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WO2024206707A1 - Azyariv et conjugués azyariv - nouveaux outils pour visualiser et caractériser des protéines d'arabinogalactane - Google Patents

Azyariv et conjugués azyariv - nouveaux outils pour visualiser et caractériser des protéines d'arabinogalactane Download PDF

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WO2024206707A1
WO2024206707A1 PCT/US2024/022083 US2024022083W WO2024206707A1 WO 2024206707 A1 WO2024206707 A1 WO 2024206707A1 US 2024022083 W US2024022083 W US 2024022083W WO 2024206707 A1 WO2024206707 A1 WO 2024206707A1
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phenyl
azyariv
glucosyl
small molecule
chemical composition
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Amit Basu
Sebastian RUEDA
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Brown University
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Brown University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/20—Carbocyclic rings
    • C07H15/203—Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/23—Heterocyclic radicals containing two or more heterocyclic rings condensed among themselves or condensed with a common carbocyclic ring system, not provided for in groups C07H19/14 - C07H19/22
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56961—Plant cells or fungi
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label

Definitions

  • the embodiments of the present invention relate to Yariv reagents with improved functions, fluorescence, and chromophores to expand the study of arabinogalactan proteins, galactans, and glycoproteins in general.
  • Yariv reagents with improved functions, fluorescence, and chromophores to expand the study of arabinogalactan proteins, galactans, and glycoproteins in general.
  • an intelligent synthesis and design of Yariv reagents is presented and expanded methods are developed and tested.
  • Arabinogalactan proteins are cell wall proteoglycans implicated in essential functions such as cell signaling, plant growth, and programmed cell death.
  • the P-glucosyl Yariv reagents e.g., 1 ,3,5-tris (4-p-D-glucopyranosyloxyphenylazo)-2,4,6- trihydroxy-benzene] are red dyes which can specifically bind to and precipitate this class of plant AGPs.
  • a p-glucosyl Yariv reagent is dissolved in ⁇ 1 mL of 0.15M NaCI. This solution is applied to a tissue section of the plant tissue for about one hour at room temperature (about 25°C).
  • the tissue section, with the solution, is then examined by bright field microscopy.
  • the AGPs will precipitate in the Yariv reagent to give a red stain, but the exact color can vary from brown-red to bright red depending on the plant tissue.
  • the AGP's are known to be water-soluble, they may be lost during these procedures for tissue embedding.
  • Several other problems occur with these methods including general sensitivity and signal to noise issues under the microscope.
  • Another major issue is the method described above can be impossible to practice on live plants. What is urgently needed are new compounds and methods that enable sensitive studies of AGPs and glycoproteins in general for a variety of biomass, including living plants.
  • the present invention solves many problems inherent in the prior art Yariv reagents that are currently in use.
  • AGPs are known to be water-soluble, and they may be lost during the procedures for tissue embedding. This loss is detrimental to limit of detection (LOD) Sensitivity, variance, and signal to noise issues are found in the prior art (i.e., in view of this disclosure) Yariv reagents.
  • LOD limit of detection
  • New reagents are urgently needed with new compounds and methods that enable sensitive studies of AGPs and glycoproteins in general for, example, using highly sensitive fluorescent and absorbance techniques.
  • the technology disclosed herein enables implementation with modern analytical instrumentation and computers, along with intricate visualization of the data.
  • new synthetic methods applied to produce functionalizable analogs of various Yariv Reagents can be used for AGP Imaging using fluorescence microscopy.
  • the present invention discloses a chemical composition
  • a chemical composition comprising a molecular structure of formula 1 below: or a tautomer and/or an E/Z isomer thereof of formula 1 ; wherein each instance of R in formula 1 above independently comprises: wh (-0-) in formula 1 ; wherein Y comprises: -N3; ; wherein X' is an anion; wherein • above represents a single bond attached to any -OH or -0- of R; or wherein Y comprises a click chemistry functional group, near-IR dye, an IR dye, a fluorescent small molecule, a small molecule with a chromophore, a Raman active small molecule, or a small molecule with a maximum absorbance band in a range from about 200 nm to about 100 pm; and wherein a small molecule has a molecular weight in a non-salt form of less than about 1000 daltons (Da).
  • a method for performing a fluorescence imaging of an arabinogalactan protein (AGP) or a glycosylated protein (glycoprotein) comprising the steps of: (1 ) contacting a material known to contain an AGP or a glycoprotein or suspected to contain an AGP or a glycoprotein with a reagent including AzYariv-Cy5, AzYariv-Cy5-X [3GlcYariv, or a combination thereof, whereby a binding between the AGP or glycoprotein and the reagent occurs; (2) directing an incident light or an incident electromagnetic radiation towards the binding, whereby a fluorescent emission occurs; and (3) gathering, observing, measuring, or acquiring the emission.
  • AGP arabinogalactan protein
  • glycosylated protein glycosylated protein
  • a method for performing an absorbance imaging of an arabinogalactan protein (AGP) or a glycosylated protein (glycoprotein) comprising the steps of: (1 ) contacting a material known to contain an AGP or a glycoprotein or suspected to contain an AGP or a glycoprotein with a reagent including AzYariv-Cy5, AzYariv-Cy5-X‘, [3GlcYariv, or a combination thereof, whereby a binding between the AGP or glycoprotein and the reagent occurs; (2a) directing an incident light or an incident electromagnetic radiation towards the binding, whereby an electromagnetic absorbance occurs; and (3a) gathering, observing, measuring, or acquiring the absorbance.
  • AGP arabinogalactan protein
  • Glycoprotein glycosylated protein
  • a method for performing a fluorescence imaging of an arabinogalactan protein (AGP) or a glycosylated protein (glycoprotein) can include any of the steps of: obtaining a material known to contain an AGP or a glycoprotein or suspected to contain an AGP or a glycoprotein; contacting the material with a reagent disclosed herein or including AzYariv-Cy5, AzYariv-Cy5-X [BGIcYariv, or a combination thereof, whereby a binding between the AGP or glycoprotein and the reagent occurs. Next is directing an incident light or an incident electromagnetic radiation towards the binding, whereby a fluorescent emission occurs.
  • a gathering, observing, measuring, or acquiring the emission is executed, typically including a digitization using a computer and software.
  • Computer graphics or other visualization can provide a decision whether or not to proceed with the data or to repeat the experiments using a different area of plant tissue.
  • a method for performing an absorbance measurement on an arabinogalactan protein (AGP) or a glycosylated protein (glycoprotein) including the steps of: obtaining a material known to contain an AGP or a glycoprotein or suspected to contain an AGP or a glycoprotein; optionally placing the material on a suitable mount for absorbance wavelengths to pass through the sample; contacting the material with a binding reagent disclosed herein including a chromophore; and directing an incident light or an incident electromagnetic radiation towards the binding area, whereby an electromagnetic absorbance occurs.
  • AGP arabinogalactan protein
  • glycoprotein glycosylated protein
  • FIG. 1A shows examples of small molecule plant cell wall probes Sirofluor, Calcofluor White, and [BGIcYariv.
  • FIG. 1 B shows synthesis of compound 3 (shown at right).
  • FIG. 10 shows synthesis of compound 7 (middle).
  • FIG. 1D shows synthesis of AzYariv Reagent (shown at right).
  • FIG. 1E shows example ATR IR spectra of AzYariv reagent and PGIcYariv reagent.
  • FIG. 2A shows CD spectra of AzYariv and PGIcYariv.
  • FIG. 2B shows an absorbance spectrum of AzYariv. All spectra obtained in water at [300 pM],
  • FIG. 3B shows Table 1 ; Optimization of Click Reaction Conditions Between AzYariv and Cy5-DBCO.
  • FIG. 3C shows a high- resolution mass spectrum for AzYariv-Cy5 acquired using +ESI (positive electrospray ionization).
  • FIG. 3D shows a high-resolution mass spectrum for AzYariv (top) acquired using +ESI and predicted isotopic distribution for [M]+ (bottom).
  • FIGs. 4A-4H show representative fluorescence images of AzYariv-Cy5 binding in agarose reverse gels with gum arabicAGP. Concentrations of GIcYariv and aGalYariv are 1.03 mM.
  • FIG. 4A AzYariv-Cy5 (5 pM)/pGlcYariv
  • FIG. 4B AzYariv-Cy5 (100 nM)/pGlcYariv
  • FIG. 4C AzYariv-Cy5 (5 nM)/pGlcYariv
  • FIG. 4D AzYariv-Cy5 (5 pM)/pGlcYariv Lyophilized
  • FIG. 4A AzYariv-Cy5
  • FIG. 4B AzYariv-Cy5 (100 nM)/pGlcYariv
  • FIG. 4C AzYariv-Cy5 (5 nM)/pGlcYariv
  • FIG. 4D AzYariv-C
  • FIGs. 5A-5F shows representative confocal images of fixed maize leaves treated with Yariv reagents (FIGs. 5A-5D, imaged with 651 nm laser) and antibodies (FIG. 5E, FIG. 5F, imaged with 495 nm laser).
  • FIG. 5A AzYariv-Cy5/pGlcYariv
  • FIG. 5B zoom of AzYariv-Cy5/pGlcYariv.
  • UE Upper epidermal cells
  • P phloem
  • X xylem
  • BS bundle sheath
  • LE lower epidermal cells
  • FIG. 5A-5F shows representative confocal images of fixed maize leaves treated with Yariv reagents
  • FIG. 5E imaged with 495 nm laser.
  • FIG. 5A AzYariv-Cy5/pGlcYariv
  • FIG. 5B zoom of AzYariv-Cy5/pGlcYariv.
  • FIG. 5C shows a representative confocal image of fixed maize leaves imaged for fluorescein (conjugated secondary antibody used in immunofluorescence) A) autofluorescence of tissue and non-specific secondary antibody staining (no primary antibody control);
  • FIG. 5H shows a representative confocal image of fixed maize leaves imaged for fluorescein (conjugated secondary antibody used in immunofluorescence) B) MAC207;
  • FIG. 51 shows a representative confocal image of fixed maize leaves imaged for fluorescein (conjugated secondary antibody used in immunofluorescence)
  • C) JIM13 C
  • Scale bar 50 pm.
  • Merged brightfield and confocal images are shown of (FIG. 5J) fixed maize leaves treated with pGIcYariv and (FIG. 5K) Fresh maize leaves treated with [3GlcYariv. Green signal (or lighter greyscale) is chlorophyll autofluorescence.
  • Scale bar 50 pM.
  • FIG. 6A, FIG. 6B, FIG. 60, and FIG. 6D show representative confocal images of fresh maize leaves treated with AzYariv-Cy5/[3GlcYariv (top, FIG. 6A, FIG. 6B) and AzYariv-Cy5/aGalYariv (bottom, FIG. 6C, FIG. 6D) imaged with 651 nm laser.
  • UE Upper epidermal cells
  • P phloem
  • X xylem
  • BS bundle sheath
  • LE lower epidermal cells.
  • Scale bar 50 pm.
  • FIG. 7A shows an example synthesis of compound 4 (shown at right).
  • FIG. 7B shows an example synthesis of compound 5 (shown at right).
  • FIG. 7C shows an example synthesis of compound 6 (shown at right).
  • FIG. 7D shows an example synthesis of compound 7 (shown at right).
  • FIG. 8A shows example (prior art) 2 tautomer interconversions of formula 1
  • FIG. 8B shows a continuation of the tautomer interconversions.
  • FIG. 9 shows a major tautomer found and established by NMR data in DMSO.
  • FIG. 10 shows an example of a novel formula 1 (left) and some examples of ‘R’ that were attached at right (prior art) from previous work 3 .
  • FIG. 11 shows additional examples of ‘R’.
  • FIG. 12 shows an example method 500 for performing a fluorescence imaging of an arabinogalactan protein (AGP) or a glycosylated protein (glycoprotein).
  • AGP arabinogalactan protein
  • glycoprotein glycosylated protein
  • FIG. 13 shows an example method 600 for performing an absorbance measurement on an arabinogalactan protein (AGP) or a glycosylated protein (glycoprotein).
  • AGP arabinogalactan protein
  • glycoprotein glycosylated protein
  • the term “approximately” or “about” in reference to a value or parameter are generally taken to include numbers that fall within a range of 5%, 10%, 15%, or 20% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).
  • reference to “approximately” or “about” a value or parameter includes (and describes) embodiments that are directed to that value or parameter. For example, description referring to "about X” includes description of "X”.
  • the term “or” means “and/or.”
  • the term “and/or” as used in a phrase such as "A and/or B” herein is intended to include both A and B; A or B; A (alone); and B (alone).
  • the term “and/or” as used in a phrase such as "A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
  • the term “consisting essentially of'” refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.
  • the term “consisting essentially of” can also be exemplified by plain language provided in the claims.
  • the technology can be described in reference to plants or living plants; however, the technology disclosed herein can be applied to any living thing, any material, or any substance in any physical form.
  • the term “statistically significant” or “significantly” refers to statistical significance and generally means a two-standard deviation (2SD) or greater difference.
  • the terms: “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount.
  • “reduce,” “reduction” or “decrease” or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g., the absence of a given treatment or agent) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more.
  • “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to
  • the terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount.
  • the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5- fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.
  • a “increase” is a statistically significant increase in such level.
  • a small molecule is less than 1000 MW and that is not including a cation or anion for a salt form.
  • a large molecule is not less than 1000 MW including biologies, oligonucleotides, peptides, oligosaccharides, and larger molecules. Any of the methods or compositions disclosed herein can be used as or in combination with small molecules and/or large molecules as discussed herein. [0045] As discussed above, unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs.
  • the p-glucosyl Yariv reagents of the past art are red to brown-red dyes which can specifically bind to and precipitate plant AGPs.
  • a solution of the Yariv reagent is typically mixed with sodium chloride and the solution is applied to a tissue section of the plant tissue at room temperature (about 25°C). The tissue section, with the solution, is then examined under a microscope to observe brown-red or red staining. Under the microscope, the AGPs will precipitate in the Yariv reagent to give a red stain, but the exact color can vary from brown-red to bright red depending on the plant tissue.
  • the AGPs are known to be water-soluble, they may be lost during these procedures for tissue embedding.
  • the technology herein provides small molecule fluorescent probes that bind selectively to plant cell wall polysaccharides.
  • the new small molecule fluorescent probes have been instrumental in elucidating the localization and function of these glycans.
  • Arabinogalactan proteins (AGPs) are cell wall proteoglycans implicated in essential functions such as cell signaling, plant growth, and programmed cell death.
  • AGPs Arabinogalactan proteins
  • Yariv reagents are the only small molecules that bind AGPs and have been used to study AGP function and isolate AGPs via precipitation of an AGP-Yariv complex.
  • the Yariv reagents of the past are not fluorescent, rendering them ineffective for localization studies using fluorescence microscopy.
  • a fluorescent version of a Yariv reagent that is capable of both binding as well as imaging AGPs would provide a powerful tool for studying AGPs in planta.
  • the modified reagent binds gum arabic in in vitro binding assays when used in conjunction with the pGIcYariv reagent.
  • Fluorescent imaging of AGPs in fixed maize leaf tissue enables localization of AGPs to cell walls in the leaf. Significantly, imaging can also be carried out using fresh tissue. This represents the first small molecule probe that can be used to visualize AGPs using fluorescence microscopy.
  • Plant cell walls are comprised of an intricate assembly of polysaccharides including cellulose, hemicelluloses, and pectins, along with crosslinked lignins and proteoglycans such as arabinogalactan proteins (AGPs). 7 ’ 10 There is a pressing need for the development of tools that can image the complex and dynamic plant cell wall. 11 ’ 14 Fluorescence microscopy is a powerful method for visualizing components of the plant cell wall because of its high sensitivity and spatial resolution. Small molecule fluorescent dyes are vital compounds in elucidating the distribution of these components. For example, Sirofluor and Calcofluor White (FIG. 1A) are commonly used as fluorescent stains for callose and cellulose, respectively. FIG.
  • AGPs are proteoglycans found throughout the plant, including in leaves, roots, stems, pollen tubes, and pollen grains. 15 AGPs are implicated in myriad functions ranging from plant growth, cellular signaling, programmed cell death, and many others. 16 ’ 18 AGPs contain protein domains rich in hydroxyproline residues that are glycosylated with long [3(1 - ⁇ 3)-linked galactan chains that make up the bulk of AGP mass. 1920 Shorter branching [3(1 — >6) galactan side chains are linked from the galactan main chains and are commonly decorated with arabinofuranose residues and other monosaccharides, with variations in the specific composition of the branches depending on the species and plant tissue.
  • MAbs monoclonal antibodies
  • Yariv reagents the set of dyes known as the Yariv reagents (vide infra). 15 While MAbs can provide bright images showing AGP decoration, they are highly epitope specific, and due to their large size, the antibodies may not be able to access all parts of the cell wall. 12 ’ 14 On the other hand, the Yariv reagents, dyes based on a tri-glycosylated phloroglucinol core (FIG. 1 A), bind AGPs but can only be visualized using the less sensitive brightfield microscopy.
  • the AGP binding ability of the Yariv reagents depends on the structure of the sugar moieties attached to the aromatic core. 43 Yariv reagents bearing (3-D linked glucose ([3GlcYariv) or galactose bind strongly to AGPs and have been used to isolate AGPs from plants by precipitation of an AGP-Yariv complex. 21-23 Yariv reagents comprised of a-linked sugars or L sugars do not bind to AGPs. The Yariv reagents aggregate in solution and bind to the core [3(1 —> 3)- linked galactan backbone, which is postulated to be helical.
  • FIG. 1 D Diazotization of 7, subsequent coupling with 3, followed by isolation and purification using a redissolution and reprecipitation protocol afforded AzYariv in 18% yield.
  • the 1 H NMR spectrum of AzYariv indicates the presence of distinct anomeric and OH resonances derived from the glucosyl and 6-azido glucosyl moieties.
  • the IR spectrum of AzYariv displays the characteristic azide stretch at 2100 cm -1 (FIG. 1 E). The IR spectrum was acquired using ATR IR, and FIG. 1 E shows example ATR IR spectra of AzYariv reagent and [3GlcYariv reagent.
  • the CD spectrum of AzYariv exhibits two positive bisignate Cotton effects similar to other [3- D glycosyl Yariv reagents, with a lower intensity than the parent pGIcYariv (FIG. 2A).
  • FIG. 2A To confirm that installation of the azide does not disrupt the AGP binding of AzYariv, we carried out reverse gel assays to determine binding.
  • the precipitate halos formed by AzYariv indicate that it still binds gum arabic, although the halos are smaller than those observed for pGIcYariv, indicative of weaker binding (FIG. 3A, black boxes).
  • FIG. 3C shows a high-resolution mass spectrum for AzYariv-Cy5 acquired using +ESI (positive electrospray ionization).
  • FIG. 3D shows a high-resolution mass spectrum for AzYariv (top) acquired using +ESI and predicted isotopic distribution for [M]+ (bottom).
  • the AzYariv-Cy5 conjugate was not further purified and was used as a 1 :4 mixture of AzYariv-Cy5/AzYariv in subsequent mixtures.
  • FIGs. 4A-4H show representative fluorescence images of AzYariv-Cy5 binding in agarose reverse gels with gum arabic AGP. Concentrations of pGIcYariv and aGalYariv are 1.03 mM.
  • FIG. 4A AzYariv-Cy5 (5 pM)/pGlcYariv
  • FIG. 4B AzYariv-Cy5 (100 nM)/pGlcYariv
  • FIG. 4A AzYariv-Cy5 (100 nM)/pGlcYariv
  • the technology herein provides a novel lyophilized product.
  • staining with LM2 which binds to oligogalactans terminated with glucuronic acid, shows weaker staining of the vasculature than staining with JIM4, for which the trisaccharide ([3-D-GlcA- (1 3)-a-D-GalA-(1 — > 2)-a-D-Rha is a strong binding epitope. 2930 Staining with
  • AzYarivCy5/pGlcYariv is stronger in the inner wall of the upper epidermal cellsthan any of the other antibodies tested. Additionally, the AzYarivCy5/ [BGIcYariv exhibits a more consistent stronger staining pattern throughout the leaf, perhaps reflecting the fact that it preferentially binds to the conserved [3(1 —3) galactan backbone of AGPs.
  • FIGs. 5A-5F shows representative confocal images of fixed maize leaves treated with Yariv reagents (FIGs. 5A-5D, imaged with 651 nm laser) and antibodies (FIG. 5E, FIG. 5F, imaged with 495 nm laser).
  • FIG. 5A AzYariv-Cy5/
  • FIG. 5B zoom of AzYariv-Cy5/pGlcYariv.
  • UE Upper epidermal cells
  • P phloem
  • X xylem
  • BS bundle sheath
  • LE lower epidermal cells;
  • FIG. 5A-5F shows representative confocal images of fixed maize leaves treated with Yariv reagents (FIGs. 5A-5D, imaged with 651 nm laser) and antibodies (FIG. 5E, FIG. 5F, imaged with 495 nm laser).
  • FIG. 5A AzYariv-Cy5/
  • FIG. 5C shows a representative confocal image of fixed maize leaves imaged for fluorescein (conjugated secondary antibody used in immunofluorescence) A) autofluorescence of tissue and non-specific secondary antibody staining (no primary antibody control);
  • FIG. 5H shows a representative confocal image of fixed maize leaves imaged for fluorescein (conjugated secondary antibody used in immunofluorescence) B) MAC207;
  • FIG. 5I shows a representative confocal image of fixed maize leaves imaged for fluorescein (conjugated secondary antibody used in immunofluorescence)
  • C) JIM13 shows a representative confocal image of fixed maize leaves imaged for fluorescein (conjugated secondary antibody used in immunofluorescence)
  • C) JIM13 shows a representative confocal image of fixed maize leaves imaged for fluorescein (conjugated secondary antibody used in immunofluorescence)
  • FIG. 5K Fresh maize leaves treated with [SGIcYariv. Green signal (or lighter greyscale) is chlorophyll autofluorescence.
  • Scale bar 50 pM.
  • An advantage of using the Yariv reagents for visualization is the ability to perform staining experiments in fresh plant tissue. 31-33 These experiments are critical to determining AGP function in live cell assays and cannot be replicated by MAbs, which do not precipitate AGPs and require chemical fixation prior to imaging.
  • FIG. 6A, FIG. 6B, FIG. 6C, and FIG. 6D show representative confocal images of fresh maize leaves treated with AzYariv-Cy5/[3GlcYariv (top, FIG. 6A, FIG. 6B) and AzYariv-Cy5/aGalYariv (bottom, FIG. 6C, FIG. 6D) imaged with 651 nm laser.
  • UE Upper epidermal cells
  • P phloem
  • X xylem
  • BS bundle sheath
  • LE lower epidermal cells.
  • Scale bar 50 pm.
  • the utility of the AzYariv-Cy5 reagent as a tool for plant biology is seen in the results of imaging experiments in fixed maize leaves, which show distinct fluorescence emanating from the cell walls of phloem, epidermal, and bundle sheath cells. Imaging experiments with fresh leaves also show expected binding patterns as well as bright fluorescence from cell wall staining, providing the opportunity to use the AzYariv-Cy5 reagents for real-time imaging experiments.
  • the present invention discloses a chemical composition comprising a molecular structure of formula 1 below: or a tautomer and/or an E/Z isomer thereof of formula 1 ; wherein each instance of R in formula 1 above independently comprises:
  • a-D-galactosyl P-D-mannosyl, or a-D-mannosyl; wherein as shown above represents a bond from R to an oxygen atom (-O-) in formula 1 ;
  • X' is an anion; wherein • above represents a single bond attached to any -OH or -0- of R; or wherein Y comprises a click chemistry functional group, near-IR dye, an IR dye, a fluorescent small molecule, a small molecule with a chromophore, a Raman active small molecule, or a small molecule with a maximum absorbance band in a range from about 200 nm to about 100 pm; and wherein a small molecule has a molecular weight in a non-salt form of less than about 1000 daltons (Da).
  • formula 1 above is further comprising each instance of R independently comprises: phenyl(4)-O-p-D-glucosyl, phenyl(4)-O-a-D-glucosyl, phenyl(4)-O-p-L-glucosyl, phenyl(4)-O-p-D-galactosyl, phenyl(4)-O-a-D-galactosyl, phenyl(4)-O-p-D-xylosyl, phenyl(4)-O-a-D-xylosyl, phenyl(4)-O-a-D-xylosyl, phenyl(4)-
  • the chemical composition is wherein a major tautomer of formula 1 is comprising a following tautomeric state:
  • tautomer D1 of formula 1 (tautomer D1 of formula 1 ); or wherein the tautomer D1 comprises about more than 50% of the molecular structure, out of all formula 1 related structures, in the composition.
  • the chemical composition disclosed above is further comprising wherein a tautomer and/or an E/Z isomer of formula 1 comprises:
  • B1 (B1 ), (B2), or a combination of B1 and B2 thereof; wherein each instance of • in B1 and B2 represents -O-R or -O-R-Y in formula 1 disclosed above.
  • the chemical composition disclosed above is further comprising wherein a tautomer and/or an E/Z isomer of formula 1 comprises:
  • the chemical composition disclosed above is wherein the molecular structure comprises:
  • the chemical composition disclosed above is made wherein X' comprises tetrafluoroborate [BF4] ", perchlorate [CICU]", Br", C“ (carbide), Cl", F", H” (hydride), I", N3", P3", O2", S2", Se2", acetate, formate, oxalate, cyanide/cyanate, carbonate, chlorate, chromate, dichromate, dihydrogen phosphate, hydrogen carbonate, hydrogen sulfate, hydrogen sulfite, hydroxide, hypochlorite, mono-hydrogen phosphate, nitrate, nitrite, perchlorate, permanganate, peroxide, phosphate, sulfate, sulfite, superoxide, thiosulfate, silicate, metasilicate, aluminum silicate, or a combination thereof.
  • the chemical composition or the formula 1 disclosed above is wherein Y further comprises a
  • the chemical composition or formula 1 disclosed above is wherein Y comprises an azide; and further comprising wherein the azide is A) operative for one or more click chemistry reactions with one or more cyclooctynes, wherein the azide B) can be modified using one or more other alkynes in a presence of a copper catalyst, wherein the azide, C) can be modified with one or more specialized phosphines via a Staudinger ligation, or a combination of A), B), and C).
  • the chemical composition is wherein the composition with a [BGIcYariv or wherein the composition without a [SGIcYariv is operative to provide a selective association with or a selective binding to an arabinogalactan protein (AGP) or a glycosylated protein (glycoprotein) found in a cell wall of a plant; and wherein the composition does not substantially bind to non-AGP or to non-glycosylated proteins found in the cell wall of the plant.
  • AGP arabinogalactan protein
  • Glycoprotein glycosylated protein
  • the chemical composition is wherein the molecular structure is operative to provide a fluorescence absorption maximum in the range from about 800 nm to about 10 pm or in a near infrared (NIR) range. According to some aspects, the chemical composition is wherein the molecular structure is operative to provide a fluorescence emission maximum in the range from about 800 nm to about 10 pm or in a near infrared (NIR) range. In some embodiments, the chemical composition is wherein the molecular structure is operative to provide a fluorescence absorption maximum in the range from about 200 nm to about 800 nm. According to some aspects, the chemical composition is wherein the molecular structure is operative to provide a fluorescence emission maximum in the range from about 200 nm to about 800 nm.
  • a method of making AzYariv or other compositions herein comprising the steps of:
  • the method of making above is further comprising the step of:
  • the method of making is further comprising the step of: (4): whereby AzYariv-Cy5-X- is produced.
  • X' comprises tetrafluoroborate [BF4] ", perchlorate [CIO4]", Br", C” (carbide), Cl", F", H” (hydride), I", Na", Pa", O2", S2", Se2", acetate, formate, oxalate, cyanide/cyanate, carbonate, chlorate, chromate, dichromate, dihydrogen phosphate, hydrogen carbonate, hydrogen sulfate, hydrogen sulfite, hydroxide, hypochlorite, mono-hydrogen phosphate, nitrate, nitrite, perchlorate, permanganate, peroxide, phosphate, sulfate, sulfite, superoxide, thiosulfate, silicate, metasilicate, aluminum silicate, or a combination thereof.
  • the method of making is further comprising attaching a click chemistry functional group, near-IR dye, an IR dye, a fluorescent small molecule, a small molecule with a chromophore, a Raman active small molecule, or a small molecule with a maximum absorbance band in a range from about 200 nm to about 100 pm; and wherein the small molecule has a molecular weight in a non-salt form of less than about 1000 daltons (Da); from any atom on the small molecule to an -N3 of AzYariv, whereby a functionalized or a fluorescent AzYariv product is produced.
  • a click chemistry functional group near-IR dye, an IR dye, a fluorescent small molecule, a small molecule with a chromophore, a Raman active small molecule, or a small molecule with a maximum absorbance band in a range from about 200 nm to about 100 pm
  • the small molecule has a molecular weight in a non
  • the method is wherein the attaching comprises a click chemistry reaction.
  • the example method of making above is wherein the fluorescent small molecule comprises coumarin, biotin, a proximity labeling probe, 1 ,8-naphthalimide, a cyanine dye, fluorescein, rhodamine, a cyanine fluorophore, or a boron dipyrromethene difluoride (BODIPY).
  • a method for performing a fluorescence imaging of an arabinogalactan protein (AGP) or a glycosylated protein (glycoprotein) comprising the steps of: (1 ) contacting a material known to contain an AGP or a glycoprotein or suspected to contain an AGP or a glycoprotein with a reagent including AzYariv-Cy5, AzYariv-Cy5-X _ , [SGIcYariv, or a combination thereof, whereby a binding between the AGP or glycoprotein and the reagent occurs; (2) directing an incident light or an incident electromagnetic radiation towards the binding, whereby a fluorescent emission occurs; and (3) gathering, observing, measuring, or acquiring the emission.
  • AGP arabinogalactan protein
  • glycosylated protein glycosylated protein
  • a method for performing an absorbance imaging of an arabinogalactan protein (AGP) or a glycosylated protein (glycoprotein) comprising the steps of: (1 ) contacting a material known to contain an AGP or a glycoprotein or suspected to contain an AGP or a glycoprotein with a reagent including AzYariv-Cy5, AzYariv-Cy5-X _ , [SGIcYariv, or a combination thereof, whereby a binding between the AGP or glycoprotein and the reagent occurs; (2a) directing an incident light or an incident electromagnetic radiation towards the binding, whereby an electromagnetic absorbance occurs; and (3a) gathering, observing, measuring, or acquiring the absorbance.
  • the absorbance can comprise a wavelength in a range or including a maximum absorbance band in the range from about 200 nm to about 100 pm.
  • the material comprises a cell, a plant cell, a plant cell wall, or any combination thereof.
  • the analysis methods are wherein the method begins an execution at any step on a living plant cell or a living plant cell wall.
  • the methods are wherein a fluorescence absorption maximum wavelength is in a range from about 200 nm to about 10 pm; and a fluorescence emission maximum wavelength is in a range from about 200 nm to about 10 pm.
  • the analysis methods are executed wherein a fluorescence absorption maximum wavelength is in a range from about 630 nm to about 650 nm; and a fluorescence emission maximum wavelength is in a range from about 655 nm to about 675 nm.
  • the methods of analysis are executed further comprising a fluorescent microscope and/or slides are obtained, whereby the method further comprises a method of fluorescent microscopy.
  • kits suitable for sale comprising any composition herein, comprising AzYariv, AzYariv-Cy5, AzYariv-Cy5-X- or any combination thereof.
  • the kit is further comprising instructions in any media format.
  • EXAMPLE 1 EXAMPLE EXPERIMENTAL PROCEDURES FOR SYNTHESES, BINDING ASSAYS, AND TISSUE STAINING
  • the sodium nitrite solution was added to a 1 mL syringe fitted with a 4' 22- gauge needle clamped above the stirring reaction to perform a gravity -assisted dropwise addition.
  • the reaction was stirred at 0 °C for 2 h.
  • Phloroglucinol (939 mg, 7.45 mmol, 5 equiv) was added to a 5 mL Eppendorf tube and dissolved in 5 M NaOH (3 mL) and cooled in an ice bath.
  • the phloroglucinol solution was added in one portion to the stirring diazotized sugar solution.
  • the pH was adjusted to 10 and maintained by the addition of 5 M NaOH as necessary and stirred overnight.
  • the reaction was acidified to pH 6 by the addition of 12 M HCI. After addition of HCI, a thin layer of precipitated material was present along the walls of the flask.
  • the solution was filtered through a Hirsch funnel under reduced pressure and a white solid was collected. The solid was washed with several portions of water. The solid was allowed to dry on the filter paper and then discarded. The filtrate was charged with another 2 mL portion of 12 M HCI and the solution reached pH 2.
  • Cold ethanol (200 mL) was added to the solution and a red precipitate was formed; the flask was placed in a freezer for 24 h to promote further precipitation from the solution. After 24 h, the red precipitate that was obtained was filtered through a Hirsh funnel to yield a red powder.
  • the resulting dark red solid was transferred to a preweighed 1 dram vial and placed into a vacuum oven set to 75 °C.
  • the R1 3 (360 mg, 90%) was obtained as a dark red powder.
  • a second redissolution and reprecipitation was performed on the R1 material with 75 mL of methanol and 150 mL of diethyl ether to yield R2 3 (259 mg, 0.38 mmol, 72%) as a dark red powder.
  • the nitrite solution was added to a syringe fitted with a 4" 22-gauge needle clamped above the stirring solution and allowed to drip into the reaction solution.
  • the reaction was allowed to stir at 0 °C for 2 h.
  • a 2 mL Eppendorf tube was charged with 3 (180 mg, 0.26 mmol, 1 equiv) and dissolved in 2 M NaOH (1.5 mL), the tube was then cooled to 0 °C.
  • the Yariv solution was added dropwise to the stirring diazotized azido sugar solution over a period of 10 min.
  • the solution which was at pH 5 after the addition was brought to pH 10 by the addition of 5 M NaOH.
  • the reaction was allowed to reach room temperature and stirred overnight. After 18 h of stirring, the dark red reaction read pH 10.
  • the solution was transferred to a 250 mL Erlenmeyer flask and acidified to pH 2 by the addition of 12 M HCI. 95% Ethanol (60 mL) was then added to the flask and a fine dark red precipitate was formed in the solution.
  • the flask was covered with parafilm and placed in a freezer for 24 h to promote further precipitation from the solution.
  • the solution was filtered under reduced pressure through a Hirsch funnel.
  • the resulting dark red solid was allowed to dry for an hour in the filter before being crushed into a fine powder and transferred to a pre-weighed 20 mL scintillation vial.
  • the vial was then placed into a vacuum oven next to a beaker with Drierite and dried overnight at room temperature.
  • the crude AzYariv reagent (290 mg) was obtained as a fine dark red powder contaminated with EtOH and water.
  • a custom-made 7-pin well mold was then affixed into the gel to create wells for the Yariv reagent to be added.
  • the well molds were removed after 10 min once the gels were set.
  • Solutions of the Yariv reagent were added to the wells using a 2.5 pL Hamilton Microlite PCG Syringe.
  • Wells were charged with the appropriate volume of Yariv reagent (1 pL for most solutions, 1.5 pL for pure AzYariv reagent) and the gels were placed on a platform in a water basin to prevent the gels drying out. The gels were incubated overnight for 16 h before imaging. Gels were imaged on the Keyence All-in- One Fluorescence Microscope (BZ-X810) using the Texas Red filter (red channel) (Ex: 560/40; Em: 630/75).
  • Lyophilized AzYariv-Cy5 was charged with DMSO (20 pL), a 2.06 mM solution of pGIcYariv in water (103 pL), and water (83 pL). The samples were then apportioned out into 23 PCR tubes (17 pL each). The PCR tubes were fixed into a holder and the holder was placed into a large lyophilizer flask. The samples were lyophilized overnight and removed, capped, and placed into a 50 mL centrifuge tube containing Drierite. The centrifuge tube was then sealed with parafilm and wrapped in aluminum foil to protect them from light. According to some aspects, a lyophilized product is provided by the technology herein.
  • Cy5-labeled AzYariv was diluted to 1 pm in a 0.1 mM solution of either pGIcYariv (binding) or aGalYariv as a non-binding fluorescent control.
  • a solution of 0.1 mM pGIcYariv without the Cy5 conjugated AzYariv spike-in was used as an additional control for these experiments.
  • Electrospray ionization (ESI) mass spectra were obtained using a Thermo LCQ Deca XP Max ion trap mass spectrometer. Purified water was obtained from an EMD Millipore Direct-Q 3 Tap to Pure and Ultrapure Water Purification system.
  • Circular dichroism (CD) and UV/vis measurements were performed in triplicate using a Jasco J815 spectropolarimeter. Temperature was controlled by a JASCO Peltier temperature control unit. Unless otherwise noted the concentration and volume of the Yariv reagent samples was 300 pM and 600 pL, respectively. The sample cell was kept at 20 °C. CD/UV-vis spectra were obtained in a Hellma analytics 2mm pathlength stoppered cuvette and corrected against a purified water standard. Wavelength readings ranged from 200 to 700 nm and were obtained at a speed of 100 nm/minute.
  • N-Boc-p-am inophenyl-6-O-tosyl-p-D-glucopyranoside (see FIG. 7B) procedure adapted from Angew. Chem. Int. Ed. 2004. 43, 5338-5342.
  • a 50 mL round bottom flask was charged with 4 (474 mg, 1.28 mmol, 1 equiv.) and the powder was dried by rotary evaporation with toluene. The process was performed three times before placing the flask under high vacuum for 1 hour. The flask was capped with a septum and placed under N2.
  • the solution was charged with activated carbon ( ⁇ 10 mg) then filtered through a pad of celite to remove orange-colored impurities, then dried over anhydrous sodium sulfate.
  • the solution was filtered through a Buchner funnel into a 1000 mL round bottom flask and concentrated in-vacuo to ca. 30 mL.
  • the solution was then transferred to a pre-weighed 100 mL round bottom flask and concentrated to yield a fine white crystalline powder.
  • the flask was then placed on high vacuum overnight.
  • the crude tosyl sugar 5 (627 mg, 1.19 mmol, 94%) was obtained as a white powder and used in subsequent reaction without further purification.
  • N-Boc-p-aminophenyl-6-deoxy-6-azido-[3-D-glucopyranoside (see FIG. 7C) procedure adapted from Angew. Chem. Int. Ed. 2004. 43, 5338-5342.
  • a 50 mL round bottom flask was charged with a stir bar and N-Boc-p-aminophenyl-6-O-tosyl-[3D- glucopyranoside 5 (502 mg, 0.956 mmol, 1 equiv.).
  • the flask was capped with a septum and N2 was delivered to the flask via a needle.
  • Sodium azide is a highly toxic potentially explosive shock sensitive solid. Sodium azide forms toxic hydrazoic acid in the presence of mineral acid and explosive diazido or triazido methane in the presence of methylene chloride and chloroform respectively. All reactions involving sodium azide were performed behind a blast shield and solid sodium azide was weighed out using a plastic spatula. All waste generated from reactions with sodium azide were collected separately from other waste streams and kept basic by the addition of sodium bicarbonate. Care was taken to avoid evaporating solutions containing unreacted sodium azide to dryness.
  • Trifluoroacetic acid (770 pL, 10 mmol, 40 equiv.) was added to a syringe and the air was expelled from the syringe.
  • the needle was pierced through a rubber stopper and the syringe was placed into an ice container for 10 minutes.
  • triethylsilane (0.1 mL, 0.6 mmol, 2.5 equiv.) was added via a syringe to the reaction flask and subsequently the trifluoroacetic was added through the septum in one addition.
  • the reaction was stirred for 10 minutes at 0 °C and monitored by 1 H NMR spectroscopy.
  • a PCR tube was charged with AzYariv-Cy5 stock solution (0.485 pL), a 2.06 mM stock solution of [3GlcYariv in water (5 pL) and water (4.515 pL) to a final concentration of 5 pM AzYariv-Cy5, 20 pM AzYariv, and 1 .03 mM pGIcYariv.
  • 100 nM and 5 nM solutions of AzYariv-Cy5/[3GlcYariv were prepared by serial dilution of the 5 pM stock using a 2.06 mM solution of [SGIcYariv in water to maintain the concentration at 1 .03 mM.
  • FIG. 12 shows an example method 500 for performing a fluorescence imaging of an arabinogalactan protein (AGP) or a glycosylated protein (glycoprotein).
  • AGP arabinogalactan protein
  • Glycoprotein glycosylated protein
  • Step 505 shows obtaining a material known to contain an AGP or a glycoprotein or suspected to contain an AGP or a glycoprotein.
  • Step 510 shows contacting the material with a reagent including AzYariv-Cy5, AzYariv-Cy5-X pGIcYariv, or a combination thereof, whereby a binding between the AGP or glycoprotein and the reagent occurs.
  • Step 515 shows directing an incident light or an incident electromagnetic radiation towards the binding, whereby a fluorescent emission occurs.
  • Step 520 shows gathering, observing, measuring, or acquiring the emission.
  • Step 525 shows digitization using a computer and software (not shown).
  • Step 526 shows a computer or other visualization and a decision whether or not to proceed with the data or to repeat 527 the experiment.
  • Step 535 shows an optional step of corresponding or analyzing locations in the starting material, and step 540 shows an optional step of corresponding the data to biological data (for larger picture findings).
  • FIG. 13 shows an example method 600 for performing an absorbance measurement on an arabinogalactan protein (AGP) or a glycosylated protein (glycoprotein).
  • AGP arabinogalactan protein
  • Glycoprotein glycosylated protein
  • Step 630 is contacting the material with a binding reagent disclosed herein including a chromophore.
  • Step 640 is directing an incident light or an incident electromagnetic radiation towards the binding, whereby an electromagnetic absorbance occurs.
  • the absorbance is gathered, observed, measured, or acquired, and at step 660, the data is digitized and visualized.
  • FIG. 8A A variety of tautomeric states are possible for the Yariv reagents (e.g., FIG. 8A, FIG. 8B).
  • Yariv reagents are depicted in the literature as tautomer A, (FIG. 8A) a set of closely related structures reported by Lee, 2 et al. (Lee 2010) have been shown to adopt the D (FIG. 8B) form, with D1 as the major tautomer.

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Abstract

L'invention concerne des synthèses chimiques permettant de produire de nouvelles compositions de réactifs de Yariv ayant des fonctions, une fluorescence et des chromophores améliorés. Les nouvelles compositions démontrent la possibilité d'étendre les études sur les protéines arabinogalactanes (AGP), les galactanes et les glycoprotéines à une plus grande variété d'échantillons, y compris des échantillons frais de tissus, et offrent des avantages supplémentaires. Les compositions et les procédés permettent des études auparavant inaccessibles dans la coloration et l'imagerie des tissus végétaux, y compris la liaison hautement spécifique des AGP, fournissant un rapport signal/bruit supérieur, de meilleures limites de détection, ainsi qu'une spécificité élevée pour les AGP ciblés.
PCT/US2024/022083 2023-03-30 2024-03-28 Azyariv et conjugués azyariv - nouveaux outils pour visualiser et caractériser des protéines d'arabinogalactane Pending WO2024206707A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101691450A (zh) * 2009-07-24 2010-04-07 华东师范大学 β-葡萄糖基-Yariv试剂的制备方法
US20210128660A1 (en) * 2017-01-26 2021-05-06 Amorepacific Corporation Composition for enhancing immunity, containing ginseng berry polysaccharides

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101691450A (zh) * 2009-07-24 2010-04-07 华东师范大学 β-葡萄糖基-Yariv试剂的制备方法
US20210128660A1 (en) * 2017-01-26 2021-05-06 Amorepacific Corporation Composition for enhancing immunity, containing ginseng berry polysaccharides

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
RUEDA SEBASTIAN, MCCUBBIN TYLER J., SHIEH MEG, HOSHING RAGHURAJ, BRAUN DAVID M., BASU AMIT: "A Functionalizable Analog of the Yariv Reagent for AGP Imaging using Fluorescence Microscopy", BIOCONJUGATE CHEMISTRY, AMERICAN CHEMICAL SOCIETY, US, vol. 34, no. 8, 16 August 2023 (2023-08-16), US , pages 1398 - 1406, XP093219909, ISSN: 1043-1802, DOI: 10.1021/acs.bioconjchem.3c00184 *
ZHOU LI HONG, WEIZBAUER RENATE A., SINGAMANENI SRIKANTH, XU FENG, GENIN GUY M., PICKARD BARBARA G.: "Structures formed by a cell membrane-associated arabinogalactan-protein on graphite or mica alone and with Yariv phenylglycosides", ANNALS OF BOTANY, ACADEMIC PRESS, LONDON., GB, vol. 114, no. 6, 1 October 2014 (2014-10-01), GB , pages 1385 - 1397, XP093219908, ISSN: 0305-7364, DOI: 10.1093/aob/mcu172 *

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