Composition of carbon dots comprising a core formed by carbohydrates and a surface comprising carboxylic acid or terminal alkyne functional groups, method of preparation thereof and use thereof

Abstract

The present invention describes a composition comprising carbon dots, which are luminescent, biocompatible, water-soluble carbon nanoparticles that can be obtained from carbohydrates. The invention also provides a method of preparing said composition and use of said composition to increase the productivity of a vegetable crop by using abundant and easily accessible raw materials, via a low-cost method and on a large scale.

Description

COMPOSITION OF CARBOHYDRATE-FUNCTIONALIZED CARBON DOTS, THEIR METHOD OF PREPARATION AND USE THEREOF FIELD OF THE INVENTION

[0001] The invention belongs to the field of agrochemistry, with particular emphasis on nanotechnology and organic chemistry. It involves a composition of nanocomposites based on carbon dots, with luminescent and nutrient-carrying properties that enhance photosynthesis. It also addresses the method for preparing said composition. Finally, the present invention involves the use of said composition in crop improvement, more specifically, sugarcane crops. BACKGROUND OF THE INVENTION

[0002] With unfavorable climate change and a growing global population, there is a great need for more productive and stress-tolerant crops. Climate change has led to a scenario of decreasing rainfall and increasing global radiation. The last two years have seen the worst accumulated water deficit values ​​from January to December in over 60 years of history in the Ribeirão Preto region of São Paulo state, reaching 619 mm in 2021 and 600 mm in 2020. This situation leads to significant declines in sugarcane productivity, requiring greater use of photosynthetic yield. According to the United Nations (UN), the global population will surpass the 9 billion mark by 2050, and it will be necessary to increase food production by 50-70%. In parallel, the demand for water and energy will grow proportionally (KEATING et al., 2014).

[0003] The Food and Agriculture Organization of the United States The United Nations Food and Agriculture Organization (FAO) has expressed serious concerns in light of the depletion and degradation of agricultural fields, the drastic reduction of water sources and their effects on food security and ecosystem preservation (PRASAD; BHATTACHARYYA; NGUYEN, 2017). For example, the demand for cereals, for both food and animal feed, could reach 3 billion tons by 2050, up from the current 2.1 billion (KEATING et al., 2014).

[0004] This pessimistic scenario spurs a global effort to develop alternative ways to meet the growing global demand for food, energy, and water without increasing pressure on natural resources. Many strategies, such as genetic improvement, gene manipulation, and environmental modeling, have been developed to interfere with and increase photosynthetic efficiency in crops. These genetic engineering modifications through biotechnology have proven effective in several plant species, specifically in expanding development opportunities for determining high-quality traits. However, there are some disadvantages of genetic technology: the cost of computational tools to develop a variety of genetic approaches, which are expensive and not accessible for all crops due to the requirements of a high-quality genome sequence (NUCCIo et al., 2017).

[0005] Thus, the development of sustainable agricultural techniques has been gaining prominence, and in this context, nanotechnology emerges as a potential solution to overcome the drawbacks of conventional agriculture, since it would be able to increase productivity while simultaneously reducing environmental impact (BHATTACHARYYA; NGUYEN, 2017).

[0006] Among the nanostructures already used in the agricultural sector, inorganic nanomaterials (metal oxide and metal nanoparticles NP) represent 55% of the total application in this sector, while nanoencapsulates represent 26%, nanocomposites correspond to 7% and carbon-based nanomaterials (carbon nanotubes, carbon black and fullerene) are explored in only 6% of the reported technologies (PETERS et al., Trends in Food Science & Technology, 54, 155-164 2016)

[0007] Nanoparticles and quantum dots, conjugated to biomolecules, have They have been used in diagnostics, therapy, targeting, and programmed drug release due to their optical properties, high degree of functionality, biocompatibility, and valence. In recent years, there has been a gradual insertion of nanomaterials in agriculture for controlled-release fertilizers, pesticides and herbicides, sensors, and plant germination and growth (GOGOS; KNAUER; BUCHELI, 2012; VISHWAKARMA et al., 2017; MULLEN, 2019). However, quantum dots can present high toxicity for in vivo use, for example, due to the presence of heavy metals, such as cadmium (THOMAS et al., 2020).

[0008] Carbon dots (CDs), or carbonaceous nanomaterials, are a class of paracrystalline spheroidal nanoparticles, with dimensions smaller than 10 nm, composed of a carbon core and polyaromatic content on the amorphous surface (CHOI et al., 2018). Carbon dots were first isolated in 2004 during the purification process of single-walled carbon nanotubes. They have been described as an alternative to traditional quantum dot semiconductors, preserving their main advantages, such as nanoparticles with lower toxicity, mainly for applications in imaging diagnostics, nanomedicine, photochemistry and electrocatalysis, catalysis, optoelectronic devices, lubrication, and biosensing (XU et al., 2004). In particular, Giraldo and collaborators described that the functionalization of the glycan surface of NPs reduces the toxicity of the biomaterial, in addition to increasing bioavailability, and allowing a less complex and efficient synthesis (GIRALDO et al., 2014).

[0009] Additionally, the carbohydrates that make up carbon dots are chiral molecules, devoid of toxicity to humans and the environment, biocompatible, readily available in nature, and can be used as an abundant, structurally diverse, and low-cost material, providing carbon dots with nearly spherical geometry and diameters generally less than 10 nm. These nanoparticles are composed of carbon core and its surface has functional groups containing oxygen / nitrogen atoms, with the fluorescence observed due to the generation of nanoscale fluorophores (poly-aromatic surface functions) during the preparation processes (BAKER & BAKER, 2010).

[0010] Therefore, there is great interest in the development of carbon dots due to their specific properties, related to greater solubility in water, low toxicity, photoluminescence and photoprotection, photochemical and chemical stability, resistance to photobleaching (loss of fluorescence), broad absorption of visible and ultraviolet light, ease of preparation, in addition to large-scale production (MANDANI, SHARMA, SARMA, 2017).

[0011] Plants have mechanisms to protect against excess solar energy and prevent harmful losses from photochemical reactions. This mechanism is called non-photochemical quenching (NPQ). Although essential for plant survival, NPQ is known to reduce photosynthetic performance, the process by which plants convert sunlight into biomass, across different species. Some studies show that plant biomass increases by up to 15% with the suppression of specific NPQ components. Consequently, increasing the rate of photosynthesis has the potential to improve agricultural productivity and crop nutritional value. Technologies to extract greater value from available light and enhance production viability are critically needed in this climate change scenario (SWIFT et al., 2019).

[0012] Thus, the inherent light interaction capabilities of quantum dots have emerged as a promising new approach for producing high-performance crops to meet the needs of a growing global population (MUELLER et al., 2012).

[0013] From this perspective, carbon nanomaterials emerge as promising for the development of a sustainable agricultural alternative, They are low-toxic, biocompatible, and non-bioaccumulative. These characteristics mean that when applied to plants, this product enhances development and productivity. Carbon nanomaterials have shown beneficial effects on seed germination, increased plant growth, and fertilizer performance (Zheng et al., 2017).

[0014] Carbon dot nanoparticles can be obtained with distinct compositions and properties, depending on the proportion and type of starting materials, additives, solvent, temperature, pH, heating time, preparation method, and passivation and surface functionalization. For example, longer heating times can result in carbon dots with larger diameters. Depending on the conditions employed, carbon dots containing carboxylic, hydroxylic, and carbonyl groups on their surfaces allow the incorporation of subsequent functionalization with organic, polymeric, inorganic agents, or biological species to enhance their properties, in a process called passivation. Passivation of carbon dots can produce products with enhanced photoluminescence. These nanomaterials are purified by centrifugation, dialysis, electrophoresis, and exclusion chromatography (BAKER & BAKER, 2010; MANDANI, SHARMA, SARMA, 2017).

[0015] Other steady-state absorption and fluorescence spectroscopy techniques revealed that it is possible to assign absorption bands associated with the crystalline core or surface aromatic layers and that, for this, the selection of the carbohydrate for functionalization of carbon dots can drastically alter the electronic structure of its surface (SWIFT et al., 2018).

[0016] Additionally, other comprehensive literature reviews describe the development of synthetic strategies, properties, photoluminescence mechanisms, and the various promising applications of dots. carbon (PENG & TRAVAS-SEJDIC, ​​2009; HUMAERA et al., 2021 ; LIM, SHEN, GAO, 2021; MIAO et al., 2015).

[0017] The synthesis and analysis techniques developed in previous studies, and the data on bioavailability, low toxicity and cellular internalization of functionalized carbon dots, allowed the University of Bristol group to apply their use to increase photosynthesis, growth and plant production, as a sustainable and low-cost way (SWIFT et al., 2019).

[0018] Thus, it was demonstrated that the functionalization of carbon dots with glucose allowed greater uptake and internalization in plant mesophyll cells than the corresponding unfunctionalized carbon dots, with a higher rate of photosynthesis, photoprotection, pigment production, and an 18% higher grain yield when tested in wheat (Triticum aestivum 'Apogee'). Although other studies have already described these photosynthesis-enhancing properties using carbon nanotubes (SWNTs) and unfunctionalized carbon dots (review), this was the first report of the impact of this modification on plant production yield (SWIFT et al., 2019).

[0019] In the same sense, the use of carbon dots functionalized with l-aminoglucose was essential to reduce NPQ mechanisms by decreasing lutein production and pigment formation, increasing chlorophyll production and reducing reactive oxygen species. Comparative results of carbon dots, functionalized or not with glucose, were obtained by measuring carbon dot uptake in plants; photosynthetic efficiency and chlorophyll fluorescence kinetics (fluorescence microscopy); and infrared gas analysis (IRGA) as a function of photosynthetic photon flux density (PPFD); and the effect on pigment composition by extraction and quantification of light absorption and photoprotective pigments by HPLC chromatographic techniques; in addition to changes in biomass production (SWIFT et al., 2019).

[0020] The development of non-toxic, water-soluble, fluorescent carbon dot nanoparticles for biological applications has been explored by a research group at the University of Bristol, using low-cost, easily functionalized materials. Using glucosamine hydrochloride as the starting material and TTDDA for subsequent surface passivation, it was possible to synthesize fluorescent carbon dots with a high proportion of sp3 carbon and surface functionalized with a layer of polyaromatic groups, with diamond-like properties, on a gram-scale (fluorescent NH2-CD), using microwave irradiation.For the subsequent functionalization of NH2-CDs with lactose disaccharide, a reactive spacer group of succinic acid was introduced by reaction with succinic anhydride, exposing the carboxylic acid function on the surface of the nanoparticle (fluorescent COOH-CDs), which were treated with 1-aminolactose (Lac), in the presence of a condensation reagent CDI (1,1-carbonyldiimidazole), to provide Lac-CDs with the maintenance of fluorescent properties in relation to the precursor NH2-CD. Several analytical techniques were used to characterize the obtained nanoparticles, such as: 1H and 13C Nuclear Magnetic Resonance (NMR) (HSQC and HMBC) and 1H-15N HMBC NMR, Fourier Transform Infrared (FTIR), UV-Visible and X-ray photoelectron spectroscopy (XPS), Dynamic Light Scattering (DLS) and Zeta analysis, and the morphology and structure of carbon dots analyzed by Transmission Electron Microscopy (TEM).Studies involving Lac-CDs in tumor cells showed their rapid cellular internalization and low toxicity when used in high concentrations and long exposure times (HILL et al., 2016).

[0021] Swift and collaborators demonstrate the use of glycan-functionalized carbon dots to increase grain yield in dwarf wheat species by up to 18%. They also demonstrated that functionalization with Glucose increases nanoparticle absorption. The mechanism by which carbon dots interact with photosynthesis involves altering NPQ and pigment production, promoting the reduction of oxidizing compounds in the plant (SWIFT et al., 2020).

[0022] Tan and collaborators (2021) explored the acid-free microwave extraction of carbon dots from palm oil waste biomass biochar. The carbon dots were applied via foliar spray to corn and rice crops. As a result, photosynthesis increased in the species exposed to the carbon dots through a simple, easy, and low-cost method using natural and renewable materials (TAN et al., 2021).

[0023] Patent documents CN110330973 and CN110278860 produce carbon dots to promote the growth of bean sprouts. The dots are produced hydrothermally by refluxing silicon dioxide in an oil bath. The resulting nanomaterial has good dispersibility and stability in water.

[0024] Patent document CN106675557 describes the preparation of fluorescent carbon dots produced from chitosan by hydrothermal reaction. Their application is to stimulate plant growth with low toxicity and high biocompatibility.

[0025] Patent document BR 102020002172-9 describes the development of nanocomposites based on carbon dots from glucose and ammonium hydroxide, with luminescent and nutrient-carrying properties, which, when applied via foliar and / or root application to the plant, enhance photosynthesis, in addition to being capable of promoting improvements in physiological responses, development and productivity.

[0026] In patent document US 2020 / 0392525 relates to the production of 0.5-20 nm carbon dots from PEG and a binding agent, with the aim of promoting plant photosynthesis by transporting nucleic acids to plant cells, altering gene expression using CRISPR / Cas9.

[0027] Based on the evidence described, the present invention aims, in general, to synthesize carbohydrate-functionalized fluorescent CD nanoparticles using precursors and methods distinct from those described in the literature. The work was directed at obtaining water-soluble fluorescent functionalized CDs that are non-toxic to humans and the environment, using renewable sources, and capable of optimizing the photosynthesis process of a crop during seasonal periods of water scarcity and high exposure to sunlight. The reactions used in the present invention do not require the use of catalysts, avoiding purification steps, can be accelerated with the use of microwaves or heating, and provide products in high yields and with a high degree of purity. The triazole groups generated are highly stable against hydrolysis, oxidation, and reduction reactions, etc. They can be performed in the presence of water, making them environmentally friendly in terms of Green Chemistry.Furthermore, the triazole ring generated introduces a certain solubility in lipids into the nanoparticles due to the aromatic heterocyclic system, which, associated with the high water solubility of the carbohydrates present, can further balance the partition coefficient of the product and improve absorption by plant cells.

[0028] In this context, the invention is based on the unprecedented identification of a CD composition capable of increasing the productivity of a plant crop, particularly sugarcane, using abundant and readily available raw materials, using a low-cost method, and on a large scale. The present invention surpasses technologies that use metal nanoparticles because it is based on carbon nanoparticles, carbon dots, which are fully metabolized by the plant and are not transferred within the food chain, meaning there is no trophic transfer.

[0029] To this end, the invention uses strategies that can (1) penetrate the plant; (2) cause photoprotection under stress conditions (high exposure to sunlight and water scarcity during winter); (3) increase the rate of photosynthesis; (4) increase pigment production and (5) alter the mechanisms called “Non-photochemical quenching (NPG)”, related to the production of reactive oxygen species with an impact on photosynthesis.

[0030] Several factors guided the studies on the preparation of nanoparticles, among which it is worth mentioning (i) standardization of methods that are easily executed, (ii) use of low-cost starting materials and solvents, (iii) preservation of the environment with the minimum generation of toxic waste, (iv) possibility of being scaled from the bench to larger-scale production; (v) capable of increasing the agricultural production of sugarcane. SUMMARY OF THE INVENTION

[0031] In a more general aspect, the invention provides a composition of carbon dots, comprising a carbon core formed by carbohydrates, precursors and, optionally, spacers, and a surface comprising carboxylic acid or terminal alkyne functional groups.

[0032] In another aspect, a method of preparing said composition is provided, comprising the steps of: a. preparing a carbohydrate and precursor solution; b. obtaining carbon dots by treatment with passivating agents; c. optionally, adding spacers to the carbon dots; d. coupling glucose units to the carbon dots; and e. purifying the functionalized carbon dots.

[0033] In another aspect, there is provided the use of said composition for the improvement of a crop. BRIEF DESCRIPTION OF THE FIGURES

[0034] The invention can be better understood based on Figures 1 to 31, the description of which follows below:

[0035] Figure 1 illustrates the general scheme for obtaining the desired final compounds. The proposed preparation of four products is presented: the first, the positive control described in the literature, obtained from glucosamine DOT CORE functionalized with a succinic acid spacer for the inclusion of aminoglucose units. The other three are novel products: one similar to the one in the literature but using hydrolyzed sucrose DOT CORE, and the last two, obtained from either glucosamine or hydrolyzed sucrose DOT CORE but introducing a different propiolic acid spacer for functionalization with azidoglucose, using the Click Chemistry strategy.

[0036] Figure 2 illustrates (A) an example TEM image of glucose dots and (B) a high-resolution image of glucose DOT topography made by AFM: the core height is 3.55 nm and the radius is 5.69 nm, the functionalization extends 15.42 nm from the core, and the height scale is 0.5–5 nm. Source: SWIFT, Thomas A. et al. 2018. In these studies, the carbon dot structures without functionalization presented a characteristic sp3 crystal structure of about 1 nm containing a layer of aromatic groups of approximately 1.4 nm, identified by TEM and SAXS (small-angle X-ray scattering), due to the low electron densities associated with the surface carbohydrates. In contrast, the glycan surface layer was only characterized using Atomic Force Microscopy (AFM), with carbon dots containing glucose, mannose and lactose units being quite uniform, while galactose and maltose gave rise to less homogeneous surface layers.

[0037] Figure 3 presents the detailed flowchart of all the steps until obtaining the proposed DOT COREs.

[0038] Figure 4 shows (A) Acid hydrolysis reaction of sucrose, a non-reducing sugar, and formation of glucose and fructose (reducing sugars). (B) Qualitative confirmation of hydrolysis of sucrose in acidic medium through the color change of Benedict's reagent from blue to red. Over time, Cu +1 , insoluble in H2O, precipitates after the addition of Benedict's reagent and heating, a color change was observed for the hydrolyzed sucrose and glucose samples, which indicated the effectiveness of the disaccharide cleavage.

[0039] Figure 5 shows the comparison of 1H NMR spectra (D2O, 300 MHz) of sucrose (A), hydrolyzed sucrose (B), glucose (C) and fructose (D) to confirm sucrose cleavage.

[0040] Figure 6 illustrates the comparisons between the masses obtained from all steps under the different conditions evaluated for the formation of DOT COREs.

[0041] Figure 7 illustrates the comparisons between the fluorescences (UV: 365 nM) of all steps in the different conditions evaluated for the formation of DOT COREs.

[0042] Figure 8 shows the comparison of the 1H NMR spectra (D20, 300 MHz) of the glucosamine DOT COREs obtained after dialysis (retained on the membrane and the one that passed through the 1.0 KDa membrane) and without dialysis and the spectrum from the literature. Source: SWIFT et al, 2019.

[0043] Figure 9 shows the comparison of the infrared (IR) spectra of the glucosamine DOT COREs obtained after dialysis (retained in the 1.0 KDa membrane and what passed to the medium) and the carbon DOT obtained without dialysis.

[0044] Figure 10 shows the comparison of the 1H NMR spectra (D2O, 300 MHz) of the hydrolyzed sucrose DOT COREs obtained after dialysis (retained in the membrane and what passed through the 1.0 KDa membrane) and without dialysis.

[0045] Figure 11 shows the comparison of the infrared (IR) spectra of the glucosamine DOT COREs obtained after dialysis (retained in the 1.0 KDa membrane and what passed to the medium) and the carbon DOT obtained without dialysis.

[0046] Figure 12 illustrates the functionalization of the DOT COREs obtained with carboxylic acid (A) and terminal alkyne (B) groups.

[0047] Figure 13 shows a comparison of the 1H NMR spectra (D20, 300 MHz) of glucosamine DOT CORE. (A) Carbon dots retained after dialysis and with the product functionalized with carboxylic acid groups. (B) Carbon dots obtained without dialysis and with the product functionalized with carboxylic acid groups. (C) Spectrum of glucosamine DOT functionalized with the same carboxylic acid groups derived from succinic anhydride, described in the literature (standard). Source: HILL et al, 2016.

[0048] Figure 14 illustrates the comparison of the 1H NMR spectra (D2O, 300 MHz) of glucosamine DOT COREs. (A) Carbon dots retained after dialysis and with the product functionalized with alkyne groups. (B) Carbon dots obtained without dialysis with the product functionalized with alkyne groups.

[0049] Figure 15 shows the comparison of the 1H NMR spectra (D2O, 300 MHz) of sucrose DOT COREs. (A) Carbon dots retained after dialysis and with the product functionalized with carboxylic acid groups. (B) Carbon dots obtained without dialysis and with the product functionalized with carboxylic acid groups.

[0050] Figure 16 shows the comparison of the 1H NMR spectra (D2O, 300 MHz) of the DOT CORE of hydrolyzed sucrose. (A) Carbon dots retained after dialysis and with the product functionalized with alkyne groups. (B) Carbon dots obtained without dialysis and with the product functionalized with alkyne groups.

[0051] Figure 17 illustrates the general scheme of the synthesis of the precursors 1-amino glucose (A), and 1-azido glucose (B), for the introduction of glucose units into the carbon dots, via the formation of amide or 1,2,3-triazole bonds (Click Chemistry), respectively.

[0052] Figure 18 represents the qualitative confirmation of the conversion of glucose into 1-amino glucose through the color change of the medium from brown to purple when using the ninhydrin test.

[0053] Figure 19 shows a comparison of the 1H NMR spectra (D2O, 300 MHz) of the products obtained under different conditions for the synthesis of 1-amino glucose. The best reaction condition so far is highlighted in orange.

[0054] Figure 20 shows the comparison of the 1H NMR spectra (D2O, 300 MHz) of the mixture of the desired product 1-amino glucose (signals highlighted with blue arrows) with the glycosyl ammonium carbamate salt (signals highlighted with green arrows) obtained following protocol C before and after treatment with basic resin.

[0055] Figure 21 illustrates the synthesis of the Cl-functionalized azido-glucose block via two distinct routes.

[0056] Figure 22 shows the comparison of the 1H NMR spectra (CDC13, 300 MHz) of the peracetylated bromide MFU 02 and azide MFU 03 derivatives, in addition to the 1H NMR spectrum (D2O, 300 MHz) of the precursor with deprotected azide MFU 04.

[0057] Figure 23 shows a comparison of the infrared (IR) spectra of the derivatives MFU 03 and MFU 04, which demonstrate the introduction and maintenance of the azido group in Cl (2100 cm' 1 ).

[0058] Figure 24 shows a comparison of the 1H NMR spectra (D20, 300 MHz) of the 1-azidoglucose derivatives obtained by two different synthetic routes (via bromide: 4 steps; via DMC: 1 step). The signals of the methyl groups of the DMC reagent are highlighted in purple, evidencing their presence next to the desired product when using the 1-step route.

[0059] Figure 25 shows the infrared (IR) spectra of the 1-azido glucose derivative MFU 04 obtained through the use of the DMC salt, which shows the introduction of the azido group at the Cl carbon of glucose (2100 cm' 1 ).

[0060] Figure 26 shows the comparison of the 1H NMR spectra (D2O, 300 MHz) of the products obtained after the amide function formation strategy between the DOT CORE SUC derivatives of glucosamine (A: 1.2 - retained in dialysis and B: 1.3 - without dialysis) and hydrolyzed sucrose (C: 4 - retained in dialysis and D: 4.2 - without dialysis) and 1-amino glucose. (E) Spectrum of the glucose-functionalized glucosamine DOT described in the literature (standard). Source: SWIFT et al, 2018.

[0061] Figure 27 shows the comparison of the 1H NMR spectra (D20, 300 MHz) of the products obtained after the Click Chemistry strategy between the DOT CORE Ac Prop derivatives of glucosamine (A: 1.2 - retained in dialysis and B: 1.3 - without dialysis) and hydrolyzed sucrose (C: 4 - retained in dialysis and D: 4.2 - without dialysis) and 1-azido glucose MFU 04.

[0062] Figure 28 presents the Net CO2 assimilation (A), Water Use Efficiency (WUE) (B), Stomatal Conductance (C) and Transpiration Rate (D) of sugarcane plants (cultivar 5476) in response to the application of 5 mg ai plant-1 of MFU 06 8.

[0063] Figure 29 presents the Net CO2 assimilation (A), Water Use Efficiency (WUE) (B), Stomatal Conductance (C) and Transpiration Rate (D) of sugarcane plants (cultivar 5476) in response to the application of 5 mg ai plant-1 of MFU 08 6

[0064] Figure 30 presents the Net CO2 assimilation (A), Water Use Efficiency (WUE) (B), Stomatal Conductance (C) and Transpiration Rate (D) of sugarcane plants (cultivar 5476) in response to the application of 1 mg ai plant-1 of MFU 09 4.

[0065] Figure 31 shows the Net CO2 assimilation (A), Water Use Efficiency (WUE) (B), Stomatal Conductance (C), and Transpiration Rate (D) of sugarcane plants (cultivar 5476) in response to the application of 5 mg ai plant-1 of MFU 07 8. DETAILED DESCRIPTION OF THE INVENTION

[0066] It is believed that a person skilled in the art can, based on the description contained in the present invention, use it to its fullest extent. The specific embodiments below should be interpreted as merely illustrative and not as limiting, in any respect, the remainder of the description.

[0067] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as understood by one skilled in the art to which the present invention belongs. The specification also provides definitions of terms to aid in the interpretation of what is described herein and the claims. The terminology used in the description of the invention is intended to describe preferred embodiments only and is not intended to limit the scope of its teachings.

[0068] Unless otherwise indicated, all numbers expressing quantities, percentages, and proportions, and other numerical values ​​used in the specification and claims, should be understood as being modified, in all cases, by the term "about." Therefore, unless otherwise indicated, the numerical parameters shown in the specification and claims are approximations that may vary depending on the properties to be obtained.

[0069] The present invention, in its most general aspect, provides a composition of carbon dots comprising a carbon core formed by carbohydrates, precursors, the presence of optional spacer groups of carboxylic acid or terminal alkyne and a surface comprising glucose units linked to the spacers by an amide function or triazole ring, respectively.

[0070] For the purposes of the present invention, the term “Carbon Dots” refers to fluorescent, biocompatible, water-soluble carbon nanoparticles that can be obtained from various precursors, including organic molecules.

[0071] When used herein, the term "carbohydrate" refers to a monosaccharide, disaccharide, or even polysaccharide selected from the group of glucose, fructose, galactose, lactose, sucrose, maltose, mannose, glucosamine, 1-amino-glucose, 1-amino-lactose, glycerol, 1-amino-fructose, 1-azido-glucose, 1-azido-fructose, 1-azido-galactose, cellulose, dextran, and their polymeric derivatives. In one embodiment, the carbohydrate is glucosamine hydrochloride (0.75-1.00-25 g). In another embodiment, the carbohydrate is sucrose. Preferably, the sucrose is hydrolyzed sucrose (glucose + fructose) (0.66-1.10 g).

[0072] As used herein, the term "precursor" refers to compounds containing sugar units (1 to 5 Equivalents-Eq). Examples of possible precursors can be seen in Figures 17 and 21, including ethanol, carbohydrates such as glycosylammonium carbamate, amino acids, citric acid, ascorbic acid, acetic anhydride, or even biomass, providing carbon dots with amorphous characteristics and lower sp2 character. In one embodiment, the synthesized precursor is 1-amino glucose (2.85-4.75 g, 3 Eq). In another embodiment, the precursor is 1-azido glucose (3.75-6.25 g, 1.2 Eq).

[0073] When used herein, the term “spacer” is an organic molecule, more specifically an organic anhydride or acid, such as succinic anhydride and propiolic acid. In one embodiment, the spacer is succinic anhydride (0.5 Eq wt, 6.00-7.35 g). In another embodiment, the spacer is propiolic acid (0.5 Eq wt, 4.09-6.80 g).

[0074] When used herein, the term “functionalization” is a reaction of the carbon dots containing the spacer with organic functional groups selected from 1-amino glucose and 1-azido glucose. In a In one embodiment, the functionalization is performed with a carboxylic acid, Figures 13 and 15. In another embodiment, the functionalization is performed with a terminal alkyne. Such functionalization is illustrated in Figures 14 and 16.

[0075] In another aspect, a method of preparing said composition is provided, comprising the steps of: a. preparing a carbohydrate and precursor solution; b. obtaining carbon dots by treatment with passivating agents; c. optionally, adding spacers to the carbon dots; d. coupling glucose units to the carbon dots; and e. purifying the functionalized carbon dots.

[0076] Thus, the specific objectives of the present invention are: A) Prepare carbohydrate-functionalized fluorescent carbon dot nanoparticles using Click Chemistry strategies; B) Purify the nanoparticles obtained by filtration methods associated with centrifugation, dialysis in low molecular weight membranes and exclusion chromatography; C) Characterize nanoparticles using Nuclear Magnetic Resonance (NMR), Infrared (IR) by Fourier Transform and spectrometric methods, such as ultraviolet (UV) and Fluorescence.

[0077] Initially, milligram-scale nanoparticles are prepared following protocols described in articles and patents so that they can serve as a standard and guide in relation to purifications and analyses in the preparation of novel carbon DOTs.

[0078] Several factors will guide studies on the preparation of nanoparticles, among them, it is worth mentioning (i) standardization of methods that are easily executed, (ii) that use low-cost starting materials and solvents, (iii) preserve the environment with minimal generation of toxic waste, (iv) can be scaled from the bench to larger-scale production; (v) are capable of increasing sugarcane agricultural production.

[0079] In one embodiment, the method of preparing carbon dots is carried out by the bottom-up approach, from simple carbohydrates using the Click Chemistry method, with subsequent functionalization with azido-glucose using a Click Chemistry strategy, using chemical precursors under specific conditions, such as the precursors described here.

[0080] In an alternative embodiment of the invention, carbon dots can be obtained by a top-down approach using bulky carbon substrates. Examples of possible substrates to be used are multi-walled nanotubes, graphite, graphene, or candle soot, subjected to electrochemical methods, chemical oxidation, arc discharge (single-walled arc discharge chemical oxidation of carbon nanotubes (SWCNTs)), and laser ablation, producing carbon dots with high sp2 character and crystalline characteristics.

[0081] In chemical synthesis, Click Chemistry is a class of biocompatible small-molecule reactions commonly used in bioconjugation, enabling the union of substrates of choice with specific biomolecules. Click Chemistry reagents include, but are not limited to, azides, alkynes, catalysts, and ligands. In one embodiment, the incorporation of glucose units via triazole formation is performed by Click Chemistry.

[0082] When mentioned herein “condensation reagent”, it can be understood as a chemical compound responsible for combining two molecules into a single chemical molecule, such as hydroxybenzotriazole (HOBt), benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PYBOP), (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), l-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and N,N-diisopropylethylamine (DIPEA), 1,1-carbonyldiimidazole (CDI). In one embodiment, the condensation reagent is selected from dichlorohexylcarbodiimide (DCC) (2 Eq, 12.0-14.7 g)-

[0083] The term “passivating agent” includes, but is not limited to, TTDDA (1.35 mL), ADH (hexanedioic acid 1,6-dihydrazide), PDHA (O,O'-1,3-propanediylbis(hydroxylamine), 3,3'-(ethane-1,2-diylbis(oxy))bis(propan-1-amino) or even modified TTDDA (4,7,10-trioxa-1,13-tridecanediamine) such as: N-(3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)prop-2-yn-1-amino, 3-(2-(2-(3-azidopropoxy)ethoxy)ethoxy)propan-1-amino, N-(3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)propiolamide.

[0084] "Physicochemical methods and treatments" for obtaining the requested composition can be among the most diverse already disclosed in the literature, including liquid or solid-phase pyrolysis, hydrothermal oxidation, microwave irradiation, chemical oxidation, reflux in an acidic medium, ultrasonic irradiation, or reaction with coated silica nanoparticles. In one embodiment, the physicochemical method used is microwave irradiation.

[0085] "Separation and purification methods" described herein refer to conventional analytical techniques that include physicochemical separation processes selected from filtration, centrifugation, dialysis, electrophoresis, affinity chromatography, gel exclusion chromatography, and membrane separation. These methods aim to obtain purified carbon dots free of monosaccharide units not bound to carbon nanostructures and other undesirable byproducts. In one embodiment, the obtained nanoparticles are purified by filtration combined with centrifugation, dialysis on low molecular weight membranes, and exclusion chromatography. Characterization methods of nanoparticles include, but are not limited to, Nuclear Magnetic Resonance (NMR), Fourier Transform Infrared (IR) techniques and spectrometric methods such as ultraviolet (UV) and Fluorescence.

[0086] For the purposes of the present invention, the introduction of succinic acid to generate a terminal carboxylic acid function in carbon DOTS formed from hydrolyzed glucosamine or sucrose occurs spontaneously by the simple presence of succinic anhydride and stirring at room temperature for several hours (from 12 to 24 hours). However, after the introduction of this succinic acid spacer, it is necessary to use a coupling reagent, such as DCC, to condense the generated carboxylic acid function with 1-aminoglucose, providing the glucose-functionalized carbon DOTS via an amide bond. In this case, the final functionalized carbon DOTS must undergo purification methods to remove byproducts of the final coupling reaction, such as dicyclohexylurea.

[0087] On the other hand, the introduction of propiolic acid to generate a terminal acetylene functional group in carbon DOTS formed from hydrolyzed glucosamine or sucrose requires the use of a coupling agent, such as DCC, with stirring at room temperature for 12 to 24 hours. This reaction occurs between the carboxylic groups of propiolic acid and the terminal amino group of the carbon DOTS, generated after the reaction of hydrolyzed glucosamine or sucrose with the passivation agent TTDDA. This is due to the lower reactivity of propiolic acid relative to succinic anhydride. The intermediate formed must be purified to eliminate byproducts such as dicyclohexylurea.However, after the addition of the propiolic acid spacer and exposure of the terminal acetylene group (triple bond), the coupling reaction with 1-azidoglucose does not require the addition of any other reagent and occurs spontaneously during conventional heating between 80-100 °C, periods of 24 to 48 h, or can be accelerated by microwave irradiation in 10 min, 80 °C. This final step. directly provides glucose-functionalized carbon DOTS via the 1,4- and 1,5-disubstituted 1,2,3-triazole function, i.e., glucose attached at the N-1 position of the triazole ring and acetylene from the carbon DOTS spacer attached at the C-4 or C-5 positions of the corresponding ring, and do not require further purification.

[0088] In another aspect, the use of said composition for the improvement of a crop is provided.

[0089] When used here, the term "crop" refers to a plant crop. In one embodiment, the plant crop is sugarcane.

[0090] When mentioned here “improvement” refers to changes in the physiological properties of a plant, selected from the group of photoprotection under stress conditions, increased rate of photosynthesis, increased production of pigments, increased photosynthesis and changes in the mechanism of production of reactive oxygen species in said crop.

[0091] Although various embodiments incorporating the teachings of the present invention have been shown and described in detail herein, those skilled in the art will readily envision many other varied embodiments that may further incorporate these teachings. Any equivalent aspects are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to be within the scope of the appended claims.

[0092] The present invention will now be described by means of examples, which are intended to be merely illustrative of the present invention and are not, in any way, limiting the scope and scope of the present invention. EXAMPLES Materials and Methods Method for obtaining CORE dots (samples 1-1.12, 2-2.4, 3-3.4 and 4-4.4)

[0093] Glucosamine HCl (1.0 g), glucose, fructose, or hydrolyzed sucrose (0.883 g), separately contained in a 100 mL flask and diluted in double distilled water (20 mL), were treated with 4,7,10-trioxa-1,13-tridecanediamine (TTDDA) (1.35 mL). This mixture was heated in a CEM laboratory microwave apparatus with a reflux condenser for 3.0-3.5 min, at 300 W, so that the internal temperature reached approximately 100 °C (90 to 95 °C). The resulting brown oil was dissolved in H2O (10 mL) and centrifuged through an AMICON filter (10,000 KDa) for 30 min at 4000 rpm and then washed with another 10 mL of H2O and centrifuged again for 30 min at 4000 rpm. The aqueous solution was then concentrated on a rotary evaporator to calculate the yield, yielding a brown oil. This, in turn, was washed with CHCl3 (3 x 10 mL) using a vortex and sonicator until the supernatant became clear.The CHCl3 supernatant was collected and disposed of in accordance with chemical waste disposal regulations or distilled for reuse. The brown oil obtained was concentrated again on a rotary evaporator (removing CHCl3 residues), rediluted in 15 mL of double-distilled water, and filtered through a syringe containing a 200 nm filter (washing subsequently with an additional 5 mL). The obtained solutions were dialyzed overnight in water on a 1 kDa MWCO membrane, replacing the water in the container during the first hours of dialysis. The resulting aqueous solutions, retained on the membrane and that outside the membrane, were concentrated on a cold-finger rotary evaporator to yield approximately 2.0 g of carbon dots. Method for functionalizing DOT cores with -COOH

[0094] Glucosamine or hydrolyzed sucrose DOT COREs (100 mg), contained in a 25 mL flask, were diluted in MeOH (10 mL) to reach a final concentration of 10 mg / mL. Then, succinic anhydride (0.5 Eq wt, 50 mg) was added and the mixture was shaken vigorously overnight. The solvent was then removed under vacuum and the mixture was washed with THF (2x 10 mL) to remove any trace of succinic anhydride. After removal of THF, the resulting brown oil was resolubilized in MeOH, dried under vacuum, weighed, and analyzed. DOT COREs functionalization method with -C=CH

[0095] Glucosamine or hydrolyzed sucrose DOT COREs (100 mg) contained in a 25 mL flask were diluted in MeOH (10 mL) to reach a final concentration of 10 mg / mL. Next, propiolic acid (0.5 Eq wt, 50 mg, 45 mL, 0.71 mmol) and DCC (N,N'-dicyclohexylcarbodiimide) (0.71 mmol, 146.5 mg) were added. The mixture was stirred vigorously overnight. The solvent was then removed under vacuum and the mixture was added with ice-cold H2O, in which the formation of a white precipitate (DCU or dicyclohexylurea, reaction byproduct) was observed. This was removed by filtration. After removal of the precipitate, the solvent was removed under vacuum and the resulting brown oil was weighed and analyzed. Method for obtaining 1-amino glucose MFU 05

[0096] Glucose (500 mg, 2.78 mmol) was added to a microwave vial with ammonium carbonate (5.0 Eq in excess of sugar, 1.4 g, 13.89 mmol) and DMF (5 mL). The vial containing the mixture was sealed and placed in a microwave at 60 °C and 10 watts of power for 90 minutes (2x 45 min). The solvent was removed in a cold-finger rotary evaporator, simultaneously removing the excess ammonium carbonate. Thus, the amino sugar was obtained as orange and hygroscopic solids (497 mg, 2.78 mmol, 100%). The conversion to aminoglycoside was also confirmed by the Ninhydrin test (presenting purple coloration after heating). Method of obtaining 1-azido glucose MFU 04

[0097] Peracetylated glucose MFU 02 (4.3 g, 1.11 mmol) was dissolved in a solution of HBr (10 mL, 33 wt % in acetic acid) and the mixture was stirred at room temperature for 5 h and refrigerated overnight. The resulting solution was then diluted in DCM (20 mL) and subsequently washed with saturated NaHCO3 solution. The organic phase was concentrated under reduced pressure, and then NaN3 (1.3 Eq, 14.4 mmol, 910 mg) and DMF (15 mL) were added. The mixture was stirred vigorously at room temperature for 18 h and then purified by column chromatography. After concentrating the fractions containing the desired compound under reduced pressure, the product MFU 03 was obtained as a slightly yellowish oil (1.9 g, 5.11 mmol, 46%). MFU 03 (1 g, 2.78 mmol) was dissolved in methanol (30 mL), and sodium methoxide solution (1.0 M in MeOH) was added until pH 9.0 was reached. After stirring at room temperature for 1 h, the mixture was neutralized with DOWEX® 50WX4-50 ion exchange resin (H+), filtered, and concentrated under reduced pressure. The resulting product MFU 04 was obtained in 100% yield (549 mg, 2.68 mmol).Method of incorporating glucose units via formation of amide bonds.

[0098] To the DOT COREs SUCs (1 Eq) were added DCC (N,N'-dicyclohexylcarbodiimide) (2 Eq wt), 1-amino glucose MFU 05 (3 Eq wt) and distilled H2O (5 mL). The solution was vortexed and sonicated for 15 min and stirred vigorously for 22 h. The resulting mixture was diluted with a higher proportion of water (5 mL) to precipitate the by-product (dicyclohexylurea), filtered and concentrated in vacuo to yield a brown oil. Method of incorporating glucose units via triazole ring formation

[0099] A solution of each DOT CORE Ac Prop (1 Eq), 1-azidoglucose MFU 04 (1.2 Eq) in DMF (0.8 mL) was placed in a microwave tube. Then, each tube was sealed and subjected to four microwave irradiations (10 min, 80°C, 150 W). Each reaction was followed by TLC (EtOAc / MeOH 8:2 v / v) by taking samples at 10 min intervals. After completion, the reaction mixture had the solvent removed under reduced pressure. and the major excess of azidoglucose was removed by washings with THF (5x4 mL). The resulting mixture was reduced in vacuo to produce a brown oil. Example 1. Getting DOT COREs

[0100] Sucrose hydrolysis was performed with 0.1 M HCl solution and stirred for 48 h at room temperature (pH ~1.0) at a concentration of 20 mg / mL. Cleavage and consequent formation of equivalent amounts of fructose and glucose were confirmed by a qualitative method (Benedict's reagent) and by spectrophotometric technique (1H NMR). The Benedict test is an alkaline solution with a bluish color due to the presence of Cu+2 cations and is used to indicate the presence of reducing sugars such as glucose, galactose, lactose, maltose, and mannose in a sample. A positive result for the presence of some of these saccharides with reducing terminals results in a visual change resulting from the reduction of water-soluble Cu+2 (blue) to insoluble, reddish Cu+1. Their absence does not cause a color change.Thus, in addition to the test performed with hydrolyzed sucrose, sucrose and glucose samples were also tested as negative and positive controls, respectively. After the addition of Benedict's reagent and heating, a color change was observed for the hydrolyzed sucrose and glucose samples, which indicated the effectiveness of the disaccharide cleavage (Figure 4 AB).

[0101] The 1H NMR spectrum of hydrolyzed sucrose, when compared with the spectra of pure sucrose, glucose, and fructose samples, confirmed the effectiveness of hydrolysis in acidic media. This conclusion was obtained by the disappearance of typical disaccharide (sucrose) signals, such as the doublets at 5 5.27, 4.07, and the triplet at 5 3.32, combined with the appearance of characteristic glucose signals, such as the doublet at 5 4.46 and the triplet at 5 3.05 (Figures 5 A-D). Thus, the hydrolyzed sucrose sample (glucose + fructose) could be tested for the formation of carbon DOTs. Example 2. Standardization of reaction conditions for the formation of DOT COREs

[0102] Initially, the method for obtaining DOT COREs as already described in the literature was investigated. This process was necessary mainly due to the difference in irradiation mode between the literature (domestic microwave, 3.0 min, 700 W) and that used (laboratory microwave - CEM). Therefore, the influence of irradiation time and power on the formation of carbon dots from hydrolyzed glucosamine, glucose, fructose, and sucrose was analyzed, as shown in Figures 6 and 7. Figure 6 shows the quantities obtained (starting from approximately 1 g of carbohydrate) after the first centrifugal filtration (crude product), the residue isolated in the chloroform extraction (e.g., related to excess TTDDA), and the product after dialysis, divided into what was "retained" in the membrane and what "passed" to the aqueous phase.The selection criteria for determining the best method were the masses obtained after each process (mainly the mass retained during dialysis), in addition to the intensity of the fluorescent activity throughout each stage. Thus, the sum of these factors determined the best conditions for use in the subsequent reactions with glucosamine (1.4 and 1.11 3.0 or 3.5 min, 300 W) and for hydrolyzed sucrose (4, 3.0 min, 300 W).

[0103] It is worth mentioning that a test was also performed under the same conditions described in the literature, using a domestic microwave (1.12 - Figures 6 and 7), but this did not show any significant difference regarding the calculated masses or the observed fluorescence in relation to the other samples obtained. Furthermore, a test reaction of non-hydrolyzed sucrose with TTDDA was also performed in a microwave apparatus to determine whether there could be cleavage of this disaccharide during the irradiation process. The result was the absence of formation of a brown oil after microwave irradiation, as observed in all other reactions, in addition to the mixture not presenting any fluorescence, which indicates the non-formation of dots. carbon and reinforces the need for the presence of reducing terminals in the sugars used to form these nanostructures. Example 3. Evaluation of the influence of dialysis during the production of functionalized DOT COREs (samples 1.29 / DOT-CORE of glucosamine and 4.6 / DOT-CORE of hydrolyzed sucrose)

[0104] The reduced proportion of DOT CORE retained by the dialysis membrane indicated that the nanoparticles obtained in the laboratory microwave were not retained on the 1.0 kDa membrane. In fact, the cited references did not clarify the exact porosity of the dialysis membrane used. Thus, it appears that the porosity should be 0.5 to 0.1 kDa (the reference describes 0.5 to 0.1 kDa). Since only the 1.0 kDa dialysis membrane was available, two additional tests (using glucosamine and hydrolyzed sucrose) were performed without dialysis to determine the actual need for this process during the preparation of non-functionalized carbon dots (intermediate step). Hydrogen Nuclear Magnetic Resonance (1H NMR) and Infrared (IR) spectra were obtained and compared to those in the literature and to the fractions that were retained or passed through the membrane as shown in Figures 8-11.

[0105] It was observed that the 1H NMR spectra between the samples that passed through the membrane, without dialysis, and that from the literature (for glucosamine, using a domestic microwave, 1.12) showed greater similarity, mainly in the multiplicity of each signal (5 1.67-1.78 - signal 1; 5 2.69-2.91 - signal 2 and 5 3.51 - signal 3) (Figure 8). The 1H NMR spectrum of the carbon dots obtained from the hydrolyzed sucrose also showed 3 signals, with a similar multiplicity pattern and chemical shifts, as shown in Figure 6. The IR spectra, on the other hand, exhibit, mostly, bands characteristic of OH and / or NH bonds (3320 cm' 1 ), C=O (1645cm' 1 ), C-0 of primary and secondary alcohols (1082cm' 1 ) for the carbon dots obtained from both glucosamine and hydrolyzed sucrose (Figures 9 and 11). Example 4. Functionalization of the surface amino groups of DOT COREs

[0106] At this stage of the work, the obtained DOT COREs were functionalized with carboxylic acid (succinic anhydride) and terminal alkynes (propiolic acid), Figure 12. In order to keep the protocol as close as possible to that described in the literature, it was decided to use the fractions that remained retained in the dialysis membrane for both glucosamine (DOT CORE 1.4 Retained) and hydrolyzed sucrose (DOT CORE 4 Retained). However, as discussed previously, tests of these reactions with carbon dot samples obtained without dialysis were also performed with succinic anhydride and propiolic acid for comparative purposes (DOT CORE 1.29 and DOT CORE 4.6). Example 5. Functionalization of glucosamine DOT COREs

[0107] Figure 13 shows the results obtained with the functionalization of glucosamine-retained DOT CORE 1.4 with succinic acid, compared to DOT CORE 1.29 and that described in the literature. The introduction of the spacer group with two methylene carbons was confirmed by the appearance of new signals in the 1 H NMR spectra. The three signals present in the spectrum of each DOT CORE starting material were changed to eight signals with chemical shifts 5 3.61-3.55, 5 3.49-3.42, 5 3.18-3.11, 5 3.04, 5 2.58-2.46, 5 2.44-2.35, 5 1.88-1.82, 5 1.70-1.63). Additionally, the same retained DOT CORE 1.4 and DOT CORE 1.29 were functionalized with propiolic acid to obtain terminal alkyne groups. The coupling of propiolic acid to the amino groups of DOT COREs was carried out in the presence of the condensation reagent dichlorohexylcarbodiimide (DCC) in methanol, and the removal of the generated by-product, dicyclohexylurea, was carried out by precipitation in water and filtration.

[0108] The 1H NMR spectra (Figure 14) show the differences in the signals of the products in relation to those of the starting materials, mainly in relation to the chemical shift. Thus, the change in signals from 5 1.67 and 5 2.79 to 5 1.81 and 5 2.85 (DOT CORE 1.4 retained) and from 5 1.85 and 5 2.96 to 5 1.90 and 5 3.06 (DOT CORE 1.29). Example 6: Functionalization of hydrolyzed sucrose DOT COREs

[0109] The same change in the number of signals was observed in the 1H NMR spectra between the hydrolyzed sucrose starting materials (DOT_CORE_4_Retained) and (DOT CORE 4.6, without dialysis) and the products (DOT CORE SUC 4) and (DOT_CORE_SUC_4.2), when treated with succinic anhydride to functionalize their surfaces with COOH groups (Figure 15). On the other hand, the functionalization with propiolic acid in the DOTs, originating from hydrolyzed sucrose, showed changes in chemical shifts of the three signals already present in the starting materials to more deshielded regions of the spectrum, as expected, and the appearance of new signals at 5 2.99 or 3.06 and 1.91. (Figure 16). Example 7: Functionalization of DOT COREs with sugar units

[0110] The third and final step consisted of coupling glucose units to the carbon dots previously functionalized with spacers. While reproducing the method described in the literature required the synthesis of the precursor 1-amino glucose 1 (Figure 17A), the new Click Chemistry strategy proposed required the preparation of the derivative 1-azido glucose 2, as shown in Figure 17B. In this third step, further purification of the products by dialysis, similar to that described in I a step, is necessary to remove reagents not incorporated into the DOT COREs, such as 1-amino-glucose or 1-azido-glucose used in excess. Example 8: Obtaining 1-amino glucose MFU 05

[0111] The 1-amino glucose reagent MFU 05 has the property of performing nucleophilic attack on the carbonyl of the carboxylic groups of DOT COREs SUC, containing a succinic anhydride spacer group. The reference which describes the process of functionalizing carbon dots with glucose units uses DMSO as a solvent for preparation of MFU 05, however, this solvent has a high evaporation point and is difficult to remove under normal atmospheric pressure. Therefore, the production of MFU 05 through other methods described in the literature was also evaluated, which use MeOH, a volatile and easily removed solvent.

[0112] Thus, the first attempt followed the protocol by Hill et al., 2016, to obtain spectrometric data of the product and compare it with other methods. This protocol uses glucose (250 mg, 1.39 mmol, 1 Eq) and ammonium carbonate (NH4)2CO3 (5 Eq, 96.94 mmol, 667 mg) in the presence of 2.0 mL of DMSO in a sealed tube and irradiated in a microwave (60°C, 10 W) for 1.5 h. Next, the Ninhydrin test was performed, a qualitative evaluation that indicates the presence of primary and secondary amines in a sample by the color change of the mixture to purple when heated (Figure 18). After drying in vacuum, the sample, in the form of brown oil, was characterized by 1H NMR spectrum, which indicated the presence of doublet at 54.15 and triplet at 5 3.09 together with the disappearance of doublets at 5 5.09 and 5 4.50, characteristic of the anomeric hydrogens of the glucose precursor (Figure 19A).These data, combined with the ninhydrin test performed, revealed the presence of amine in the sample, indicating the formation of the desired product.

[0113] Then, the protocol of Tang et al.18 was tested, involving glucose (250 mg, 1.39 mmol, 1 Eq) and ammonium carbonate (NH4)2CO3 (5 Eq, 6.94 mmol, 667 mg), MeOH (25 mL, final concentration of 10 mg / mL) in a volumetric flask and irradiated in a microwave (60°C, 10 W, open system) for 1.5 h. After removal of the solvent in vacuum, a white solid was obtained in high yield (256 mg). However, the 1 H NMR of this sample showed signals identical to those of glucose, indicating that the starting material did not undergo modification under the conditions used (Figure 19B). The low glucose concentration and / or the irradiation mode (open system) was possibly the cause of non-conversion and formation of 1-amino-glucose. Thus, a new reaction was performed in which 100 mg of glucose (0.56 mmol, 1 Eq) and (NH4)2CO3 (5 Eq, 2.78 mmol, 267 mg) MeOH (1.5 mL, final concentration 75 mg / mL) were added. After irradiation in a sealed system (60°C, 10 W, 1.5 h), the solvent was removed and the sample analyzed by 1H NMR and ninhydrin test. Despite the presence of a doublet at 5 4.22 and a triplet at 5 3.09, another doublet at 5 4.03 and a triplet at 5 3.17 were also observed. These new signals indicated the presence of a mixture between the desired product MFU 05 and the glycosylammonium carbonate salt, a reaction intermediate (Figure 13C) (Figure 17). The ninhydrin test also gave a positive result.

[0114] Still following the conditions described by Tang et al, the replacement of ammonium carbonate with ammonium carbamate (H2NCOONH4) was explored. The first test with this new salt was performed using 100 mg of glucose and 87.4 mg of H2NCOONH4 (2 Eq, 1.12 mmol) and MeOH (1.0 mL, final concentration of 100 mg / mL). After irradiation in a sealed system (60 °C, 10 W, 1.5 h), the solvent was removed and the sample analyzed by 1H NMR and ninhydrin test. The 1H NMR spectrum (Figure 19D) and the ninhydrin test showed great similarity with those from the previously tested condition.

[0115] Additionally, the reproduction of another protocol described by Likhosherstov et al., involving 500 mg of glucose (2.78 mmol, 1 Eq) in the presence of H2NCOONH4 (2 Eq, 5.56 mmol, 434 mg) and diluted in MeOH (6.6 mL) and NH4OH (0.4 mL). The medium was kept under stirring for 24 h at 37 °C. Then, the flask containing the mixture was stored in a freezer and kept under refrigeration for 24 h, when the formation of a white precipitate was observed. After removal of the solvent, the precipitate was treated with 8 mL of MeOH and 22 pL of DIPEA (diisopropylethylamine) and subsequently dried under vacuum, yielding a white solid (474.5 mg). The 1H NMR spectrum and the ninhydrin test were also similar to the last ones obtained, but with a lower proportion of the desired product, approximately 25% (Figure 19E).

[0116] Finally, since only DMSO as the solvent produced a direct and higher-purity product, the possibility of replacing the DMSO solvent with DMF, which is easier to remove, was investigated. Thus, the same conditions as in the first protocol were repeated, but with a different solvent. After microwave irradiation, the DMF was removed under vacuum, and the sample was analyzed by 1H NMR and the ninhydrin test. The 1H NMR spectrum shows the formation of only one product, as in the first case, indicating the feasibility of replacing DMSO with DMF (Figure 19F). The ninhydrin test also yielded a positive result. A summary of all the conditions mentioned above is presented in Table 1. Table 1 - Comparisons between different conditions for the synthesis of 1-amino glucose MFU 05. The best reaction condition so far is using DMF as solvent and ammonium carbonate as nitrogen source.

[0117] Since the protocol described by Likhosherstov treated the product after irradiation with a base (DIPEA), it was proposed to try to treat one of the product mixtures with basic resins, in an attempt to convert the intermediate salt into the compound MFU 05, containing a free amino group. Thus, a sample obtained following protocol C (10 mg) was diluted in H2O MilliQ (1.0 mL), treated with ion exchange resin [OH-] and kept under stirring at room temperature for 3 h. Subsequently, the sample was The resin was filtered, the water was removed under vacuum, and the sample was analyzed. 1H NMR shows significant reduction of one of the products, possibly the glycosyl ammonium carbonate salt (Figure 20). Thus, this procedure also demonstrates great potential for obtaining 1-aminoglucose MFU 05 in pure form from methanol, for later use in coupling with carbon DOTs. Example 9. Obtaining 1-azidoglucose MFU 04

[0118] The synthesis of the 1-azido-galactose derivative MFU 04 was carried out for use in cycloaddition reactions with carbon dots functionalized with alkyne groups (DOT CORE Ac Prop of glucosamine or hydrolyzed sucrose), using the Click Chemistry strategy. The azido-glucose product was easily obtained in four steps, from D-glucose, following previously established protocols (Figure 21).

[0119] The selected method involved, first, the acetylation of the hydroxyls of D-glucose by treatment with acetic anhydride (Ac2O) in pyridine. The peracetylated product MFU 04 was obtained in quantitative yield as a slightly yellowish oil. Subsequently, the formation of 2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl bromide MFU 02 occurred by treating the protected sugar MFU 04 with a 33% HBr solution in AcOH, maintaining stirring for 5 hours. Subsequently, the bromide intermediate MFU 02 was directly used in a nucleophilic substitution reaction using sodium azide (NaN3), which afforded compound MFU 03 in 46% yield. 21 The formation of compound MFU 03 was confirmed by 1H NMR. Additionally, the infrared spectrum showed a band at 2100 cm' 1, characteristic of an azido group (Figure 22). Finally, the last step consisted of the deacetylation of positions 2, 3, 4, and 6 of MFU 03 by treating it with MeOH and 1 M NaOMe sol. After 1 h under stirring, the solution was then neutralized with acid resin DOWEX [H+], filtered and concentrated under reduced pressure to obtain the desired precursor 1-azido glucose, completely deprotected in quantitative yield. The IR spectrum of this product confirmed the persistence of the azide group by the presence of the band at 2100 cm' 1 as well as the removal of the OAc groups due to the absence of the band before at 1750 cm' 1 , characteristic of carbonyls of the acetyl protecting group (Figure 23).

[0120] Since the starting material is glucose and the reagents used are inexpensive, this route for obtaining azido-glucose MFU 04 is low-cost. However, mainly aiming to accelerate the preparation process of this reagent, the possibility of converting the -OH group, in Cl of glucose, directly to azido in a single step was also investigated.22 Therefore, 2-chloro-1,3-dimethyl imidazolinium chloride (DMC) salt was used. Thus, D-glucose (100 mg, 0.56 mmol), NaN3 (10 Eq, 5.6 mmol, 361 mg), N,N-diisopropylethylamine (DIPEA) (9 Eq, 5.04 mmol, 880 mL) were dissolved in MilliQ H2O (6 mL) with stirring in an ice bath. After the mixture had cooled, DMC (3.0 Eq, 1.68 mmol, 284 mg) was added and kept under stirring for 3 h (Figure 21).

[0121] After concentrating the reaction mixture and adding ethanol, the solid formed was removed by filtration. The filtrate was concentrated in vacuo and the product dissolved in MilliQ H2O. The mixture was then washed with dichloromethane and the aqueous phase was concentrated in vacuo. After concentration, the product was again solubilized in MilliQ H2O and treated with ion exchange resin (activated with overnight NaOH solution and washed with MilliQ H2O). The product was concentrated and ethanol was added, and the solid was removed by filtration. The filtrate was concentrated, yielding a white solid (97.5 mg, 0.48 mmol, 85%). Although the 1H and IR NMR spectra of this product indicated the formation of the desired compound (Figures 25 and 25), the presence of two singlets at δ 3.28 and δ 2.65, consistent with the methyl groups (CH3) of the DMC salt, was observed. This indicates that the Resin treatment was not sufficient to completely remove it from the reaction mixture, so new strategies are being investigated (varying the base resin used, amount of resin used, and resin agitation time). However, it is worth remembering that, after the 'click' step with the Ac Prop DOT Cores, the mixture must be dialyzed. Therefore, it is expected that during this process, the water-soluble DMC will be removed from the remaining portion of the sample.

[0122] Once in possession of the precursors MFU 04 and MFU 05, the next steps of the project were carried out to standardize the protocols for functionalizing carbon DOTs with glucose units as shown in Figure 17. Example 10. Incorporation of glucose units via amide bond formation

[0123] Once the amino sugar MFU 05 was obtained, it was added in excess (3 Eq wt) to DCC (dicyclohexylcarbodiimide, 2 Eq wt) and to the DOT COREs SUC of glucosamine (1.2 - retained in dialysis and 1.3 - without dialysis) and hydrolyzed sucrose (4 - retained in dialysis and 4.2 - without dialysis) in milliQ H2O (final concentration of 10 mg / mL). After vigorous stirring at room temperature for 22 h, the mixture was filtered and dried under vacuum. The next step would consist of dialysis through a 0.5 KDa membrane (500 MW); however, this imported material has not yet been delivered by the supplier. Therefore, 1H NMR spectra of the crude materials were performed in order to verify any indication that the reaction had occurred as expected. Despite the presence of excess 1-amino-glucose in the crude product, the generated by-product, dicyclohexylurea, was removed by precipitation in water and filtration.

[0124] As observed in Figures 26A–D, the signals between the spectra of DOT COREs of glucosamine (Figures 26A, retained and B, without dialysis) and sucrose (Figures 26C, retained and D, without dialysis) are very similar, even comparing the raw ones using DOT COREs that were retained in the membrane (Figures 26 A and C) of those that used DOT COREs without dialysis (Figures 26 B and D). It is possible to observe the presence of signals at 5 4.30-4.26 referring to the Hl of the amino-glucose MFU 05 used in excess. However, the appearance of two new signals at 5 5.24-5.20 and at 5 4.65-4.61 is also noted, possibly referring to the Hl a and H- ip respectively, of the glucose bound to the DOT COREs. Example 11. Incorporation of glucose units via triazole formation - Click Chemistry

[0125] The procedure used for coupling DOT COREs functionalized with a terminal alkyne group and 1-azido-glucose was the one already described by the inventors. Thus, DOT COREs Ac Prop of both glucosamine (1.2 - retained in dialysis and 1.3 - without dialysis) and hydrolyzed sucrose (4 - retained in dialysis and 4.2 - without dialysis) were treated with azido-glucose MFU 04 (1.2 Eq) and DMF in a sealed microwave tube. After three 10 min irradiations (80 °C, 150 W), the solvent was removed and the crude material was washed with THF to remove the remaining excess MFU 04 (used in excess 1.2 Eq by weight). This organic phase containing azido-glucose was also dried and weighed. From these recovered starting materials, it was possible to calculate the proportion of 1-azido-glucose, MFU 04, incorporated into the DOT COREs.Thus, for the reactions with glucosamine, the incorporation rate was 7.6% and 24.5% of the initial mass added for the retained Click glucosamine and Click glucosamine without dialysis reactions, respectively. For the reactions starting from hydrolyzed sucrose DOT COREs, these values ​​were 2% (retained Click sucrose) and 17.1% (Click sucrose without dialysis), demonstrating the better efficiency in the reactions using DOT COREs without dialysis. Although the THF extraction removed much of the 1-azido-glucose precursor, a small amount was still retained in the mixture to be purified by dialysis (500 MW membrane, under acquisition).

[0126] In general, the 1H NMR spectra show that, Glucosamine Ac Prop DOT COREs obtained without dialysis (Ac Prop_1.3 DOT COREs) provided the expected product by the appearance of H-triazole signals at 5 8.47 and 5 8.06, which is not observed for the product obtained using the retained Ac Prop 1.2 DOT COREs. Furthermore, the appearance of two signals is observed at 5 6.43 and 5 5.70, probably referring to the H 1 of the incorporated glucose units (Figures 27 A and B). It is possible that one of these signals refers to the 1,4-disubstituted product and the other to the 1,5-disubstituted product since no catalyst was used to achieve regioselective products.

[0127] For the products using hydrolyzed sucrose DOT COREs, the 1H NMR spectra did not show a significant difference between the retained and non-dialyzed DOT COREs, both demonstrating signs indicative of the formation of the triazole ring due to the presence of a signal at 5 8.47 and 5 8.07 in addition to the glucose signals at 5 6.52 and 5 5.80, as evidenced by Figures 27 C and D. This result reinforces the viability of not performing dialysis (I a stage) after formation of the initial DOT COREs. Example 12. Study of the effectiveness of carbon dots as photosynthesis enhancers in sugarcane Objectives

[0128] To test the potential of products prepared by the method described to increase the photosynthetic activity of two sugarcane cultivars, depending on the dose applied.

[0129] Test the potential of the best product, in comparison with the product imported from England, in increasing the photosynthetic activity of two sugarcane cultivars, depending on the dose applied. Materials and methods Location, experiment conduct and treatments

[0130] The experiment was set up in an open area located in the Weed Laboratory (LAPDA) of the Faculty of Agricultural Sciences and Veterinary Sciences - FCAV / UNESP - Jaboticabal Campus, SP, Brazil. The altitude of the site is 590 m and the geographic coordinates are: latitude 21°15'17”S and longitude 48°19'20”W. The experiment used the sugarcane cultivar 5476, which was supplied by Usina São Martinho.

[0131] The experiment was conducted for 9 days after application (DAA) of the treatments, which were composed of four distinct products developed, hereinafter called MFU 06 8, referring to carbon dots formed from glucosamine and functionalized by Click Chemistry with azido-glucose, generating the triazole group; MFU 08 6, referring to carbon dots formed from glucosamine and functionalized with amino-glucose containing the amide bond; MFU 09 4, referring to carbon dots formed from hydrolyzed sucrose and functionalized with amino-glucose containing the amide bond; and MFU 07 8, referring to carbon dots formed from hydrolyzed sucrose and functionalized by Click Chemistry with azido-glucose, generating the triazole group, applied at three doses (1 and 5 mg ai plant) J ), in addition to the control without application (Table 2). Table 2 - Products and doses used in the composition of treatments for each sugarcane cultivar. Jaboticabal / SP, 2022, Application technology

[0132] The MPB seedlings, after planting in 5-liter pots, were sprayed with the doses and products described in Table 1. For this, a manual sprayer was used, at constant pressure, equipped with a nozzle with a TT 110.02 tip, regulated to consume a spray volume of 150 L ha' 1 .

[0133] Twenty-four hours after applying the treatments, all seedlings were placed in the experimental area to begin evaluations. Experimental design

[0134] A randomized block design was used, with three replicates, totaling thirty-nine experimental plots for each sugarcane cultivar. Each plot consisted of one sugarcane plant (MPB) planted in a 1.5 L pot. Reviews

[0135] In order to evaluate the effect of the product on the photosynthetic characteristics of sugarcane plants, evaluations of the relative content of total chlorophyll, gas exchange and quantum efficiency of photosystem II (PSII) were carried out.

[0136] Thus, from 1 to 9 days after application (DAA) of the products, always at ten o'clock in the morning, the rate of net CO2 assimilation, transpiration and stomatal conductance (in addition to the calculation of water use efficiency) were evaluated on the first fully expanded leaf, with an infrared gas analyzer (IRGA mod. LI-6400, Li-Cor®). For this purpose, working conditions of 15 mmol H2O mol' were used 1 , 398 pmol CO2 mol' 1 , the flow rate at 400 pmol s' 1 , atmospheric pressure at 1000 KPa and the photosynthetically active photon flux (PFF) at 1300 pmol m' 2 s' 1 . At these times, on the same sheet, the quantum efficiency of photosystem II - F was also determined. v / F m (fluorometer, model MINI-PAM-II, Walz®), and the relative content of total chlorophyll (chlorophyll meter, model ClorofiLog, Falker®). statistical analysis

[0137] The collected data were subjected to analysis of variance using the F test and, when significant, the means were compared using the Tukey test at a 5% probability level or subjected to regression analysis, to better illustrate the observed biological phenomenon, using Origin v. 9.0 software (Microcal®). Results Step 1 - Experiment 1 (Cultivar 5476, Lot 008) Gas exchange Product 1 - MFU 06 8

[0138] Regarding the dose of 5 mg plant' 1 , higher values ​​of A were observed, USA eg s in the applied plants, compared to the control, for all evaluation days (Figures 28-A, 28-B and 28-C). In addition, a higher transpiration rate was observed in the plants sprayed with the product, from 03 to 08 days after application (DAA). After this period, there was a tendency for reduction in values, when compared to the control (Figure 28-D). Product 2 - MFU 08 6

[0139] Regarding the dose of 5 mg plant' 1 , higher values ​​of A, USA, g were observed s and E in the applied plants, compared to the control, during the entire experimental period (09 DAA) (Figures 29-A, 29-B, 29-C and 29-D). Product 3 - MFU 09 4

[0140] For the third product tested (MFU 09 4), at a dose of 1 mg plant' 1 , an increase in net CO2 assimilation was observed in relation to the control without application (Figure 30-A). The same response pattern occurred for the stomatal conductance and transpiration rate variables (Figures 30-C and 30-D). Despite this, the plants sprayed with the product obtained greater water use efficiency (WUE) only during the first two days after the start of the experiment (Figure 30-B). Product 4 - MFU 07 8

[0141] Regarding the dose of 5 mg plant' 1, higher values ​​of net CO2 assimilation and water use efficiency were observed for the plants that received the product application, compared to the control, for all evaluation days (Figures 31-A and 31-B). The same was observed for stomatal conductance from 02 days after application (Figure 31- C). Furthermore, a slight decrease in the transpiration rate of plants treated with the product was observed after 7 days after application (DAA). Before this period, there was no difference in the values ​​observed for this treatment when compared to the control (Figure 31- D).

Claims

CLAIMS 1. CARBON DOT COMPOSITION, characterized by the fact that it comprises a carbon core formed by carbohydrates, precursors and a surface comprising carboxylic acid or terminal alkyne functional groups.

2. CARBON DOT COMPOSITION according to claim 1, characterized in that it also optionally comprises spacers.

3. COMPOSITION, according to claim 1 or 2, characterized by the fact that the core carbohydrates comprise at least one of hydrolyzed sucrose, chitosan, glucose, fructose or glucosamine.

4. COMPOSITION, according to any one of claims 1 to 3, characterized by the fact that the precursors of the nucleus comprise at least one of 1-amino-glucose or 1-azido-glucose.

5. COMPOSITION, according to any one of claims 1 to 4, characterized by the fact that the precursor is present in a concentration ranging from 2% to 24.5% of the initial mass.

6. COMPOSITION, according to any one of claims 2 to 5, characterized by the fact that the core spacers comprise at least one of succinic acid or propiolic acid.

7. METHOD FOR PREPARING CARBON DOTS, characterized by the fact that it comprises the steps of: a. preparing a carbohydrate and precursor solution; b. obtaining carbon dots by treatment with passivating agents; c. optionally, adding spacers to the carbon dots; d. coupling glucose units to the carbon dots; and e. perform the purification of functionalized carbon dots.

8. METHOD, according to claim 7, characterized by the fact that the treatment employed to obtain the carbon dots is microwave irradiation.

9. METHOD, according to claim 8, characterized by the fact that the irradiations last from 3 to 3.5 min, at a power of 300 W.

10. METHOD, according to claim 8 or 9, characterized by the fact that the incorporation of the precursor in the carbon dots consists of three irradiations of 10 min at a temperature of 80 °C, and a power of 150 W.

11. USE OF CARBON DOT COMPOSITION, characterized by the fact that it is for the improvement of a plant crop.

12. USE, according to claim 11, characterized by the fact that the improvements are selected from the group of photoprotection under stress conditions, increased photosynthesis rate, increased pigment production, increased photosynthesis and altered mechanism of production of reactive oxygen species in said plant crop.

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