Methods and compositions for the production of cyclic and caged molecules

Abstract

A chemoenzymatic process comprising contacting benzyl alcohol with one or more oxidase catalyst systems under conditions suitable for the formation of an oxidized intermediate; aminating the oxidized intermediate to form a nitrogen-containing compound; and hydrogenating the nitrogen-containing compound to form benzylamine. A chemoenzymatic process comprising contacting benzyl alcohol with a galactose oxidase catalyst system under conditions suitable for the formation of benzaldehyde;reductively aminating benzaldehyde in the presence of Rainey nickel and a nitrogen source to form benzylamine; contacting benzylamine and glyoxal in the presence of a mineral acid and solvent under conditions suitable for the formation of hexabenzylhexaazaisowurtzitane; and acetylating hexabenzylhexaazaisowurtzitane to form tetraacetyl-diaminohexaazaisowurtzitane.

Description

METHODS AND COMPOSITIONS FOR THE PRODUCTION OF CYCLIC AND CAGED MOLECULESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. provisional patent application Serial No 63 / 654,832 filed May 31 , 2024, and entitled “METHODS AND COMPOSITIONS FOR THE PRODUCTION OF CYCLIC AND CAGED MOLECULES,” which is hereby incorporated herein by reference in its entirety for all purposes.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] Not applicable.REFERENCE TO SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML file, created on April 28, 2025 is named “24FRP007-PCT_3416-20001. xml” and is 26,934 bytes in size.TECHNICAL FIELD

[0004] The present disclosure relates generally to compositions and methods for the production of value-added chemicals. More particularly, the present disclosure relates to chemoenzymatic methods for production of cyclic and caged molecules.BACKGROUND

[0005] 2, 4, 6, 8, 10,12-hexanitro-2,4,6,8, 10, 12-hexaazatetracyclo[5.5.0.05.903, 11 ]- dodecane (CL-20), also called CL-20, is a polycyclic nitroamine explosive with the formula C6H6N120i2. CL-20 has a better oxidizer-to-fuel ratio than conventional explosives such as octahydro-1 ,3,5,7-tetranitro-1 ,3,5,7-tetrazocine (HMX) or hexahydro-1 ,3, 5-trinitro-1 ,3, 5-triazine (RDX). Due to the high energy content of these cage like molecules, the detonation pressure is also very high, more than double that of trinitrotoluene.

[0006] Synthesis of CL-20 has relied on the use of chemicals sourced from fossil-based fuels, transition metal-based catalysts and environmentally-unfriendly production processes. For example, the most common process for the production of benzylamine,a reagent in the production of CL-20, involves selective hydrogenation of benzonitrile with molecular hydrogen while benzonitrile itself is most commonly produced by the ammoxidation of toluene.

[0007] Both the cost and means of generating reagents used in CL-20 production represent challenges to the large scale production of this material. Thus, an ongoing need exists for methods and compositions to address one or more of these challenges.BRIEF SUMMARY OF THE DISCLOSURE

[0008] Disclosed herein is a chemoenzymatic process comprising contacting benzyl alcohol with one or more oxidase catalyst systems under conditions suitable for the formation of an oxidized intermediate; aminating the oxidized intermediate to form a nitrogen-containing compound; and hydrogenating the nitrogen-containing compound to form benzylamine.

[0009] Also disclosed herein is a chemoenzymatic process comprising contacting benzyl alcohol with a galactose oxidase catalyst system under conditions suitable for the formation of benzaldehyde; reductively aminating benzaldehyde in the presence of Rainey nickel and a nitrogen source to form benzylamine; contacting benzylamine and glyoxal in the presence of a mineral acid and solvent under conditions suitable for the formation of hexabenzylhexaazaisowurtzitane; and acetylating hexabenzylhexaazaisowurtzitane to form tetraacetyl-diaminohexaazaisowurtzitane.

[0010] Aspects described herein comprise a combination of features and characteristics intended to address various shortcomings associated with certain prior devices, systems, and methods. The foregoing has outlined rather broadly the features and technical characteristics of the disclosed aspects in order that the detailed description that follows may be better understood. The various characteristics and features described above, as well as others, will be readily apparent to those skilled in the art upon reading the following detailed description, and by referring to the accompanying drawings. It should be appreciated that the conception and the specific aspects disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes as the disclosed aspects. It should also be realized that such equivalent constructions do not depart from the spirit and scope of the principles disclosed herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] For a detailed description of various exemplary aspects, reference will now be made to the accompanying drawings in which:

[0012] Figure 1 schematically depicts the reaction of benzyl alcohol with a plurality of oxidase systems and hydrogenation to form benzylamine.

[0013] Figure 2 schematically depicts the reaction of benzyl alcohol to form benzylamine utilizing an oxidase catalyst system, hydroxylamine / water and hydrogenation.

[0014] Figure 3 schematically depicts the reaction of benzyl alcohol to form benzylamine utilizing an oxidase catalyst system, hydroxylamine / organic solvent and hydrogenation.

[0015] Figure 4 schematically depicts the reaction of benzyl alcohol to form benzaldehyde utilizing a oxidase catalyst system and subsequent reductive amination in the presence of a Rainey Nickel catalyst to form benzylamine.

[0016] Figure 5 schematically depicts the reaction of glyoxal and benzylamine to form tetraacetyldiamino hexaazaisowurtzitane which is then acetylated in the presence of a catalyst to form tetraacetyl-diamino-hexaazaiaowurtzitane.

[0017] Figure 6 is a bar graph depicting the specific activity of the indicated galactose oxidase mutant using gluconate as the substrate.

[0018] Figure 7 is a bar graph depicting the specific activity of the indicated galactose oxidase mutant using glucose as the substrate.

[0019] Figure 8 depicts schematically the overoxidation of benzyl alcohol and a bar graph depicting the product distribution for the indicated samples.

[0020] Figure 9 depicts the product distribution in the aqueous and organic extracts of the reaction mixture from Example 2.

[0021] Figure 10 schematically depicts the reaction of glyoxal and benzylamine to form HBIW.DETAILED DESCRIPTION

[0022] The following discussion is directed to various exemplary aspects. However, one of ordinary skill in the art will understand that the examples disclosed herein have broad application, and that the discussion of any aspect is meant only to be exemplary of that aspect, and not intended to suggest that the scope of the disclosure, including the claims, is limited to that aspect.

[0023] The figures are not necessarily to scale. Certain features and components herein may be shown exaggerated in scale or in somewhat schematic form and some details of conventional elements may not be shown in interest of clarity and conciseness.

[0024] In the following discussion and in the claims, the terms “including” and “comprising” are used in an open-ended fashion, and thus should be interpreted to mean “including, but not limited to... .” As used herein, the terms “approximately,” “about,” “substantially,” and the like mean within 10% (i.e., plus or minus 10%) of the recited value. Thus, for example, a recited angle of “about 80 degrees” refers to an angle ranging from 72 degrees to 88 degrees

[0025] Disclosed herein are methods and compositions for production of CL-20. In one or more aspects, the present disclosure provides methods and compositions for the chemoenzymatic production of benzylamine. In one or more aspects, the present disclosure provides methods and compositions for the chemoenzymatic production of the CL-20 precursor, tetraacetyldiamino hexaazaisowurtzitane (HBIW).

[0026] In aspects, one or more of molecules of the present disclosure are biobased molecules characterized by equal to or greater than about 70% of the carbon atoms in the molecule originating from a renewable resource; additionally or alternatively equal to or greater than about 75%; additionally or alternatively equal to or greater than about 80%; additionally or alternatively equal to or greater than about 85%; additionally or alternatively equal to or greater than about 90%. Herein a renewable resource refers to a natural resource which will replenish to replace the portion depleted by usage and consumption, either through natural reproduction or other recurring processes in a finite amount of time on a human time scale. In one or more aspects, the renewable resource is a sugar. In one or more aspects, the renewable resource is glucose.

[0027] In one or more aspects, a method of the present disclosure comprises the production of benzylamine, designated BAM, from benzyl alcohol, designated BAL, as depicted schematically in Figure 1. With reference to Figure 1 , a chemoenzymatic method of the present disclosure comprises contacting BAL with (i) one or more oxidase enzymes under conditions suitable to produce one or more oxidized BAL products and contacting the one or more oxidized BAL products with (ii) one or more hydrogenation catalysts and (iii) a nitrogen-source under conditions suitable for the production of BAM. In Figure 1 , BAL is contacted with an oxidase catalyst system under conditions suitable for the formation of a benzaldehyde intermediate, designated BAD. Herein an oxidase catalyst system comprises (a) one or more oxidases; (b) one or more small molecule activators (SMA); and (c) one or more single electron oxidizers (SEO); each of which are described in further detail herein.

[0028] In one or more aspects, BAD is reacted with another oxidase catalyst system in the presence of a nitrogen source under conditions suitable for the formation of benzonitrile, designated BIN. Any suitable nitrogen source may be use, for example the nitrogen source may be ammonia. In some aspects, the another oxidase catalyst system is the same as the oxidase catalyst system previously used for the formation of BAL. In alternative aspects, the another oxidase catalyst system differs from the oxidase catalyst system used for the formation of BAL.

[0029] The oxidase of any of the oxidase catalyst systems disclosed herein may comprise any suitable oxidase enzyme. In some aspects, the oxidase of the oxidase catalyst system comprises a copper radical oxidase (CRO). CROs have been categorized as 'green’ small-molecule oxidation catalysts as they lack dependence on an organic cofactor and require only molecular oxygen as a cosubstrate. CROs are nonflavoprotein alcohol oxidoreductases that employ molecular oxygen as a terminal electron acceptor to generate hydrogen peroxide. CROs include glyoxal oxidases (EC 1.1.3.-, GLOX) and galactose 6-oxidases (EC 1 .1 .3.9, GAO). GLOXes typically function on aldehydes such as methylglyoxal to produce acids. One GLOX from Phanerochaete chrysosporium primarily accepts alpha-dicarbonyl and alpha-hydroxycarbonyls. Pycnoporus cinnabarinus expresses three GLOXes, one of which has been found to function on methylglyoxal while the other two of which show high catalytic efficiency for glyoxylic acid. GAOs typically function on the C6 or similar alcohols of galactose or other sugars to produce aldehydes. By far, the most characterized CRO is GAO from Fusarium graminearum. Two additional CROs capable of oxidizing aliphatic alcohols were discovered in Colletotrichum graminicola and C. gloeosporioides (CgrAlcOx and CglAlcOx). An aryl-alcohol oxidase (CgrAAO) was also discovered in C. graminicola. In one or more aspects, a CRO for use in the present disclosure is a wildtype enzyme. In one or more aspects, a CRO for use in the present disclosure is a mutated enzyme. In one or more other aspects, a CRO suitable for use in the present disclosure comprises a mutated GAO. In one or more aspects, the CRO has any of SEQ ID NO:1 through SEQ ID NO:11.

[0030] In one or more aspects, the oxidase catalyst systems disclosed herein comprise an SMA. Nonlimiting examples of SMAs suitable for use in the present disclosure include L-tryptophan, 2-mercaptobenzothiazole, L-histidine, methylchloroisothiazolinone, o-dianisidine, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 4-aminoantipyrine, L-tyrosine, (2,2,6,6-tetramethylpiperidin-1- yl)oxyl, chloromethylisothiazolinone, 4-thiazolecarboxylic acid, Sunset yellow FCF, tartrazine, p-benzoquinone, dicoumarol, phthalimide, saccharin, phthalic anhydride, erythrosine B, 2-aminobenzothiazole, thiabendazole, 2-hydroxybenzothiazole, phenothiazine, 6-aminobenzothiazole, indigo carmine, naphthalimide, 2-aminothiazole, thiazole, 2H-1 ,4-benzothiazin-3(4H)-one, 2-oxindole, beta-lapachone, menaquinone, thiamine, 4-methyl-5-thiazoleethanol, Allura Red AC, menadione, p-cresol, Fast green FCF, Brilliant Blue FCF, methylisothiazolinone, caffeine, veratryl alcohol, fluorescein, and combinations thereof

[0031] In one or more aspects, the oxidase catalyst systems disclosed herein comprise an SEO. For example, the SEO may be an enzyme such as a laccase, horseradish peroxidase, Dyp-type peroxidase, lactoperoxidase, chloroperoxidase, manganese peroxidase 1 , ascorbate peroxidase, dye-decolorizing peroxidase, unspecific peroxygenase, dehaloperoxidase, catalase-peroxidase, lignin peroxidase, soybean seed coat peroxidase, isoforms thereof and combinations thereof. In one or more aspects , the SEO has SEQ ID NO:18.

[0032] The oxidase catalyst systems disclosed herein may be utilized under reaction conditions comprising one or more of the following parameters: an amount of reactant of from about 0.1 weight per volume percent (w / v%) to about 60 w / v%; additionally or alternatively, from about 5 w / v% to about 50 w / v% ; additionally or alternatively, from about 10 w / v% to about 40 w / v% and an amount of oxidase (e.g., CRO) of from about 0.1 mg / L to about 30,000 mg / L; additionally or alternatively, from about 5 mg / L to about 500 mg / L; additionally or alternatively, from about 10 mg / L about 100 mg / L based on desired throughput of the reaction; and an oxygen pressure of from about 10 psi to about 400 psi; additionally or alternatively from about 25 psi to 300 psi; additionally or alternatively from about 50 psi to about 200 psi. Further reaction conditions may include a temperature ranging from about 1 °C to about 70 °C, additionally or alternatively from about 5 °C to about 30 °C; additionally or alternatively from about 10 °C to about 25 °C and an aqueous media such as a phosphate buffer at a pH of from about 5 to about 10; additionally or alternatively from about 6 to about 9; additionally or alternatively from about 7 to about 8.5.

[0033] In one or more aspects, the oxidase catalyst systems disclosed herein comprise GAO or a mutated GAO which is present in an amount ranging from about 0.01 g / L toabout 1 g / L; additionally or alternatively, from about 0.1 g / L to about 1 g / L; additionally or alternatively, from about 0.2 g / L to about 1 g / L; additionally or alternatively, from about 0.4 g / L to about 1 g / L; additionally or alternatively, from about 0.6 g / L to about 1 g / L; additionally or alternatively, from about 0.75 g / L to 1 g / L; additionally or alternatively, about 0.01 g / L, about 0.05 g / L, about 0.1 g / L, about 0.2 g / L, about 0.3 g / L, about 0.4 g / L, about 0.5 g / L, about 0.6 g / L, about 0.7 g / L, about 0.8 g / L, about 0.9 g / L or, additionally or alternatively, about 1 g / L. The catalytic composition may comprise an SEO in an amount ranging from about 1 mg / L to about 250 mg / L; additionally or alternatively, from about 5 mg / L to about 250 mg / L, additionally or alternatively, from about 10 mg / L to about 250 mg / L; additionally or alternatively, from about 25 mg / L to about 250 mg / L; additionally or alternatively, from about 50 mg / L to find 250 mg / L; additionally or alternatively, from about 75 mg / L to 250 mg / L; additionally or alternatively, from about 100 mg / L to 250 mg / L; additionally or alternatively, from about 150 mg / L to 250 mg / L; additionally or alternatively, about 1 mg / L, about 5 mg / L, about 10 mg / L, about 15 mg / L, about 20 mg / L, about 25 mg / L, about 30 mg / L, about 35 mg / L, about 40 mg / L, about 45 mg / L, about 50 mg / L, about 55 mg / L, about 60 mg / L, about 65 mg / L, about 70 mg / L, about 75 mg / L, about 80 mg / L, about 85 mg / L, about 90 mg / L, about 95 mg / L, about 100 mg / L, about 105 mg / L, about 110 mg / L, about 115 mg / L, about 120 mg / L, about 125 mg / L, about 130 mg / L, about 135 mg / L, about 140 mg / L, about 145 mg / L, about 150 mg / L, about 155 mg / L, about 160 mg / L, about 165 mg / L, about 170 mg / L, about 175 mg / L, about 180 mg / L, about 185 mg / L, about 190 mg / L, about 195 mg / L, about 200 mg / L, about 205 mg / L, about 210 mg / L, about 215 mg / L, about 220 mg / L, about 225 mg / L, about 230 mg / L, about 235 mg / L, about 240 mg / L, about 245 mg / L or, additionally or alternatively, about 250 mg / L.

[0034] The oxidase catalyst systems disclosed herein may comprise an SMA in an amount ranging from about 1 ppm to about 500 ppm; additionally or alternatively, from about 5 ppm to about 500 ppm; additionally or alternatively, from about 10 ppm to about 500 ppm; additionally or alternatively, from about 20 ppm to about 500 ppm; additionally or alternatively; additionally or alternatively, from about 40 ppm to about 400 ppm; additionally or alternatively, from about 50 ppm to about 350 ppm; additionally or alternatively, from about 75 ppm to about 200 ppm; additionally or alternatively, about 1 ppm, about 5 ppm, about 10 ppm, about 15 ppm, about 20 ppm, about 25 ppm, about 30 ppm, about 35 ppm, about 40 ppm, about 45 ppm, about 50 ppm, about 55 ppm,about 60 ppm, about 65 ppm, about 70 ppm, about 75 ppm, about 80 ppm, about 85 ppm, about 90 ppm, about 95 ppm, about 100 ppm, about 105 ppm, about 110 ppm, about 115 ppm, about 120 ppm, about 125 ppm, about 130 ppm, about 135 ppm, about 140 ppm, about 145 ppm, about 150 ppm, about 155 ppm, about 160 ppm, about 165 ppm, about 170 ppm, about 175 ppm, about 180 ppm, about 185 ppm, about 190 ppm, about 195 ppm, about 200 ppm, about 205 ppm, about 210 ppm, about 215 ppm, about 220 ppm, about 225 ppm, about 230 ppm, about 235 ppm, about 240 ppm, about 245 ppm, about 250 ppm, about 255 ppm, about 260 ppm, about 265 ppm, about 270 ppm, about 275 ppm, about 280 ppm, about 285 ppm, about 290 ppm, about 295 ppm, about 300 ppm, about 305 ppm, about 310 ppm, about 315 ppm, about 320 ppm, about 325 ppm, about 330 ppm, about 335 ppm, about 340 ppm, about 345 ppm, about 350 ppm, about 355 ppm, about 360 ppm, about 365 ppm, about 370 ppm, about 375 ppm, about 380 ppm, about 385 ppm, about 390 ppm, about 395 ppm, about 400 ppm, about 405 ppm, about 410 ppm, about 415 ppm, about 420 ppm, about 425 ppm, about 430 ppm, about 435 ppm, about 440 ppm, about 445 ppm, about 450 ppm, about 455 ppm, about 460 ppm, about 465 ppm, about 470 ppm, about 475 ppm, about 480 ppm, about 485 ppm, about 490 ppm, about 495 ppm, or, additionally or alternatively, about 500 ppm.

[0035] With reference to Figure 1 , BIN can then be hydrogenated to form the corresponding BAM. The hydrogenation of BIN to BAM may be carried out in the presence of a hydrogenation catalyst. Hereinafter, the hydrogenation catalyst is termed the H-cat.

[0036] In one or more aspects, a H-cat for use in the present disclosure comprises (i) a transition-metal compound or a transition-metal salt and (ii) a support material. For example, the H-cat may comprise iron (Fe), copper (Cu), rhodium (Rh), rhenium (Re), iridium (Ir), cobalt (Co), nickel (Ni), platinum *Pt), palladium (Pd), gold (Au), ruthenium (Ru), oxides thereof, or combinations thereof. In one or more aspects, the support material comprises an inert or substantially inert material such as glass, titania, silica, alumina, zirconia, ceria, ceramic, or carbon.

[0037] In one or more aspects, the H-cat comprises a Rainey Nickel catalyst. Raney Nickel catalyst refers to a fine-grained nickel also known as spongy nickel that is derived from a nickel-aluminum alloy. The metal loadings of the indicated metals onto a support material can range from about 0.1 weight percent (wt.%) to about 90 wt.%, or about 0.15 wt.% to about 80 wt.% based on the total weight of the catalyst. A metal catalyst of thepresent disclosure may be characterized the ability to convert aldehyde functionalities to carboxylic acids with productivities of greater than about 0.1 mol acid g-1active metal IT1at selectivities of greater than about 80% and conversions of greater than about 90% with steady state metal leaching of less than about 100 parts per billion by weight based on the total weight of the catalyst.

[0038] In one or more aspects, reaction conditions for the hydrogenation of BIN may include one or more of the following parameters: an amount of BIN of from about 0.1 weight per volume percent (w / v%) to about 60 w / v%, additionally or alternatively from about 5 w / v% to about 50 w / v% additionally or alternatively from about 10 w / v% to about 40 w / v%; a hydrogen pressure ranging from about 10 bar about 100 bar; additionally or alternatively from about 10 bar to about 75 bar; additionally or alternatively from about 10 bar to about 50 bar; a reaction temperature of from about 40 °C to about 120 °C; additionally or alternatively from about 40 °C to about 100 °C; additionally or alternatively from about 40°C to about 80 °C.

[0039] The yield, based on the amount of BAL, of a BAM for any method disclosed herein may range from about 20% to about 95%; additionally or alternatively from about 25% to about 90%; additionally or alternatively from about 40% to about 80% wherein the yield is the percent yield which is the ratio of the actual yield (e.g., grams, moles, etc..) to the theoretical yield multiplied by 100%.

[0040] In another aspect, a method for production of BAM comprises contacting of an oxidase catalyst system with BAL under conditions suitable for the formation of BAD, as described previously herein. In such aspects, the BAD formed is then contacted with a nitrogen source (e.g., hydroxylamine, etc.) in an aqueous solvent at a temperature between about 5 °C to about 30 °C; additionally or alternatively from about 10 °C to about 25 °C; additionally or alternatively at about room temperature under conditions suitable for formation of benzaldoxime, designated BOX. BOX can then be reacted with a hydrogenation catalyst under conditions suitable for formation of the corresponding BAM. Conditions for the hydrogenation of a BAL oxidized product have been previously described herein. In some aspects, the oxidase catalyst system can perform a one-pot conversion of alcohols into nitriles when supplied an ammonium buffer as the nitrogen source and atmospheric oxygen as the oxidant. This reaction is depicted schematically in Figure 2.

[0041] In another aspect, a method for the production of BAM comprises contacting of an oxidase catalyst system with BAL under conditions suitable for formation BAD, as described previously herein. In such aspects, the BAD formed is then contacted with hydroxylamine in an organic solvent (e.g., DMSO) at temperatures ranging from about 20 °C to about 40 °C under conditions suitable for formation of BIN. BIN can then be reacted with a hydrogenation catalyst under conditions suitable for formation of the corresponding BAM. Conditions for the hydrogenation of BIN have been previously described herein. This reaction is depicted schematically in Figure 3.

[0042] In another aspect, a method for production of a BAM comprises contacting of an oxidase catalyst system with BAL under conditions suitable for formation BAD, as described previously herein. In such aspects, the BAD formed is then contacted with a Rainey Nickel catalyst under conditions suitable for the formation of the corresponding BAM. Conditions for the hydrogenation of BAD can include any of those previously described herein with respect to the hydrogenation of BIN. This reaction is depicted schematically in Figure 4.

[0043] As will be understood by one of ordinary skill in the art with the benefit of the present disclosure, reactions of the type disclosed herein may result in the production of byproducts (e.g., hydrogen peroxide, etc.) that can detrimentally impact other components of the reaction mixture. For example, hydrogen peroxide may degrade the enzyme resulting in a loss of catalytic activity. In such aspects, mitigation of the detrimental effects of hydrogen peroxide may be carried out such as by the introduction of a catalase (E.C. 1.11.1.61), the use of a hydrogen peroxide-resistant enzyme or combinations thereof. In one or more aspects, the oxidase catalyst system further comprising a catalase having any of SEQ ID NO:12 through SEQ ID NO:17.

[0044] In an aspect, any enzyme of the type disclosed herein is a wild type enzyme, a functional fragment thereof, or a functional variant thereof. “Fragment” as used herein is meant to include any amino acid sequence shorter than the full-length enzyme, but where the fragment maintains a catalytic activity sufficient to meet some user or process goal. Fragments may include a single contiguous sequence identical to a portion of the biocatalyst sequence. Alternatively, the fragment may have or include several different shorter segments where each segment is identical in amino acid sequence to a different portion of the amino acid sequence of the enzyme but linked via amino acids differing in sequence from the enzyme. Herein, a "functional variant" of the enzyme refers to apolypeptide which has at one or more positions of an amino acid insertion, deletion, or substitution, either conservative or n on-conservative, and wherein each of these types of changes may occur alone, or in combination with one or more of the others, and / or one or more times in a given sequence but retains catalytic activity.

[0045] In the alternative or in combination with the aforementioned mutations, the enzyme may be mutated to improve the catalytic activity. Mutations may be carried out to enhance the protein or a homolog activity, increase the protein stability in the presence of substrates and products (e.g., hydrogen peroxide) and increase protein yield.

[0046] Herein, reference has been made to “sources” of enzyme. It is to be understood this refers to the biomolecule as expressed by the named organism. It is contemplated the enzyme may be obtained from the organism or a version of said enzyme (wildtype or recombinant) and provided as a suitable construct to an appropriate expression system.

[0047] In an aspect, any enzyme of the type disclosed herein may be cloned into an appropriate expression vector and used to transform cells of an expression system such as E. coli, Saccharomyces sp., Pichia sp., Aspergillus sp., Trichoderma sp., or Myceliophthora sp. A "vector" is a replicon, such as plasmid, phage, viral construct or cosmid, to which another DNA segment may be attached. Vectors are used to transduce and express a DNA segment in cells. As used herein, the terms "vector" and "construct" may include replicons such as plasmids, phage, viral constructs, cosmids, Bacterial Artificial Chromosomes (BACs), Yeast Artificial Chromosomes (YACs), Human Artificial Chromosomes (HACs), and the like into which one or more gene expression cassettes may be or are ligated. Herein, a cell has been "transformed" by an exogenous or heterologous nucleic acid or vector when such nucleic acid has been introduced inside the cell, for example, as a complex with transfection reagents or packaged in viral particles. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell.

[0048] In an aspect, the gene of an enzyme disclosed herein is provided as a recombinant sequence in a vector where the sequence is operatively linked to one or more control or regulatory sequences. "Operatively linked" expression control sequences refer to a linkage in which the expression control sequence is contiguouswith the gene of interest to control the gene of interest, as well as expression control sequences that act in trans or at a distance to control the gene of interest.

[0049] The term "expression control sequence" or "regulatory sequences" are used interchangeably and are used herein to refer to polynucleotide sequences which affect the expression of coding sequences to which they are operatively linked. Expression control sequences are sequences that control the transcription, post-transcriptional events, and translation of nucleic acid sequences. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., ribosome binding sites, etc.); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence. The term "control sequences" is intended to include, at a minimum, all components whose presence is essential for expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

[0050] The term "recombinant host cell" ("expression host cell", "expression host system", "expression system", or simply "host cell"), as used herein, is intended to refer to a cell into which a recombinant vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. A recombinant host cell may be an isolated cell or cell line grown in culture or may be a cell which resides in a living tissue or organism.

[0051] In one or more aspects, a method of the present disclosure further comprises contacting of BAM (e.g., prepared as disclosed herein) and glyoxal under conditions suitable for the formation of hexabenzylhexaazaisowurtzitane (HBIW). The yield of HBIW may be equal to or greater than about 70%, additionally or alternatively equal to or greater than about 80%; additionally or alternatively equal to or greater than about 90%. HBIW prepared as described herein may be used without further processing ormay be subjected to any suitable methodologies for purification of HBIW to provide a purity of equal to or greater than about 90%; additionally or alternatively equal to or greater than about 92%; additionally or alternatively equal to or greater than about 95%. HBIW may then, under suitable conditions, be acetylated in the presence of hydrogen and a transition metal catalyst to form TADA. The yield of TADA may be equal to or greater than about 70%, additionally or alternatively equal to or greater than about 80%; additionally or alternatively equal to or greater than about 90%. TADA prepared as described herein may be used without further processing or may be subjected to any suitable methodologies for purification of TADA to provide a purity of equal to or greater than about 90%; additionally or alternatively equal to or greater than about 92%; additionally or alternatively equal to or greater than about 95%. The overall reaction is depicted schematically in Figure 5.

[0052] In one or more aspects, the glyoxal contacted with BAM to produce TADA may be obtained from any suitable source. In alternative aspects, the glyoxal is prepared from a renewable resource such as glucose. For example, glyoxal may be obtained by chemoenzymatic methods from ethylene glycol using an oxidase system of the type disclosed herein.

[0053] A general scheme for the production of glyoxal comprises contacting ethylene glycol with an oxidase catalyst system capable of catalyzing the oxidation of ethylene glycol to glyoxal. This reaction is described in more detail in World Intellectual Property Organization Publication No. WO / 2024 / 249896 which is incorporated herein in its entirety.

[0054] Reaction conditions for the production of HBIW may include one or more of the following parameters: a ratio of benzylamine to glyoxal of from about 1 :5 to about 5:1 ; additionally or alternatively from about 1 :3 to about 3:1 ; additionally or alternatively from about 1 :2 to about 2: 1 ; additionally or alternatively about 1 : 1 ; an acid catalyst nonlimiting examples of which include sulfuric acid, hydrochloric acid, Lewis acids such as aluminum chloride phosphoric acid, toluenesulfonic acid, and zeolites; a polar organic solvent that is substantially nonreactive with glyoxal and benzylamine such as acetonitrile, nitromethane, methanol, ethanol, and tetrahydrofuran; a reaction temperature ranging from about 0°C to about 20°C ; additionally or alternatively from about 0°C to about 10°C; additionally or alternatively from about 5°C to about 10°C ; and a reaction time of from about 10 minutes to about 24 hours; additionally oralternatively from about 1 hour to about 18 hours; additionally or alternatively from about 4 hours to about 12 hours. In one or more aspects, glyoxal is included in the reaction as an aqueous mixture having a pH range from about 4 to about 7; additionally or alternatively from about 5 to about 7; additionally or alternatively from about 5 to about 6.

[0055] For example, 3 molar equivalents of glyoxal can be reacted with 6 molar equivalents of benzylamine to form HBIW. This is a self-assembly reaction under mild conditions. Rather than traditional acetonitrile solvent and formic acid as the catalyst, alcohols such as methanol may be used as the solvent and a mineral acid (e.g., sulfuric acid) as the acid catalyst. The method may further comprise the debenzylation of HBIW via heterogenous catalytic hydrogenolysis as well as 4 molar equivalents are acetylated to form tetraacetyl dibenzyl hexaazaisowurtzitane (TADBIW).ADDITIONAL DISCLOSURE

[0056] The following are additional nonlimiting exemplary aspects of the presently disclosed subject matter

[0057] A first aspect which is a chemoenzymatic process comprising contacting benzyl alcohol with an oxidase catalyst system under conditions suitable for the formation of an oxidized intermediate aminating the oxidized intermediate to form a nitrogencontaining compound; and hydrogenating the nitrogen-containing compound to form benzylamine.

[0058] A second aspect which is the process of the first aspect wherein the oxidase catalyst system comprises: (i) one or more oxidase enzymes, (ii) one or more small molecule activators, and (iii) one or more single electron oxidizers.

[0059] A third aspect which is the process of the second aspectswherein the one or more oxidase enzymes comprise one or more copper radical oxidases, mutants thereof, fragments thereof, or combinations thereof.

[0060] A fourth aspect which is the process of any of the second through third aspects wherein the one or more oxidase enzymes comprise galactose oxidase, glucose oxidase, alcohol oxidase, aryl alcohol oxidase, glyoxal oxidase, mutants thereof, fragments thereof or combinations thereof.

[0061] A fifth aspect which is the process of any of the second hrough fourth aspects wherein the one or more oxidase enzymes has any of SEQ ID NO:1 to SEQ ID NO:11 .

[0062] A sixth aspect which is the process of any of the first through fifth aspects wherein conditions suitable for formation of an oxidized intermediate further comprises a catalase.

[0063] A seventh aspect which is the process of the sixth aspect wherein the catalase has any of SEQ ID NO:12 to SEQ ID NO:17.

[0064] An eighth aspect which is the process of any of the first through seventh aspects wherein the small molecule activator is selected from the group consisting of-tryptophan, 2-mercaptobenzothiazole, L-histidine, methylchloroisothiazolinone, o-dianisidine, 2,2'- azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 4-aminoantipyrine, L-tyrosine, (2,2,6,6-tetramethylpiperidin-1-yl)oxyl, chloromethylisothiazolinone, 4- thiazolecarboxylic acid, Sunset yellow FCF, tartrazine, p-benzoquinone, dicoumarol, phthalimide, saccharin, phthalic anhydride, erythrosine B, 2-aminobenzothiazole, thiabendazole, 2-hydroxybenzothiazole, phenothiazine, 6-aminobenzothiazole, indigo carmine, naphthalimide, 2-aminothiazole, thiazole, 2H-1 ,4-benzothiazin-3(4H)-one, 2- oxindole, beta-lapachone, menaquinone, thiamine, 4-methyl-5-thiazoleethanol, Allura Red AC, menadione, p-cresol, Fast green FCF, Brilliant Blue FCF, methylisothiazolinone, caffeine, veratryl alcohol, fluorescein, and combinations thereof.

[0065] A ninth aspect which is the process of any of the first through eighth aspects wherein the single electron oxidizer is selected from the group consisting of laccase, horseradish peroxidase, Dyp-type peroxidase, lactoperoxidase, chloroperoxidase, manganese peroxidase 1 , ascorbate peroxidase, dye-decolorizing peroxidase, unspecific peroxygenase, dehaloperoxidase, catalase-peroxidase, lignin peroxidase, soybean seed coat peroxidase, isoforms thereof and combinations thereof.

[0066] A tenth aspect which is the process of the ninth aspect wherein the single electron oxidizer has SEQ ID NO: 18.

[0067] An eleventh aspect which is the process of any the first through tenth wherein an oxidized intermediate comprises benzaldehyde, benzonitrile, benzaldoxime or combinations thereof

[0068] A twelfth aspect which is the process of any of the first through eleventh aspects wherein the nitrogen-containing compound comprise ammonia.

[0069] A thirteenth aspect which is the process of any of the first through twelfth aspects wherein the hydrogenation catalyst comprises Rainey nickel.

[0070] A fourteenth aspect which is a chemoenzymatic process comprising contacting benzyl alcohol with a galactose oxidase catalyst system under conditions suitable for the formation of benzaldehyde; reductively aminating benzaldehyde in the presence of Rainey nickel and a nitrogen source to form benzylamine; contacting benzylamine and glyoxal in the presence of a mineral acid and solvent under conditions suitable for the formation of hexabenzylhexaazaisowurtzitane; and acetylating hexabenzylhexaazaisowurtzitane to form tetraacetyl- diaminohexaazaisowurtzitane.

[0071] A fifteenth aspect which is the process of the fourteenth aspect wherein the galactose oxidase has any of SEQ ID NO:1 to SEQ ID NO:6.

[0072] A sixteenth aspect which is the process of any of the fourteenth through fifteenth aspects wherein the galactose oxidase system further comprises a small molecule activator and a single electron acceptor.

[0073] A seventeenth aspect which is the process of any of the fourteenth through sixteenth aspects wherein the small molecule activator is selected from the group consisting of-tryptophan, 2-mercaptobenzothiazole, L-histidine, methylchloroisothiazolinone, o-dianisidine, 2,2'-azino-bis(3-ethylbenzothiazoline-6- sulfonic acid (ABTS), 4-aminoantipyrine, L-tyrosine, (2,2,6,6-tetramethylpiperidin-1- yl)oxyl, chloromethylisothiazolinone, 4-thiazolecarboxylic acid, Sunset yellow FCF, tartrazine, p-benzoquinone, dicoumarol, phthalimide, saccharin, phthalic anhydride, erythrosine B, 2-aminobenzothiazole, thiabendazole, 2-hydroxybenzothiazole, phenothiazine, 6-aminobenzothiazole, indigo carmine, naphthalimide, 2-aminothiazole, thiazole, 2H-1 ,4-benzothiazin-3(4H)-one, 2-oxindole, beta-lapachone, menaquinone, thiamine, 4-methyl-5-thiazoleethanol, Allura Red AC, menadione, p-cresol, Fast green FCF, Brilliant Blue FCF, methylisothiazolinone, caffeine, veratryl alcohol, fluorescein, and combinations thereof.

[0074] An eighteenth aspect which is the process of any of the fourteenth through seventeenth aspects wherein the single electron oxidizer is selected from the group consisting of laccase, horseradish peroxidase, Dyp-type peroxidase, lactoperoxidase, chloroperoxidase, manganese peroxidase 1 , ascorbate peroxidase, dye-decolorizing peroxidase, unspecific peroxygenase, dehaloperoxidase, catalase-peroxidase, lignin peroxidase, soybean seed coat peroxidase, isoforms thereof and combinations thereof.

[0075] A nineteenth aspect which is the process of any of the fourteenth through eighteenth aspects, wherein the nitrogen source comprises ammonia.

[0076] A twentieth aspect which is the process of any of the fourteenth through nineteenth aspects wherein the galactose oxidase system further comprises a catalase having any of SEQ ID NO:12 to SEQ ID NO:17.

[0077] A twenty-first aspect which is the process of any of the fourteenth through twentieth aspects wherein hexabenzylhexaazaisowurtzitane is formed at a yield of greater than about 70% and a purity of greater than about 90%.

[0078] A twenty-second aspect which is a mixture comprising benzyl alcohol; an oxidase enzyme; an oxidized intermediate; and an amination agent.

[0079] A twenty-third aspect which is the mixture of the twenty-first aspect further comprising a small molecule activator.

[0080] A twenty-fourth aspect which is the mixture of any of the twenty-second through twenty-third aspects further comprising a single electron oxidizer.

[0081] A twenty-fifth aspect which is the mixture of any of the twenty-second through twenty-fourth aspects wherein the oxidase enzyme comprises one or more copper radical oxidase, a mutant thereof, a fragment thereof, or a combination thereof.

[0082] A twenty-sixth aspect which is the mixture of any of the twenty-second through twenty-fifth aspects, wherein the oxidase enzyme comprises galactose oxidase, glucose oxidase, alcohol oxidase, aryl alcohol oxidase, glyoxal oxidase, a mutant thereof, a fragment thereof, or a combination thereof.

[0083] A twenty-seventh aspect which is the mixture of any of the twenty-second through the twenty-sixth aspects wherein the oxidase enzyme has any of SEQ ID NO:1 to SEQ ID NO:11.

[0084] A twenty-eighth aspect which is the mixture of any of the twenty-second through the twenty-seventh aspects, further comprising a catalase.

[0085] A twenty-ninth aspect which is the mixture of any of the twenty-second through the twenty-eighth aspects, wherein the catalase has any of SEQ ID NO:12 to SEQ ID NO:17.

[0086] A thirtieth aspect which is the mixture of any of the twenty-ninth aspect wherein the small molecule activator is selected from the group consisting of-tryptophan, 2- mercaptobenzothiazole, L-histidine, methylchloroisothiazolinone, o-dianisidine, 2,2'- azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 4-aminoantipyrine, L-tyrosine,(2,2,6,6-tetramethylpiperidin-1-yl)oxyl, chloromethylisothiazolinone, 4- thiazolecarboxylic acid, Sunset yellow FCF, tartrazine, p-benzoquinone, dicoumarol, phthalimide, saccharin, phthalic anhydride, erythrosine B, 2-aminobenzothiazole, thiabendazole, 2-hydroxybenzothiazole, phenothiazine, 6-aminobenzothiazole, indigo carmine, naphthalimide, 2-aminothiazole, thiazole, 2H-1 ,4-benzothiazin-3(4H)-one, 2- oxindole, beta-lapachone, menaquinone, thiamine, 4-methyl-5-thiazoleethanol, Allura Red AC, menadione, p-cresol, Fast green FCF, Brilliant Blue FCF, methylisothiazolinone, caffeine, veratryl alcohol, fluorescein, and combinations thereof.

[0087] A thirty-first aspect which is the method of any of the twenty-second through the thirtieth aspect wherein the single electron oxidizer is selected from the group consisting of laccase, horseradish peroxidase, Dyp-type peroxidase, lactoperoxidase, chloroperoxidase, manganese peroxidase 1 , ascorbate peroxidase, dye-decolorizing peroxidase, unspecific peroxygenase, dehaloperoxidase, catalase-peroxidase, lignin peroxidase, soybean seed coat peroxidase, isoforms thereof and combinations thereof.

[0088] A thirty-second aspect which is the method of any of the twenty-fourth through thirty-first aspects wherein the single electron oxidizer has SEQ ID NO: 18.

[0089] A thirty-third aspect which is the method of any of the twenty-second through thirty-second aspects wherein the oxidized intermediate comprises benzaldehyde, benzylnitirile, benzonitrile, benzaldoxime or combinations thereof.EXAMPLES

[0090] The subject matter having been generally described, the following examples are given as particular aspects of the disclosure and are included to demonstrate the practice and advantages thereof, as well as aspects and features of the presently disclosed subject matter. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the present subject matter, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific aspects which are disclosed and still obtain a like or similar result without departing from the scope of the instant disclosure. It is understood that the examples are given by way of illustration and are not intended to limit the specification of the claims to follow in any manner.

[0091] The following are nonlimiting examples of the presently disclosed subject matter: Generation of a promiscuous GAO mutant as an oxidation catalyst

[0092] Variants of GAO, most exemplary being GAO-Mut107, were engineered and demonstrated to oxidize a broad range of substrates.Directed Evolution

[0093] Directed evolution of thirty sites within 10 A of the catalytic copper was performed on a parent sequence to lower the KM of the enzyme on galactose, to enhance activity on a D-N-acetyl glucosamine substrate. Other mutations were found to have neutral or deleterious effects on glucodialdose-generating activity. The new combination sequence was designated GAO-Mut1 and is presented at SEQ ID NO:1. The full sequence of the expressed construct is given as follows:GAO-Mut1

[0094] MGHHHHHHSSGHIEGRHMASAPIGSAIPRNNWAVTCDSAQSGNECNKAIDG NKDTFWHTFYGANGDPKPPHTYTIDMKTTQNVNGLSVLPRQDGNQNGWIGRHEVY LSSDGTNWGSPVASGSWFADSTTKYSNFETRPARYVRLVAITEANGQPWTSIAEINV FQASSYTAPQPGLGRWGPTIDLPIVPAAAAIEPTSGRVLMWSSYRNDAFEGSPGGIT LTSSWDPSTGIVSDRTVTVTKHDMFCPGISMDGNGQIWTGGNDAKKTSLYDSSSD SWIPGPDMQVARGYQSSATMSDGRVFTIGGSFSGGVFEKNGEVYSPSSKTWTSLP NAKVNPMLTADKQGLYKSDNHAWLFGWKKGSVFQAGPSTAMNWYYTSGSGDVKS AGKRQSNRGVAPDAMSGNAVMYDAVKGKILTFGGSPDFEDSDATTNAHIITLGEPG TSPNTVFASNGLYFARTFHTSWLPDGSTFITGGQRRGIPFEDSTPVFTPEIYVPEQD TFYKQNPNSIVRAYHSISLLLPDGRVFNGGGGLCGDCTTNHFDAQIFTPNYLYDSNG NLATRPKITRTSTQSVKVGGRITISTDSSISKASLIRYGTATHTVNTDQRRIPLTLTNNG GNSYSFQVPSDSGVALPGYWMLFVMNSAGVPSVASTIRVTQ

[0095] Please note that the sequence above, SEQ ID NO:1 , contains a “MGHHHHHHSSGHIEGRHM” N-terminal his-tag and linker for expression and purification in E. coli. Only the sequence of the catalytic domain is given for sequences in the “Biomolecule Inventions” section as these proteins were expressed in multiple expression systems.

[0096] Selected positions in GAO-Mut1 were mutated via the QUIKCHANGE method to all 20 amino acids using primers containing NNS codons. The constructs were then screened in the following manner: Colonies were picked and used to inoculate one well each in a 96-well deepwell plate charged with LB. The grown clones were then used toinoculate autoinduction media in a separate 96-well deepwell plate for protein expression. Harvested cells were lysed with Bacterial Protein Extraction Reagent (B- PER) and the lysate was then screened for oxidase activity using a colorimetric ABTS assay which detects hydrogen peroxide. In short, lysate was assayed for activity with and without exposure to heat. To assay activity in the absence of a heat challenge, lysate was diluted 50 times. A volume of 5 pL of the diluted lysate was combined with ABTS assay solution (final concentration of 2% w / v glucose, 0.0125 mg / ml horseradish peroxidase, 50 mM sodium phosphate buffer at pH 8, 0.05% ABTS) to a final volume of 200 pL and the change in absorbance at 405 nm was monitored until the reaction was complete. To assay residual activity after a heat challenge, 50 pL lysate was incubated for ten minutes at 50 °C and 20 pL of the heat-treated lysate was added to the ABTS solution before monitoring change in absorbance at 405 nm. Specific activity was calculated from the formulas below using the linear portion of the curve to measure AA405 / min and taking the extinction coefficient of ABTS at 405 nm as 36.8 mM’1cm’1.. AA405 min-1Units mg1= -36.8 x (pathlength in cm) x (mg enzyme) / (ml reaction mixture) . AA405 min~rUnits ml = -36.8 x (pathlength in cm) x (ml enzyme) / (ml reaction mixture)

[0097] Mutant lysates exhibiting a AA405 / min greater than GAO-Mut1 were chosen for further characterization. Following identification of the mutation by DNA sequencing, hits were expressed, purified, and assayed for specific activity and thermostability as assessed by the temperature at which one half maximal activity was observed (T50). Mutants were purified by from 5ml culture with auto-induction medium in 24 well plate. Harvested cells were lysed with B-PER and the lysate was span down with 15,000 ref for 30 min at 4 °C. The lysate supernatant was used to protein purification with HISPIIR™ Ni-NTA Spin Plates. The eluted protein sample was diluted with 100 mM potassium phosphate buffer pH 7.5 with 0.5 mM CuSO4, and specific activity was measured using the ABTS assay outlined above. Tsowas measured by heating protein in the absence of substrate, cooling, and then measuring residual activity using the ABTS assay. Heating was accomplished by diluting the protein to a concentration of 2.5 mg / L in a volume of 100 mM phosphate buffer at pH 7.5, aliquoting 50 pL into a row of a 96-well PCR plate, and incubating over a temperature gradient sufficient to capturemaximal and minimal enzyme performance for ten minutes. Promptly after heating, the mixture was cooled on ice and the AA405 / min of 20 pL of enzyme solution in 200 pL of final volume of ABTS solution was measured as described above.

[0098] Hits were purified, tested for activity and Tso, and recombined to generate a final best mutant from the directed evolution step. Promising point mutants that could beneficially be combined in the Mut1 background included A193R D404H F441Y A172V (Table I). These mutations were combined into a single combination mutant named GAO-Mut47 which exhibited a specific activity of 27.3 U mg'1and a Tso of 56.8 °C.Rational Engineering

[0099] Rational engineering of GAO to further accept a glucose substrate and identify stabilizing mutations was accomplished with a combination of computational methods based on structural and multiple sequence alignment data (MSA). Previously, identified GAO-M-RQW-S, the GAO-Mut1 mutants without the Y405F and Q406E mutations could accept both glucose and gluconate as substrate, Figure 6 and Figure 7, respectively. As efforts were underway to produce a GAO active on both substrates, rational design was performed on the GAO-M-RQW-S sequence rather than GAO- Mut1. Structural methods employed included applying FoldX38 (40 predicted mutations) and PROSS39 (80 mutations) to a modified form of the PDB structure 2WQ8 to contain the GAO-M-RQW-S mutations. MSA-based predictions were made using and (28 mutations) applied to a 185-member MSA. This MSA was generated from an initial set of 1000 sequences curated with JALVIEWto remove sequences with 98% redundancy and retain only sequences experimentally verified as carbohydrate oxidases. 30 mutations identified by Merck in designing a GAO for synthesizing an intermediate of the HIV drug ISI-ATRAVIR were also added to the panel.

[0100] In total, 202-point mutants were screened using the same methods described above for screening the directed evolution clones. Thirty-nine hits were identified from an initial screen and sixteen were reidentified from a second round of screening. Upon generation of combo mutants in the best combination mutant from the directed evolution step (GAO-Mut47), the mutations N66S, S306A, S311 F, and Q486L were identified as complementary and beneficial while N28I, Y189W, S331 R, A378D, and R459Q were deemed detrimental in this background (Table II). The final GAQ-Mut107 construct containing the Mut47 mutations and N66S, S306A, S311 F, and Q486L exhibits a specific activity of 34.96 U mg-1on 2% glucose and a Tso of 60.56 °C.Table IIBolded mutations are beneficial in a Mut47 background A193R D404H F441Y A172V *Data collected in a separate experiment from other data. Fold improvement is calculated compared to an internal Mut47 control.EXAMPLE 2

[0101] The oxidation of benzyl alcohol to benzaldehyde using the compositions and methods disclosed herein was investigated. Enzymatic oxidation of benzyl alcohol to benzaldehyde was performed by first charging separate wells of a 24 deep-well plate with 4.5 mL of a reaction master mix containing 54 mM sodium phosphate pH 8.0, 45 pM CuSC>4, 12 pM MnSC 7.3 ppm of a small molecule, activator, 0.09% antifoam, 87mg / L GAO at 6.3 mg / mL, 6 mg / L horseradish peroxidase, and 100 mg / L Katalase-S in MilliQ water. To each of these wells was added 400 p.L of benzyl alcohol, pipetted directly to the bottom of the well. This reaction plate was incubated at 11 °C, 250 rpm for 66 hr. The cloudy white mixtures were analyzed over the reaction time course by combining 400 pL 100 mM maleic acid with 100 gL reaction and measured by H-NMR with maleic acid as the internal standard for quantitation. The reaction is depicted schematically in Figure 8 in addition to a bar graph depicting the product distribution for the indicated samples.

[0102] Computed yield was 76%, with 1 .6% of the mass balance being residual benzyl alcohol. The remaining mass balance was lost to evaporation over the reaction. The crude reaction mixture could be taken directly to the next step. The molecule distribution in the aqueous and organic layers is presented graphically in Figure 9.EXAMPLE 3

[0103] The conversion of benzaldehyde to benzylamine was investigated using the methods and compositions disclosed herein. Specifically, 5 mL of solution from the enzymatic reaction (Example 2) was mixed with 10 mL of 30 wt.% NH3 and Raney Ni was added. The reactor was pressurized (60 bar of H2) and heated to 105 °C for 4 hours. Yield of the reductive amination reaction was 82%. The final product was isolated as the HCI salt after extracted with ether to remove any unreacted materials followed by vacuum distillation to remove the water.HBIW Synthesis

[0104] During a 1 hour period, glyoxal (72.5g, 40% aqueous solution, 0.50 mol) was added dropwise to a solution of benzylamine (117.9g, 1.10 mol), water (100 mL), and formic acid (88%, 5.76g, 0.110 mol) in acetonitrile (1100 mL) while keeping the temperature below 20°C. The addition funnel was rinsed with 10 mL of water. After standing at 25°C overnight (16-18h), the precipitated product was removed by filtration and washed with cold acetonitrile. The crude product was resuspended twice in cold acetonitrile with stirring and filtered. Product was recrystallized from a MeCN / acetone mixture. The yield was 75 g (63%) of hexabenzylhexaazaisowurtzitane. The synthesis was repeated to increased scale as summarized in Table III.Table III

[0105] The yield was 31.5% (270 g of material). The quality of the final product is comparable to the 1 L scale according to H-NMR.

[0106] A 10 g scale synthesis was carried out. The following reagents were poured into the reactor: methanol (75mL), water (6.7mL), benzylamine (19.33mL, 0.177 mol), and 99% formic acid (0.8 mL, 0.02 mol). While stirring, the reaction mixture was cooled to 0°C. Then, a 40% aqueous glyoxal solution (11 .76 mL, .08 mol) was introduced into the reactor using a syringe pump over 2 h. The vessel content was allowed to warm to 20 °C and then heated and maintained at 50 °C for 4 h. Next, the reaction mixture was cooled to 20 °C and then filtered. The precipitate was washed with 100 mL of methanol. Upon drying, a total of 8.2 g of HBIWwas obtained with a crude yield of 44%.

[0107] TADBIW synthesis was carried out. This step involves the debenzylation of four of the benzyl groups of HBIW to get to TADBIW. Considerations for this reaction are mass transfer phenomena (good stirring) and catalyst selection. Bad stirring or poor catalyst performance can lead to degradation of HBIW which in turn creates side products that can poison the catalyst decreasing the activity even further to the point of obtaining no product during this reaction. Absence of water is also monitored as it can hydrolyze the acetic anhydride which can catalyze the degradation of HBIW. The reaction is depicted schematically at Figure 10.

[0108] The effectiveness of different hydrogenation catalysts were evaluated. The catalysts evaluated were palladium hydroxide (Pd(OH)2) and the EVONIK NOBLYST® P1173 10% Pd on carbon commercially available from Millpore Sigma.

[0109] Catalyst screening was carried out using a 500 mL Parr bomb was loaded with twelve 8-mL vials to test 12 catalysts at a time under the same conditions. The standard method was carried out at 400 rpm, 5 bar of pure hydrogen gas, and at 35 °C. Typically, 2.5 mL of DMF, 1.5 mL of AC2O, 0.02 mL of PhBr (please confirm this compound), 1.0 g of HBIW, and 0.05 g of catalysts were placed into each vial in the reactor. Catalystscreening was carried out with good stirring: Specifically, the reaction was carried out without heat to have direct contact with the stir plate and the yields drastically improved. Additionally, 20-mL vials were used to be able to place larger stirbars and the pressure set to 10 bar of pure hydrogen. All reactions had colorless supernatants which is indicative of a successful synthesis. The results of this screening is presented in Table IV.Table IV

[0110] The final step of the synthesis involves the debenzylation of the last two benzyl groups in the presence of water and acetic acid. The TADBIW intermediate is known to have a very stable shelf life and be pretty sturdy under the reaction conditions. As long as the catalyst has not lost all activity in the previous step, this reaction should proceed to the desired product with high yield.Small scale reaction screening:[oom] Small scale reactions were carried over from the previous step in 20 mL vials at 1 atm using balloons to keep a hydrogen reservoir at the correct pressure during the course of the reaction at 400 rpm. The solids from the first step were suspended in 1 .6 mL of acetic acid and 0.4 mL of water and flushed with pure hydrogen. The heating block was set to 50 °C and the reaction was allowed to proceed for 20 hours. The product was filtered, washed with ethanol, and H-NMR in D2O was performed to confirm the product. The results are presented in Table V.Table V

[0112] The subject matter having been shown and described, modifications thereof can be made by one skilled in the art without departing from the spirit and teachings of the subject matter. The aspects described herein are exemplary only and are not intended to be limiting. Many variations and modifications of the subject matter disclosed herein are possible and are within the scope of the disclosed subject matter. Where numerical ranges or limitations are expressly stated, such express ranges or limitations should be understood to include iterative ranges or limitations of like magnitude falling within the expressly stated ranges or limitations (e.g., from about 1 to about 10 includes, 2, 3, 4, etc.; greater than 0.10 includes 0.11 , 0.12, 0.13, etc.). Use of the term "optionally" with respect to any element of a claim is intended to mean that the subject element is required, or alternatively, is not required. Both alternatives are intended to be within the scope of the claim. Use of broader terms such as comprises, includes, having, etc. should be understood to provide support for narrower terms such as consisting of, consisting essentially of, comprised substantially of, etc.

[0113] Accordingly, the scope of protection is not limited by the description set out above but is only limited by the claims which follow, that scope including all equivalents of the subject matter of the claims. Each and every claim is incorporated into the specification as an aspect of the present disclosure. Thus, the claims are a further description and are an addition to the aspects of the present invention. The discussion of a reference herein is not an admission that it is prior art to the presently disclosed subject matter, especially any reference that may have a publication date after the priority date of this application. The disclosures of all patents, patent applications, and publications cited herein are hereby incorporated by reference, to the extent that they provide exemplary, procedural or other details supplementary to those set forth herein.

Claims

CLAIMSWhat is claimed is:1 . A chemoenzymatic process, comprising: contacting benzyl alcohol with one or more oxidase catalyst systems under conditions suitable for the formation of an oxidized intermediate; aminating the oxidized intermediate to form a nitrogen-containing compound; and hydrogenating the nitrogen-containing compound to form benzylamine.

2. The process of claim 1 , wherein the one or more oxidase catalyst systems comprise: (i) one or more oxidase enzymes, (ii) one or more small molecule activators, and (iii) one or more single electron oxidizers.

3. The process of claim 2, wherein the one or more oxidase enzymes comprise one or more copper radical oxidases, mutants thereof, fragments thereof, or combinations thereof.

4. The process of claim 2, wherein the one or more oxidase enzymes comprise galactose oxidase, glucose oxidase, alcohol oxidase, aryl alcohol oxidase, glyoxal oxidase, mutants thereof, fragments thereof or combinations thereof.

5. The process of claim 2, wherein the one or more oxidase enzymes has any of SEQ ID NO:1 to SEQ ID NO:11.

6. The process of claim 1 , wherein conditions suitable for formation of an oxidized intermediate further comprise a catalase.

7. The process of claim 6, wherein the catalase has any of SEQ ID NO: 12 to SEQ ID NO:17.

8. The process of claim 2, wherein the small molecule activator is selected from the group consisting of-tryptophan, 2-mercaptobenzothiazole, L-histidine,methylchloroisothiazolinone, o-dianisidine, 2,2'-azino-bis(3-ethylbenzothiazoline-6- sulfonic acid (ABTS), 4-aminoantipyrine, L-tyrosine, (2,2,6,6-tetramethylpiperidin-1- yl)oxyl, chloromethylisothiazolinone, 4-thiazolecarboxylic acid, Sunset yellow FCF, tartrazine, p-benzoquinone, dicoumarol, phthalimide, saccharin, phthalic anhydride, erythrosine B, 2-aminobenzothiazole, thiabendazole, 2-hydroxybenzothiazole, phenothiazine, 6-aminobenzothiazole, indigo carmine, naphthalimide, 2-aminothiazole, thiazole, 2H-1 ,4-benzothiazin-3(4H)-one, 2-oxindole, beta-lapachone, menaquinone, thiamine, 4-methyl-5-thiazoleethanol, Allura Red AC, menadione, p-cresol, Fast green FCF, Brilliant Blue FCF, methylisothiazolinone, caffeine, veratryl alcohol, fluorescein, and combinations thereof.

9. The process of claim 2, wherein the single electron oxidizer is selected from the group consisting of laccase, horseradish peroxidase, Dyp-type peroxidase, lactoperoxidase, chloroperoxidase, manganese peroxidase 1 , ascorbate peroxidase, dye-decolorizing peroxidase, unspecific peroxygenase, dehaloperoxidase, catalase- peroxidase, lignin peroxidase, soybean seed coat peroxidase, isoforms thereof and combinations thereof.

10. The process of claim 2, wherein the single electron oxidizer has SEQ ID NO: 18.

11. The process of claim 1 , wherein an oxidized intermediate comprises benzaldehyde, benzonitrile, benzaldoxime or combinations thereof.

12. The process of claim 1 , wherein the nitrogen-containing compound comprise ammonia.

13. The process of claim 1 , wherein the hydrogenation catalyst comprises Rainey nickel.

14. A chemoenzymatic process, comprising: contacting benzyl alcohol with a galactose oxidase catalyst system under conditions suitable for the formation of benzaldehyde;reductively aminating benzaldehyde in the presence of Rainey nickel and a nitrogen source to form benzylamine; contacting benzylamine and glyoxal in the presence of a mineral acid and solvent under conditions suitable for the formation of hexabenzylhexaazaisowurtzitane; and acetylating hexabenzylhexaazaisowurtzitane to form tetraacetyl- diaminohexaazaisowurtzitane.

15. The process of claim 14, wherein the galactose oxidase has any of SEQ ID NO:1 to SEQ ID NO:6.

16. The process of claim 14, wherein the galactose oxidase system further comprises a small molecule activator and a single electron acceptor.

17. The process of claim 14, wherein the small molecule activator is selected from the group consisting of-tryptophan, 2-mercaptobenzothiazole, L-histidine, methylchloroisothiazolinone, o-dianisidine, 2,2'-azino-bis(3-ethylbenzothiazoline-6- sulfonic acid (ABTS), 4-aminoantipyrine, L-tyrosine, (2,2,6,6-tetramethylpiperidin-1- yl)oxyl, chloromethylisothiazolinone, 4-thiazolecarboxylic acid, Sunset yellow FCF, tartrazine, p-benzoquinone, dicoumarol, phthalimide, saccharin, phthalic anhydride, erythrosine B, 2-aminobenzothiazole, thiabendazole, 2-hydroxybenzothiazole, phenothiazine, 6-aminobenzothiazole, indigo carmine, naphthalimide, 2-aminothiazole, thiazole, 2H-1 ,4-benzothiazin-3(4H)-one, 2-oxindole, beta-lapachone, menaquinone, thiamine, 4-methyl-5-thiazoleethanol, Allura Red AC, menadione, p-cresol, Fast green FCF, Brilliant Blue FCF, methylisothiazolinone, caffeine, veratryl alcohol, fluorescein, and combinations thereof.

18. The process of claim 14, wherein the single electron oxidizer is selected from the group consisting of laccase, horseradish peroxidase, Dyp-type peroxidase, lactoperoxidase, chloroperoxidase, manganese peroxidase 1 , ascorbate peroxidase, dye-decolorizing peroxidase, unspecific peroxygenase, dehaloperoxidase, catalase- peroxidase, lignin peroxidase, soybean seed coat peroxidase, isoforms thereof and combinations thereof.

19. The process of claim 14, wherein the nitrogen source comprises ammonia.

20. The process of claim 14, wherein the galactose oxidase system further comprises a catalase having any of SEQ ID NO:12 to SEQ ID NO:17.21 . The process of claim 14, wherein hexabenzylhexaazaisowurtzitane is formed at a yield of greater than about 70% and a purity of greater than about 90%.

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