6 Benzylaminopurine Synthesis Essay

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1. Introduction

The phytohormones termed cytokinins regulate numerous aspects of plant development including cell division, leaf senescence, apical dominance and nutrient translocation, and appear to be essential for plant growth. Cytokinins are N6-substituted adenines and the naturally occurring compounds are of two types: compounds with an isoprenoid substituent (e.g., zeatin, 6-(4-hydroxy-3-methylbut-2-enylamino)purine) and those with an N6-aromatic ring (e.g., 6-benzylaminopurine (BAP)) [1]. Three membrane-located histidine kinase cytokinin receptors, termed AHK2, AHK3 and AHK4, have been characterised in Arabidopsis thaliana [2] and are located on the plasma membrane and/or endoplasmic reticulum [3]. Similar receptors necessary for nodule organogenesis have been detected in roots of Lotus japonicas plants [4]. Studies of binding of cytokinin-active BAP derivatives to the histidine kinase receptors indicated that another receptor able to bind BAP derivatives occurs in plants and it was proposed that diverse recognition systems may operate for cytokinins [5].

In addition to the membrane-bound histidine kinase receptors, numerous cytokinin-binding soluble proteins (CBPs) have been detected in higher plants [6,7]. Most have been partially characterised and some exhibit receptor-like properties including a correlation between affinities for different cytokinins and their biological activities. However, in no case has the encoding gene for the CBP been identified and cloned and the physiological significance of the CBPs remains uncertain. The very few which do appear to exert a regulatory role include 67 kDa CBPs from barley and Arabidopsis leaves. These proteins bind to zeatin to promote in vitro transcription directed by RNA polymerase I and also appear to be nuclear CBPs [8,9]. Further significant CBPs are a 70 kDa protein involved in cytokinin regulation of transcription in plastids [10] and HOG1 protein of Arabidopsis which appears to play a role in modulating cytokinin signal transduction [11]. It is also possible that some CBPs may have a passive role by simply sequestering cytokinin to stabilise and protect the hormone.

In addition to their role as plant hormones, cytokinins may influence the normal growth and activity of some human cells in vitro [12,13,14,15]. Kinetin (6-furfurylaminopurine) suppressed free radical formation associated with platelet aggregation and appeared to have antithrombotic activity in vivo [16]. Considerable interest now focuses on the ability of cytokinin 9-ribosides to inhibit growth of cancer cells and there are many reports in this regard, including some in the early cytokinin literature, e.g., [17,18]. While cytokinin bases induced differentiation of human leukaemia HL-60 cells into granulocytes [19], cytokinin ribosides caused apoptosis in these cells [20] and in HeLa and mouse melanoma cells [21]. While the mass of melanoma tumours on mice was very markedly reduced by injection of kinetin riboside [21], this riboside also induced G2/M arrest and death in human heptamoa cells in vitro [22] and inhibited cyclin expression in myeloma cells causing cell cycle arrest [23]. In a recent study, the cytotoxic activities of almost all known naturally occurring cytokinins (over 40 compounds) were determined towards a diverse range of human tumour cell lines [24,25]. Cytotoxicity and cytokinin activity in plant cell division bioassays did not correlate. iPA and the ribosides of kinetin, BAP and o-hydroxy-BAP (o-topolin) were the most effective inhibitors of tumour growth, exhibiting cytotoxicity at about 0.5–5 μM in 7 of 9 tumour cell lines. Small structural changes in the N6-substituent altered inhibitory activity markedly, suggesting the receptors/binding proteins involved recognised this structural feature on which growth promotion in plants also depends.

Cytokinins may also be important in the development of insects. Larvae have been found to synthesise isoprenoid cytokinins, which are secreted to induce gall formation [26]. In newly laid eggs of the insect Locusta migratoria, the cytokinin N6-isopentenyl adenosine 5′-monophosphate occurs in ester linkage to the steroid hormone ecdysone [27]. Important contributions from cytokinins are suggested by the capacity of BAP at less than 10−6 M in culture medium to replace foetal calf serum in stimulating growth of Drosophila cells [28] and the ability of kinetin added at low levels to the diet of fruit flies (Zaprionus paravittiger) to prolong their life span [29].

Further analysis of cytokinin action at the molecular level in higher plants, mammalian cells and insects requires characterisation of cytokinin receptors/binding proteins in diverse organisms, a difficult biochemical problem. Two new approaches that could be developed for this task are based on yeast cells expressing cDNA libraries from target organisms. One is based on the yeast three hybrid (Y3H) system [30] and the other on the fluorescence activated cell sorting technique [31,32]. The proposed former system is designed on the detection of a specific reporter gene signal triggered by the interaction between DNA-binding and activation domains of the yeast GAL4 transcription factor upon cytokinin binding to a receptor in yeast cells. It would utilise three key components, namely, (1) a fusion protein with a dexamethasone receptor linked to the DNA-binding domain of GAL4; (2) a second fusion protein in which a target CBP expressed by the cDNA library is fused to the activation domain of GAL4; and (3) a synthetic hybrid ligand in which dexamethasone (regarded as the ‘anchor’ molecule [30]) is linked to BAP through a C10 spacer. Once the hybrid ligand is bound by both dexamethasone and BAP receptors produced in yeast, the respectively fused DNA-binding and activation domains will join together to trigger the expression of the reporter gene controlled by GAL4. This allows selection of yeast cells that harbour the cytokinin-binding protein. The methodology for producing the two fusion proteins in yeast has been established [30] and only the ligand molecule requires development to make the system relevant to CBPs. This paper therefore details the first synthesis of compounds in which a cytokinin is linked at one of three positions through a spacer of 10 carbon atoms to a dexamethasone moiety. In some studies unrelated to cytokinins, this has been replaced by alternative “anchor” molecules, e.g., methotrexate and FK506 [30].

Since individual yeast cells, in a population that has been exposed to a gene library, receives only a single introduced gene for expression, this can be readily recovered and the encoded protein characterised. The Y3H method is a powerful system and has been used to identify the targets of several inhibitors and drugs in mammalian cells and target proteins for cucurbic acid methyl ester and for salicylic acid in plant cell libraries [30]. This system has advantages over earlier biochemical strategies in that the interaction between receptor and ligand is determined in vivo. Furthermore the method is independent of the quantity of biological material available and can lead directly to the sequencing and identification of hormone-binding protein.

However, the Y3H method as currently employed is limited to the identification of soluble proteins that move to the yeast nucleus, while proteins located on membranes and organelles will not be recognised. To test for such proteins, we here also show that cytokinins can be chemically modified to conjugates that retain biological properties of cytokinin but are also targets for fluorescence detection. With such reagents, fluorescence activated cell sorting (FACS) [31,32] can be used to recover individual yeast cells that express a membrane-bound CBP, as discussed below.

This system utilising FACS is based on BAP-biotin conjugates. If any yeast cell expresses a membrane-bound CBP, FACS would be able to recognise it due to binding BAP-biotin conjugate detectable by streptavidin-phycoerithrin. The sorted cell would be recovered by growth on agar medium leading to characterisation of the introduced gene for a cytokinin-binding protein and study of its cellular location by fluorescent microscopy. Hence, in this paper we report the synthesis and properties of compounds in which BAP is linked to either dexamethasone or biotin through a C10 spacer moiety for use in the Y3H and FACS methods respectively.

2. Results and Discussion

2.1. Synthesis of Dexamethasone Conjugates

Dexamethasone does not have a functional group suitable for conjugation to a cytokinin moiety. To confer such a group, dexamethasone was oxidised with periodate to convert the hydroxymethyl ketone moiety into a carboxyl group and the resulting compound is referred to herein as “dexamethasone acid”. Conversion to a succinimidyl ester (1; Figure 1) by reaction with N-hydroxy-succinimide in the presence of dicyclohexylcarbodiimide yielded an activated ester. This reacted at 20–25 °C with the free amino group of amino-alkyl amino adenines to yield the corresponding amides with a dexamethasyl (DM) moiety (2). This approach based on activated ester and then amide formation appears to be the only convenient conjugation procedure for dexamethasone.

The amine 2-(10-aminodecylamino)-6-benzylaminopurine (3a) was readily prepared by reacting 1,10-diaminodecane with 6-benzylamino-2-chloropurine at 156 °C in isoamyl alcohol. However, the synthesis of N-9 alkyl derivatives of BAP and adenine terminating in a free amino group was more problematic, but was achieved after initial reaction with ethyl acrylate in ethanol containing sodium ethoxide (a known Michael type reaction at N-9; Figure 2). This introduced a CH2CH2COOC2H5 moiety at N-9 of adenine and BAP and further reaction with 1,10-diaminodecane at 110 °C yielded the amines 3-(6-benzylaminopurin-9-yl)propionamido-decylamine (4a) and 3-(6-aminopurin-9-yl)propionamido-decylamine (7a).

To facilitate synthesis of BAP linked at position C-8 to an aminoalkyl moiety, 8-bromoadenosine was benzylated with benzyl bromide (Figure 2). Alkylation of a 9-substituted adenine is known [33] to yield a 1-substituted derivative and a 1-benzyl product was obtained above. This was then converted by the Dimroth rearrangement [34] to 6-benzylamino-8-bromopurine 9-riboside. The latter was separated from 6-benzylamino-9-benzyl-8-bromopurine, which also formed in the benzylation reaction. The benzylamino riboside and 8-bromo-adenosine were then reacted with 1,10-diamino-decane yielding 8-(10-aminodecylamino)-6-benzylamino-9-β-d-ribofuranosylpurine (5a) and 6-amino-8-(10-aminodecylamino)-9-β-d-ribofuranosylpurine (8a). The ribose moiety of 5a was cleaved at 37 °C in methanol containing HCl and 2,2-dimethoxypropane yielding 6a.

The above 6 amines (3a8a) reacted readily with the dexamethasone active ester (1) yielding the corresponding amide conjugates (3b8b) with the dexamethasyl moiety (DM, 2). These compounds were characterised by UV and mass spectral data (Table 1). The mass spectral data confirm the structures assigned to compounds 3b to 8b. Further confirmation was provided by the analysis of their UV spectra, which shows that each contains a dexamethasone acid moiety linked to BAP (3b6b) or adenine (7b, 8b). Both dexamethasone acid and the purine moieties in the conjugates exhibit strong UV absorption but the λmax values for the two are quite different (dexamethasone acid, λmax 240 nm; the substituted purines typically show absorption at 200–240 nm and at 265–300 nm but the spectrum depends on the site of substitution (i.e., linkage). Pure crystalline model purine compounds were synthesised for UV spectral comparison with the conjugates. Each synthesised compound and the purine part of the corresponding conjugate would exhibit an identical UV spectrum. These compounds were 6-benzylamino-2-n-hexylaminopurine (cf.3b), ethyl 3-(6-benzylaminopurin-9-yl)propionate (cf.4b), 6-benzylamino-8-n-hexylaminopurine (cf.6b), ethyl 3-(6-aminopurin-9-yl)propionate (cf.7b) and 8-n-pentylaminoadenosine (cf.8b). The UV spectrum of each compound differed considerably from that of the corresponding conjugate in Table 1, but when dexamethasone acid was added in an equimolar amount to the model compound solution, the spectrum became identical to that of the conjugate. This confirms the purity and identity of the conjugates listed in Table 1.

2.2. Synthesis of Biotin Conjugates

To synthesise the biotin-cytokinin conjugates 5c and 6c (Figure 1) for FACS, biotin was converted to the succinimidyl ester and reacted with the amines 5a and 6a respectively. Both 5c and 6c contain a biotin moiety linked through a C10 spacer to the C-8 of the purine ring, but 5c has at N-9 a ribofuranosyl moiety, a frequent feature of naturally occurring cytokinins. The biotin structure does not exhibit significant UV absorption and the UV spectrum of the biotin conjugates closely match those of the corresponding BAP-amines used in their synthesis.

In order to prepare biotin conjugates, suitable for FACS, from purines with short amino- alkyl sides chains, an amidocaproyl spacer moiety was added to the C4 side chain of biotin. Formation of the succinimidyl active ester and reaction with, for example, 9-(3-aminopropyl)-6-benzylaminopurine yielded 9 with a nine carbon linkage between the biotin and N-9 of BAP. Addition of a second spacer moiety would yield a biotin derivative to which BAP could be conjugated directly through an amino group inserted on the benzene ring.

2.3. Cytokinin Activity of Conjugates

The activities of the dexamethasone- and biotin-BAP conjugates were determined using a bioassay based on the ability of cytokinins to induce betacyanin pigment formation in the excised cotyledons of Amaranthus seedlings when cultured in darkness [35,36]. The bioassay shows high sensitivity and specificity for cytokinins.

The dexamethasone conjugates 3b6b all exhibited cytokinin activity when tested alone, but this was much less than that of BAP. They also significantly/ reduced the activity of BAP and 6b was the most active (Table 2). These results suggest that the dexamethasone conjugates possess certain low cytokinin activity but can compete with BAP for receptor binding. The 6-aminopurine-dexamethasone conjugates 7b and 8b (data not shown) were inactive indicating that the response observed for 3b6b required the benzyl substituent. Substitution of the 6-amino group is required for cytokinin activity in plants. The biotin-BAP conjugates 5c and 6c exhibited very similar activity to that of the corresponding dexamethasone compounds 5b and 6b (data not shown).

The activity of BAP in the presence of compounds 3b6b is particularly relevant. Compounds 3b6b (Table 2) and also 5c and 6c (data not shown) reduced the response observed for BAP, 6b with a purine C-8 linkage being the most effective. This is consistent with the view that the above conjugates compete with BAP for binding at a receptor. Hence the conjugates reported above, especially 6b, appear to be suitable for a Y3H system for cytokinin receptors in plants.

Unlike BAP, the above conjugates (3b6b) do not appear to function effectively after binding to the receptor resulting in reduced response in the bioassay. It is relevant that cytokinin analogues are now recognised that bind to characterised cytokinin receptors competing with active cytokinins, but the analogues evoke a negligible cytokinin response [37,38,39].

Relevant results were obtained with three simple alkyl derivatives of BAP. The BAP derivatives 2-hexylamino-BAP (A), 9-undecenyl-BAP (B) and 8-hexylamino-BAP (C) which resemble 3b, 4b and 6b respectively, but lack a dexamethasone moiety, showed cytokinin activity very similar to that of the above three dexamethasone conjugates (data not shown). Hence the reduced cytokinin activity of 3b6b, relative to that of BAP, is probably largely attributable to the long chain alkyl substituents present. While the compounds A, B and C all reduced the activity of BAP in the bioassay, C with a C-8 substituent appeared to be the most effective. The increment in betacyanin absorbance induced by BAP (5 μM) was reduced by A, B and C (each at 20 μM) by 23%, 15% and 39% respectively. Based on this result and the data of Table 2, it can be concluded that C-8 of BAP is the preferable linkage position for synthesis of conjugates for cytokinin binding studies.

Finally, it should be noted that the betacyanin response was used as a convenient bioassay to characterise the synthesised conjugates, but the relevant receptor is probably one of the identified membrane-associated receptors (compare [37,39]). The proposed Y3H system is very unlikely to detect this receptor or similar membrane-bound receptors that cannot translocate to the yeast nucleus. Soluble non-membrane bound CBPs have been detected in plants [8,9]. The proposed Y3H system has the potential to identify and clone these soluble proteins and thus define new regulatory CBPs in plants.

Abstract

Yeast cells expressing cDNA libraries have provided two new approaches to facilitate further identification of cytokinin-binding proteins and receptors. These are the yeast three hybrid (Y3H) system and fluorescence activated cell sorting (FACS). The Y3H system requires a synthetic hybrid ligand comprising an “anchor” moiety (e.g., dexamethasone) linked to a cytokinin via a spacer. In the yeast nucleus, this ligand by binding connects two fusion proteins leading to a reporter gene activation and detection and characterisation of cytokinin binding proteins. Herein is reported the first synthesis of dexamethasone-cytokinin ligands with a spacer linkage. This was attached to the purine ring of 6-benzylaminopurine (BAP) at positions 2, 8 or 9. To achieve this, dexamethasone was modified by periodate oxidation yielding a carboxylic group used for conjugation to the spacer by amide formation. Biotinyl derivatives of cytokinins for FACS included those synthesised by reaction of an activated ester of biotin with 8-(10-amino-decylamino) derivatives of BAP and BAP 9-riboside. Properties of the conjugates and some biological situations where they could be applicable are discussed briefly. View Full-Text

Keywords: 6-benzylaminopurine (BAP); BAP-dexamethasone conjugates; BAP-biotin conjugates; yeast three hybrid; cytokinin-binding proteins6-benzylaminopurine (BAP); BAP-dexamethasone conjugates; BAP-biotin conjugates; yeast three hybrid; cytokinin-binding proteins

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MDPI and ACS Style

Wang, Y.; Letham, D.S.; John, P.C.L.; Zhang, R. Synthesis of a Cytokinin Linked by a Spacer to Dexamethasone and Biotin: Conjugates to Detect Cytokinin-Binding Proteins. Molecules2016, 21, 576.

AMA Style

Wang Y, Letham DS, John PCL, Zhang R. Synthesis of a Cytokinin Linked by a Spacer to Dexamethasone and Biotin: Conjugates to Detect Cytokinin-Binding Proteins. Molecules. 2016; 21(5):576.

Chicago/Turabian Style

Wang, You; Letham, David S.; John, Peter C.L.; Zhang, Ren. 2016. "Synthesis of a Cytokinin Linked by a Spacer to Dexamethasone and Biotin: Conjugates to Detect Cytokinin-Binding Proteins." Molecules 21, no. 5: 576.

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