1. Introduction
As society advances, there is an escalating focus on the ecological ramifications of agricultural practices—particularly the safety and environmental impact of pesticides used in crop production [
1]. Traditional synthetic pesticides, which have long been the backbone of pest control in agriculture, are increasingly under scrutiny for their potential to harm non-target species, pollute water bodies, and contribute to soil degradation [
2]. This growing concern has propelled the development of green pesticides into the limelight [
3]. The shift toward green pesticides is a challenging task; fortunately, nature provides an array of natural products (NPs) that serve as sources for many food additives, medicines, materials and others [
4]. With emerging ecological issues, the appeal of NP-derived strategies extends beyond medicines and food additives to encompass agricultural pest management [
2]. For example, ethylicin (ET) is a bionic agrochemical with proven inhibitory effects toward several pathogenic bacteria and fungi (
Fig. 1(a)). As an ethyl homologue derived from the natural compound allicin in garlic, ET exhibits several advantages, including high control efficiency, quick degradation, and ecological sustainability. These qualities have led to its widespread application in the field for phytomicrobicidal purposes. Similarly, the strategic exploration of structures from nature’s effective designs holds promise for the discovery of environmentally friendly pesticides.
Vanillin, a natural flavoring agent, is widely used across an array of food products such as baked goods, confectionery, and dairy products [
5]. Importantly, vanillin is classified by the US Food and Drug Administration (FDA) as a substance that is generally recognized as safe (GRAS) for use in food; it even appears as an ingredient in some infant formulas [
6]. Along with its use in the food industry, vanillin has been found to have potential biological activity, including antioxidant [
7], [
8], [
9] and anti-inflammatory activity [
10], [
11], [
12], [
13], [
14], and has been studied for its potential use in treating neurological disorders associated with Alzheimer’s disease [
15], [
16], [
17]. In addition, vanillin is an exceptionally versatile compound that facilitates the synthesis of numerous crucial chemicals. This adaptability allows for its transformation into a variety of molecules, such as vanillyl alcohol, vanillic acid, and acylvanillins, among others (
Fig. 1(b)).
Typically, vanillyl alcohol is employed as an intermediate in the production of flavors and fragrances, as well as in the synthesis of bioactive pharmaceutical compounds [
18], [
19], [
20]. Considering both safety and price (at 3-4 USD∙kg
−1), vanillin undoubtedly stands out as a hopeful choice for developing ecofriendly pesticides. This is because an ideal pesticide must embody efficiency, sustainability, and affordability. Should the production costs for a safe and highly effective pesticide candidate prove to be prohibitively expensive, the likelihood of it reaching the market diminishes significantly. However, despite vanillin’s popularity in the food industry and the field of medicine, this substance has only recently been explored for agrochemical development, showing promising capacity. Therefore, there is a pressing need for a comprehensive review of the literature pertaining to reported vanillin derivatives within pesticide science.
This review summarizes the progress that has been achieved in developing a series of agro-active compounds against agricultural pests based on the structural modification and optimization of vanillin—from the design and synthesis of the compounds to their activity evaluation and mechanism of action studies, along with a perspective on future directions (
Fig. 1(c)). It is worth noting that the scope of this review does not extend to the use of vanillin and its derivatives as food preservatives and plant growth regulators. Although the synthesis of vanillin and its applications in the pharmaceutical and food sectors have received extensive coverage [
21], [
22], [
23], this review presents—for the first time—a comprehensive summary of vanillin’s role in the development of pesticides. Our aim is to encourage the exploration of NPs as a means to identify valuable molecular structures for the advancement of green pesticides, thereby protecting food security in a sustainable manner.
2. Vanillin and its structural characteristics
Vanillin is an aromatic compound that was first isolated from vanilla extract in relative purity through evaporation and recrystallization in 1858 [
24]. As one of the most popular and extensively used food additives across the globe, the sources of vanillin are abundant and diverse; this compound can not only be extracted from vanillin-containing plants but also be synthesized artificially [
25], [
26]. General pathways to obtain vanillin are shown in
Fig. 2. The most commonly used method for synthesizing vanillin in industry is through chemical synthesis from lignin or guaiacol. The lignin-based process involves breaking down lignin, a natural polymer found in wood, into smaller components, one of which can be converted into vanillin [
27], [
28], [
29], [
30]. Alternatively, the guaiacol process starts with guaiacol as a precursor, which then undergoes chemical reactions to produce vanillin. Both methods are favored for their cost-effectiveness and the ability to produce vanillin on a large scale [
31], [
32], [
33].
Currently, increasing health awareness among consumers is fueling the demand for natural vanillin, making its extraction and purification a subject of keen interest [
4]. Zhu et al. [
34] have provided a comprehensive summary of recent advancements in the extraction, purification, and applications of vanillin derived from diverse sources, including vanilla pods, lignin, and fermentation broth, offering valuable insights into the diverse uses and ecological production of natural vanillin. Despite the sustainability and preference for extracting and purifying vanillin from natural raw materials, this approach presents challenges, including high costs, low efficiency, and a reliance on organic solvents [
4]. Recently, biosynthesis has emerged as a promising strategy for achieving vanillin. This approach employs microbial genetic and metabolic engineering to convert biomass-based substrates into vanillin [
35], thus offering a sustainable alternative. However, challenges persist, including low conversion efficiency, high production costs, and difficulties in separating target components from the fermentation broth [
36], [
37]. Consequently, chemical synthesis remains the predominant method for producing vanillin. Nonetheless, with ongoing refinements in genetic engineering and improvements to extraction and purification techniques, the proportion of vanillin acquired through biosynthesis and botanical extraction is expected to grow progressively, better aligning with sustainable practices.
The complex structure of NPs significantly affects their subsequent modification, either cost-wise or chemically. Fortunately, vanillin benefits from an extremely straightforward skeleton. Its structure contains two functional groups with high reactivity and modifiability (
Fig. 3). The hydroxyl group (-OH) can generally undergo deprotonation under basic conditions, affording a hydroxyl anion that behaves as a nucleophile. Subsequent reaction with an electrophile enables the splicing of a series of bioactive moieties via an ether bridge. This strategy not only permits the introduction of a myriad of units into the vanillin skeleton in an effortless manner—as it is only necessary to prepare the halides of introduced groups for nucleophilic substitution—but also simultaneously builds an ether linkage. Many chemical fragments, including thienopyrimidine [
38], coumarin [
39], and even the recently popular mesoionic structure [
40], [
41], have been spliced into vanillin with this method and proved to be compatible with the compound. Ether bonds play a crucial role in drug design, particularly in the context of medicinal chemistry and the synthesis of bioactive compounds [
42], [
43], [
44], [
45], [
46]. Such bonds can be introduced or substituted in various regions of a drug molecule to alter its physicochemical properties, such as its lipophilicity, solubility, and metabolic stability [
47], [
48], [
49], [
50]. Moreover, ethers can participate in hydrogen bonding interactions with target proteins, enzymes, or receptors. These interactions can contribute to drug-target binding affinity, specificity, and molecular recognition.
Regarding the aldehyde region in vanillin, it is well known that aldehydes are versatile functional groups that can react with diverse substances through a variety of chemical transformations. However, only three types of modifications to this functional group in vanillin have been implemented. The first approach involves the synthesis of imines by mixing vanillin with amines, followed by conversion of the aldehyde group into an amino group, facilitated by the presence of reducing agents for subsequent transformations. This reductive amination has been employed to introduce mesoionic substituents into the vanillin backbone [
41]. Thioacetalization is another modification and the most commonly used method, by which vanillin’s carbonyl group reacts with a series of thioalcohols or dimercaptans to give thioacetal products in a usually irreversible way [
51], [
52], [
53], [
54]. In comparison with alcohol-participant acetalization reaction, this strategy endows products with additional stability, as acetals are normally labile and easily return to aldehydes via a reverse reaction. A β-cyclodextrin-SO
3H-mediated catalytic strategy serves as the third approach. This three-component methodology allows the formation of imidazo[1,2-
a]pyridin-3-amines via the intermolecular annulation of isocyanides, pyridin-2-amines, and aldehydes [
55].
It is worth noting that alteration of the benzene ring of vanillin seems to be a feasible strategy. Nonetheless, many factors, such as steric effects, electronic effects, and regioselectivity, must be taken into account when installing atoms or groups on substituted aromatic rings. Also, exchange of the vanillin benzene ring with other heterocycles (e.g., pyrazine or pyridine) is not considered optimal, as it deviates from the premise of utilizing a NP as a starting point. Hence, it is our contention that maximizing the optimization and derivatization of vanillin’s intrinsic reaction sites—namely, its aldehyde and hydroxyl groups—offers significant benefits in terms of both reaction efficiency and cost-effectiveness.
3. Recent advancement in vanillin-derived pesticides
In 2017, Song et al. [
39], [
56], [
57], [
58], [
59] pioneered an exploration of the possibility of vanillin being developed into agrochemicals. They designed and synthesized a series of novel vanillin derivatives featuring dithioacetal moieties. In this synthesis, a variety of vanillin compounds are accessed via merely a two-step transformation. As shown in
Fig. 4, the reaction occurs with the nucleophilic substitution of the vanillin hydroxy to haloalkane (
1), affording 4-(alkoxy)-3-methoxybenzaldehyde (
2). As an intermediate of the whole conversion,
2 subsequently undergoes a catalytic thioacetalization to provide the target compound (
3). The selected instances show the wide scope this synthetic method is capable of, with the possibility of installing a wide variety of thioacetals including aryl, alkyl, or cyclic thioacetals. Compound
3a is the so-called “xiangcaoliusuobingmi,” which is spelled based on Mandarin pinyin, while its formal English name is “vanisulfane.”
Notably, this work not only achieves innovation in molecular design but establishes a facile way of synthesis. Zirconium tetrachloride (ZrCl
4) acts as a key accelerator and selective regulator in the facilitation of this reaction. In the researchers’ postulated mechanism, 2-mercaptoethanol first links with ZrCl
4 to form complex
A, in which the hydroxy group shows preferential affinity to zirconium (Zr) and allows only the sulfhydryl group to react with the aldehyde substrate. This ingenious design significantly enhances the chemoselectivity and regioselectivity, as both -OH and -SH can undergo acetalization with the carbonyl. The resulting intermediate
B is then dehydrated, generating intermediate
C. After the release of ZrCl
4 to rejoin the catalytic cycle, the final product is obtained in turn (
Fig. 5). All the pathways involved in this transformation have been verified by
1H nuclear magnetic resonance spectra and density functional theory calculations.
In addition to the successful accomplishment of this molecular design and synthetic route, the researchers found that these target compounds exhibit promising bioactivities in combating agricultural pests. For example, bioassays conducted using the half-leaf method indicated that vanisulfane presents broad-spectrum and superior antiviral activities against potato virus Y (PVY) and cucumber mosaic virus (CMV). The half-maximal effective concentration (EC
50) of vanisulfane was significantly lower than those of commercially available ribavirin, ningnanmycin (NNM), and dufulin (
Table 1 [
59]). This groundbreaking work by Song and colleagues led to the development of vanisulfane, a highly effective antiviral agent derived from natural vanillin—an achievement that has significantly heightened interest in vanillin’s potential for pesticide development, prompting further studies aimed at its derivatization or structural modification.
Since the pioneering development of vanisulfane, the exceptional performance of vanillin in the design of pesticides has garnered widespread attention, leading to the report of thousands of derivatives that exhibit resistance to agricultural pests [
60], [
61]. In this context, we present a selection of notable instances showcasing advancements in antiviral species and other potential bioactivities. Strobilurins encompass a collection of naturally occurring compounds and their artificially produced analogues [
62]. Many strobilurins are applied in the field of agriculture for their fungicidal properties [
63], [
64], including azoxystrobin [
65], pyraclostrobin, trifloxystrobin, and fluoxastrobin [
66]. Moreover, it has been documented in several studies that strobilurins exhibit the ability to augment the resistance of crops against diseases caused by plant viruses [
67], [
68], [
69], [
70], [
71]. On this basis, in 2018, Song et al. [
72] undertook the task of designing and synthesizing a collection of novel dithioacetalized vanillin derivatives. This was accomplished by swapping the 4-chlorophenyl in vanisulfane with the key fragment
β-methoxyacrylate in strobilurins (
Fig. 6). More specifically, methoxyacrylate-containing bromides (
5) were synthesized through the bromination of the methyl group in substrate
4, with acetonitrile and KI serving as the solvent and catalyst, respectively. The subsequent reaction involved stirring the corresponding substituted 4-hydroxybenzaldehydes and
5 under reflux conditions to yield intermediates (
6); finally, the target compounds (
7a-
7d) were synthesized by employing ZrCl
4 as a catalyst and condensing
6 with different mercaptans.
The further optimization of vanisulfane has resulted in an important advancement in the suppression of plant viruses. As presented in
Table 2 [
72], product
7b shows improved performance against PVY and CMV, compared with the lead compound vanisulfane. Significantly, this study broadened the range of antiviral activity exhibited by vanillin derivatives and unlocked their potential application in combating tobacco mosaic virus (TMV). It is important to emphasize that natural vanillin was not used in the design of all compounds. Compound
7b, for instance, was made by employing artificially synthesized 3-chloro-4-hydroxybenzaldehyde as a substrate. However, this research does pave the way for more broadly effective antiviral drugs; it also suggests that altering the 3-methoxy of vanillin is both acceptable and promising for discovering enhanced anti-viral substances.
A glycoside unit is commonly observed in various natural sources; it serves as a means to increase the physicochemical characteristics of drugs and improve the biological efficacy of substances by their integration into the molecular framework [
73], [
74]. Thus, glycosides hold considerable importance in the domain of drug design. In 2019, Song et al. [
73] attempted to introduce glycosides into vanillin and synthesized a series of vanillin derivatives containing glucopyranoside (
Fig. 7). The key synthetic step employed click chemistry, which involves combining azide-functionalized glucoses (
8) with vanillin alkynes (
9) upon the addition of a CuSO
4·5H
2O and sodium ascorbate solution in distilled water. This forms 1,2,3-triazole intermediates (
10) [
74], which can condense directly with substituted thiols to give products
11a and
11b; alternatively, it can react with sodium methoxide in anhydrous methanol to result in deacetylation intermediates (
12), which are subsequently converted to target compounds
13c and
13d. Significantly, this study concerns the use of NaHSO
4·SiO
2 as a catalyst for the condensation reaction, offering an economical alternative to the previously used ZrCl
4.
These target compounds exhibited excellent antiviral activity against tomato chlorosis virus (ToCV), even surpassing NNM and chitosan oligosaccharide (COS), which also include glycoside fragments. Notably, the researchers also established a coat protein (CP)-based screening approach for the bioactivity evaluation of compounds against ToCV (
Table 3 [
73]). This strategy can be practical, since the characteristics of ToCV do not cause model plants such as
Chenopodium amaranticolor and
Nicotiana glutinosa to show visible spot symptoms; therefore, the evaluation of drug activity against ToCV in the laboratory was not previously available. The employment of the established activity-screening strategy led to the identification of compound
13c, which exhibited remarkable biological activity. This was evidenced by a significant reduction in the expression level of the
ToCV-CP gene, as determined through the implementation of reverse-transcription polymerase chain reaction. More specifically, the
ToCV-CP gene expression level in the
13c-treated group was substantially lower compared with the control group, with a decrease of 92%.
The first application of vanillin derivatives in the control of tomato spotted wilt orthotospovirus (TSWV) was reported in 2021 by Hu et al. [
75]. These derivatives are structurally designed by linking quinazolinone to thioacetalized vanillin through an ether bond (
Fig. 8). Concretely, compounds
18a−18d were synthesized through a five-step reaction sequence, taking anthranilic acid and vanillin as the starting materials. Initially, a quinazoline (
14) was prepared via the ring-closure of anthranilic acids with formamide. Subsequently, a hydroxymethyl species (
15) emerged from the reaction between
14 and formaldehyde [
76]. The third step involved the oxidation of
15 with SOCl
2, giving rise to corresponding chlorides (
16) [
77]. The penultimate process entailed a substitution reaction in which
16 was combined with vanillin to yield intermediates (
17). Finally, the desired compounds
18a−18d were synthesized by the catalytic action of NaHSO
4·SiO
2.
Compound
18c exhibited remarkable
in vivo anti-TSWV performance, with an EC
50 value of 188 μg∙mL
−1 (
Table 4 [
75]). This is significantly superior to the observed EC
50 values for ribavirin (642 μg∙mL
−1), vanisulfane (420 μg∙mL
−1), and NNM (257 μg∙mL
−1). In addition, four π-alkyl connections were formed between compound
18c and the TSWV CP at the ARG
94 and ARG
95 locations. Among the tested concentrations for binding affinity to the TSWV CP, compound
18c (9.4 mol∙L
−1) outperformed ribavirin (67.8 mol∙L
−1), vanisulfane (33.8 mol∙L
−1), and NNM (24.3 mol∙L
−1). It can be seen that the results obtained by performing microscale thermophoresis (MST) are consistent with what was tested in
in vivo experiments, indicating that compounds with better antiviral activity have better binding affinity to CP. Based on these results, compound
18c has become a lead compound for the discovery of new antiviral agents for the management of TSWV.
Diverse biological properties are often shared by several fragments or functional groups of pesticide molecules. For example, amides are prevalent not only in insecticides [
78], [
79], [
80], [
81], [
82], [
83], [
84], [
85] but also in drugs targeting viruses [
86], [
87], [
88], [
89], [
90], [
91] and bacteria [
92], [
93], [
94], [
95], [
96]. Since DuPont took the lead in the research of mesoionic insecticides, accompanied by the two outstanding products triflumezopyrim and dicloromezotiaz [
97], [
98], the potential of these substances in the field of insecticide development has received extensive attention. Considering that mesoionic structures should have broader application prospects beyond their insecticidal properties, Gan et al. [
99] introduced the mesoionic pyrido[1,2-
a]pyrimidinone into the skeleton of vanillin for the first time, designing and synthesizing a series of novel pyrido[1,2-
a]pyrimidinone mesoionic compounds containing vanillin moieties (
Fig. 9 [
99]). To obtain the first building block for building mesoionic structures, 2-methylmalonic acid was reacted with 2,4,6-trichlorophenol in the presence of phosphorus oxychloride to generate bis(2,4,6-trichlorophenyl) 2-methylmalonate (
19) [
100]. Another unit (
21) was prepared by treating etherified vanillins (
20) with 2-aminopyridine to carry out a reductive amination [
101]. Finally, combining the two blocks
19 and
21 via a ring-closure reaction allowed the construction of the mesoionic products
22a-
22d.
These newly developed molecules exhibited promising anti-bacterial properties against phytopathogenic bacterium; in particular, compound
22c showed better activities against
Xanthomonas oryzae pv.
oryzae (
Xoo) and
Xanthomonas axonopodis pv.
citri (
Xac) (
Table 5 [
99]) than the control agents bismerthiazol and thiodiazole copper via
in vitro bio-evaluation. This work not only contributed a feasible method for structurally altering vanillin based on reductive amination but laid a solid foundation for the future application of mesoionic compounds to control plant bacterial diseases.
Another attempt to splice vanillin with the 1-position of the mesoionic pyrido[1,2-
a]pyrimidinone was carried out by Hu et al. [
40]. The preparation of intermediate
25 involved a sequence of reactions, including substitution, protection, and deprotection, utilizing vanillin as the initial material. 2-substituted bis(2,4,6-trichlorophenyl) malonates (
26) were produced by performing an esterification reaction. The subsequent cyclization of
25 with
26 under reflux conditions led to the formation of the intermediates
27 [
100], [
102], which were further subjected to different thiols to produce the desired compounds
28a-
28d.
In addition, the researchers constructed a three-dimensional (3D) quantitative structure-activity relationship model based on the EC
50 results of the antimicrobial activity of the compounds (
Fig. 10). This model allowed for further improvement of the drug structure, resulting in the birth of the optimum,
28d. Compound
28d was found to have significant antibacterial activity in a biological activity assay against
Xoo and
Xac (
Table 6 [
40]). More specifically, against
Xoo and
Xac, the EC
50 values for compound
28d were determined to be 10.9 and 17.5 μg∙mL
−1, respectively. These concentrations were less effective than those of bismerthiazol (29.3 and 39.8 μg∙mL
−1) and thiadiazole copper (64.8 and 78.1 μg∙mL
−1). Moreover, evaluation of the
in vivo antibacterial efficacy toward bacterial leaf blight (BLB) and bacterial leaf streak (BLS) indicated that compound
28d had protective activities of 43.9% and 41.7%, respectively. Also, the protective efficacy of this activity was found to be superior to that of thiodiazole copper, as thiodiazole copper showed protective activities of 40.6% and 35.0% versus BLB and BLS, respectively (
Table 7 [
40]).
Overall, the aforementioned studies offer various synthetic approaches that can be employed for the derivatization of vanillin, and a broader spectrum of antiviral activity has been achieved. Most importantly, they offer a solid foundation for further exploration of the vanillin framework in the realm of antibacterial research. In terms of design and synthesis, vanillin can be easily connected to many other active fragments to derive new compounds with biological activity. The synthesis method of vanisulfane is short and efficient, while the pathways of some other derivatization examples, such as compounds 18 and 28, require relatively longer steps (> five steps), which may increase the cost of synthesis and the use of organic solvents despite their acceptable bioactivities.
4. Anti-phytoviral mechanism of vanillin-derived pesticides
As these are new structures, the anti-viral mechanisms of action of vanillin-derived pesticides have rarely been reported. Nevertheless, most current available studies indicate that these substances may serve as plant immune activators [
103], [
104], [
105], [
106]. In contrast, only limited reports have shown that some vanillin derivatives can act on functional proteins involved in plant virions, resulting in inactivation of virus behavior [
107].
Given the outstanding protective performance of vanisulfane against CMV, in 2018, Song et al. [
103] investigated the vanisulfane-induced immune response of capsicum to CMV infection. They found that vanisulfane possessed the ability to enhance the defensive enzyme activities of peroxidase (POD), phenylalanine ammonia lyase (PAL), superoxide dismutase (SOD), and catalase (CAT). In addition, some relative defensive gene expressions were up-regulated after treatment with vanisulfane, which was confirmed by real-time reverse transcription polymerase chain reaction assay. The findings from label-free quantitative proteomics, in conjunction with bioinformatics analysis, suggested that the administration of vanisulfane leads to an up-regulation of DEAD-box ATP-dependent RNA helicase, which is mostly located in the mitochondria. This up-regulation subsequently results in an increased accumulation of reactive oxygen species (ROS), ultimately triggering the abscisic acid (ABA) response. The insufficient stimulation of ABA repressor 1 (ABR1) was found to enhance the biosynthesis of ABA while simultaneously suppressing the generation of salicylic acid (SA) and ROS, ultimately resulting in the development of disease resistance in pepper leaves. Thus, the study revealed through plant physiology, biochemistry analyses, and investigations at the gene and protein levels that vanisulfane has the capability to confer resistance to the CMV virus by augmenting the host’s innate resistance mechanisms (
Fig. 11).
Pepper mild mottle virus (PMMoV) is a pathogen in the Virgaviridae family that primarily affects pepper plants worldwide. The virus causes symptoms such as mild leaf mottling and puckering, reduced growth, and hampered fruit development. Managing PMMoV largely involves preventative approaches like using virus-free or resistant seeds, practicing good sanitation, crop rotation, pest management, and application of immune inducer. As a degradation product of chitosan, COS has gained significant attention in agricultural sectors owing to its ability to induce plant immune responses and confer resistance against various plant diseases. In 2022, Song et al. [
104] discovered that vanisulfane exhibited unexpected protective activity (59.4% ± 1.9%, at 500 μg∙mL
−1) against PMMoV, which contrasts with the control agent COS (36.9% ± 2.8%, at 500 μg∙mL
−1). Moreover, following the application of vanisulfane treatment, an increase in the levels of chlorophyll, flavonoids, and total phenols was observed, which would help remove harmful free radicals from plants. Concurrently, there was a notable augmentation in the activities of some enzymes associated with defensive mechanisms, namely POD, CAT, SOD, and PAL. Quantitative real-time polymerase chain reaction (RT-qPCR) and Western blotting techniques were used to provide evidence supporting the enhancement of UspA expression in
Capsicum annuum L. leaves following treatment with vanisulfane, at both the gene and protein levels. Based on the findings of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, it was determined that the differentially abundant proteins (DAPs) mostly participate in several biological processes, including starch and sucrose metabolism, photosynthesis, the MAPK signaling pathway, and the oxidative phosphorylation pathway.
Very recently, the phytohormone-dependent signaling pathway regarding vanisulfane-driven plant resistance to PVY was successfully discovered by Yu et al. [
105]. The initial step included in their study was the adoption of physiological and biochemical analysis to establish that vanisulfane has the capacity to not only raise the activities of CAT, SOD, POD, and PAL in
Nicotiana benthamiana L. (
N. benthamiana L.) but also induce the accumulation of ROS and the influx of Ca
2+. In this case, they performed transcriptome and proteome analysis to identify and quantify the genes and proteins associated with the PVY + vanisulfane and PVY treatments. The SA content in the plants was analyzed using liquid chromatography quadrupole-time-of-flight mass spectrometry (LC−QTOF−MS). RT-qPCR analysis was conducted to study the expression of key genes involved in the SA signaling pathway. The combined transcriptome and proteome analysis identified 30 genes that were differentially expressed in both the transcriptome and proteome levels in the PVY + vanisulfane and PVY treatments. Among these genes, 12 were found to be associated with both levels of analysis (
Table 8 ([
105])). The functional analysis revealed that these genes were involved in plant defense, hemicellulose and cell wall metabolism, and respiratory metabolism. The SA content analysis showed an increase in SA levels in the PVY + vanisulfane treatment compared with the PVY treatment. The RT-qPCR analysis confirmed the upregulation of key genes in the SA signaling pathway in the PVY + vanisulfane treatment. In particular, vanisulfane increased
POD52,
APX, and
PR-1 genes and proteins in the SA signaling pathway.
In transgenic Arabidopsis plants, vanisulfane was shown to increase SA accumulation, increase gene expression of
ICS1 and
PR-1, and impart resistance to PVY. Thus, vanisulfane treatment induces the expression of key defense proteins and activates the SA signaling pathway, leading to enhanced plant resistance against PVY (
Fig. 12). This study enhances our knowledge of the SA signaling pathway, thereby contributing to a more comprehensive understanding of the antiviral mechanisms in plants.
Certain vanillin viricides have the potential to manifest their antiviral properties through the inhibition of functional proteins associated with plant viruses. For example, CP holds significant importance inside the Closteroviridae virus, as it encapsulates approximately 95% of the viral DNA [
108]. Abnormal CPs might result in the failure of the virus to successfully generate intact and live virions. Consequently, an extensive amount of research has been dedicated to the development of drugs that specifically target the CP and impede its functionality for antiviral purposes [
53]. As an example, compound
13d achieved a superior anti-ToCV activity in comparison with NNM, COS, ribavirin, and its lead compound
7b. Hu et al. [
73] successfully expressed and purified ToCVCP in
Escherichia coli. Using MST technology, they were able to determine the binding affinity of compound
13d to ToCVCP. The results indicated that compound
13d exhibited a notable interaction with ToCVCP, as seen by its
Kd value of 0.12 μmol∙L
−1, which was considerably greater than the
Kd values of NNM (0.26 μmol∙L
−1), COS (0.82 μmol∙L
−1), ribavirin (37.26), and
7b (2.05). The anti-ToCV activity of these compounds was assessed
in vivo using quantitative reverse transcription polymerase chain reaction, which demonstrated that compound
13d, as well as the commercial antiviral agent NNM, effectively lowered the expression level of the
ToCV-CP gene in tomato.
Aside from CPs, there is evidence that the nucleocapsid (N) protein of TSWV may act as a molecular target for vanillin derivatives. The N protein plays a central role in multiple vital processes during the viral life cycle, serving as the primary multifunctional protein responsible for maintaining the structural integrity of the virus. Compound
29 demonstrates a high level of efficacy in inactivating TSWV. On this basis, Song et al. [
109] effectively introduced a prokaryotic expression vector, pET-32a-TSWV N, into
Escherichia coli. Subsequently, they proceeded with the expression and purification processes, resulting in the successful isolation of the purified N protein. With the purified N protein in hand, they proceeded to assess the binding affinity of the compound to TSWV N using MST (
Fig. 13). The results indicated that compound
29 exhibited the highest binding capacity to TSWV N (
Table 9 [
109]), with a binding affinity of 10.22 μmol∙L
−1, surpassing those of NNM (16.55 μmol∙L
−1) and ribavirin (126.56 μmol∙L
−1). The obtained data revealed a correlation between the inactivation activities of the compounds and their bonding strength, indicating that compounds with stronger bonding had superior inactivating properties. Furthermore, utilizing molecular docking techniques on the N protein and compound
29 showed that the compound has the capability to establish a stable complex with the N protein via several amino acid sites. These sites are of the utmost importance in the interaction between TSWV N and nucleic acids. Hence, it is plausible that the vanillin derivative under investigation possesses the ability to impede the dissemination and duplication of the virus through its capacity to obstruct the interaction between TSWV N and viral RNA.
In general, vanillin-derived anti-phytoviral agents have the potential to enhance plant resistance against viral infections. It is also possible for them to engage in direct interactions with the functional proteins of the virus, resulting in their inactivation. This, in turn, impacts several aspects of the virus biology, such as reproduction, assembly, and movement, ultimately serving as a means to achieve antiviral objectives.
5. Biosafety of vanillin-derived pesticides
Vanillin is usually considered safe for consumption by most people when used in normal food and drink quantities. It is widely used globally as a flavoring agent in various foods, beverages, and some medications [
110], [
111], [
112], [
113]. As mentioned earlier, the US FDA classifies vanillin as a substance that is GRAS for use in food. However, as with any chemical, excessive or inappropriate use can have negative effects. Thus, it is always advised to consume vanillin (like any other food additive) within moderation and recommended limits.
Although the safety of vanillin obtained from
Vanilla planifolia is generally acknowledged, there is no guarantee that pesticides derived from the vanillin skeleton are safe. In 2020, a detailed investigation of the hydrolysis degradation kinetics of vanisulfane in water was initially conducted by Hu et al. [
114] in situations involving the presence of exogenous chemicals. The results indicated that vanisulfane possesses considerable stability in the context of Ni
2+, Zn
2+, Pb
2+, and Fe
3+ aqueous solutions. However, the degradation of vanisulfane was substantially accelerated by Cu
2+ and fulvic acid (FA)—a common acid present in soil that is widely recognized for its capacity to augment the uptake of nutrients in plants. In addition, vanisulfane has the potential to undergo chemical transformations, including thioether cleavage, reverse thioacetalization, ether bond cleavage, demethylation, and intramolecular dehydration, resulting in the formation of compounds
D,
E,
F,
G, and
H, respectively (
Fig. 14). The study demonstrated that vanisulfane exhibits quick degradation when subjected to comparable soil conditions involving the presence of copper ions and FA.
In 2021, Ye et al. [
115] revealed the tissue distribution, excretion, and metabolism of vanisulfane in rats, specifically with regards to sex-related characteristics (
Fig. 15). Their investigation, which was conducted using
14C labeling, indicated that a significant proportion (83.30%-87.51%) of the administered
14C activity was eliminated by urine and feces during the 24-h period following oral ingestion. The liver and kidney exhibited the highest levels of
14C accumulation in both males and females. Unexpectedly, it was revealed that males exhibited lower body clearance compared with females 24 h after treatment, and male rats showed a preference for biliary excretion of the pesticide, whereas female rats exhibited a preference for renal excretion. Furthermore, together with two oxidized metabolites
A1 and
A2, the glucuronide conjugate
A3 was identified through the utilization of LC-QTOF-MS in conjunction with
14C labeling.
The aforementioned tests provided empirical evidence regarding the fate of vanisulfane, particularly in relation to its distribution and excretion pathways. Moreover, these investigations resulted in the identification of potential metabolites that may undergo metabolic processes. Nevertheless, there has been a lack of research examining the specific metabolic pathways by which these compounds can be obtained. Thus, soon after, Ye et al. [
107] conducted further investigations into the probable metabolic routes of vanisulfane in rats of both sexes. Their work employed two chemicals labeled at distinct places to discern potential variations in the metabolic pathways of distinct benzene rings in rat organisms. Through high-resolution mass spectrometry analysis, a total of eight metabolites (
30-37) of vanisulfane were successfully identified in the rat population (
Fig. 16). Both phase I and phase II metabolism were observed in both genders. Phase I metabolism mostly involves oxidative processes, while phase II metabolism features binding reactions to produce glucuronides, sulfates, and amino acid conjugates. It is worth noting that gender differences may have implications for the timeframe in which these metabolites are formed. Later, investigations involving the accumulation and biotransformation of vanisulfane in livestock following food exposure, as well as the destiny of vanisulfane originating from chicken manure in plant-soil systems, were carried out by Ye et al. [
107], [
115], [
116].
To summarize, the existing biosafety assessment cases for vanillin-derived pesticides have predominantly focused on vanisulfane. Hence, further investigation may be warranted regarding other types of vanillin-based derivatives. 14C labeling in conjunction with LC-QTOF-MS serves as a powerful and valuable research methodology for investigating the processes of drug accumulation, metabolic pathways, and metabolite analysis. It is posited that further exploration of potential applications for this amalgamation will continue. Moreover, while the available evidence suggests that vanisulfane is a readily degradable pesticide and is relatively safe for rats and livestock, more stringent pesticide registration tests may be needed for further toxicological evaluation.
6. Perspectives
In this work, we have summarized the progression from the initial development of an antiviral compound derived from vanillin to the subsequent production of other derivatives with enhanced activity against a wider range of agricultural pests. The mechanisms by which these compounds exert their antiviral effects and evaluate their biosafety were also discussed. Although vanillin itself does not possess strong inhibitory effects against harmful organisms, its unique structural framework facilitates straightforward, efficient, and low-cost conjugation with various efficacious pesticidal groups. Moreover, its guaranteed safety and low selling price make vanillin an ideal candidate for pesticide development. A considerable range of modifications have been carried out on the -OH moiety of vanillin, resulting in the creation of several compounds with high levels of bioactivity. The vanillin-derived pesticides that are now documented have demonstrated notable antiviral activities and promising bacteriostatic activity properties. However, it is advisable for further research to be directed toward the aldehyde component of this substance, encouraging unique chemical transformations to generate a diverse array of innovative structures in the future. A series of possible reactions of aldehydes can be attempted, such as the aldol reaction and benzoin condensation, or vanillin can be given an α,β-unsaturated structure through the Perkin reaction. By using asymmetric catalytic strategies, it may even be possible to implement asymmetric versions of these approaches to develop chiral vanillin-based pesticides. Furthermore, it is expected that the next generation of vanillin-based agrochemicals will be explored for development as insecticides and herbicides by further modifying the aldehyde moiety, such as introducing common insecticidal active units (e.g., 2-chlorothiazole, 2-chloropyridine, and cyano) or herbicidal pharmacophores (e.g., pyrimidinedione and triazinone).
As another direction, studying the solid chemistry—including polymorphs, cocrystals, and salts—of pesticides is of great significance for improving their physical properties and bioavailability and thereby enhancing their overall performance in the field. Given the ongoing reports of solid chemistry studies involving vanillin [
117], [
118], [
119], there is a compelling case for extending these investigations to encompass vanillin-based pesticides. This extension could unlock crucial insights, potentially leading to the development of more effective agrochemicals. Mechanically, many vanillin derivatives have demonstrated the ability to function as plant immune activators. This feature is highly advantageous in the practical management of plant viral infections, as it operates by stimulating the plant’s inherent defense mechanisms rather than directly targeting the pathogen. Consequently, the development of resistance to phytoimmune inducers poses a considerable challenge for pathogens, in contrast to their response to conventional chemical pesticides.
Despite these promising findings, studies on the mechanism of action of vanillin-derived immune activators are still insufficient. The use of proteomics coupled with bioinformatics analysis only allows preliminary elucidation of the relevant resistance pathways, which does not directly help to screen for drug target proteins. Moreover, how target proteins mediate plant resistance to viruses is largely unknown. Fortunately, due to ongoing advancements in molecular biology techniques, a variety of sophisticated methods such as activity-based protein profiling, co-immunoprecipitation, yeast two-hybrid screening, immunoblotting, and others can potentially be employed to identify the target proteins of highly active molecules and acquire a deeper understanding of their mediated functions. Few biosafety evaluations have been carried out for vanillin-derived pesticides. However, there is hope that vanisulfane—as a novel phytoimmune inducer—will gain formal registration in China in the near future, followed by commercialization. Before that, it has been subjected to stringent toxicological and environmental safety assessments. We expect vanisulfane to be applicable to a range of virus pathogens.
7. Summary
The inherent safety of the natural food additive vanillin makes derivation based on this substance highly promising for the development of green pesticides. Overall, this review summarized and provided insight into recent advancements in pesticide engineering based on natural vanillin concerning molecule design, synthetic methodology, bio-evaluation, mechanisms of action, and bio-security assessment. We hope this review will introduce researchers to valuable empirical findings. Undoubtedly, this is an evolving story, and it is anticipated that a proliferation of environmentally friendly pesticides derived from natural compositions will be developed in future, thereby catering to the needs of sustainable agricultural practices.
Acknowledgments
The authors are grateful to the National Natural Science Foundation of China (32330087 and 32272590) and the National Key Research and Development Program of China (2022YFD1700300) for financial support.
Compliance with ethics guidelines
Mingshu Lou, Sha Li, Fangru Jin, Tangbing Yang, Runjiang Song, and Baoan Song declare that they have no conflict of interest or financial conflicts to disclose.
PDF
(4342KB)
