homologous recombinant plasmid (as indicated by the dashed line). The resulting strain p.ananatis MMG2010 expressed the entire hvr operon of the Ptac promoter (operon). The position of the native hvr promoter (promoles) is shown as unlabeled curved arrow.
FIG. 3 production of phosphonates (phosphonates) is associated with expression of the hvr operon. Ananatis MMG2010, expressing the hvr operon from IPTG-induced promoter (promoter), grown in glycerol minimal medium with (grown with) and without (grown with out) IPTG. The spent medium is then concentrated and passed through 31 P NMR was performed. Phosphonic acids (phosphonics) generally produce peaks at 5-30ppm, while the chemical shift of the peaks of phosphates (phosphonates) and esters and anhydrides thereof is generally<5ppm。
Fig. 4. Chemical complementation of the Δ hvr onion rot phenotype by ananatis phosphonate. Surface sterilized onion bulbs were inoculated as shown in each picture, then cultured at 30 ℃ for 14 days, and then sectioned to show a heart rot phenotype. Panels a and B show sterile water control and inoculation of the Δ hvr mutant. Panels C and D show inoculation of hvr mutants supplemented with IPTG-induced p.ananatis MMG2010 or purified pantaphos spent medium producing phosphonate. Panels E-F show the inoculation of onions with sterile used medium or purified panaphos in the absence of bacteria.
Fig. 5. Phytotoxicity of pantaphos compared to known herbicides. Mustard seedlings were treated as indicated and incubated for 7 days at 23℃for 16 hours with light cycling. Panel A shows the phenotype observed after incubation with the indicator compound. Panels B and C show root length and dry weight of each replicate after culture. Welch's t test was performed to determine significant differences between treatments (< 0.001, <0.01, <0.05, < n=6 per treatment). Error bars represent standard error of the mean.
FIG. 6 phytotoxicity study of Panthahos on Arabidopsis Col-0. Arabidopsis Col-0 seedlings were treated as indicated and incubated at 23℃and 60% humidity for 7 days under 16 hours of light cycle. The Welch's t test was used to calculate the mean of the different treatments (×p-value <0.001, ×p-value <0.01, ×p-value <0.05; n=6 per treatment). Error bars represent standard error of the mean.
Hvr biosynthetic gene cluster and biosynthetic pathway in FIG. 7.P.ananatis LMG 5342. Based on BLAST search and conserved protein domain analysis, the protein function of the Hvr BGC gene was proposed.
Cytotoxicity of pantaphos against various cancer cell lines. Measurement was performed under the conditions shown (mBio.2021 Feb2;12 (1): e 03402-20), using the Alamar Blue method for 72 hours treatment, using Raptinal (50 mM) as a dead cell control; n=3. Cell seeding density CT26 (colon cancer) 2000c/w; HOS (osteosarcoma) 2500c/w; ES-2 (ovarian cancer), HCT-116 (carcinoma of large intestine), A-549 (lung cancer), A-172 (glioblastoma), D54 (glioblastoma), U87 (glioblastoma), T98G (glioblastoma), SK-ML-28 (melanoma), MCF-7 (breast cancer), AM38 (glioblastoma) and MDA-MB-231 (breast cancer) 3000c/w; u118MG (glioblastoma) 4000c/w; hepG2 (liver cancer) 8000c/w. c/w = number of cells per well. IC shown in Table 6 50 And E is max The values are extracted from the raw data plotted in fig. 8.
Detailed Description
Pantoea ananatis (Pantoea ananatis) is the main cause of onion heart rot. Genetic data indicate that a phosphonic acid natural product is a necessary condition for onion heart rot disease; however, the nature of such molecules is not yet clear. Here we show that p.ananatis produces at least three phosphonates, two of which are purified and structurally characterized. The first, designated pantaphos (Pan Dalin), was demonstrated to be 2-hydroxy (phosphonomethyl) maleate (2-hydroxy (phosphonomethyl) maleate); the second, one possible biosynthetic precursor, proved to be 2- (phosphonomethyl) maleate (2- (phosphomethyl) maleate). Purified pantaphos are both necessary and sufficient for the marker lesions of onion heart rot. In addition, when mustard seedlings are tested, the phytotoxic activity of pantaphos is comparable to that of the widely used herbicides glyphosate and phosphinothricin. Panthahos is also active against a variety of human cell lines, but is significantly more toxic to glioblastoma cells. The pantaphos showed little activity when tested against various bacteria and fungi.
Definition of the definition
The following definitions are provided to provide a clear and consistent understanding of the specification and claims. The terms used herein have the following meanings. Other terms and phrases used in this specification have the ordinary meaning as will be understood by those skilled in the art. Such common meanings may be obtained by reference to the technical dictionary, e.g. Hawley's Condensed Chemical Dictionary 14 th Edition,by R.J.Lewis,John Wiley&Sons,New York,N.Y.,2001。
Reference in the specification to "one embodiment," "an embodiment," or the like, means that a particular aspect, feature, structure, portion, or characteristic is included in the described embodiment, but that not every embodiment necessarily includes the particular aspect, feature, structure, portion, or characteristic. Furthermore, these phrases may, but do not necessarily, apply to the same embodiments mentioned elsewhere in this specification. Furthermore, when a particular aspect, feature, structure, portion, or characteristic is described in connection with an embodiment, it is submitted that it is within the knowledge of one skilled in the art to effect or relate such particular aspect, feature, structure, portion, or characteristic to other embodiments whether or not explicitly described.
The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a compound" may be intended to include a plurality of such compounds, such that compound X includes a plurality of compound X. It is further noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as antecedent basis for use of exclusive terminology such as "solely," "only," and the like, in association with any element described herein and/or as a definition of or as a "negative" limitation on the element recited in the claims.
The term "and/or" means any one, any combination, or all of the relevant items. The phrases "one or more" and "at least one (at least one)" are well understood by those skilled in the art, especially in light of the context. For example, these phrases may refer to one, two, three, four, five, six, ten, one hundred, or any upper value that is about 10, 100, or 1000 times higher than the lower limit already recited. For example, one or more substituents on the phenyl ring refer to one to five, or one to four, for example, if the phenyl ring is disubstituted.
Those skilled in the art will appreciate that any number including the terms of component content, properties such as molecular weight, reaction conditions, etc., may be approximated and modified in various circumstances using the term "about". These values may vary depending on the properties desired by the person skilled in the art by the methods described herein. It should also be appreciated that these values necessarily lead to inherent variability due to the standard deviation that occurs in the test. When values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value without the modifier "about" also forms another aspect.
The terms "about" and "about" are used interchangeably. Both represent that a particular value may have a variation of + -5%, + -10%, + -20%, or + -25%. For example, "about 50" percent may vary from 45 to 55 percent in some embodiments. For an integer range, the term "about" can include one or two integers less or greater than the listed integers at each end of the range. The term "about" is intended to include values approaching the listed range that are equivalent for the function of the ingredient, combination, or embodiment unless the context indicates otherwise. The terms "about" and "approximately" can also be used to adjust the endpoints of the ranges previously described in this paragraph.
It will be appreciated by those skilled in the art that any range provided herein also includes all possible sub-ranges and combinations of these sub-ranges, as well as individual values, particularly integer values, that make up the range, for any of a variety of purposes, particularly for providing a written description. Thus, it should be understood that each unit between two particular units is disclosed. For example, if 10 to 15 are disclosed, 11, 12, 13 and 14, respectively, are also disclosed as part of the scope. The recited ranges (e.g., weight percent or carbon groups) include each specific value, integer, fraction, or characteristic within the recited range. It should be readily understood that any of the ranges recited are fully described and that the same ranges can be broken down into equal halves, thirds, quarters, fifths, or tenths. As a non-limiting example, each of the ranges set forth herein can be readily broken down into a lower third, a middle third, an upper third, and so forth. It will also be understood by those skilled in the art that terms such as "up to", "at least", "above", "below", "more than", "above" or the like include the recited numbers, and that such terms also mean that the range may be subsequently divided into the sub-ranges recited above. Likewise, any ratio listed herein also encompasses sub-ratios that fall within the broader ratio. Accordingly, the specific values of radicals, substituents, and ranges are for illustration only; it does not exclude other specified values or other specified ranges of values for radicals and substituents. It will also be understood that the endpoints of each of the ranges are related to the other endpoint, and independently of the other endpoint.
Provided herein are ranges, limits, and deviations for variables such as volume, mass, percent, ratio, and the like. Those of ordinary skill in the art will appreciate that ranges such as "number1" to "number2" are meant to include continuous numerical ranges of integers and fractions. For example, 1 to 10 represent 1,2,3,4,5, …,9, 10. It also means 1.0,1.1,1.2.1.3, …,9.8,9.9,10.0, 1.01,1.02,1.03, etc. If the disclosed variable is a number less than "number10," then this is meant to encompass a continuous range of integers and decimal numbers less than number10, as described above. Similarly, if the disclosed variable is a number greater than "number10," it means a continuous range including integers and fractions greater than number 10. These ranges may be modified by the term "about," which has been described above.
It should also be readily understood by those skilled in the art that when units are combined in the usual manner, for example in a markush group, the invention includes not only the totality of the combinations of units listed, but also each unit of the group individually as well as any possible subgroup of the basic group. In addition, for all purposes, the present invention includes not only a basic group, but also a group in which the basic group excludes one or more units. It is to be understood that the present invention may include any one or more elements that explicitly exclude the recited groups. Thus, relative constraints may be appended to any disclosed category or embodiment from which any one or more units, species or embodiments may be excluded, for example, for explicit negative definition purposes.
The term "contacting" refers to touching, contacting, or abutting or in close proximity, including, for example, at the cellular or molecular level, such as in a solution or reaction mixture, in which a physiological, chemical, or physical change occurs in vitro or in vivo.
The term "effective amount" refers to an amount that is effective to treat a disease, disorder, and/or condition, or to cause an effect such as activation or inhibition of the described effect. The disease, disorder and/or condition occurs in a living body, such as an animal, plant or crop. For example, an effective amount may be a dose effective to slow the progression or extent of the condition or symptom being treated. Therapeutically effective amounts are well within the ability of those skilled in the art. The term "effective amount" is meant to include the amount of a compound described herein, or the amount of a combination of peptides described herein, e.g., for use in treating or preventing a disease or disorder, or treating a symptom of a disease or disorder, in a subject. Thus, an "effective amount" generally refers to an amount that provides the desired effect.
Alternatively, the term "effective amount" or "therapeutically effective amount (therapeutically effective amount)", as used herein, refers to a sufficient amount of an administered formulation or composition or combination of compositions that will alleviate one or more symptoms of the disease or disorder being treated to some extent. The result may be a reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, an "effective amount" for therapeutic use is that amount of a composition comprising a compound disclosed herein that is required to reduce the symptoms of a disease clinically significantly. The appropriate "effective" amount in any individual case can be determined by techniques such as dose escalation studies. The dose may be used in one or more administrations. However, the precise determination of what is considered an effective dose may be based on individual factors of each patient, including, but not limited to, the age, size, type or extent of disease, disease stage, route of administration of the composition, type or extent of supplemental treatment used, ongoing course of disease, and type of treatment required (e.g., active versus conventional treatment).
The compounds disclosed herein may be used as active ingredients in pharmaceuticals for the treatment of animals, or as active ingredients in herbicide formulations for the treatment of plants or crops. The active ingredient in the agent or herbicide is applied in an amount effective to treat a disease, disorder, and/or condition in an animal, plant, or crop.
The term "treatment" includes (i) preventing the occurrence of a disease, pathology or medical condition (e.g., preventing); (ii) Inhibiting or arresting the development of a disease, pathology, or medical condition; (iii) alleviating a disease, pathology, or medical condition; and/or (iv) alleviating symptoms associated with a disease, pathology, or medical condition. Thus, the terms "treat," "treating" and "treatment" are intended to extend to prophylaxis and may include preventing, reducing, halting or reversing the progression or severity of the condition or symptom being treated. Thus, where appropriate, the term "treatment" may include medical, therapeutic and/or prophylactic administration.
The term "subject" or "patient" as used herein refers to an individual having symptoms of a disease or other malignancy or a risk thereof. The patient may be a human or a non-human, and may include, for example, an animal strain or species used as a "model system" for research purposes, such as a mouse model as described herein. Also, the patient may include an adult or a juvenile (e.g., a child). Furthermore, a patient may refer to any living organism, preferably a mammal (e.g., human or non-human), that may benefit from administration of the compositions contemplated herein. Examples of mammals include, but are not limited to, any member of the mammalian class: a human being; non-human primates, such as chimpanzees; other apes and monkey species; livestock, such as cattle, horses, sheep, goats, and pigs; domestic animals such as rabbits, dogs and cats; laboratory animals, including rodents, such as rats, mice, guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish, and the like. In one embodiment of the methods described herein, the mammal is a human.
The terms "providing", "administering", "introducing" and "introducing" are used interchangeably herein and refer to placing a composition of the invention in a subject by a method or route that at least partially places the composition in a desired location. The composition may be administered by any suitable route of administration that delivers to a desired location in a subject.
The compositions described herein may be administered with other compositions to extend the stability and activity of the compositions, or in combination with other therapeutic agents.
The terms "inhibit" and "inhibit" refer to slowing, preventing or reversing the growth or progression of a disease, infection, condition or cell population. Inhibition may be greater than about 20%,40%,60%,80%,90%,95% or 99%, for example, as compared to the extent of growth or progression that occurs without treatment or management.
The term "effective amount" refers to an amount effective to produce the effect, e.g., the amount required to form a product in a reaction mixture. Determination of an effective amount is generally within the ability of one skilled in the art, particularly in light of the detailed disclosure provided herein. The term "effective amount" is intended to include the amount of a compound or agent described herein, or the amount of a combination of compounds or agents described herein, e.g., an amount effective to form a product in a reaction mixture. Thus, an "effective amount" generally refers to an amount that provides the desired effect.
The term "substantially" as used herein is a broad term and is used in its ordinary sense, including, but not limited to, substantially, but not necessarily entirely, as specified. For example, the term may refer to a value that may not be 100% of the full value referred to. The complete value may be less than about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, or about 20%.
Herein, whenever the term "comprising" is used, it is considered that the term "consisting of … …" or "consisting essentially of … … (consisting essentially of)" is used alternatively. As used herein, "comprising" and "including", "containing" or "characterized by" and are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. As used herein, "consisting of … …" excludes any element, step or component not specified in the described aspects. Herein, "consisting essentially of … … (consisting essentially of)" does not exclude materials or steps that have no substantial effect on the essential and novel features of the aspect. In each case herein, any of the terms "comprising," "consisting essentially of … … (consisting essentially of)" and "consisting of … … (collocation of)" may be substituted with any of the other two terms. The disclosure illustratively disclosed herein suitably may be practiced in the absence of any element or elements or limitations which are not specifically disclosed herein.
The formulas and compounds described herein may be modified with protecting groups. Suitable amino and carboxyl protecting groups are known to those skilled in the art (see, e.g., protecting Groups in Organic Synthesis, second Edition, greene, T.W., and Wutz, P.G.M., john Wiley & Sons, new York, and references therein; philip J.Kocienski; protecting Groups (Georg Thieme Verlag Stuttgart, new York, 1994), and references therein); and Comprehensive Organic Transformations, larock, r.c., second Edition, john Wiley & Sons, new York (1999), and references therein.
The term "halo" or "halogenated" refers to fluoro, chloro, bromo or iodo. Also, the term "halogen" refers to fluorine, chlorine, bromine and iodine.
The term "alkyl" refers to a straight or branched chain alkyl group, for example having 1 to 20 carbon atoms, typically 1 to 12,1 to 10,1 to 8,1 to 6 or 1 to 4 carbon atoms; or for example in the range between 1 and 20 carbon atoms, for example 2 to 6, 3 to 6, 2 to 8 or 3 to 8 carbon atoms. As used herein, the term "alkyl" also includes "cycloalkyl" as defined below. Examples include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl (isopropyl), 1-butyl, 2-methyl-1-propyl (isobutyl), 2-butyl (sec-butyl), -2-methyl-2-propyl (tert-butyl), 1-pentyl, 2-pentyl, 3-pentyl, -2-methyl-2-butyl, 3-ethyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 2-methyl-3-pentyl, 2, 3-dimethyl-2-butyl, 3-dimethyl-2-butyl, hexyl, octyl, decyl, dodecyl, and the like. The alkyl group may be unsubstituted or optionally substituted, for example by the substituents described below. The alkyl groups may also optionally be partially or fully unsaturated. Thus, in certain embodiments, an alkyl group optionally contains an alkenyl or alkynyl group. The alkyl group may be a monovalent hydrocarbon group as listed above, or may be a divalent hydrocarbon group (i.e., alkylene group), depending on the context of its use.
An alkylene (alkylene) is an alkyl group having two free valences on one carbon atom or on two different carbon atoms of the carbon chain. Similarly, alkenylene and alkynylene are alkenyl and alkynyl groups, respectively, having two free valencies on two different carbon atoms.
The term "cycloalkyl" refers to a cyclic alkyl group having a single ring or multiple condensed rings, e.g., consisting of 3 to 10 carbon atoms. For example, cycloalkyl includes a monocyclic structure such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or a polycyclic structure such as adamantyl and the like. Cycloalkyl groups may be unsubstituted or substituted. Cycloalkyl groups may be monovalent or divalent and may optionally be substituted as described for alkyl groups. Cycloalkyl groups may optionally include one or more unsaturated groups, for example, cycloalkyl groups may include one or more carbon-carbon double bonds, for example, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, and the like.
The term "heterocycloalkyl" or "heterocyclyl" refers to a saturated or partially saturated monocyclic, bicyclic or polycyclic ring radical containing at least one heteroatom selected from nitrogen, sulfur, oxygen, preferably 1 to 3 heteroatoms in at least one ring. Each ring preferably has 3 to 10 members, more preferably 4 to 7 members. Suitable heterocycloalkyl substituents include pyrrolidinyl, tetrahydrofuranyl, tetrahydrothiofuranyl, piperidinyl, piperazinyl, tetrahydropyranyl, morpholinyl, 1, 3-diazapentanyl, 1, 4-oxaproyl, and 1, 4-oxathianyl. The groups may be terminal groups or bridging groups.
The term "aryl" refers to an aromatic hydrocarbon group obtained by removing at least one hydrogen atom from a single carbon atom of a parent aromatic ring system. The radical attachment site may be located at a saturated or unsaturated carbon atom of the parent ring system. The aryl group may have 6 to 30 carbon atoms, for example about 6-10 carbon atoms. Aryl groups may have a single ring (e.g., phenyl) or multiple condensed (fused) rings, wherein at least one ring is aromatic (e.g., naphthyl, dihydrophenanthryl, fluorenyl, or anthracenyl). Typical aryl groups include, but are not limited to, groups derived from benzene, naphthalene, anthracene, biphenyl, and the like. Aryl groups may be unsubstituted or optionally substituted with substituents described below.
The term "heteroaryl" refers to a monocyclic, bicyclic or tricyclic ring system containing one, two or three aromatic rings, and at least one nitrogen, oxygen or sulfur atom is contained in an aromatic ring. As described in the definition of the term "substituted", heteroaryl groups may be unsubstituted or substituted with one or more substituents, in particular with one to three substituents. Typical heteroaryl groups contain 2 to 20 carbon atoms in addition to one or more heteroatoms in the ring backbone, wherein the ring backbone comprises a 5-membered ring, a 6-membered ring, two 5-membered rings, two 6-membered rings, or one 5-membered ring fused to one 6-membered ring. Examples of heteroaryl groups include, but are not limited to, 2H-pyrrolyl, 3H-indolyl, 4H-quinolinyl, acridinyl, benzo [ b ] thiophenyl, benzothiazolyl, beta-carbolinyl, carbazolyl, chromen-yl, xin Nuoji, dibenzo [ b, d ] furanyl, imidazolyl, imido, indazolyl, indolinyl, indolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthyridinyl, oxazolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenylarsinyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolinyl, quinoxalinyl, thiadiazolyl, thianthrenyl, thiazolyl, thienyl, triazolyl, tetrazolyl, and xanthinyl. In one embodiment, the term "heteroaryl" refers to a monocyclic aromatic ring containing 5 or 6 ring atoms, wherein the carbon atoms and 1, 2, 3 or 4 heteroatoms are independently selected from the group consisting of non-peroxide oxygen, sulfur and N (Z), wherein Z is absent or H, O, alkyl, aryl or (C1-C6) alkylaryl. In some embodiments, heteroaryl refers to ortho-fused bicyclic heterocycles derived therefrom having about 8 to 10 ring atoms, particularly benzene ring derivatives or heterocycles derived from the fusion of propylene, trimethylene or tetramethylene diyl.
As used herein, the term "substituted" or "substituent" is used to denote that one or more (e.g., 1-20 in some embodiments, 1-10 in other embodiments, 1, 2, 3, 4, or 5 in some embodiments, 1, 2, or 3 in other embodiments, and 1 or 2) hydrogens on the group denoted by "substituted" (or "substituent") are taken to be selected from the group specified or the appropriate groups known to those of skill in the artSubstitution, provided that the normal valency of the atom shown is not exceeded, and substitution results in a stable compound. Suitable such specified groups include, for example, alkyl, alkenyl, alkynyl, alkoxy, haloalkyl, hydroxyalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, alkylamino, dialkylamino, carboxyalkyl, alkylthio, alkylsulfinyl and alkylsulfonyl. Substituents for the groups shown may be those described in the specific list of substituents described herein, or may be selected from alkyl, alkenyl, alkynyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, alkylamino, dialkylamino, trifluoromethylthio, difluoromethyl, amido, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thio, alkylthio, alkylsulfinyl, alkylsulfonyl and cyano as will be appreciated by those skilled in the art. Suitable substituents for the groups shown may be bonded to substituted carbon atoms, including F, cl, br, I, OR ', OC (O) N (R') 2 ,CN,CF 3 ,OCF 3 R ', O, S, C (O), S (O), methylenedioxy, ethylenedioxy, N (R') 2 ,SR',SOR',SO 2 R',SO 2 N(R') 2 ,SO 3 R',C(O)R',C(O)C(O)R',C(O)CH 2 C(O)R',C(S)R',C(O)OR',OC(O)R',C(O)N(R') 2 ,OC(O)N(R') 2 ,C(S)N(R') 2 ,(CH 2 ) 0-2 NHC(O)R',N(R')N(R')C(O)R',N(R')N(R')C(O)OR',N(R')N(R')CON(R') 2 ,N(R')SO 2 R',N(R')SO 2 N(R') 2 ,N(R')C(O)OR',N(R')C(O)R',N(R')C(S)R',N(R')C(O)N(R') 2 ,N(R')C(S)N(R') 2 ,N(COR')COR',N(OR')R',C(＝NH)N(R') 2 C (O) N (OR ') R ', OR C (=nor ') R ', wherein each R ' may independently be hydrogen OR a carbon moiety (e.g., (C) 1 -C 6 ) Alkyl), and wherein the carbon moiety itself may be further substituted. When the substituent is monovalent, for example F or Cl, it is bonded to the atom it is substituted for by a single bond. When a substituent is divalent, such as O, it is double bonded to the atom it is substituted for; for example, carbon atoms substituted by OCarbonyl is formed, c=o.
The stereochemical definitions and conventions used herein generally follow S.P. Parker, ed., mcGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, new York; and Eliel, e.and Wilen, s., "Stereochemistry of Organic Compounds", john Wiley & Sons, inc., new York,1994. The compounds of the invention may contain asymmetric or chiral centers and thus exist in different stereoisomeric forms. All stereoisomeric forms of the compounds of the invention, including but not limited to diastereomers, enantiomers and diastereomers, and mixtures thereof, such as racemic mixtures, form part of the present invention. Many organic compounds exist in optically active form, i.e. they have the ability to rotate plane-polarized light planes. In describing compounds having optical activity, the prefixes D and L or R and S are used to represent the absolute configuration of the molecule with respect to its chiral center. The prefixes d and l or (+) and (-) are used to represent the sign that plane polarized light is rotated by the compound, (-) or l represents that the compound is left-handed and the compound prefixed with (+) or d is right-handed. For a given chemical structure, these stereoisomers are identical except that they are mirror images of each other. Certain stereoisomers may also be referred to as enantiomers and mixtures of such isomers are commonly referred to as enantiomeric mixtures. The 50:50 enantiomeric mixture is referred to as a racemic mixture or racemic enantiomer (defined below), which may occur in the absence of stereoselectivity or stereospecificity in a chemical reaction or process.
The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomers, without optical activity.
The term "IC 50 "generally defined as the concentration required to kill 50% of the cells within 24 hours.
The term "genome" or "genome DNA (genomic DNA)" refers to heritable genetic information of a host organism. The genomic DNA includes all genetic material of cells or organisms, including bacterial chromosomal DNA and prokaryotic plasmids, and includes eukaryotic nuclear DNA (chromosomal DNA), extrachromosomal DNA, and organ DNA (e.g., mitochondria). Preferably, the term genomic or genomic DNA refers to chromosomal DNA of the nucleus.
In the case of eukaryotic cells, the term "chromosome DNA (chromosomal DNA)" or "chromosomal DNA sequence (chromosomal DNA sequence)" is understood to mean the genomic DNA of the cell nucleus independent of the cell cycle state. Thus, chromosomal DNA may be organized in chromosomes or chromatids, which may be compressed or expanded. Insertion of chromosomal DNA can be demonstrated and analyzed by various methods known in the art, such as Polymerase Chain Reaction (PCR) analysis, salsep blot analysis, fluorescence In Situ Hybridization (FISH), in situ PCR, and Next Generation Sequencing (NGS).
The term "promoter" refers to a polynucleotide that directs transcription of a structural gene to produce mRNA. Typically, the promoter is located in the 5' region of the gene, near the start codon of the structural gene. If the promoter is an inducible promoter, the transcription rate will be responsive to the inducer. In contrast, if the promoter is a constitutive promoter, the transcription rate is not regulated by the inducer. The term "enhancer" refers to a polynucleotide. Enhancers increase the efficiency of transcription of a particular gene into mRNA regardless of the distance or direction of the enhancer relative to the transcription initiation site. Typically, the enhancer is located near the promoter, 5' -untranslated sequence, or intron.
The polynucleotide is "heterologous" to the organism, and is a second polynucleotide if it is from a foreign species, or if it is from the same species, modified from its original form. For example, a promoter operably linked to a heterologous coding sequence refers to a coding sequence from a species other than the source of the promoter, or, if from the same species, a coding sequence not naturally associated with the promoter (e.g., a genetically engineered coding sequence or allele from a different genotype or variety).
"Transgene", "transgenic" or "recombinant" refers to a polynucleotide manipulated by a human or a copy or complement of a polynucleotide manipulated by a human. For example, a transgenic expression cassette comprising a promoter operably linked to a second polynucleotide may comprise a promoter heterologous to the second polynucleic acid as a result of human manipulation of an isolated nucleic acid comprising the expression cassette (e.g., sambrook et al, molecular Cloning-A Laboratory Manual, cold Spring Harbor Laboratory, cold Spring Harbor, new York, (1989) or Current Protocols in Molecular Biology Volumes 1-3,John Wiley&Sons,Inc (1994-1998)). In another example, a recombinant expression cassette may comprise polynucleotides combined in such a way that the polynucleotides are highly unlikely to be found in nature. For example, a restriction site or plasmid vector sequence that is manipulated by a human may flank or isolate the promoter from the second polynucleotide. Those skilled in the art will recognize that polynucleotides may be manipulated in a number of ways, and are not limited to the above examples only.
If the term "recombinant" is used to designate an organism or cell, such as a microorganism, it is used to indicate that the organism or cell comprises at least one "transgenic", "transgenic" or "recombinant" polynucleotide, which is generally specified later.
A polynucleotide "exogenous" to a single organism refers to a polynucleotide that is introduced into the organism by any means other than sexual hybridization.
The term "operably linked" or "operably linked" is generally understood to mean an arrangement in which a genetic control sequence (e.g., a promoter, enhancer or terminator) is capable of functioning with respect to a polynucleotide (e.g., a polynucleotide encoding a polypeptide) to which it is operably linked. The function, in this case, may be referred to, for example, as controlling the expression, i.e.transcription and/or translation, of the nucleic acid sequence. The control, in this case, includes, for example, the initiation, increase, control or inhibition of expression, i.e.transcription, translation (if appropriate). Accordingly, the control may be, for example, tissue and/or time specific. It may also be inducible by, for example, certain chemicals, stress, pathogens, etc. Preferably, an operable linkage is understood as a sequential arrangement of, for example, a promoter, a nucleic acid sequence to be expressed and, if appropriate, further regulatory elements such as terminators, in such a way that each of the regulatory elements is able to fulfill its function when the nucleic acid sequence is expressed. An operable linkage does not necessarily require a direct linkage in a chemical sense. For example, genetic control sequences such as enhancer sequences can also function to a target sequence from a location remote from the polynucleotide to which the polynucleotide is operably linked. The preferred arrangement is that the nucleic acid sequence to be expressed is located after the sequence which acts as a promoter, so that the two sequences are covalently linked to one another. The distance between the promoter and the amino acid sequence of the coding polynucleotide in the expression cassette is preferably less than 200 base pairs, particularly preferably less than 100 base pairs, more preferably less than 50 base pairs. Those skilled in the art are familiar with various methods to obtain such expression cassettes. However, the expression cassette may also be constructed such that the nucleic acid sequence to be expressed is placed under the control of endogenous genetic control elements, such as endogenous promoters, for example, by homologous recombination or random insertion. For the purposes of the present invention, such constructs are likewise understood as expression cassettes.
The term "expression cassette (expression cassette)" or "expression vector (expression vector)" refers to those constructs in which a nucleic acid sequence encoding an amino acid sequence to be expressed is operably linked to at least one gene control element capable of or regulating its expression (i.e., transcription and/or translation). For example, the expression may be stable or transient, constitutive or generalized. Examples of expression vectors are well known in the art and are described, for example, in U.S. Pat. No.7,416,874.
The term "expression" refers to the expression of a gene product (e.g., a biosynthetic enzyme of a gene of a pathway or reaction defined and described herein) at a level that is the activity of the enzyme produced encoded by the protein or the pathway or reaction to which it refers that allows metabolic flux through the pathway or reaction in an organism expressing the gene/pathway. Such expression may be achieved by genetic alteration of the microorganism used as the starting organism. In some embodiments, the microorganism may be genetically altered (e.g., by genetic engineering) to express a gene product at an increased level relative to the level of production by the starting microorganism or in a comparable microorganism that is not altered. Genetic alterations include, but are not limited to, altering or modifying regulatory sequences or sites associated with expression of a particular gene (e.g., by adding a strong promoter, an inducible promoter or promoters, or by removing regulatory sequences that make up expression), modifying the chromosomal location of a particular gene, altering nucleic acid sequences in the vicinity of a particular gene, such as ribosome binding sites or transcription terminators, increasing the copy number of a particular gene, modifying proteins involved in transcription of a particular gene and/or translation of a particular gene product (e.g., regulatory proteins, suppressors, enhancers, transcriptional activators, etc.), or any other conventional means for relieving expression of a particular gene using methods conventional in the art (including, but not limited to, the use of antisense nucleic acid molecules, e.g., to prevent expression of a suppressor protein).
In some embodiments, the microorganism may be physically or environmentally altered to express the gene product at a level that is increased or decreased relative to the level of expression of the unaltered microorganism. For example, the microorganism may be treated with or cultured in the presence of an agent known or perceived to increase transcription of a particular gene and/or translation of a particular gene product, thereby enhancing or increasing transcription and/or translation. Alternatively, the microorganism may be cultured at a temperature selected to increase transcription of a particular gene and/or translation of a particular gene product, thereby enhancing or increasing transcription and/or translation.
The term "vector" preferably includes phage, plasmid, egg, viral vectors, and artificial chromosomes, such as bacterial or yeast artificial chromosomes. Furthermore, the term also relates to targeting constructs that allow random or site-directed integration of the targeting construct into genomic DNA. Such target constructs, preferably, comprise DNA of sufficient length for homologous or heterologous recombination as detailed below. Preferably, the vector comprising the polynucleotide of the invention further comprises a selectable marker for propagation and/or selection in a recombinant microorganism. The vector may be incorporated into a recombinant microorganism by a variety of techniques well known in the art. If introduced into a recombinant microorganism, the vector may reside in the cytoplasm or may be incorporated into the genome. In the latter case, it is to be understood that the vector may also comprise nucleic acid sequences which allow homologous recombination or heterologous insertion. The vector may be introduced into a prokaryotic or eukaryotic cell by conventional transformation or transfection techniques.
The terms "transformation" and "transfection", coupling and transduction ", as used herein, are intended to include a variety of prior art processes for introducing exogenous nucleic acid (e.g., DNA) into a recombinant microorganism, including calcium phosphate, rubidium chloride or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipid modification, native activity, carbon-based clusters, chemical-mediated transfer, electroporation or particle bombardment. Methods for many kinds of microorganisms are readily available in the literature.
Unless otherwise indicated, the nucleic acid sequences recited herein are written in the 5 'to 3' direction. The term "nucleic acid" refers to DNA or RNA or modified forms thereof, including purine or pyrimidine bases present in DNA (adenine "a", cytosine "C", guanine "G", thymine "T") or RNA (adenine "a", cytosine "C", guanine "G", uracil "U"). The interfering RNA provided herein can comprise a "T" base, for example at the 3' end, even though the "T" base does not naturally occur in the RNA. In some cases, these bases may be present in the form of "dT" to distinguish deoxyribonucleotides present in a ribonucleotide chain.
The term "sequence identity (sequence identity)" between two nucleic acid sequences is understood to mean the percentage of identity of the nucleic acid sequences over the entire sequence length in each case, calculated by the alignment of the program algorithm GAP (Wisconsin Package Version10.0,University of Wisconsin,Genetics Computer Group (GCG), madison, USA), the following parameters being set, for example: gap weight: 12, length weight: 4, a step of; average matching: 2,912, average mismatch: -2,003.
The term "sequence identity (sequence identity)" between two amino acid sequences is understood as meaning the percentage of identity of the amino acid sequences over the entire sequence length in each case, calculated by the alignment of the program algorithm GAP (Wisconsin Package version10.0, university of Wisconsin, genetics Computer Group (GCG), madison, USA), the following parameters being set, for example: gap weight: 8, 8; length weight: 2; average matching: 2,912; average mismatch: -2003.
The term "hybridization" as defined herein is a process in which substantially homologous complementary nucleotide sequences anneal to each other. The hybridization process may occur entirely in solution, i.e., both complementary nucleic acids are in solution. Hybridization can also occur with one of the complementary nucleic acids immobilized on a matrix such as magnetic beads, agarose gel beads, or any other resin. The hybridization process can also fix one of the complementary nucleic acids to a solid support, such as nitrocellulose or nylon membrane, or to a siliceous glass support, for example, by photolithography (the latter being referred to as a nucleic acid array or microarray or nucleic acid chip). To allow hybridization to occur, the nucleic acid molecule is typically thermally or chemically denatured, fusing the double strand into two single strands and/or removing hairpins or other secondary structures from the single stranded nucleic acid.
The term "stringency" refers to the condition under which hybridization occurs. The stringency of hybridization is affected by conditions such as temperature, salt concentration, ionic strength, and hybridization buffer composition. In general, for a particular sequence, at a defined ionic strength and pH, the low stringency conditions are selected to be the specific thermal melting point (T m ) Is lower by about 30 ℃. Moderately stringent conditions are below T m 20 ℃ and the high stringency conditions are lower than T m 10 ℃. High stringency hybridization conditions are typically used to isolate hybridization sequences that have a high degree of sequence similarity to the target nucleic acid sequence. However, due to the degeneracy of the genetic code, nucleic acids may deviate in sequence but still encode substantially the same polypeptide. Thus, hybridization conditions of moderate stringency may sometimes be required to identify such nucleic acid molecules.
T m Is the temperature at which 50% of the target sequence hybridizes to a perfectly matched probe at a defined ionic strength and pH. T (T) m Depending on the solution conditions and the probeBase composition and length of the needle. For example, longer sequences hybridize specifically at higher temperatures. At T m The maximum hybridization rate is obtained between about 16℃and 32℃below. The presence of monovalent cations in the hybridization solution reduces electrostatic repulsion between the two nucleic acid strands, thereby promoting hybridization formation; this effect is evident at sodium concentrations below 0.4M (this effect is negligible for higher concentrations of sodium). Formamide lowers the melting temperature of the DNA-DNA and DNA-RNA duplex, 0.6 to 0.7 ℃ per percent formamide, and the addition of 50% formamide allows hybridization to occur at 30 to 45 ℃, although the hybridization rate will decrease. Base pair mismatches reduce the hybridization rate and duplex thermal stability. For large probes, average per% base mismatch, T m Reduced by about 1 deg.c. T, depending on the type of hybrid m The following formula can be used for calculation:
1) DNA-DNA hybridization (Meinkoth and Wahl, anal. Biochem.,138:267-284,1984):
T m ＝81.5℃+16.6xlogio[Na + ] a +0.41x％[G/C b ]-500x[L c ]- 1 -0.61x% formamide
2) DNA-RNA or RNA-RNA hybridization:
T m ＝79.8℃+18.5(logio[Na + ] a )+0.58(％G/C b )+1 1.8(％G/C b ) 2 -820/L c
3) oligo-DNA or oligo-RNA d :
For the following<20 nucleotides T m ＝2(l n )
For 20-35 nucleotides T m ＝22+1.46(l n )
a or other monovalent cation, but only in the range of 0.01-0.4M.
b is accurate only for% GC in the range of 30% -75%.
c L = length of duplex in base pair.
d oligo oligonucleotides; l (L) n (effective length of primer = 2x (number of G/C) + (number of a/T).
Nonspecific binding can be controlled using any of a number of known techniques, for example, blocking the membrane with a solution containing the protein, adding heterologous RNA, DNA, and SDS to the hybridization buffer, and treatment with RNAse. For non-homologous probes, a series of hybridizations can be performed by changing one of (i) a gradual decrease in annealing temperature (e.g., from 68 ℃ to 42 ℃) or (ii) a gradual decrease in formamide concentration (e.g., from 50% to 0%). Those skilled in the art will be aware of various parameters that may be altered during hybridization, which will maintain or alter stringency conditions.
In addition to hybridization conditions, the specificity of hybridization is generally dependent on the function of post-hybridization washes. To remove background from non-specific hybridization, the samples were washed with dilute saline. Key factors for such washing include the ionic strength and temperature of the final wash solution, the lower the salt concentration, the higher the wash temperature and the higher the severity of the wash. Washing conditions are generally carried out at or below hybridization stringency. Positive hybridization produces a signal at least twice that of the background signal. In general, suitable stringent conditions for the nucleic acid hybridization assay or gene amplification detection procedure are as follows. More or less stringent conditions may also be selected. Those skilled in the art will be aware of various parameters that may change during the wash process that will maintain or change the stringency conditions.
For example, typical high stringency hybridization conditions for DNA hybridization that is more than 50 nucleotides in length include hybridization in 1 XSSC at 65℃or hybridization in 1 XSSC at 42℃and 50% formamide, followed by washing in 0.3 XSSC at 65 ℃. Examples of moderately stringent hybridization conditions for DNA hybridization of more than 50 nucleotides in length include hybridization in 4 XSSC at 50℃or hybridization in 6 XSSC at 40℃and 50% formamide, followed by washing in 2 XSSC at 50 ℃. The length of the hybrid is the desired length of the hybrid nucleic acid. When hybridizing nucleic acids of known sequences, the length of hybridization can be determined by aligning the sequences and identifying the conserved regions described herein. 1 XSSC is 0.15M NaCl and 15mM sodium citrate; the hybridization solution and the washing solution can also be added with 5x Denhardt's reagent, 0.5-1.0% SDS, 100 mug/mL denatured fragmented salmon sperm DNA, 0.5% sodium pyrophosphate.
For definition of the degree of stringency, reference may be made to Sambrook et al (2001)Molecular Cloning:a laboratory manual,3 rd Edition, cold Spring Harbor Laboratory Press, CSH, new York or Current Protocols in Molecular Biology, john Wiley&Sons, n.y. (1989 and yearly updates).
Embodiments of the present technology
The present disclosure provides a composition comprising a compound of formula I:
Or a salt thereof; wherein:
represents a single bond or a double bond;
represents a double bond or a single bond, wherein->And->Not both double bonds;
g is X A CHOR 5 ,O,C(＝O),C(＝CH 2 ),CHP(＝O)(R 6 ) 2 Or CX (CX) B 2 ；
X A Is a defect or O;
each X is B Each independently is H or halogen;
R 1 and R is 2 Each independently is OR A Or an amino acid;
R 3 is-C (=O) R 7 Or triazole or tetrazole;
R 4 is-C (=O) R 8 Or triazole or tetrazole;
R 5 is H, - (C) 1 -C 6 ) Alkyl (C)Radical- (C) 3 -C 6 ) Cycloalkyl, aryl or heteroaryl;
each R 6 Each independently is OR B Or an amino acid;
R 7 and R is 8 Each independently is OR C Or an amino acid; and
R A 、R B and R is C Each independently is H, - (C) 1 -C 6 ) Alkyl, - (C) 3 -C 6 ) Cycloalkyl, aryl or heteroaryl; and liquids (aqueous or non-aqueous), additives (non-naturally occurring), or combinations thereof.
In various embodiments, R 1 、R 2 、R 6 、R 7 And R is 8 Each independently is NR X R Y Wherein R is X And R is Y Each independently is H, - (C) 1 -C 6 ) Alkyl, - (C) 3 -C 6 ) Cycloalkyl, aryl or heteroaryl. In further embodiments, R 3 And R is 4 Each independently is NO 2 、CO 2 R X 、P(＝O)(OR X ) 2 、S(＝O) 2 OR X Or S (=O) R X Wherein each R is X Each independently is H, - (C) 1 -C 6 ) Alkyl or- (C) 1 -C 6 ) Cycloalkyl groups. In further embodiments, the compound is a prodrug. In further embodiments, the compounds are appropriately substituted (e.g., forming esters) that are metabolized or cleaved to release the active form of the compounds (e.g., pantaphos).
In some embodiments, the fluid is water or an aqueous solution, a non-aqueous fluid or solution, an oil, an organic solvent, a liquid, or a combination thereof. In other embodiments, the composition is formulated as a powder, fine powder, granule, or pellet. The formulation may include additives, salts, emulsifiers, nanoparticles, surfactants, buffers, wetting agents, colloids, lipids, phospholipids, biodegradable polymers, secondary actives, one or more actives, or combinations thereof. In other embodiments, the compounds in the composition may be encapsulated in microcapsules or nanocapsules or extended release capsules.
In various embodiments, G is CHOR 5 . In various embodiments, the compound is the (S) -enantiomer. In various embodiments, the compound is the (R) -enantiomer. In various embodiments, R 1 And R is 2 Is OR (OR) A . In various embodiments, R 3 And R is 4 is-CO 2 R C . In various embodiments, R 3 And R is 4 When (when)In the case of double bonds, cis-configuration is present.
In some embodiments, the compound of formula I is represented by formula II:
or a salt thereof.
In various embodiments, the compounds are pantaphos:
in various embodiments, the compound is compound 2:
In some embodiments, the composition comprises pantaphos and compound 2.
The present disclosure also provides compounds of formula I:
or a salt thereof; wherein:
represents a single bond or a double bond;
represents a double bond or a single bond, wherein->And->Not both double bonds;
g is X A CHOR 5 ,O,C(＝O),C(＝CH 2 ),CHP(＝O)(R 6 ) 2 Or CX (CX) B 2 ；
X A Is a defect or O;
each X is B Each independently is H or halogen;
R 1 and R is 2 Each independently is OR A Or an amino acid;
R 3 is-C (=O) R 7 Or triazole or tetrazole;
R 4 is-C (=O) R 8 Or triazole or tetrazole;
R 5 is H, - (C) 1 -C 6 ) Alkyl, - (C) 3 -C 6 ) Cycloalkyl, aryl or heteroaryl;
each R 6 Each independently is OR B Or an amino acid;
R 7 and R is 8 Each independently is OR C Or an amino acid; and
R A 、R B and R is C Each independently is H, - (C) 1 -C 6 ) Alkyl, - (C) 3 -C 6 ) Cycloalkyl, aryl or heteroaryl.
In some embodiments, the compound is not a natural product. In some embodiments, the compound is not 2- (hydroxy (phosphonomethyl) maleic acid or 2- (phosphonomethyl) maleic acid. At each ofIn one embodiment, G is CHOH. In various embodiments, R 1 And R is 2 Is OH. In various embodiments, R 3 And R is 4 is-CO 2 H。
In some other embodiments, the compound is represented by one of the following structures:
/>
( And (3) injection: Δshift=Δshift; Δreduction = Δreduction; alkyl = alkyl; ester = Ester; alcohol manipulation = ethanol operation; difluoromethylene phosponate = difluoromethylene phosphonate; phosphate = phosphate; di-phosphate = bisphosphonate; tetrazoles=tetrazole )
In some embodiments, the compound is represented by:
or an enantiomer thereof, or a salt thereof,
wherein R is 1 、R 2 And R is 3 Each independently is a carboxylate, phosphonate, nitrate, sulfonate, sulfoxide, or a combination thereof;
wherein R is 1 、R 2 、R 3 And R is 4 Each independently is H, alkyl, aryl, or a combination thereof;
wherein R is 1 And R is 2 Each independently is H, F,Cl, br, I, or a combination thereof;
or an enantiomer thereof, or a salt thereof,
wherein R is 1 、R 2 、R 4 And R is 5 Each independently is an amino acid, wherein the amino acids form an amide or a phosphoramide linkage.
In some embodiments, the aqueous composition includes any one or more of the structures disclosed herein. In some embodiments, the compositions include adjuvants and surfactants known to those of ordinary skill in the art for herbicide formulations. In various embodiments, the compound is the (R) -or (S) -enantiomer. In various embodiments, the compound is left-handed or right-handed. In various embodiments, the compound is a salt.
In addition, the present disclosure provides a method of inhibiting the growth or development of weeds comprising contacting the weeds and/or soil in which the weeds can develop with a herbicidally effective amount of a composition or compound as described herein, thereby inhibiting the growth or development of weeds. In some embodiments, the weeds are killed. In some embodiments, weed germination is killed or prevented or inhibited without significant damage to other plants, vegetation, or crops.
In some embodiments, the weeds are controlled, wherein the control is to destroy unwanted weeds or destroy them to such an extent that they are no longer competitive with crops, other plants or vegetation. In some other embodiments, weeds are inhibited, wherein the inhibition is not fully controlled but provides an economic benefit, such as reducing competition with crops, other plants, or vegetation.
In some embodiments, the composition or compound contacts vegetation and/or soil where vegetation may be produced, thereby selectively inhibiting the growth or production of weeds.
In addition, the present disclosure provides a method of inhibiting the growth of a cancer cell comprising contacting the cancer cell with an effective amount of a composition or compound of the present disclosure, thereby inhibiting the growth of the cancer. In some embodiments, the cancer cell is a glioblastoma cell.
Also, the present disclosure provides a method for treating cancer in a subject in need thereof, comprising administering a therapeutically effective amount of a compound or composition of the present disclosure, wherein the cancer is treated.
In various embodiments, the compounds disclosed herein, e.g., pantaphos, are effective in an amount of about 0.01mg/m for an animal or crop 2 Or less than about 100g/m 2 Or more. In other embodiments, the effective amount is about 0.1mg/m 2 To about 10g/m 2 . In other embodiments, the effective amount is about 0.05mg/m 2 ,0.1mg/m 2 ,0.2mg/m 2 About 0.5mg/m 2 ,1mg/m 2 About 2mg/m 2 About 5mg/m 2 ,10mg/m 2 About 15mg/m 2 ,20mg/m 2 About 50mg/m 2 About 100mg/m 2 ,200mg/m 2 About 300mg/m 2 ,500mg/m 2 About 750mg/m 2 About 1000mg/m 2 ,1500mg/m 2 About 2000mg/m 2 ,3000mg/m 2 About 5000mg/m 2 About 7500mg/m 2 ,10000mg/m 2 About 20000mg/m 2 ,50000mg/m 2 About 75000mg/m 2 About 100000mg/m 2 Or any number in between.
In various embodiments, the compounds in the herbicide compositions or pharmaceutical compositions disclosed herein act synergistically with the second active agent to control weeds or treat cancer.
Further, the present disclosure provides a method of forming 2- (hydroxy (phosphono) methyl) maleic acid:
or a salt thereof, comprising:
a) Phosphoenolpyruvic acid (PEP); isomerization of 2- (phosphooxy) acrylic acid to 3-phosphonopyruvic acid (3-phosphopyruvate, pnPy;2-oxo-3-phosphonopropanoic acid);
b) Condensing acetyl and PnPy to form phosphonomethyl malate (PMM); 2-hydroxy-2- (phosphomethyl) succinic acid);
c) Dehydrating the PMM to 2-phosphonomethyl maleate (2- (phosphonomethyl) maleic acid); and
d) Oxidizing 2-phosphonomethyl maleate (2-phosphonomethyl maleate) to pantaphos (2- (hydroxy (phosphono) methyl) maleic acid);
wherein each of steps a) -d) is accomplished in vitro, in a vessel or in a reactor, wherein the vessel or reactor is artificial.
In some embodiments, the isomerization is catalyzed by PEP mutase (HvrA); the condensation is catalyzed by phosphonomethyl malate synthase ((HvrC) and acetyl is acetyl-coa, the dehydration is catalyzed by large isopropyl malate dehydratase (HvrD) and/or small isopropyl malate dehydratase (HvrE), and the oxidation is catalyzed by flavin-dependent monooxygenase (HvrB) and optionally flavin reductase (HvrK).
In some embodiments, the compounds pantaphos can be prepared by sequential biosynthetic processes using purified enzymes HvrA, hvrC, hvrDE and HvrBK (see scheme 1), starting with phosphoenolpyruvate (PEP) and HvrA in the order shown, with the reaction product contacted by the next enzyme in the order.
In another embodiment, the compounds pantaphos can be prepared by a sequential biosynthesis process using the purified enzymes HvrA (SEQ ID NO: 14), hvrC (SEQ ID NO: 16), hvrD (SEQ ID NO: 17), hvrE (SEQ ID NO: 18), hvrB (SEQ ID NO: 15) and HvrK (SEQ ID NO: 24) (see scheme 1), in the order shown, starting with the contacting of phosphoenolpyruvate (PEP) and HvrA (SEQ ID NO: 14), wherein the reaction product is contacted by the next enzyme in the sequence.
Furthermore, the present invention provides a nucleic acid molecule comprising the hvr operon of Pantoea Sp. (hvrA-hvrL). In other embodiments, the nucleic acid molecule comprises one or more genes selected from the genes hvrA, hvrB, hvrC, hvrD, hvrE and hvrK of Pantoea Sp.. In another embodiment, the nucleic acid molecule comprises the genes hvrA, hvrB, hvrC, hvrD, hvrE and hvrK of Pantoea Sp..
In other embodiments, the nucleic acid molecules include the gene hvrA (SEQ ID NO: 1), hvrB (SEQ ID NO: 2), hvrC (SEQ ID NO: 3), hvrD (SEQ ID NO: 4), hvrE (SEQ ID NO: 5), hvrF (SEQ ID NO: 6), hvrG (SEQ ID NO: 7), hvrH (SEQ ID NO: 8), hvrI (SEQ ID NO: 9), hvrJ (SEQ ID NO: 10), hvrK (SEQ ID NO: 11) and hvrL (SEQ ID NO: 12) of Pantoea ananatis.
In some embodiments, the nucleic acid molecule comprises one or more genes selected from the group consisting of the genes hvrA (SEQ ID NO: 1), hvrB (SEQ ID NO: 2), hvrC (SEQ ID NO: 3), hvrD (SEQ ID NO: 4), hvrE (SEQ ID NO: 5), hvrF (SEQ ID NO: 6), hvrG (SEQ ID NO: 7), hvrH (SEQ ID NO: 8), hvrI (SEQ ID NO: 9), hvrJ (SEQ ID NO: 10), hvrK (SEQ ID NO: 11) and hvrL (SEQ ID NO: 12) of Pantoea ananatis.
In other embodiments, the nucleic acid molecules include the genes hvrA (SEQ ID NO: 1), hvrB (SEQ ID NO: 2), hvrD (SEQ ID NO: 4), hvrE (SEQ ID NO: 5) and hvrK (SEQ ID NO: 11) of Pantoea ananatis. In other embodiments, the nucleic acid molecules include the gene hvrA (SEQ ID NO: 1), hvrB (SEQ ID NO: 2), hvrC (SEQ ID NO: 3), hvrD (SEQ ID NO: 4), hvrE (SEQ ID NO: 5) and hvrK (SEQ ID NO: 11) of Pantoea ananatis.
In other embodiments, the nucleic acid molecule comprises one or more genes selected from the genes hvrA (SEQ ID NO: 1), hvrB (SEQ ID NO: 2), hvrC (SEQ ID NO: 3), hvrD (SEQ ID NO: 4), hvrE (SEQ ID NO: 5) and hvrK (SEQ ID NO: 11) of Pantoea ananatis.
In other embodiments, the disclosure provides a nucleic acid molecule comprising the hvr operon of Pantoea Sp. (hvrA-hvrL) operably linked to an inducible promoter sequence, wherein induction of the promoter and expression of the gene of the hvr operon results in production of the phosphonate compound of formula I or formula II. In various embodiments, the phosphonate compound is 2- (hydroxy (phosphono) methyl) maleic acid (pantaphos).
Inducible promoters suitable for use in various embodiments include, but are not limited to, T7lac, rpoS, prhabAD, mmsA, trc,tetA,tac,lac,tacM,P L ,araBAD,cspA,cspB,phyL,NBP3510,P43,Pspac,P 170 pgraph and trp. In some embodiments, the inducible promoter is a lac or tac promoter. In some embodiments, the inducible promoter is a tac promoter having a nucleic acid sequence according to SEQ ID NO. 13.
In other embodiments, the nucleic acid molecule comprises an inducible promoter and one or more genes selected from the genes hvrA, hvrB, hvrC, hvrD, hvrE and hvrK of Pantoea Sp, wherein the one or more genes are operably linked to the inducible promoter.
In some embodiments, the nucleic acid molecule comprises an inducible promoter and the genes hvrA (SEQ ID NO: 1), hvrB (SEQ ID NO: 2), hvrC (SEQ ID NO: 3), hvrD (SEQ ID NO: 4), hvrE (SEQ ID NO: 5), hvrF (SEQ ID NO: 6), hvrG (SEQ ID NO: 7), hvrH (SEQ ID NO: 8), hvrI (SEQ ID NO: 9), hvrJ (SEQ ID NO: 10), hvrK (SEQ ID NO: 11) and hvrL (SEQ ID NO: 12) of Pantoea ananatis, wherein the genes are operably linked to the inducible promoter.
In some embodiments, the nucleic acid molecule comprises an inducible promoter and one or more genes selected from the genes hvrA (SEQ ID NO: 1), hvrB (SEQ ID NO: 2), hvrC (SEQ ID NO: 3), hvrD (SEQ ID NO: 4), hvrE (SEQ ID NO: 5), hvrF (SEQ ID NO: 6), hvrG (SEQ ID NO: 7), hvrH (SEQ ID NO: 8), hvrI (SEQ ID NO: 9), hvrJ (SEQ ID NO: 10), hvrK (SEQ ID NO: 11) and hvrL (SEQ ID NO: 12).
In other embodiments, the nucleic acid molecule comprises an inducible promoter and the gene hvrA (SEQ ID NO: 1), hvrB (SEQ ID NO: 2), hvrD (SEQ ID NO: 4), hvrE (SEQ ID NO: 5) and hvrK (SEQ ID NO: 11) of Pantoea ananatis, wherein the gene is operably linked to the inducible promoter. In another embodiment, the nucleic acid molecule comprises an inducible promoter and the gene hvrA (SEQ ID NO: 1), hvrB (SEQ ID NO: 2), hvrC (SEQ ID NO: 3), hvrD (SEQ ID NO: 4), hvrE (SEQ ID NO: 5) and hvrK (SEQ ID NO: 11) of Pantoea ananatis, wherein the gene is operably linked to the inducible promoter.
In other embodiments, the nucleic acid molecule comprises an inducible promoter and one or more genes selected from the genes hvrA (SEQ ID NO: 1), hvrB (SEQ ID NO: 2), hvrD (SEQ ID NO: 4), hvrE (SEQ ID NO: 5) and hvrK (SEQ ID NO: 11) of Pantoea ananatis, or one or more genes selected from the genes hvrA (SEQ ID NO: 1), hvrB (SEQ ID NO: 2), hvrC (SEQ ID NO: 3), hvrD (SEQ ID NO: 4), hvrE (SEQ ID NO: 5) and hvrK (SEQ ID NO: 11) of Pantoea ananatis.
In another embodiment, the nucleic acid molecule comprises an inducible promoter according to the nucleic acid sequence of SEQ ID NO. 13 and nucleic acid sequences encoding the genes hvrA, hvrB, hvrC, hvrD, hvrE and hvrK according to SEQ ID NO. 1,2,3,4,5 and 11, respectively, wherein the genes are operably linked to the inducible promoter.
In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least 60%, preferably at least 65,70,75,80,85,90,95,96,97,98 or 99%, identical to SEQ ID NOs 1,2,3,4,5,6,7,8,9,10,11 and 12.
Furthermore, the present disclosure provides a recombinant cell for producing a phosphonate compound comprising a nucleic acid molecule (e.g., an expression vector) disclosed herein according to formula I or formula II. In some embodiments, the recombinant cells express one or more proteins selected from the group consisting of SEQ ID NOs 14,15,16,17,18,19,20,21,22,23,24 and 25. In some embodiments, the recombinant cells express one or more proteins having an amino acid sequence at least 60% (preferably at least 65,70,75,80,85,90,95,96,97,98 or 99%) identical to one or more proteins selected from the group consisting of SEQ ID NOs 14,15,16,17,18,19,20,21,22,23,24 and 25.
In various embodiments, the nucleic acid molecule is inserted into a cell and maintained as a plasmid or integrated into the chromosome of the cell. In various embodiments, the cell is selected from the group consisting of Pantoea (Pantoea), clostridium, zymomonas (Zymomonas), escherichia coli (Salmonella), salmonella, serratia (Serratia), equisqualis (Erwinia), klebsiella (Klebsiella), shigella (Shigella), rhodococcus, pseudomonas (Pseudomonas), bacillus, lactobacillus, lactococcus (Lactobacter), enterococcus (Enterococcus), alcaligenes, bacillus (Paenibacillus), arthrobacter
(Arthrobacter), corynebacteria (Corynebacterium), brevibacterium (Brevibacterium), schizosaccharomyces (Schizosaccharomyces), kluveromyces (Kluveromyces), yarrowia (Yarrowia), pichia (Pichia), saccharomyces (Zygosaccharomyces), decore yeast (Debaryomyces), candida (Candida), brettanomyces (Brettanomyces), clostridium thickii (Pachysolen), hansen (Hansenula), issatchenkia (Issatchenkia), trichosporon (Trichosporon), sub Ma Daci (Yamadazyma) and Saccharomyces (Saccharomyces).
In various embodiments, the cell belongs to the genus Pantoea, escherichia coli or Saccharomyces. Species belonging to the genus Pantoea include, but are not limited to, pantoea agglomerans (Pantoea agglomerans), pantoea ananatis, pantoea stonecrop (Pantoea stewartii), pantoea citrifolia (Pantoea citruses), pantoea dispersion (Pantoea distrosa), pantoea punctifolia (Pantoea pubete), pantoea terrea (Pantoea terrea), deluxe (Pantoea delley i), pantoea variabilis (Pantoea anthophila), pantoea anii (Pantoea alii) and Pantoea eucalypti (Pantoea eucalypti). In various embodiments, the cell is Pantoea ananatis (Pantoea ananatis), escherichia coli (Escherichia coli) or Saccharomyces cerevisiae (Saccharomyces cerevisiae).
Methods for preparing recombinant cells and nucleic acid molecules are known in the art and their description can be found herein and e.g. in International Pat.Pub.No. WO/2020/090940 and WO2014129898, and U.S. Pat.Pub.No. U.S. 20190256838 and U.S. 20140134689.
The present disclosure also provides a process for preparing a phosphonate compound of formula I or formula II, comprising the steps of:
a) Providing a cell culture of recombinant cells described herein (see above), wherein the recombinant cells produce phosphonate and the cell culture has a volume of about 1L to about 10L;
b) Mixing an inducing molecule with the recombinant cell culture;
c) Culturing the induced recombinant cell culture under constant oxygenation conditions for 96 hours;
d) Pelletizing the cells of the recombinant cell culture and collecting the supernatant;
e) Concentrating the supernatant;
f) Extracting phosphonate from the concentrated supernatant by methanol extraction to form an extracted supernatant; and
g) The phosphonate is purified from the methanol soluble portion of the extracted supernatant.
In various embodiments, the phosphonate is 2- (hydroxy (phosphono) methyl) maleic acid. In some embodiments, step g comprises iron-IMAC purification followed by flash chromatography and HILIC HPLC. In some embodiments, the cell culture comprises Pantoea ananatis (Pantoea ananatis), escherichia coli (Escherichia coli) or saccharomyces cerevisiae (Saccharomyces cerevisiae) cells. In some embodiments, the cell culture is recombinant Pantoea ananatis (Pantoea ananatis). In some embodiments, the constant oxygenation conditions have a flow rate of 5L/min and the cell culture is maintained at a temperature of 30 ℃.
Another embodiment of the present disclosure provides a compound comprising a hydroxylated allylphosphonic acid of formula a, wherein R 1 And R is 2 Is independently selected from the group consisting of carboxylic acids and derivatives thereof (ketones, esters, carboxylic acids); a hydroxyl group; an amine group; an ether group; halogen; alkyl or aryl; or an alkyl or aryl group containing the above functional group.
Another embodiment of the present disclosure provides a compound comprising a hydroxylated allylphosphonic acid of formula B, wherein each R is independently selected from the group consisting of halogen, amino acid, carboxylic acid, and derivatives thereof (ketone, ester, carboxylic acid); a hydroxyl group; an amine group; an ether group; halogen; alkyl or aryl; or an alkyl or aryl group containing the above functional group.
Another embodiment of the present disclosure provides a compound of formula a, wherein R 1 And R is 2 Has a (Z) configuration.
Another embodiment of the present disclosure provides a compound of formula a, wherein R 1 And R is 2 Has the configuration (E).
Another embodiment of the present disclosure provides a compound of formula a or formula B, wherein the chiral orientation is R.
Another embodiment of the present disclosure provides a compound of formula a or formula B, wherein the chiral orientation is S.
Another embodiment of the present disclosure provides a compound of formula a, wherein R 1 And R is 2 COOH or COO-.
Another embodiment of the present disclosure provides a compound of formula a, wherein R 1 And R is 2 Is COOH or COO-, and one or both of the carboxylates are salts selected from the group comprising monovalent and divalent counter ions.
Another embodiment of the present disclosure provides salts of compounds of formula a and/or formula B wherein the monovalent and divalent counter ions are selected from the group consisting of sodium, potassium, calcium, lithium, magnesium, manganese, and mixtures thereof.
Another embodiment of the present disclosure provides herbicide formulations, including compounds of formula a and/or formula B, that are effective on plants.
Another embodiment of the present disclosure provides salts of compounds of formula a and/or formula B, which are effective against monocotyledonous plants.
Another embodiment of the present disclosure provides a salt of a compound of formula a and/or formula B, which is effective against phyllanthus niruri.
Another embodiment of the present disclosure provides salts of compounds of formula a and/or formula B, comprising water, one or more emulsifiers, one or more surfactants, one or more wetting agents and/or one or more pH buffering ingredients, wherein the pH of the composition is from 5 to 9.
Another embodiment of the present disclosure provides salts of compounds of formula a and/or formula B that include other bioactive agents, including other pesticides, adjuvants, or macro-or micronutrients.
Another embodiment of the present disclosure provides a method for biosynthesis of compounds of formula a and/or formula B using pantoea bacteria.
Another embodiment of the present disclosure provides a method for biosynthesis of a compound of formula a and/or formula B using a bacterium, wherein the strain of the bacterium is Pantoea ananatis.
Another embodiment of the present disclosure provides a method of biosynthesizing a compound of formula a and/or formula B using Pantoea bacteria, wherein the expression of the hvr gene cluster is modified.
Another embodiment of the present disclosure provides a method of biosynthesizing a compound of formula a and/or formula B using Pantoea bacteria that has been genetically modified to biosynthesize a compound of formula a and/or formula B, wherein the titer is >3mg/L.
Another embodiment of the present disclosure provides a genetically modified bacterium in which expression of the hvr gene cluster has been modified to produce a compound of formula a and/or formula B.
Another embodiment of the present disclosure provides a herbicide composition comprising the bacterium, bacterium-derived isolate, or supernatant of the bacterium of any of the above embodiments effective on plants.
Another embodiment of the present disclosure provides a herbicide composition of the above embodiments that is effective against monocotyledonous plants.
Another embodiment of the present disclosure provides a herbicide composition of the above embodiments that is effective against phyllanthus niruri.
Another embodiment of the present disclosure provides a method of killing or inhibiting a plant comprising contacting the plant with the herbicide composition of the above embodiments.
Another embodiment of the present disclosure provides a method of treating or preventing a fungal infection comprising administering to a patient in need thereof an antifungal amount of a compound of formula a and/or formula B.
Another embodiment of the present disclosure provides a method of treating or preventing cancer comprising the step of administering to a patient in need thereof a therapeutic amount of a compound of formula a and/or formula B.
Another embodiment of the present disclosure provides a method of treating or preventing cancer comprising administering to a patient in need thereof a therapeutic amount of a compound of formula a and/or formula B, wherein the patient is a mammal and the cancer is glioblast cells
Phosphonate natural products from Pantoea ananatis are essential and sufficient for the characteristic lesions of onion heart rot
The involvement of biologically active phosphonates in the pathogenesis of p. Here we demonstrate that the hvr operon does encode the enzyme responsible for the production of small molecule phosphonates, which we show is 2- (hydroxy (phosphono) methyl) maleate. The purified molecule, we named pantaphos (Pan Dalin), has remarkable herbicidal activity and is able to produce the characteristic lesions of onion center rot without p. Thus, this novel phosphonate natural product is both necessary and sufficient in the pathogenesis of onion rot. Furthermore, pathogenicity was enhanced in strains lacking phosphonate catabolism, suggesting that the endogenous catabolism of pantaphos attenuated virulence.
Results
Phosphonate metabolism plays a role in onion center decay. To investigate the broader role of phosphonate metabolism in onion pathogenesis, we identified p.ananatis LMG 5342 carrying hvr, pgb and phn sites using p.ananatis B-14773, which does not encode any phosphonate biosynthesis genes, as a control. Since the pathogenicity of these strains has not been established, we have conducted preliminary experiments to assess their ability to cause onion decay. Consistent with the correlation between the presence of the hvr locus observed before and the pathogenesis, we observed central decay of onion in bulbs inoculated with strain LMG 5342, but not in bulbs inoculated with B-14773 (fig. 1).
To verify that hvr was required for the observed pathogenic phenotype and to examine whether additional phosphonate metabolizing genes play a role in pathogenesis, we constructed a series of LMG 5342 mutants lacking hvr, pgb and phn sites and scored their ability to cause onion decay (fig. 1). A central rot phenotype was observed in all mutants retaining the hvr locus, but not in all the delta hvr mutant strains. Thus, it was observed in p.ananatis OC5a that the hvr locus was essential for onion pathogenicity of strain LMG 5342. In contrast, the pgb locus did not contribute significantly to pathogenicity, as the onion rot phenotype of mutants lacking these genes was the same as each other isogenic strain. Interestingly, strains with intact hvr and phn sites had reduced toxicity relative to the Δphn mutant strains, indicating that endogenous phosphonate catabolism minimized toxicity (fig. 1).
Production of phosphonic acid in p.ananatis LMG 5342. To examine whether P.ananatis LMG 5342 was truly capable of producing phosphonate, we cultured wild-type strains in various liquid and solid media. The spent medium is then concentrated and used 31 P Nuclear Magnetic Resonance (NMR) screening for the presence of phosphonates allows relatively sensitive detection of molecules containing carbon-phosphorus (C-P) bonds, even in complex mixtures containing phosphates and phosphates. In any case, we did not observe a signal consistent with the presence of phosphonate. We suspected that our inability to detect phosphonate in spent medium was due to poor expression of the hvr operon in our medium used. Thus, we performed similar experiments in the medium supplemented with onion extract, we considered that plant metabolites were required to induce expression at the hvr locus; however, we also failed to detect phosphonate in these media.
To avoid the problems caused by the natural gene regulation, we constructed a strain expressing the hvr operon, which was derived from a strong isopropyl- β -D-1-thiogalactose-pyriproxyfen (IPTG) -inducible promoter (fig. 2). To avoid complications caused by other phosphonate metabolizing genes, the strain also carries Δphn and Δ pgb mutations. Three different strains were observed after growth of the recombinant strain in IPTG medium 31 P NMR peak (FIG. 3). Chemistry of these peaksDisplacement (delta) P 17.6, δ15.0, and δ10.4 ppm) are consistent with the presence of molecules containing c—p bonds. No such peaks were observed after growth in IPTG-free medium. Thus, putative phosphonates were only produced when expressed by the hvr operon. The IPTG-induced strain catalyzes almost complete conversion of phosphate to biomass and phosphonate with final concentrations of compound 1 of about 0.653, 0.080, 0.039mM, respectively, through optimization of medium and growth conditions; 31 p NMR (δ17.6), 2 (δ15.0) and 3 (δ10.4 ppm).
Structural identification of phosphonate related hvr. Under these optimized conditions, we were able to isolate 8.9mg of pure compound 1 and small amounts of [. Sup.1 ] from 3.2 liter of culture<600 μg) of pure compound 2. Compound 3 (delta) P 10.42 ppm) was not obtained because it was unstable at the low pH values used in affinity chromatography (data not shown). The structure of compounds 1 and 2 was elucidated by a series of one-and two-dimensional carbon, phosphorus and proton NMR experiments (see table 1 for a summary; complete data set is described in supplemental data set 2). Compound 1, we named pantaphos, was demonstrated to be 2- (hydroxy (phosphono) methyl) maleate (2- (hydroxy (phosphono) methyl) maleate). Compound 2, i.e. (2-phosphonomethyl maleate), has nearly the same structure but lacks a hydroxyl group, indicating that it may be an intermediate in pantaphos biosynthesis (table 1).
Table 1, NMR data summaries supporting the structures of Compound 1 (Compound 1) and Compound 2 (Compound 2). A detailed description of the preliminary data and structural description is given in the examples.
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Lesions of onion heart rot are caused by pantaphos. To investigate the role of hvr-related phosphonates in pathogenesis, we repeated the onion heart rot assay (fig. 4) using concentrated culture supernatants or purified pantaphos in the presence and absence of the p.ananatis Δ hvr mutant (a sufficient amount of pure compound 2 was not obtained for the bioactivity assay). As described above, onions vaccinated with the p.ananatis Δ hvr mutant showed minimal damage. However, when concentrated spent medium from strains of IPTG-induced phosphonate overproducing cultures was co-inoculated with the Δ hvr mutant, central decay was again observed. Also co-inoculation with purified pantaphos can lead to onion decay. Notably, in the absence of bacteria, onions injected with concentrated spent medium or purified pantaphos bacteria showed severe onion rot lesions. The occurrence of central rot is dose-dependent, and characteristic lesions can be observed with 90. Mu.g (0.40. Mu. Mol) of pantaphos alone. Thus, pantaphos is a necessary and sufficient condition to cause a rotten lesion in the center of onion.
The phytotoxic effect of Pantaphos treatment is comparable to that of known herbicides. To test whether the phytotoxicity observed in onion bulbs can be extended to growing plants, we treated freshly germinated mustard seedlings (Brassica sp.) and arabidopsis Col-0 seedlings with purified panaphos, two well-characterized phosphonate herbicides (glyphosate) and phosphinothricin) were used as controls (fig. 5 and 6). After seven days of growth, all three compounds resulted in a significant reduction in root length and total dry weight of seedlings relative to water (water) treated controls, the effect of pantaphos was significantly higher than that of phosphinothricin (phosphinothricin) in root length (root length) test and was significantly higher than that of Yu Caogan phosphine (glyphosate) in dry weight (dry weight) test. The virulence of Panthahos is dose-dependent, and the activity is remarkable when the concentration is 1.95 mu M or more in root length test and 31.3 mu M or more in dry weight test.
Cytotoxicity, antibacterial and antifungal activity of Pantaphos. To investigate whether the biological activity of pantaphos is characteristic for plants, we also performed a series of bioassays on human cell lines, bacteria and fungi. Panthahos exhibits moderate cytotoxicity against several human cell lines Sex (table 2). IC for each test cell line except that one ovarian cancer cell line (ES-2) was unaffected at the maximum dose 50 The levels were substantially similar in the range of 6.0 to 37.0 μm. Glioma cell line (A-172) was particularly sensitive to pantaphos (IC 50 1.0 μm). In contrast, this molecule had no effect on fungal growth in rich or minimal medium, including candida albicans, aspergillus fumigatus and two strains of saccharomyces cerevisiae (table 2). Also, various gram negative and gram positive bacteria, including all so-called ESKAPE pathogens, are insensitive to pantaphos in basal and rich media (table 2). To examine whether the insensitivity of E.coli was due to lack of transport, we also tested the biological activity using phosphonate specific bioassay strain E.coli WM6242, which carries two copies of the IPTG-induced broad substrate-specific phosphonate transporter. The strain is insensitive to pantaphos, and whether IPTG induction exists or not, which indicates that the lack of bioactivity in E.coli is not due to poor molecular transport.
Table 2, panthahos have biological activities on human cells and microorganisms.
| Human cell lines | IC 50 (μM) a |
| HOS (human osteosarcoma) | 36.98±6.28；E max 58％ |
| ES-2 (human ovarian cancer) | >100 |
| HCT-116 (colon cancer) | 10.42±2.00；E max 59％ |
| A-549 (human lung cancer) | 14.73±0.61；E max 66％ |
| HFF-1 (human fibroblast) | 6.69±0.29；E max 85％ |
| A-172 (human glioma carcinoma) | 1.01±0.06；E max 99％ |
a 50% inhibitory concentration as determined by the Amara-Mulan method. Emax = percent cell death.
b Minimum Inhibitory Concentration (MIC) determined after 48 hours of growth according to CLSI guidelines. Biological activity in enriched media measured with RPMI 1640 media. Bioactivity in minimal medium measured using M9 minimal medium.
c The organism was not tested because it was unable to grow in minimal medium.
d Minimum Inhibitory Concentration (MIC) determined according to CLSI guidelines. Biological activity in rich medium as determined using Mueller-Hinton 2 medium, and biological activity in minimal medium as determined using glucose-MOPS minimal medium.
hvr encodes the putative function of the protein and proposed pantaphos biosynthetic pathway. Combining the above-identified structure with the proposed function of the Hvr protein, a rational P.ananatis phosphonate biosynthetic pathway was proposed (FIG. 7). As with most phosphonate natural products, the pathway begins with the PEP mutant enzyme catalyzing the rearrangement of phosphoenolpyruvate (PEP) to phosphopyruvate (PnPy). In p. ananatis, this reaction will be catalyzed by the HvrA protein, which is highly homologous to the known PEP mutant enzymes. Since the PEP mutase reaction is highly energy-absorbing (ΔG-125 kJ/mol), the subsequent steps must be very favorable to drive the synthesis of net phosphonates. In the proposed pathway, this thermodynamic driving force is provided by the homoginic condensation of acetyl-CoA and phosphopyruvate (PnPy) catalyzed by the HvrC protein, a homolog of methylethyl malate (PMM) synthase which is a biochemical feature involved in FR-900098 biosynthesis. The PMM is then dehydrated by HvrD and HvrE to form 2-phosphonomethyl maleate. These proteins are homologues of the small and large subunits, respectively, of isopropyl malate dehydratase, which catalyze the isomerization of isopropyl 3-malate to isopropyl 2-malate via the dehydration intermediate (isopropyl 2-malate) during leucine biosynthesis. We expect that HvrDE does not catalyze the entire reaction but stays in the dehydrated intermediate product. The precedent for this partial reaction is found in the propionic acid catabolic pathway of some bacteria, which uses one member of the isopropyl malate dehydratase family to catalyze the dehydration of 2-methyl citrate to 2-methyl-cis-aconitate. The conversion of 2-phosphonomethyl maleate to pantaphos is probably catalyzed by HvrB, a homolog of the flavin-dependent monooxygenases NtaA and ScmK (48% and 47%, respectively). Consistent with the belief that this is an oxygen dependent reaction, we observed that poorly aerated cultures accumulated 2-phosphonomethyl maleate instead of pantaphos. Flavin-dependent monooxygenases typically require a separate flavin reductase to provide the electrons needed to reduce oxygen to water. We believe that this function is provided by HvrK, a member of the flavin reductase family, 30% identical to NtaB, which functions in a similar NtaA catalytic reaction. Finally, we consider the HvrI protein, a member of the major facilitator superfamily, responsible for the export of phosphate products.
The proposed pantaphos biosynthetic pathway uses only 7 of the 11 genes in the hvr operon (scheme 1). Based on homology to known functional proteins, the remaining 3 genes were predicted to encode O-methyltransferase (HvrF), N-acetyltransferase (HvrG) and ATP-Grasp family protein (HvrH). The last unassigned protein (HvrJ) has no characteristic homolog and therefore, we cannot predict its function. The twelfth protein may or may not be part of the hvr operon, and also encodes an ATP-Grasp family protein (HvrL). Members of the ATP-Grasp enzyme family often catalyze the formation of peptide bonds. Therefore, we suspected that the peptide derivatives of pantaphos might be produced by p. Given the lack of nitrogen in pantaphos, peptide derivatives may also help to explain the putative presence of the N-acetyltransferase HvrG, which may act as a self-resistance gene similar to the pat gene, conferring self-resistance during the biosynthesis of the phosphinothricin tripeptide. Finally, the putative O-methyltransferase HvrF is highly homologous to trans-aconitate methyltransferases, which are believed to be involved in resistance to the trans-isomer spontaneously formed by this TCA cycle (TCA cycle) intermediate. Similar effects of HvrF can be envisaged if similar trans-isomer byproducts are produced during pantaphos biosynthesis. Scheme 1, the biosynthetic pathway proposed based on the biosynthetic logic of the p.ananatis phosphonate structure and a similar reaction catalyzed by the Hvr protein homolog (analogous reactions) as determined in this study. Pep=phosphoenolpyruvate; pnp=phosphopyruvate; pmm=phosphomethyl malate (methylethyl malate); ipms= isopropylmalate synthase (isopropyl malate synthase); ipmd= isopropylmalate dehydratase (isopropyl malate dehydratase); ace-coa= acetyl coenzyme A (acetyl CoA).
Homologs of the hvr operon in other bacteria. Aselin et al note the presence of a cluster of genes similar to the hvr operon in many bacterial genomic sequences. It is considered necessary to re-analyze the structure. A total of 33 related gene clusters were identified in the NCBI genome sequence database using bioinformatics methods. Based on the arrangement and presence of homologous genes, a total of 9 hvr-like biosynthetic gene clusters were observed. The putative homolog of HvrA-F is conserved in all of these groups except for type VIII, which replaces the HvrDE protein with putative aconitase. As shown in FIG. 6, aconitase catalyzed reactions were essentially identical to the putative HvrDE reactions. Thus, we predicted that the biosynthetic pathway encoded by the type VIII hvr-like gene cluster had the same intermediate produced by a parahomologous enzyme. One feature distinguishing the nine hvr-like clusters is the presence or absence of one or more ATP-Grasp proteins, which suggests that various peptide derivatives of pantaphos may be produced in nature. However, type IX lacks ATP-gripping (ATP-grasp) family proteins, which may have very different structural modifications compared to other types, based on the presence of homologs of several additional enzyme families. Finally, these clusters also differ by the presence/absence of their putative export proteins and putative flavin reductase. The latter is not unexpected as many flavin-dependent enzymes can utilize universal reductases encoded by non-linked genes. Interestingly, the hvr-like clusters found in our study were found in only a few lineages of the Proteobacteria and actinomycetes.
Discussion of the invention
The phosphonate natural product produced by p.ananatis LMG 5342 is the main virulence factor responsible for the central decay of onion. In fact, our data indicate that the same lesions are produced using purified pantaphos in the absence of bacteria. Although bioactive phosphonate natural products are well known, little data is available to support the direct role of these molecules in pathogenesis. The only known example to date is a complex phosphonate modified polysaccharide produced by bacteroides fragilis (Bacteroides fragilis), which has been shown to promote the formation of intestinal abscess in mammals. Pantpaphos is both necessary and sufficient for onion center rot, which proves that a second phosphonate class of natural products was added to this short list and demonstrated the predictive function of the P.ananatis hvr locus proposed by Asselin et al (Mol Plant Microbe Interact 2018, 31:1291).
Although unmodified pantaphos are phytotoxic, it appears that modified derivatives of this compound may also be produced by p. As described above, the hvr locus encodes two ATP-grasp proteins. Members of this family of proteins often catalyze the formation of ATP-dependent peptide bonds, including those found in the peptide phosphonate natural products rhizopus, plumbum mould and phosphoramides. The addition of amino acid substituents often facilitates the absorption of biologically active compounds. For example, a phosphinothricin tripeptide is an effective antimicrobial compound, whereas unmodified phosphinothricin is less active. However, it should be noted that these transport-mediated effects are species-specific. Thus, phosphinothricin and phosphinothricin tripeptides are equally effective herbicides. One particularly notable example of the specificity conferred by amino acid substituents is seen in the spectrum of biological activity of rhizopus and plumbum mycotoxins. These natural products have the same bioactive phosphate warhead attached to different amino acids, which may determine their uptake by a particular peptide delivery system. Thus, rhizopus is a potent antifungal agent, but lacks antibacterial activity; plumbum mould is a powerful antimicrobial agent, but lacks antifungal activity. Based on these precedents, we suspected that peptide derivatives of pantaphos might be produced during plant infection, increasing their potency or specificity for a particular target species, which might be the cause of unstable compound 3 we observed in used media.
Interestingly, panaphos is not the only phosphonate produced by the p.ananatis species. At least four different phosphonate biosynthesis gene clusters can be found in the p.ananatis genome currently sequenced. Except for the hvr operon, the structure and biological function of the molecules produced by these clusters are currently not determinable; however, our data clearly indicate that the pgb cluster is not necessary for onion center rot. Notably, when analyzing large amounts of Pantoea genome, the strict correlation between plant pathogenicity and the presence of the hvr operon was broken. Thus, the Banana subtilis strains PA4, PNA 14-1 and PNA 200-3 lack the hvr operon, but still cause onion diseases; while strains PANS 04-2, PNA 07-10 and PNA 200-7 carry the hvr locus, but do not cause disease. These data indicate that additional virulence traits are important in onion pathogenesis. They also demonstrated that caution was required when using the presence/absence of the hvr cluster as a marker of plant pathogenicity.
Gene clusters similar to hvr are relatively common in sequenced bacterial genomes; however, their phylogenetic distribution is rather limited. Interestingly, many bacteria encoding these hvr-like gene clusters are associated with plant or insect hosts. homologs of the hvr operon are particularly abundant in closely related members of the genera Photorhabdus and Xenorhabdus. These bacteria have a unique lifestyle that depends on infection of the insect host by a nematode vector. Similarly, the hvr-site is also found in the intracellular pathogen, serpentis Barballs OS02 in mammals. In view of the cytotoxic effects we observe in human cells, we easily believe that pantaphos-like compounds may be involved in bacterial pathogenesis in insects and humans. homologs of the hvr operon can also be found in some members of actinomycetes, including the species Streptomyces. These bacteria are common epiphytes and endophytes, well known for the production of a wide range of secondary metabolites, including antibacterial, antifungal and phytotoxic compounds. Thus, the biologically active panaphos-like molecules appear to be important for actinomycete interactions as well.
The idea that Pantaphos and its putative derivatives are involved in a variety of reciprocal interactions circumvents the problem of biological targets of these molecules. Of the organisms tested to date, only plant and human cells show a marked sensitivity to pantaphos molecules, whereas bacteria and fungi are completely insensitive to such molecules. A simple explanation for these observations is that both plants and animals have this target, whereas bacteria and fungi do not. Another possibility is that all organisms have this target, but bacterial and some fungi are very different targets and therefore insensitive to this molecule. It is also possible that both bacteria and some fungi have the ability to inactivate pantaphos, or they cannot transport molecules into their cells. A lot of experimental work outside the scope of this preliminary report will be required to distinguish between these possibilities; however, at least for E.coli, the data indicate that lack of transport is not responsible for lack of biological activity. The structures of pantaphos are similar to some common metabolites including citrate, isocitrate, aconitate, isopropyl malate and maleate. Since most typical phosphonates are molecular mimics of normal cellular metabolites, this suggests that pantaphos might be targeted to the TCA cycle or leucine biosynthesis. Since maleic acid compounds are known to inhibit transaminases, this molecule may also inhibit the synthesis of another essential amine-containing metabolite.
Finally, the studies described herein have important agricultural implications, providing a powerful impetus for future studies of the biosynthesis and molecular targets of pantaphos, as well as the potential resistance mechanisms to this compound. Identification of Pantoea ananatis as the primary virulence factor for onion rot suggests a number of methods to deal with agricultural infestations of Pantoea ananatis. Because this molecule is necessary for toxicity, it seems possible to prevent plant infection by inhibitors of the pantaphos biosynthetic pathway. The use of purified or synthetic pantaphos also makes it possible to determine the molecular targets of the compounds in plants, which will pave the way for the development of crops with resistant alleles that are immune to the disease. Plants expressing putative pantaphos-modifying enzymes encoded by the hvr locus, or unrelated phosphonate catabolic genes, are also expected to be specifically resistant to pantaphos bacterial infection. Finally, there is an urgent need to develop new treatments effective against the dramatic growth of herbicide-resistant weeds. The strong phytotoxicity of pantaphos suggests that it may have agricultural uses similar to the widely used phosphinic herbicides glyphosate and phosphinothricin. The true potential for developing pantaphos-resistant crops has enhanced this view, as has the lack of biological activity against bacteria and fungi, suggesting that the effect of the molecule on the soil microbiome is very small. However, taking into account the moderate cytotoxicity we observe in human cell lines, care should be taken that this is appropriate.
General synthetic method
Methods of preparing the compounds and compositions of the invention are also described herein. The compounds and compositions may be prepared by any suitable organic synthesis technique, such as the techniques described herein. Many such techniques are well known in the art. However, many known techniques have been described in detail in Compendium of Organic Synthetic Methods (John Wiley&Sons, new York), vol.1, ian t.harrison and Shuyen Harrison,1971; vol.2, ian T.Harrison and Shuyen Harrison,1974; vol.3, louis S.Hegedus and Leroy Wade,1977; vol.4, leroy G.Wade, jr.,1980; vol.5, leroy G.Wade, jr.,1984; and vol.6, michael b.smith; standard organic reference books such as March's Advanced Organic Chemistry: reactions, mechanics, and Structure,5 th Ed.by M.B.Smith and J.March(John Wiley&Sons,New York,2001),Comprehensive Organic Synthesis；Selectivity,Strategy&Efficiency in Modern Organic Chemistry,in 9Volumes,Barry M.Trost,Ed.-in-Chief(Pergamon Press,New York,1993printing))；Advanced Organic Chemistry,Part B:Reactions and Synthesis,Second Edition,Cary and Sundberg(1983)；Protecting Groups in Organic Synthesis,Second Edition,Greene,T.W.,and Wutz,P.G.M.,John Wiley&Sons,New York；and Comprehensive Organic Transformations,Larock,R.C.,Second Edition,John Wiley&Sons,New York(1999)。
Several exemplary methods for preparing the compounds of the present invention are provided below. These methods are intended to illustrate the nature of such formulations and are not intended to limit the scope of applicable methods.
In general, the reaction conditions, such as temperature, reaction time, solvents, treatment procedures, etc., will be common conditions for the particular reaction to be performed in the art. The cited reference materials and the materials cited therein contain detailed descriptions of such conditions. Typically, the temperature is from-100 ℃ to 200 ℃, the solvent is aprotic or protic, and depending on the desired conditions, the reaction time is from 1 minute to 10 days. The treatment process typically involves quenching any unreacted reagents, then partitioning (extraction) between the water/organic layer systems, and separating the product-containing layers.
The oxidation and reduction reactions are typically carried out at temperatures near room temperature (about 20 ℃) but for the reduction of metal hydrides, the temperature is typically reduced to 0 ℃ to-100 ℃. Heating may also be used where appropriate. The solvent is typically aprotic for the reduction reaction and may also be aprotic for the oxidation reaction. The reaction time can be adjusted to achieve the desired transition.
The condensation reaction is usually carried out at a temperature close to room temperature, although kinetically controlled condensation reduced temperatures (0 ℃ to-100 ℃) are also common for non-equilibrium. The solvent may be either protic (as is common in equilibrium reactions) or aprotic (as is common in kinetic control reactions). Standard synthetic techniques such as azeotropic removal of reaction byproducts and the use of anhydrous reaction conditions (e.g., inert gas environments) are common in the art and will be applied where applicable.
Protecting group: the term "protecting group" refers to any group that, when bound to a hydroxyl group or other heteroatom, prevents unwanted reactions from occurring on that group and can be removed by conventional chemical or enzymatic steps to reconstruct the hydroxyl group. The particular removable protecting group used is not always critical, and preferred removable hydroxyl end capping groups include conventional substituents such as allyl, benzyl, acetyl, chloroacetyl, thiobenzene, benzyl, benzoyl, methylmethoxy, silyl ethers (e.g., trimethylsilyl (TMS), t-butyldiphenylsilyl (TBDPS), or t-butyldimethylsilyl (TBS)) and any other groups, can be chemically introduced to the hydroxyl function and then selectively removed by chemical or enzymatic means under mild conditions compatible with the nature of the product.
Suitable hydroxy protecting groups are known to those skilled in the art and are disclosed in more detail in t.w. greene, protecting Groups In Organic Synthesis; wiley: new York,1981 ("Greene") and references therein, kocienski, philip J.; protecting Groups (Georg Thieme Verlag Stuttgart, new York, 1994), both of which are incorporated herein by reference.
Protecting groups, commonly known and used, are available and optionally used to prevent side reactions with the protecting groups during the synthesis, i.e., the route or process by which the compounds are prepared by the methods of the present invention. The nature of which groups are protected, when protected, and the chemical protecting group "PG" will depend, to a large extent, on the chemical nature of the reaction to be protected (e.g., acidic, basic, oxidative, reductive, or other conditions) and the intended direction of synthesis.
Herbicide formulation
In general, agrochemical formulations, in particular in liquid form, include inorganic solvents or organic solvents. Most organic solvents known in the art are non-biodegradable and highly flammable. Organic solvent-based agrochemical formulations typically use solvents, preferably water-immiscible, to completely dissolve the active ingredient and produce a transparent homogeneous liquid free of extraneous substances. In addition, organic solvents generally have low flash points, are non-biodegradable, are irritating to the skin, have moderate or high evaporation rates, etc., but provide a transparent, uniform liquid. The known agrochemical composition further comprises at least one surfactant, wherein the properties and amount of the surfactant depend on the active content and solvent in the formulation, the type of active ingredient, the solubility of the active ingredient in the solvent and the desired emulsion properties of the final product.
Pharmaceutical formulation
The compounds described herein may be used to prepare therapeutic pharmaceutical compositions, for example, by combining the compounds with a pharmaceutically acceptable diluent, excipient or carrier. The compounds may be added to the support in the form of salts or solvates. For example, where the compound has sufficient basicity or acidity to form a stable non-toxic acid or base salt, it may be appropriate to administer the compound as a salt. Examples of pharmaceutically acceptable salts are organic acid addition salts with acids forming physiologically acceptable anions, such as tosylate, mesylate, acetate, citrate, malonate, tartrate, succinate, benzoate, ascorbate, alpha-ketoglutarate and beta-glycerophosphate. Suitable inorganic salts may also be formed, including hydrochlorides, halides, sulphates, nitrates, bicarbonates and carbonates.
Pharmaceutically acceptable salts can be obtained using standard procedures well known in the art, for example, by reacting a sufficiently basic compound such as an amine with a suitable acid to provide a physiologically acceptable ionic compound. Alkali metal (e.g., sodium, potassium, or lithium) or alkaline earth metal (e.g., calcium) carboxylates can also be prepared in a similar manner.
The compounds of the formulations described herein may be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient, in a variety of forms. These forms may be particularly suitable for the chosen route of administration, for example, oral or parenteral, by intravenous, intramuscular, topical or subcutaneous routes of administration.
The compounds described herein may be administered systemically in combination with a pharmaceutically acceptable carrier, e.g., an inert diluent or an edible carrier that may be assimilated. For oral administration, the compound may be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or added directly to the patient's diet. The compounds may also be combined with one or more excipients and used in the form of edible tablets, troches, lozenges, capsules, long-acting drugs, suspensions, syrups, wafers and the like. Such compositions and formulations typically comprise at least 0.1% active compound. The percentage of the compositions and formulations may vary, and may conveniently be from about 0.5% to about 60%, from about 1% to about 25%, or from about 2% to about 10% by weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions can be such that an effective dosage level can be obtained.
The tablets, troches, pills, capsules and the like may also contain one or more binders such as xanthan gum, acacia gum, corn starch or gelatin; adjuvants, such as dicalcium phosphate; disintegrants such as corn starch, potato starch, alginic acid and the like; and also lubricants such as magnesium stearate. Optionally adding sweetener such as sucrose, fructose, lactose or aspartame; or flavoring agent such as peppermint, wintergreen oil or cherry flavoring. When the unit dosage form is a capsule, it may contain, in addition to materials of the type described above, a liquid carrier, such as a vegetable oil or polyethylene glycol. Various other materials may be present as coatings or otherwise modify the physical form of the solid unit dosage form. For example, tablets, pills or capsules may be coated with gelatin, waxes, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylhydroxybenzoates as preservatives, a dye and flavoring agent such as cherry or orange flavor. Any material used to prepare any unit dosage form should be pharmaceutically acceptable and the amount used is substantially non-toxic. In addition, the active compounds can be incorporated into slow release formulations and devices.
The active compounds may be administered by intravenous or intraperitoneal injection. Solutions of the active compounds or salts thereof may be prepared in water, optionally mixed with non-toxic surfactants. The dispersion may be prepared in glycerol, liquid polyethylene glycol, triacetic acid or mixtures thereof, or in pharmaceutically acceptable oils. Under ordinary conditions of storage and use, the formulation may contain a preservative to prevent the growth of microorganisms.
Pharmaceutical dosage forms suitable for injection or infusion may comprise sterile aqueous solutions, dispersions or sterile powders containing the active ingredient which are suitable for the extemporaneous preparation of sterile injectable or insoluble solutions or dispersions, optionally entrapped in liposomes. The final dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or carrier may be a solvent or liquid dispersion medium including, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), vegetable oils, non-toxic glycerides, and suitable mixtures thereof. For example, proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersion, or by the use of surfactants. Various antibacterial and/or antifungal agents can prevent the action of microorganisms, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is desirable to include isotonic agents, for example, sugars, buffers, or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by agents which delay absorption, for example, aluminum monostearate and/or gelatin.
Sterile injectable solutions may be prepared by incorporating the active compound in the required amount in the appropriate solvent with various other ingredients as required and then optionally filter sterilized. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation may include vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient present in the solution thereof.
When administered topically, the compounds may be used in pure form, for example, when they are liquids. However, it is generally desirable to apply the active agent to the skin as a composition or formulation, for example, in combination with a dermatologically acceptable carrier, which may be a solid, liquid, gel or the like.
Suitable solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Suitable liquid carriers include water, dimethyl sulfoxide (DMSO), alcohols, glycols, or water-alcohol/glycol mixtures, wherein the compounds can be dissolved or dispersed at an effective level with the aid of non-toxic surfactants. Adjuvants, such as fragrances and additional antimicrobial agents, may be added to optimize the characteristics of a particular application. The resulting liquid composition may be applied from an absorbent pad for impregnating bandages and other dressings, or sprayed onto the affected area using a pump or aerosol sprayer.
Thickeners, such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials, may also be used with the liquid carrier to form spreadable pastes, gels, ointments, soaps, and the like, for direct application to the skin of the user.
Examples of skin compositions for delivering active agents to the skin are known in the art; see, for example, U.S. patent nos.4,992,478 (Geria), 4,820,508 (worth), 4,608,392 (Jacquet et al), and 4,559,157 (Smith et al). Such skin compositions may be used in combination with the compounds described herein, wherein the ingredients of such compositions may optionally be replaced with the compounds described herein, or the compounds described herein may be added to the composition.
Useful dosages of the compositions described herein can be determined by comparing their in vitro activity to in vivo activity in animal models. Methods for extrapolating effective dosages of mice and other animals to humans are known in the art; see, for example, U.S. patent No.4,938,949 (Borch et al). The amount of the compound or active salt or derivative thereof required for treatment will depend not only on the particular compound or salt selected, but also on the route of administration, the nature of the condition being treated and the age and condition of the patient and will ultimately be at the discretion of the attendant physician or clinician.
Generally, however, a suitable dosage will be in the range of about 0.5 to about 100mg/kg, for example, about 10 to about 75mg/kg body weight per day, such as 3 to about 50mg per kg body weight per day, preferably in the range of 6 to 90 mg/kg/day, and most preferably in the range of 15 to 60 mg/kg/day, of the recipient.
The compounds are conveniently formulated in unit dosage form; for example, it contains 5 to 1000mg, conveniently 10 to 750mg, more conveniently 50 to 500mg of the active ingredient per unit dosage form. In one embodiment, the present invention provides a composition comprising a compound of the present invention formulated in such unit dosage form.
The compounds may conveniently be administered in unit dosage form, e.g. containing from 5 to 1000mg/m per unit dosage form 2 Conveniently from 10 to 750mg/m 2 More conveniently 50 to 500mg/m 2 Is effective in the composition. The required dose may conveniently be presented in a single dose or may be administered in divided doses at appropriate intervals, for example, two, three, four or more doses per day. Sub-doses themselves may be further divided into discrete loose doses.
The required dose may conveniently be presented in a single dose or may be administered in divided doses at appropriate intervals, for example, two, three, four or more doses per day. Sub-doses themselves may be further divided into discrete, loosely spaced administrations; for example, by multiple inhalation with a nebulizer or by instilling multiple drops of the drug solution into the eye.
The present invention provides a method of treatment of cancer in a mammal, which involves administering to a mammal suffering from cancer an effective amount of a compound or composition of the present invention. Mammals include primates, humans, rodents, canines, felines, bovines, ovines, equines, porcines, ovines, bovines, and the like. Cancer refers to various types of malignant tumors, such as brain cancer, colon cancer, breast cancer, melanoma, leukemia, etc., and is generally characterized by poor cell proliferation, such as irregular growth, lack of differentiation, local tissue infiltration, metastasis, etc.
The ability of the compounds of the invention to treat cancer can be determined by using assays well known in the art. For example, the biological significance of treatment regimen design, toxicity assessment, data analysis, quantification of tumor cell killing, and screening using transplantable tumors is known.
The following examples are intended to illustrate the above invention and should not be construed as narrowing the scope thereof. Those skilled in the art will readily recognize that the examples illustrate that many other methods of practicing the invention are possible. It should be understood that many variations and modifications may be made while remaining within the scope of the invention.
Examples
Example 1 methods and materials
We constructed a recombinant strain of P.ananatis LMG 5342 that was capable of producing large amounts (> 3mg per liter) of native phosphonate small molecules produced by biosynthetic gene clusters within the pathogenic island of the bacterium (also known as "HiVir" or hvr). By nuclear magnetic resonance and mass spectrometry analysis, we isolated and purified this small molecule, which was found to be (2E) -3-carboxy-4-hydroxy-4-phosphobut-2-enoate, compound 1). Based on our data on herbicide bioassays for onion and mustard seedlings, this small molecule acts as a herbicide. The exact mechanism of action is not known, but it produces toxicity similar to the known herbicides glyphosate and glyphosate. Banana DNA and protein sequences can be found in ATGC databases, such as Kristensen et al, nucleic Acids res.2017jan 4;45 D1D 210-D218. The ATGC database is held in combination with University of Iowa at dmk-brain. Ech. Uiowa. Edu/ATGC/NCBI at ftp. NCBI. Nlm. Nih. Gov/pub/kristensen/ATGC/ATGC/atgc_home. Html or at ftp. NCBI. Lm. Nih. Gov/pub/kristensen/ATGC/atgc_list. Html. The hvr operator gene nucleic acid sequence database accession numbers of p.ananatis are hvrA (wp_ 013027161.1), hvrB (wp_ 041455823.1), hvrC (wp_ 013027159.1), hvrD (wp_ 014605075.1), hvrE (wp_ 013027157.1), hvrG (wp_ 013027156.1), hvrH (wp_ 013027154.1), hvrI (wp_ 013027153.1), hvrJ (wp_ 014605079.1) hvrK (wp_ 013027151.1), hvrL (wp_ 013027150.1). The whole genome sequence of p.ananatis can also be searched using Genbank accession No. HE617160.1, nc_016816.1, HE617161.1 or nc_016817.1.
Bacteria, plasmids and growth conditions: the strains, plasmids and primers used in this study are shown in tables 3 and 4. General chemical and molecular biological reagents were purchased from Sigma-Aldrich or New England Biolabs. Phosphofacillin (glufosinate) was purchased from GoldBio (CAS# 77182-82-2). Standard bacterial growth media (Current Protocols in Molecular Biology 2019,125: e81, e82, e 83) were prepared as described. Coli strains are typically grown at 37 ℃; the P.ananatis strain is usually grown at 30 ℃. Phosphonate-induced Medium (PIM) was prepared by mixing 8.37g/L MOPS,0.72g/L Tricine,0.58g/L NaCl,0.51g/L NH 4 Cl,1.6g/LKOH,0.1g/L MgCl 2 ·6H 2 O,0.05g/L K 2 SO 4 ,0.2g/L K 2 HPO 4 Dissolving in water, and steam sterilizing at 121deg.C for 20min. After cooling, 1X sterile trace element solution (1000X stock solution: 1.5g/L trisodium nitrate, 0.8g/LFe (NH) 4 ) 2 (SO 4 ) 2 ·7H 2 O,0.2g/L Na 2 SeO 3 ,0.1g/L CoCl 2 ·6H 2 O,0.1g/L MnSO 4 ·H 2 O,0.1g/LNa 2 MoO 4 ·2H 2 O,0.1g/L Na 2 WO 4 ·2H 2 O,0.1g/L ZnSO 4 ·7H 2 O,0.1g/L NiCl 2 ·6H 2 O,0.01g/L H 3 BO 3 ,0.01g/L CuSO 4 ·5H 2 O) 1% (v/v) sterile glycerol and 50. Mu.g/mL sterile kanamycin were added. Culture of Candida (Candida) and Saccharomyces (Saccharomyces) strains, rosewell Park Memorial Institute 1640 Medium (RPMI; sigma-Aldrich R)6504 For liquid culture and Sabouraud Dextrose Agar (SDA; difco TM 210950 For solid electroplating unless otherwise indicated. For Aspergillus strain Rosewell Park Memorial Institute 1640 medium (RPMI) was used for liquid culture and potato dextrose agar (PDA; difcoTM 213400) for solid plating unless otherwise indicated. YMM medium (6.7 g/L yeast nitrogen base, free of amino acids [ BD-Difco ] TM 291940]20g/L glucose, 1 Xtrace element solution) as the minimum medium for the fungal strain. .
TABLE 3 microorganisms used in this study
a Cell 1978,15(4):1199-208
b Proc Natl Acad Sci U S A 1990,87(12):4645-4649
c Nat Chem Biol 2007,3:480–485
d Chem Biol 2008,15(8):765-770
Bioinformatics analysis of phosphonate metabolism in p.ananatis strain: the p.ananatis genome was downloaded using HMMER version 3.2.1 from NCBI database and Hidden Markov Model (HMM) retrieval. HMMs used for analysis are phosphoenolpyruvate phosphomutase (pepM, TIGR 02320), phosphoacetaldehyde hydrolase (phnX, TIGR 01422), phosphoacetate hydrolase (phnA, TIGR 02335), 2-AEP aminotransferase (phnW, TIGR 02126 and TIGR 03301), phosphopyruvate hydrolase (palA, TIGR 02321), phosphoacetaldehyde dehydrogenase (phnY, TIGR 03250), HD-phosphohydrolase (phnZ, TIGR00277 and PF 01966), C-P lyase (phnJ, PF 06007). Genomes with HMM matches are considered to have associated metabolism. Using geneous
2020.2.1 software maps the pepM-containing genome to the p.ananatis LMG 5342 gene cluster to screen for the presence of the hvr locus. For operations not corresponding to hvrThe pepM gene of the indulger can infer the cluster boundaries around pepM based on flanking integration and the presence of conjugated elements or genes that occur in the operon with pepM. BLASTP is used to assign functions to genes in a gene cluster based on homology of known functional proteins.
Genomic sequencing of Pantoea NRRL strain: the genomes of P.ananatis NRRL B-14773 and P.ananatis B-133 (renamed P.stewartii based on NCBI mean nucleotide profile) were sequenced in this study. High molecular weight genomic DNA useDNeasy ulclean Microbial Kit purification was performed. Purified genomic DNA was prepared using Shotgun Flex DNA library and sequenced by Roy j.carver Biotechnology Sequencing Center, UIUC using Illumina MiSeq v2 platform (250 nt paired end read). Genome reads were trimmed using BBDuk software, assembled using spades3.14.1, and annotated using RAST Server. Assembled reads (reads) are submitted to NCBI Whole Genome Shotgun (WGS) database. The WGS project has been stored in DDBJ/ENA/GenBank under the numbers JACETZ000000000 (for P.steweriti NRRL B-133) and JACEUA000000000 (for P.ananatis NRRL B-14773). Genomic analysis was performed on phosphonate metabolism as described above.
Table 4, plasmids used in this study
d Chem Biol 2008,15(8):765-70
e J Bacteriol 2001,183(21):6384-93
f J Biol Chem 2011,286(25):22283-90
Genetic method for preparing marker-free deletion mutations: DNA approximately 1kb upstream and downstream of the region to be deleted was cloned into pHC001A (see Table 4 for a complete list of plasmid constructs). The resulting plasmid was introduced into E.coli DH 5. Alpha./λpir and stored in LB medium +50. Mu.g/mL kanamycin (LB-kan). The plasmid construct was transferred by electroporation-mediated transformation into the conjugate strain WM6026 and then onto the p.ananatis receptor by conjugation. WM 6026-derived donor strains, which are DAP dystrophies, were grown by combining isolated colonies of donor and recipient in small (2 cm) plaques on agar-solidified LB medium containing 60. Mu.M Diaminodipentaerythritol (DAP). After overnight incubation at 30 ℃, plaques were taken, re-crushed on LB-Kan without DAP to select p.ananatis recombinants carrying the deletion plasmid for insertion into the target site by homologous recombination. The deleted plasmid was unable to autonomously replicate in p. The isolated colonies were decolorized on LB-kan at 30℃and then on LB without antibiotics to isolate the integrative plasmid. Recombinants that lost the integration plasmid were then band isolated on LB medium without 5% sucrose, which was selected for the sacB gene encoded on the integration deletion plasmid. Deletion of the integrative plasmid was verified by the sensitivity of the purified recombinants to kanamycin. Finally, recombinants carrying the desired deletions were identified by PCR-based screening (Table 5), the primers used being described in Polidore et al, mBio.2021Jan-Feb;12 (1) e03402-20, which is incorporated herein by reference in its entirety.
TABLE 5 primers used in this study
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g Mol Plant Microbe Interact 2018,31(12):1291-1300
Introduction of IPTG-inducible Ptac systems. Plasmid pAP01 was introduced into WM6026 by electroporation and LB+50. Mu.g/mL kanamycin (LB-kan) was selected. The plasmid was then transferred to P.ananatis MMG1988, described in the previous section. Recombinants carrying a complete copy of pAP01 inserted with the hvrA gene were selected on LB-Kan. Using aph and lacI q The primer and Hvr marker gene hvrI were used to perform plasmid integration screening on the colonies obtained. The resulting p.ananatis MMG2010 strain was stored in LB-Kan to prevent loss of the integrating plasmid.
NMR and MS: recording on an Agilent DD2 600MHz spectrometer 1 H-NMR, 13 C-NMR 31 P-NMR Spectroscopy [ ] 1 The H is 600MHz and the frequency of the H is, 13 c is 150MHz, and the frequency of the catalyst is equal to or higher than the frequency of the catalyst, 31 p is 243 MHz). The sample is 20 to 100 percent D 2 O was prepared as a locking solvent. Quantification of 31 PNMR was performed using internal standards of 0.5mM dimethylphosphonate and 0.9mM EDTA and was collected using 5 x T1 measurements (relaxation time) of the samples. Phosphonate peak integration was calculated using MestReNova v11.0.1 software and normalized to internal standard. The concentration was calculated from the ratio of the normalized phosphonate peak integral to the known internal standard concentration. Mass spectrometry was used by the School of Chemical Sciences Mass Spectrometry LaboratoryQ-TOF Ultima ESI was performed in which 10. Mu.L of sample was injected at a concentration of 10. Mu.g/mL in methanol.
IPTG-induced expression of the hvr operon in p. Frozen glycerol stocks of P.ananatis MMG2010 were revived on LB+50. Mu.g/mL kanamycin and incubated at 30℃for 24 hours. The individual colonies were then transferred to 5mL Phosphonate Induction Medium (PIM). The culture was incubated at 30℃for 48 hours, then 0.5mL of the culture was transferred to 50mL of PIM, and incubated at 30℃for 24 hours. The next day, 8mL of culture was transferred to four flasks with 800mL PIM medium and 1mM IPTG. These 800mL cultures were incubated at 30℃for 72 hours with shaking at 175 rpm. After the completion of the culture, the cells and chips were removed by centrifugation at 8000rpm for 20min, and the supernatant was concentrated by freeze-drying. Quantifying the concentrated supernatant 31 P NMR analysis to determine phosphonate yield levels after addition of dimethyl phosphonate (0.5 mM final) as internal standard.
Pan Dalin (pantaphos) and compound 2 (compound 2). 3.2L of P.ananatis MMG2010 was grown in PIM medium containing 1mM IPTG as described in the previous section. Cell removal by centrifugationAfter freeze-drying the spent medium, the dried material was resuspended in 300mL H 2 O. 1200mL of 100% cold methanol was then added and the final concentration was 75% methanol and incubated overnight at-20 ℃. Using The grade 42 ashless filter was vacuum filtered to remove precipitated material and stored, and the methanol soluble fraction was dried to completion using initial rotary evaporation followed by freeze drying. 48.0g of the dried material obtained by this process (sample A) was saved for further purification. The precipitated material saved from the above methanol extraction was subjected to a second 75% methanol extraction as described. The methanol soluble fraction was then dried to completion using an initial rotary evaporation followed by freeze drying. 1.60g of the dried material obtained by this process (sample B) was saved for further purification. Sample A was purified by Fe 3+ IMAC decontamination is as follows. 10g of Chelex resin (sodium form) was incubated in 1M HCl for 30min, then washed with 5 Column Volumes (CV) of water and converted to H + Form of the invention. Next, the resin was dissolved in 100mL of 300mM FeCl 3 ·6H 2 Re-suspending in O, charging Fe at 4deg.C +3 Then washed with 100mL of 0.1% acetic acid and incubated overnight at 4℃in 100mL of 0.1% acetic acid. Sample A was acidified to pH 3 with concentrated acetic acid and incubated with Fe-IMAC resin at 4deg.C for 2 hours. The solution was separated from the resin using a gravity column and the stream containing unbound phosphonic acid (sample stream) was stored. Using 100mL NH 4 HCO 3 (1,5,25,50,100,250,500 and 1000 mM) the bound phosphonic acid was eluted from the Fe-IMAC resin and the fractions were collected and neutralized to neutral pH with acetic acid. The 8 fractions were concentrated using initial rotary evaporation followed by lyophilization. The sample stream from above, containing any unbound phosphonic acid, was bound to sample B and subjected to a second round of Fe-IMAC purification as described above. The fractions were concentrated using initial rotary evaporation followed by lyophilization. From each Fe-IMAC purification, these components were combined in 2-3mL H as follows 2 In O500-1000 mM NH 4 HCO 3 Component (sample 1), 1-5mM NH 4 HCO 3 Component (sample 2) and 25-50-100-250mM NH 4 HCO 3 Component (sample 3). Samples 1, 2 and 3 were concentrated and dried by freeze drying to obtain 45.1mg, 30.6mg and 6.9mg of dry matter, respectively. Phosphorus NMR was quantified, based on a 0.5mM dimethylphosphonate standard reference, and each sample was evaluated for purity using proton NMR. The phosphonate content in samples 1 and 3 was 0.551mmol and 0.215mmol, respectively. However, sample 2 was found to contain only 0.0165mmol phosphonate and contained residual phosphate not present in samples 1 and 3, and thus was not used for further purification steps.
Sample 1 was further purified using a Teledyne ISCO CombiFlash RF + UV-Vis system and using a RediSep SAX anion exchange resin. Sample 1 was lyophilized and then reconstituted in 75% methanol. Insoluble material was removed by centrifugation at 1mL100% D 2 Resuspension in O and pass 31 P NMR detection. The sample containing residual phosphate was subjected to an additional drying cycle and 75% methanol until all phosphate compounds were dissolved. The methanol soluble components were then mixed and subjected to CombiFlash purification. For this purpose, a 5.7g RedieSep SAX column was used with 20 Column Volumes (CV) of H 2 O5% NH 4 OH balance followed by 20CV H 2 O, then 20CV of 90% methanol. 1mL of a sample was taken from the methanol-soluble fraction, loaded onto the column by direct injection, and then: 3.3min 100% A (90% methanol), linear gradient to 100% B (5% NH) 4 OH is at H 2 O) for more than 15min, followed by 100% B for 6min, then 3.5min 100% A, flow rate of 18mL/min. The components were monitored using UV 250nm and 210nm and passed through 31 P NMR combined and analyzed the components. Preservation of delta-containing P 18 and 15ppm phosphorus chemical shift fractions and dried by rotary evaporation followed by lyophilization.
Sample 3 and Combiflash purified sample 1 were each subjected to HPLC purification. Dried sample in 1mL H 2 O was reconstituted and then 50-100. Mu.L of the sample was diluted in solvent B (90% acetonitrile+10 mM NH) 4 HCO 3 pH 9.20) to a final concentration of 75% solvent B. The sample was then filtered through a 0.45 μm filter and purified using an Atlantis HILIC silica gel column (10X 250 mm) 2 Particle size of 5 μm) is eluted by gradientAnd (5) purifying. Chromatography was performed at a flow rate of 4mL/min using H 2 O+10mm NH 4 HCO 3 At pH 8.50 (solvent A) and 90% acetonitrile+10mm NH 4 HCO 3 At pH 9.20 (solvent B). The operating gradients were as follows: 8min in 90% solvent B, then a linear gradient to 70% solvent B for 20min, then 50% solvent B for 1min, holding 50% solvent B for 8min, then back to 90% solvent B for 1min, then holding in 90% solvent B for 8min. Fractions were collected and uv absorbance was monitored at 210 and 250 nm. The components that absorb at these wavelengths are combined, dried by rotary evaporation, and analyzed using phosphorus NMR. Obtaining the product containing delta P Fractions of pure phosphate compounds (corresponding to pantaphos) with chemical shifts of 15ppm, and containing delta P A fraction of 18ppm of chemically displaced purified phosphate compound (corresponding to compound 2). In addition, a mixture fraction containing pantaphos and compound 2 was obtained. The purified compound was dried and stored at 4 ℃ for MS and NMR structural analysis as described above.
Onion bioactivity assay: yellow onions purchased from the local market were surface sterilized in a laminar flow biosafety hood as follows. First, the damaged or yellowing outermost layer is removed and then soaked with 10% bleach for 10min. Then using sterile dH 2 Washing onion three times with O, soaking in 70% ethanol for 10min, and sterilizing with dH 2 O was rinsed four times. The onion was placed in a biosafety laminar flow hood until the water and ethanol were completely evaporated. To test the virulence of microbial strains, onion seed was pricked with a previously sterilized wood toothpick dipped into a bacterial cell suspension (1X 103 CFU/mL in 1 XPBS buffer). 100. Mu.L of the sterilized sample was added to the wells formed during toothpick inoculation for chemical complementation studies. To test the biological activity of crude and purified chemical samples without bacteria, sterilized onions were perforated with sterile toothpicks and 100 μl of filter sterilized samples were then added. Following inoculation and/or treatment with filter sterilized compounds, onions were placed in zipper-locked plastic bags and incubated in the dark at 30 ℃ for the indicated number of days. After incubation, onions were sectioned at the inoculation/sample application site for visual inspection A central rot phenotype.
Mustard seedlings and Arabidopsis Col-0 bioactivity assay: all seed preparations were carried out in laminar flow biosafety hood.
The method for cleaning the light green leaf mustard seeds comprises the following steps: 10% bleaching agent for 1min, sterile dH 2 O is washed 3 times, 70 percent ethanol is washed for 1min, and then sterile dH is carried out 2 O was flushed 5 times. The cleaned seeds were transferred to sterilized paper towels and placed into a sterile container. Then, 5mL of sterile tap water was added to the paper towel, and the container was incubated in the dark for 48 hours to allow the seeds to germinate. Germinated seedlings were transferred to 24-well cell culture plates, each well containing 1mL Murashige and Skoog agar (1% agar). The sterile seeds of Arabidopsis Col-0 (accession number: CS 1092) were purchased from Arabidopsis Biological Resource Center (ABRC) and cultured at 4℃for 2-3 days. The cold-cultivated seeds were then transferred to 24-well cell culture plates, each well containing 1mL of Murashige and Skoog agar (1% agar), and cultivated in the dark until seed germination was observed. For mustard and arabidopsis germinated seedlings, 20 μl of the appropriately diluted filter sterilizing compound is then added to each well to achieve the desired concentration. Negative control, apply 20. Mu.l sterile dH on agar 2 O. The 24-well plates were incubated in a 60% humidity controlled growth chamber for 16 hours. Light was cycled for one week at 23 ℃. After cultivation, the plants are extracted from the growing agar by gentle pulling, which results in plants that are essentially agar free. Measuring root length immediately; the dry weight was determined after 24 hours of drying. At 150 ℃. The conditions were statistically analyzed using a standard Welch's t assay in GraphPad Prism 8.4.1 software to determine significance.
Cell culture cytotoxicity screening: these compounds were evaluated for their ability to kill cancer cell lines in culture, against HOS (osteosarcoma); ES-2 (ovarian cancer); HCT 116 (colon cancer); a549 (lung cancer); a172 (glioma) cells. Human skin fibroblasts (HFF-1) were also evaluated. Cells (3000 cells per well for ES-2, HCT 116, A549 and A172; 4000 cells per well for HFF-1, 2500 cells per well for HOS) were plated in 96-well platesAnd allowed to attach overnight. Cells were treated with pantaphos in water. The concentration of the compound measured was 5nM to 100. Mu.M (final 1% water; 100. Mu.l per well). Raptin (50. Mu.M) served as a dead control. On each dish, 5 technical replicates of each compound were performed. Cell viability was assessed using the Amara blue method 72 hours after treatment (http:// www.bio-rad-anti-bodies. Com/measuring-cytotoxity-pro-duction-phosphometric-fluorescence-alamarBlue. Html). Azma blue solution (10. Mu.l of 440. Mu.M resazurin in sterile 1 XPBS) was added to each well and incubated for 3-4 hours. The Azma blue conversion plate reader (SpectraMax M3; molecular device) fluoresces (excitation wavelength: 555nm; emission wavelength: 585nm; cut-off 570nm; automatic gain). Mortality was determined by normalization to water-treated cells and Raptinal-treated cells. For IC 50 The assay, data are plotted as compound concentration versus percentage of dead cells and fitted to a logical dose response curve using an OriginPro 2019 (OriginLab). Data in triplicate reporting IC 50 Values are the average of three independent experiments, with SEM values reported.
Antibacterial bioassay: susceptibility testing of ESKAPE pathogens and salmonella enterica LT2 was performed using the cobici-pall (Kirby-Bauer) method outlined by Clinical and Laboratory Standards Institute (CLSI). For the rich medium bioassay, mueller Hinton Broth (MH-2, sigma-Aldrich, 90922) was used for all strains except enterococcus faecalis (Enterococcus faecalis) ATCC 19433, to which 1% BHI was added. For minimal medium bioassays, glucose MOPS minimal medium was used. Briefly, overnight bacterial cultures were subcultured and inoculated into 5mL of top agar (0.7% agar) at a final concentration of 5X 10 5 CFU/mL. Then, 20. Mu.L of the compound was measured in a 2-fold dilution series (200,100,50,25,12.5,6.25. Mu.M in water) on a blank tray (BD BBL) having a diameter of 6mm TM 231039 A) is provided. Control discs received 20. Mu.M of 50mg/mL kanamycin. Culturing at 35 deg.C for 20-24 hr. The Minimum Inhibitory Concentration (MIC) was recorded as the lowest concentration of compound that resulted in a distinct zone of inhibition around the disc. The sensitivity of the IPTG-induced phosphate uptake strain WM6242 to pantaphos was examined by means of a sheet diffusion method. In a plate containing growth medium The mixture was covered with 5mL of top agar (0.7% agar) and inoculated with 100. Mu.L (OD) 600 =0.8) phosphate-specific escherichia coli indicator strain WM6242 with or without 1mM IPTG. WM6242 employs an IPTG-induced nonspecific phosphate uptake system (phnCDE). After the seeding cover layer had cured, a 6mm paper tray was marked with 10. Mu.L of pantaphos dilution series (200,100,50,25,12.5,6.25. Mu.M in water) and applied to a plate and then incubated at 37℃for 24 hours. Phosphonate specific activity was queried by comparing sensitivity to 200 μm kanamycin and 200 μm fosfomycin. The Minimum Inhibitory Concentration (MIC) was recorded as the lowest concentration of compound that resulted in a distinct zone of inhibition around the disc.
Bioassay of fungi: the method used for the fungicide test was from CLSI file M27, reference broth dilution antifungal. Briefly, a stock solution of fungus at-80℃was streaked onto a suitable medium as previously indicated and incubated at 35℃for 24-48 hours. For Candida (Candida) and yeast (Saccharomyces), individual colonies were resuspended in 1ml of 1x PBS and then diluted to a final concentration of 1x10 in growth medium 4 CFU/mL. For Aspergillus, hyphal spores on growth substrates were resuspended on grass by spinning 1mL of 90% saline+0.1% Tween-20. The 1mL yeast suspension was then diluted to 1X10 in growth medium 4 Final concentration of CFU/mL. Then, 2. Mu.L of the compound was added to 198. Mu.L of the yeast suspension and placed in a sterile 96-well round bottom plate. For positive control, 2 μl of amphotericin B stock was added to the designated final concentration of 2 or 10 μΜ. 200. Mu.L of untreated yeast suspension and uninoculated medium served as controls. According to different growth media, the culture is carried out at 35 ℃ for 24-48 hours without shaking. The Minimum Inhibitory Concentration (MIC) was determined intuitively by looking for the compound concentration, which was not visually different from the uninoculated medium control.
Identification and analysis of bacterial Hvr biosynthetic homologous gene clusters. NCBI tblastn is used to identify the bacterial genome containing a Hvr-like gene cluster. The query was constructed by gene translation linked to hvrA-L. Tblastn was used for RefSeq genome and RefSeq representative genome database, using the `Organism` parameter of `bacterium [ taxi: 2]', and non-redundant nucleotide database. The results were filtered according to 45% query coverage to yield 185 unique strains. GenBank files of each strain were downloaded and the homologous Hvr gene cluster was determined using the MultiGeneBlast technique. The homologous Hvr biosynthetic gene cluster is organized by type based on the arrangement of genes within the cluster and the presence of additional gene functions.
Example 2 structural resolution of phosphonate compounds.
The structure of compound 1 is illustrated by the NMR spectrum data summarized in the text (table 1) and below; high resolution mass spectrometry data for compound 1 (purified pantaphos) are presented herein. The specified mass fragments with the specified chemical structure are as indicated above. MS chemistry formulas and mass errors were calculated using the ChemCalc workspace. Compound 1 was isolated as a white amorphous solid. Its molecular formula is derived by negative mode HRMS (calcd.for C) 5 H 6 O 8 P -1 :224.98058,observed m/z 224.9805[Δppm 2.09])。
Compound 1 was dissolved in 100% D 2 O NMR experiments were performed. Compound 1 1 H-NMR spectrum showed that at delta H At 4.31 and 5.91ppm, the two signals appear in bimodal form with coupling constants J of 15.3Hz and 6.00Hz, respectively. In phosphonic acid, the coupling constant J of the proton bound to the adjacent carbon atom to the phosphorus atom is 15.3Hz. At the position of 1 H- 31 In the P HMBC analysis, these protons are also associated with delta P 15.40ppm of the compound phosphorus atom are related, indicating close proximity to P (within 3 bond distance). In addition, delta H The lower field signal of 5.91 indicates an ethylene carbon or olefin structure, indicating that the signal corresponds to a single proton. 13 C-NMR spectrum showed delta C 71.00 (d, j= 144.00 Hz), 142.98(s), 174.62(s), 126.50 (d, j=9.05 Hz) and 175.20(s) ppm, indicating that compound 1 contains 5 carbons. Signal at delta C The large coupling constant at 71.00ppm indicates that the carbon is bound to a phosphorus atom, as this cleavage pattern has been observed in other phosphonic acid compoundsC-P bond. Thus, delta C The 71.00ppm signal is designated carbon number 1. None of the other carbon signals exhibit the typical C-P cleavage pattern, and therefore, the signals corresponding to these carbons must reflect the carbon positions adjacent to or near carbon 1 as opposed to the phosphonate moiety.
Proton-carbon HSQC and HMBC experiments show that at delta H Protons were coupled with carbon in position 1 (δC71.00 ppm) at 4.31ppm, and were observed at δ C 142.98, 174.62 and 175.20ppm are related to other carbons, supporting the positional distribution of these carbons near or near carbon 1. Delta C The carbon signal at 142.98ppm has no cleavage pattern, consistent with the chemical shift of the vinyl compound binding to the adjacent carboxylic acid and methyl group, indicating a carbon position of 2. At delta C 174.62 and 175.20ppm, similar carbon signals have no cleavage pattern, consistent with predicted chemical transfer of carboxylic acid, indicating carbon positions of 3 or 5. However, the carbon signal is at δ C Splitting at 126.50ppm (d, j=9.05 Hz) indicates the presence of adjacent protons, because 13 C-NMR analysis was not performed 1 Decoupling of H. This is due to the carbon and protons at delta H HSQC between 5.91 ppm. These data fully support delta C 126.50ppm of carbon was allocated to carbon site 4. According to delta H Protons and delta at 4.31 and 5.91ppm C 142.98, 174.62 and 175.20ppm of proton-carbon HMBC between carbons, we can confirm delta C 142.98, 174.62 and 175.20ppm of carbon were assigned to positions 2, 3 and 5, respectively. Finally, by 1 H- 1 H correlation analysis to determine delta H 4.31 and 5.91ppm protons are in cis carbon-carbon double bond configuration. Based on the agreement between the mass spectral data and these NMR assignments, the compound structure was determined to be (E) -2- (hydroxy (phosphono) methyl) -4-oxopent-2-enoic acid ester ((E) -2- (hydroxy (phosphono) methyl) -4-oxopent-2-enoate).
Structural resolution of compound 2: NMR spectroscopic data are summarized in the text (table 1); NMR spectrum and high resolution Mass Spectrometry data for Compound 2 can be obtained in this paragraphFound at the end of (c). MS chemistry formulas and mass errors were calculated using the ChemCalc workspace. Compound 2 was isolated as a white amorphous solid. Its molecular formula is derived by using negative mode HRMS (calcd.for C) 5 H 6 O 7 P -1 :208.98566,observed m/z 208.9851[Δppm-0.07]). The component containing pure compound 2 was dissolved at 100% D 2 O, and proton and phosphorus NMR analyses were performed. For carbon NMR experiments, samples containing trace amounts of compound 1 were used, without sufficient concentration of pure compound 2 for carbon-13 analysis. Compound 2 1 H-NMR spectra showed two signals of δ5.71 (d, j=6.00 Hz) and 2.43ppm (d, j=18.0 Hz). In phosphonic acid, the coupling constant J of the proton bound to the adjacent carbon atom to the phosphorus atom is 18.0Hz. At the position of 1 H- 31 In the P HMBC analysis, these protons are also associated with delta P 18.46ppm of the compound phosphorus atom is related, indicating close proximity to P (within 3 bond distance). In addition, delta H The lower field signal of 5.71 indicates an ethylene carbon or olefin structure, indicating that the signal corresponds to a single proton. 13 C-NMR analysis showed that the signal associated with Compound 2 was delta C 34.05 (d, j=123.0 Hz), δ 126.10 (d, j=10.60 Hz), δ 140.36(s), δ 174.68(s) and δ 177.02(s) ppm, indicating that compound 2 is a five carbon molecule. Carbon signal at delta C The large coupling constant at 34.05ppm indicates that the carbon is bound to a phosphorus atom, as this cleavage pattern has observed a C-P bond in other phosphonic acid compounds. Thus, delta C The signal at 34.05ppm is designated carbon number 1. None of the other carbon signals exhibit the typical C-P cleavage pattern, and therefore, the signals corresponding to these carbons must reflect the carbon positions adjacent to or near carbon 1 as opposed to the phosphonate moiety. Proton-carbon HSQC and HMBC experiments showed that at δH2.43 ppm, the proton was reacted with carbon in position 1 (δ C 34.05 ppm) coupling and observed at delta C 140.36, 174.68 and 177.02ppm protons are associated with other carbons, supporting the positional distribution of these carbons near or near carbon 1. Delta C The carbon signal at 140.36ppm has no cleavage pattern, consistent with the predicted chemical shift of the vinyl compound binding to the adjacent carboxylic acid and methyl group, indicating a carbon position of 2. At delta C 174.68 and 177.02ppm, are similarThe carbon signal has no cleavage pattern, consistent with predicted chemical transfer of carboxylic acid, indicating a carbon position of 3 or 5. However, the carbon signal is at δ C Splitting at 126.10ppm (d, j=10.60 Hz) indicates the presence of adjacent protons, because 13 C-NMR analysis was not performed 1 Decoupling of H. This is represented by the carbon and proton at delta H HSQC between 5.71 ppm. These data fully support delta C 126.10ppm of carbon was allocated to carbon site 4. According to delta H Protons and delta at 2.43 and 5.71ppm C 140.36, 174.68 and 177.02ppm of proton-carbon HMBC between carbons, we can confirm delta C 140.36, 174.68 and 177.02ppm of carbon were assigned to positions 2, 3 and 5, respectively. Finally, by 1 H- 1 H correlation analysis to determine delta H 2.43 and 5.71ppm protons are in cis carbon-carbon double bond configuration. Based on the agreement between the mass spectrum data and these NMR assignments, the compound structure was determined to be (E) -2- (phosphono) methyl) -4-oxopent-2-enoic acid ester (E) -2- (phospho) methyl) -4-oxopent-2-enoate.
Example 3. Preparation of phosphonate compound multiple grams.
The multi-gram-level preparation process flow of the phosphonate product is published as follows:
1. phosphate induction with IPTG in a 10L bioreactor
2. Freeze-dried granulosa cells and concentrated supernatant
3. Methanol extraction of phosphonic acids
4. Purification from methanol soluble fraction using iron-IMAC
5. Further purification by flash chromatography and HILIC HPLC
The detailed steps of the flow are as follows:
step 1, streaking a frozen culture solution of phosphonate-induced strains on an LB culture medium and culturing the phosphonate-induced strains at 30 ℃ for 18 hours;
step 2. Single colonies were transferred to 5mL phosphonate induction medium (PIM minus IPTG; see published reference for formulation) and incubated at 30℃for 48 hours;
step 3. Transfer 1mL of the previous culture to 200mL PIM (minus IPTG) and incubate at 30℃for 48 hours;
step 4 100mL of the previous culture was transferred to 10L PIM (containing 1mM IPTG) in New Brunswick BIOFLO fermenter/bioreactor and incubated for 96 hours as follows:
a) The oxygenation is carried out through a sterile air filter (air bubbles are formed at the bottom of the reactor), and a pressure gauge is set to be an air flow rate of 5 liters/min;
b) The bioreactor was fitted with a 30 ℃ heating jacket;
c) Growth of Pantoea ananatis and phosphonate production, manifested by development of yellow pigment;
Step 5, granulating the cells (7500 Xg is centrifuged for 20 min), collecting culture supernatant (used culture medium), and freeze-drying to completely dehydrate;
step 6, extracting phosphonic acid from the used culture medium by using 75% cold methanol;
step 7, separating the methanol soluble part by filtration or centrifugation, and concentrating the obtained solution by rotary evaporation to remove methanol;
step 8, purifying the methanol-soluble phosphonic acid from the concentrated solution by using 200 g of iron-IMAC resin affinity chromatography, wherein the gradient of ammonium bicarbonate reaches 1M. Neutralizing the obtained fraction with acetic acid, and concentrating by rotary evaporation;
step 9 the resulting concentrated fraction containing phosphonic acid was further purified by a flash chromatography system with a strong anion exchange column with a gradient of 5% ammonium hydroxide. The resulting components having UV absorbance at 250nm are brought together and neutralized with acetic acid, and then concentrated by rotary evaporation;
step 10, further purifying the fractions of the flash chromatography by a HILIC HPLC method, wherein the gradient is acetonitrile+10 mM ammonium bicarbonate, pH 8.5 (solvent B) and water+10 mM ammonium bicarbonate, pH 8.5 (solvent a); fractions that absorbed at 250nm were collected and may be further separated using HILIC HPLC, gradient acetonitrile +10mM ammonium acetate, pH4 (solvent B) and water +10mM ammonium acetate, pH4 (solvent a). Finally, the remaining ammonium acetate was removed from the pure compound using a HILIC HPLC method, gradient of acetonitrile (solvent B) and water (solvent a).
Example 4 phosphonate related gene clusters associated with various pathogenic and non-pathogenic p.ananatis strains.
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Bold text = strain other than correlation between hvr and pathogenicity
n/a= data not available (without data)
nt=non-tested (not tested)
g=strain showing plant growth promoting ability according to data of related reference materials
e = strain exhibiting an endogenous (endophytic) lifestyle based on data of related reference material
The preservation is only as virulence determining loci and not whole genome
* Loci identified based on the presence of pepM gene and related genomic regions referencing the p.ananatis LMG 5342 hvr gene cluster
* Locus identified based on phnJ Gene Presence
* Strains sequenced in this study are indicated (see materials and methods)
Example 5 cytotoxicity assay.
We have previously performed cytotoxicity assays on a panel of human cell lines, including normal fibroblast (HFF-1 cell line) and five cancer cell lines (HOS (human osteosarcoma), ES-2 (human ovarian carcinoma), a-549 (human lung carcinoma) and a-172 (human glioma), cyclophosphates showed moderate levels for several human cell linesIs shown in Table 6. IC for each test cell line except that one ovarian cancer cell line (ES-2) was unaffected at the maximum dose 50 The levels are substantially similar in the range of 6.0 to 37.0 mM. Glioma cell line (A-172) was particularly sensitive to pantaphos (IC 50 1.0 mM). To investigate whether this sensitivity is specific for glioma cell lines, we extended the study panel to another 10 human cancer cell lines (fig. 8 and table 6). Consistent with previous data, some cell lines were sensitive, while others were drug resistant, including glioma cell line TG98. Thus, pantaphos cytotoxicity is not characteristic of all glioblastomas. However, cell lines from other cancer types, including MCF-7 (breast cancer) MDA-MB-231 (breast cancer) CT26 (colon cancer) HepG2 (liver cancer) AM38 (glioblastoma), are sensitive to the compound. These data indicate that these cell lines carry mutations, conferring sensitivity to pantaphos, and paves the way for using the molecules as a treatment for cancers carrying these genetic markers.
Cytotoxicity of pantaphos against various cancer cell lines.
Example 6 nucleic acid and amino acid sequence
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Example 7: pharmaceutical dosage forms
The following formulations describe representative pharmaceutical dosage forms useful for therapeutic or prophylactic administration of the compositions of the formulae described herein, particularly disclosed herein, or pharmaceutically acceptable salts or solvates thereof (hereinafter "composition X (Composition X)"):
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These formulations may be prepared by conventional methods well known in the pharmaceutical arts. It will be appreciated that the above pharmaceutical combinations may be varied according to well known pharmaceutical techniques to accommodate different amounts and different types of active ingredient "compound X". Aerosol (vi) may be used in combination with a standard metered dose nebulizer. The specific components and proportions are for illustration only. The specific ingredients may be replaced with suitable equivalents (e.g., the ingredients described above) and the proportions may be varied, depending on the desired properties of the dosage form required.
The foregoing description of some embodiments and examples has been presented for purposes of illustration and description only, and is not intended to limit the scope of the invention. Variations and modifications are possible in light of the teachings of the present invention without departing from the broader scope of the invention as defined in the following claims.
All publications, issued patents, and patent application documents are incorporated by reference herein as if individually incorporated by reference. From these publications, no limitations should be introduced that are inconsistent with the disclosure herein. The description of the present invention refers to various specific and preferred embodiments and technical solutions. It should be understood that numerous changes and modifications could be resorted to, falling within the spirit and scope of the invention.
Claims (41)
1. A composition comprising a compound of formula I:
or a salt thereof; wherein:
-represents a single bond or a double bond;
represents a double bond or a single bond, wherein->And-not both are double bonds;
g is X A CHOR 5 ,O,C(＝O),C(＝CH 2 ),CHP(＝O)(R 6 ) 2 Or CX (CX) B 2 ；
X A Is a defect or O;
each X is B Each independently is H or halogen;
R 1 and R is 2 Each independently is OR A Or an amino acid;
R 3 is-C (=O) R 7 Or triazole or tetrazole;
R 4 is-C (=O) R 8 Or triazole or tetrazole;
R 5 is H, - (C) 1 -C 6 ) Alkyl, - (C) 3 -C 6 ) Cycloalkyl, aryl or heteroaryl;
each R 6 Each independently is OR B Or an amino acid;
R 7 and R is 8 Each independently is OR C Or an amino acid; and
R A ，R B and R is C Each independently is H, - (C) 1 -C 6 ) Alkyl, - (C) 3 -C 6 ) Cycloalkyl, aryl or heteroaryl; and a non-aqueous fluid, an additive, or a combination thereof.
2. The composition of claim 1 wherein G is CHOR 5 。
3. The composition of claim 2, wherein the compound is the (S) -enantiomer.
4. The composition of claim 2, wherein the compound is the (R) -enantiomer.
5. The combination of claim 1, wherein R 1 And R is 2 Is OR (OR) A 。
6. The composition of claim 1, wherein R 3 And R is 4 is-CO 2 R C 。
7. The composition of claim 6 wherein R 3 And R is 4 When (when)In the case of double bonds, has the cis configuration.
8. The combination of claim 1, wherein the compound of formula I is representable by formula II:
or a salt thereof.
9. The composition of claim 1, wherein the compound is pantaphos:
10. the composition of claim 1, wherein the compound is compound 2:
11. the composition of claim 10, wherein the composition further comprises pantaphos.
12. A compound having formula I:
or a salt thereof; wherein:
-represents a single bond or a double bond;
represents a double bond or a single bond, wherein->And-not both are double bonds;
g is X A CHOR 5 ,O,C(＝O),C(＝CH 2 ),CHP(＝O)(R 6 ) 2 Or CX (CX) B 2 ；
X A Is a defect or O;
each X is B Each independently is H or halogen;
R 1 and R is 2 Each independently is OR A Or an amino acid;
R 3 is-C (=O) R 7 Or triazole or tetrazole;
R 4 is-C (=O) R 8 Or triazole or tetrazole;
R 5 is H, - (C) 1 -C 6 ) Alkyl, - (C) 3 -C 6 ) Cycloalkyl, aryl or heteroaryl;
each R 6 Each independently is OR B Or an amino acid;
R 7 and R is 8 Each independently is OR C Or an amino acid; and
R A ，R B and R is C Each independently is H, - (C) 1 -C 6 ) Alkyl, - (C) 3 -C 6 ) Cycloalkyl, aryl or heteroaryl;
wherein the compound is not a natural product.
13. The compound of claim 12, wherein the compound is not 2- (hydroxy (phosphonomethyl) maleic acid or 2- (phosphonomethyl) maleic acid.
14. The compound of claim 12, wherein G is CHOH.
15. The compound of claim 12, wherein R 1 And R is 2 Is OH.
16. The compound of claim 12, wherein R 3 And R is 4 is-CO 2 H。
17. A method of inhibiting the growth or development of weeds comprising allowing the weeds and/or soil from which the weeds can develop, and a herbicidally effective amount of a composition or compound as claimed in any one of claims 1 to 16 wherein the growth or development of weeds is inhibited.
18. The method of claim 17, wherein the composition or compound is contacted with vegetation and/or soil where vegetation may be produced, and the growth or production of weeds is selectively inhibited.
19. A method of inhibiting the progression of a cancer cell comprising contacting the cancer cell with the effective amount of the composition or compound of any one of claims 1-16, wherein the progression of cancer is inhibited.
20. The method of claim 19, wherein the cancer cell is a glioblastoma cell.
21. A method of forming 2- (hydroxy (phosphono) methyl) maleic acid:
or a salt thereof, comprising:
a) Isomerising phosphoenolpyruvate (PEP) to 3-phosphopyruvate (PnPy);
b) Condensing acetyl with PnPy to form phosphonomethyl malate (PMM);
c) Dehydrating the PMM to 2-phosphonomethyl maleate; and
d) Oxidizing 2-phosphonomethyl maleate to pantaphos;
wherein each of steps a) -d) is accomplished in a container.
22. The method of claim 21, wherein isomerization is catalyzed by PEP mutant enzyme (HvrA); the condensation is catalyzed by phosphonomethyl malate synthase (HvrC), acetyl is acetyl coa; dehydration is catalyzed by large isopropyl malate dehydratase (HvrD) and/or small isopropyl malate dehydratase (HvrE); and oxidation is catalyzed by a flavin dependent monooxygenase (HvrB) and optionally a flavin reductase (HvrK).
23. A nucleic acid molecule comprising the hvr operon of Pantoea Sp. and optionally an inducible promoter operably linked to the hvr operon.
24. The nucleic acid molecule of claim 23, comprising one or more genes selected from the group consisting of hvrA, hvrB, hvrC, hvrD, hvrE and hvrK, wherein the one or more genes are operably linked to the inducible promoter.
25. The nucleic acid molecule according to claim 23, comprising the inducible promoter of SEQ ID No. 13 and the nucleic acid sequences of SEQ ID NOs 1,2,3,4,5 and 11 encoding the genes hvrA, hvrB, hvrC, hvrD, hvrE and hvrK, respectively, wherein the genes are operably linked to the inducible promoter.
26. The nucleic acid molecule of claim 23, wherein the Pantoea Sp. is Pantoea ananatis.
27. The nucleic acid molecule of claim 23, wherein the inducible promoter is a tac promoter.
28. The nucleic acid molecule of claim 23, comprising one or more genes selected from hvrA, hvrB, hvrC, hvrD, hvrE and hvrK, wherein the one or more genes are operably linked to the inducible promoter.
29. An expression vector comprising the nucleic acid molecule of any one of claims 23-28, wherein induction of the promoter and expression of the gene results in formation of a phosphonate compound of formula I:
or a salt thereof; wherein:
-represents a single bond or a double bond;
represents a double bond or a single bond, wherein->And-not both are double bonds;
g is X A CHOR 5 ,O,C(＝O),C(＝CH 2 ),CHP(＝O)(R 6 ) 2 Or CX (CX) B 2 ；
X A Is a defect or O;
each X is B Each independently is H or halogen;
R 1 and R is 2 Each independently is OR A Or an amino acid;
R 3 is-C (=O) R 7 Or triazole or tetrazole;
R 4 is-C (=O) R 8 Or triazole or tetrazole;
R 5 is H, - (C) 1 -C 6 ) Alkyl, - (C) 3 -C 6 ) Cycloalkyl, aryl or heteroaryl;
each R 6 Each independently is OR B Or an amino acid;
R 7 and R is 8 Each independently is OR C Or an amino acid; and
R A ，R B and R is C Each independently is H, - (C) 1 -C 6 ) Alkyl, - (C) 3 -C 6 ) Cycloalkyl, aryl or heteroaryl.
30. The expression vector of claim 29, wherein the phosphonate compound is 2- (hydroxy (phosphono) methyl) maleic acid (pantaphos).
31. A recombinant cell for producing a phosphonate compound comprising the nucleic acid molecule of any one of claims 23-28, wherein the phosphonate compound is represented by formula I:
or a salt thereof; wherein:
-represents a single bond or a double bond;
represents a double bond or a single bond, wherein->And-not both are double bonds;
g is X A CHOR 5 ,O,C(＝O),C(＝CH 2 ),CHP(＝O)(R 6 ) 2 Or CX (CX) B 2 ；
X A Is a defect or O;
each X is B Each independently is H or halogen;
R 1 and R is 2 Each independently is OR A Or an amino acid; the method comprises the steps of carrying out a first treatment on the surface of the
R 3 is-C (=O) R 7 Or triazole or tetrazole;
R 4 is-C (=O) R 8 Or triazole or tetrazole;
R 5 is H, - (C) 1 -C 6 ) Alkyl, - (C) 3 -C 6 ) Cycloalkyl, aryl or heteroaryl;
each R 6 Each independently is OR B Or an amino acid;
R 7 and R is 8 Each independently is OR C Or an amino acid; and
R A ，R B and R is C Each independently is H, - (C) 1 -C 6 ) An alkyl group, a hydroxyl group, - (-) aC 3 -C 6 ) Cycloalkyl, aryl or heteroaryl; a kind of electronic device with a high-pressure air-conditioning system.
32. The recombinant cell of claim 31, wherein the nucleic acid molecule is integrated into the chromosome of the cell.
33. The recombinant cell of claim 31, wherein the cell is selected from the group consisting of pantoea, clostridium, zymomonas, escherichia coli, salmonella, serratia, escherichia, klebsiella, shigella, rhodococcus, pseudomonas, bacillus, lactobacillus, lactococcus, enterococcus, alcaligenes, bacillus, arthrobacter, corynebacterium, brevibacterium, schizosaccharomyces, kluyveromyces, yarrowia, pichia, saccharomyces, stonecrop, candida, budabove, clostridium, hansen, isja, trichosporon, hypocrellina, and saccharomyces.
34. The recombinant cell of claim 33, wherein the cell is of the genus pantoea, escherichia coli or saccharomyces.
35. The recombinant cell of claim 34, wherein the cell is pantoea ananatis, escherichia coli, or saccharomyces cerevisiae.
36. A process for producing a phosphonate compound of formula I comprising the steps of:
a) Providing a cell culture of the recombinant cell of claim 31, wherein the recombinant cell produces the phosphonate, the cell culture having a volume of about 1L to about 10L;
b) Mixing an inducing molecule with the cell culture;
c) Culturing the induced cell culture under constant oxygenation conditions for 96 hours;
d) Pelletizing the cells of the cell culture and collecting supernatant;
e) Concentrating the supernatant;
f) Extracting phosphonate from the concentrated supernatant by methanol extraction to form an extracted supernatant; and
g) The phosphonate is purified from the methanol soluble portion of the extracted supernatant.
37. The process of claim 36, wherein the phosphonate is 2- (hydroxy (phosphono) methyl) maleic acid.
38. The process of claim 36, wherein step g comprises iron-IMAC purification followed by flash chromatography and HILIC HPLC.
39. The process of claim 36, wherein the cell culture comprises pantoea, escherichia coli or saccharomyces cerevisiae.
40. The process of claim 39, wherein the cell culture is Pantoea.
41. The process of claim 40, wherein the constant oxygenation flow rate is 5L/min and the cell culture is maintained at a temperature of 30 ℃.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US63/075,138 | 2020-09-05 | ||
| US202163181745P | 2021-04-29 | 2021-04-29 | |
| US63/181,745 | 2021-04-29 | ||
| PCT/US2021/048904 WO2022051527A1 (en) | 2020-09-05 | 2021-09-02 | Phosphonate products and methods |
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| Publication Number | Publication Date |
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| CN116529254A true CN116529254A (en) | 2023-08-01 |
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