Synthesis of a cytokinin linked by a spacer to dexamethasone and biotin: Conjugates to detect cytokinin-binding proteins

Article abstract of DOI:10.3390/molecules21050576

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.

Full text of DOI:10.3390/molecules21050576

molecules
Article
Synthesis of a Cytokinin Linked by a Spacer to
Dexamethasone and Biotin: Conjugates to Detect
Cytokinin-Binding Proteins
You Wang 1, David S. Letham 2, Peter C. L. John 2 and Ren Zhang 1,
*
1
School of Biological Sciences, University of Wollongong, Wollongong, NSW 2522, Australia;
wyou@uoguelph.ca
Research School of Biology, Australian National University, Canberra, ACT 0200, Australia;
david.letham@bigpond.com (D.S.L.); peteandjenny.john@gmail.com (P.C.L.J.)
Correspondence: rzhang@uow.edu.au; Tel.: +61-2-42213427
2
*
Academic Editor: Derek J. McPhee
Received: 19 February 2016; Accepted: 25 April 2016; Published: 30 April 2016
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.
Keywords: 6-benzylaminopurine (BAP); BAP-dexamethasone conjugates; BAP-biotin conjugates;
yeast three hybrid; cytokinin-binding proteins
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)) [
AHK4, have been characterised in Arabidopsis thaliana [
and/or endoplasmic reticulum [ ]. Similar receptors necessary for nodule organogenesis have been
detected in roots of Lotus japonicas plants [ ]. Studies of binding of cytokinin-active BAP derivatives to
1]. Three membrane-located histidine kinase cytokinin receptors, termed AHK2, AHK3 and
2] and are located on the plasma membrane
3
4
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
Molecules 2016, 21, 576; doi:10.3390/molecules21050576
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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
formation associated with platelet aggregation and appeared to have antithrombotic activity in vivo
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].
[
12
–15]. Kinetin (6-furfurylaminopurine) suppressed free radical
[16].
,
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 µ
M 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 ecdyso´ne6 [27]. Important contributions from cytokinins are
suggested by the capacity of BAP at less than 10 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

Molecules 2016, 21, 576
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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

Molecules 2016, 21, 576
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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.
1
2 (DM)
3a R = H; 3b R = DM
4a R = H; 4b R = DM
5a R = H; 5b R = DM; 5c R = biotinyl
6a R = H; 6b R = DM; 6c R = biotinyl
7a R = H; 7b R = DM
8a R = H; 8b R = DM
9
Figure 1. Structures of compounds used and synthesised in this study.

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Figure 2. The sequence of reactions used to synthesise BAP-dexamethasone conjugates with purine
ring linkage at C-8 (5b 6b) or N-9 (4b). (A) 8-bromoadenosine; (B) 1-benzyl-8-bromoadenosine;
(C) 6-benzylamino-8-bromopurine 9-riboside; (D) 6-benzylamino-9-benzyl-8-bromopurine;
,
(E) 6-benzylaminopurin; (F) ethyl 3-(6-benzylaminopurine-9-yl)propionate. The reactions are:
(i) benzylbromide in DMF at 37 ˝C; (ii) 0.25 M NH4OH at 37 ˝C; (iii) 1,10-diaminodecan in n-PrOH at
100 ˝C; (iv) methanolysis with HCl at 37 ˝C; (v) activated ester of “dexamethasone acid” at 23 ˝C;
(vi) ethyl acrylate/NaOEt; (vii) 1,10-diaminodecane in toluene at 110 ˝C.
The above 6 amines (3a
–
8a) reacted readily with the dexamethasone active ester (1) yielding the
corresponding amide conjugates (3b
–
8b) with the dexamethasyl moiety (DM, ). These compounds
2
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 (3b
or adenine (7b, 8b). Both dexamethasone acid and the purine moieties in the conjugates exhibit strong
UV absorption but the max values for the two are quite different (dexamethasone acid, max 240 nm;
–6b)
λ
λ
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.
)

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Table 1. List of purine-dexamethasone conjugates showing numbers assigned to compounds and molecular formulae, position and structure of linkages, other purine
substituents present and mass spectral and UV characteristics of compounds. UV spectra were determined in 95% (v/v) ethanol containing HCl (0.1 M). Accurate m/z
values for M + H molecular ions determined by ESI-MS are listed in a footnote. Calc. denotes the calculated value A
.
Conjugate
Number
Purine Location and
Structure of Linkage
Other Purine
Substituents
MS
m/z
UV λ
(log ε)
nm
UV λmin nm
(log ε)
max
FAB (rel.int.)
204 (4.50), 238 (4.57),
248 (sh) (4.55), 291
(4.20)
3b
Purine C-2
-NH-CH2-(CH2)8-CH2-NH-
6-benzylamino
6-benzylamino
756 [M + H]+ (100)
254 (50), 253 (97)
217 (4.34), 274 (4.11)
232 (4.23)
253 (4.16)
277 (3.91)
226
C43H58O4N7F
FAB (rel. int.)
4b
Purine N-9
-CH2-CH2-CO-NH-CH2-(CH2)8-CH2-NH-
812 [M + H]+ (37)
280 (41), 226 (100)
211 (4.45), 264 (4.37)
221 (4.48), 275 (4.24)
238 (4.38), 308 (4.25)
210, 256 (4.40)
C46H62O5N7F
ESI-MS
5b
Purine C-8
-NH-CH2-(CH2)8-CH2-NH-
6-benzylamino
9-ribofuranosyl
888 [M + H]+
910 [M + Na]+
C48H66O8N7F
ESI-MS
6b
Purine C-8
-NH-CH2-(CH2)8-CH2-NH-
6-benzylamino
6-amino
756 [M + H]+
778 [M + Na]+
C43H58O4N7F
ESI-MS
7b
Purine N-9
-CH2-CH2-CO-NH-CH2-(CH2)8-CH2-NH-
722 [M + H]+
744 [M + Na]+
C39H56O5N7F
ESI-MS
8b
Purine C-8
-NH-CH2-(CH2)8-CH2-NH-
6-amino
9-ribofuranosyl
798 [M + H]+
820 [M + Na]+
219 (4.49), 269 (4.27)
256 (4.24)
C41H60O8N7F
A
3b: 756.4602, Calc. 756.4613; 4b: 812.4883, Calc. 812.4875; 5b: 888.5023, Calc. 888.5035; 6b: 756.4606, Calc. 756.4613; 7b: 722.4416, Calc. 722.4405; 8b: 798.4580, Calc. 798.4566.

Molecules 2016, 21, 576
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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 3b–6b 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 3b–6b
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).
Table 2. Betacyanin level in Amaranthus seedlings cultured in the presence and absence of
BAP-dexamethasone conjugates and of BAP. Betacyanin level was determined by the difference in
absorbance of seedling extract at 542 and 620 nm. Each replicate combined 15 cotyledons.
A542nm ´ A620nm ˘ SE A
Conjugates (20 µM)
BAP (0.0 µM)
BAP (2.5 µM)
BAP (5.0 µM)
None
3b
4b
5b
6b
0.023 ˘ 0.004
0.061 ˘ 0.004
0.067 ˘ 0.003
0.062 ˘ 0.003
0.077 ˘ 0.003
0.283 ˘ 0.001
0.242 ˘ 0.001
0.241 ˘ 0.003
0.204 ˘ 0.002
0.192 ˘ 0.002
0.308 ˘ 0.002
0.259 ˘ 0.003
0.267 ˘ 0.002
0.245 ˘ 0.002
0.230 ˘ 0.006
A
The overall least significant difference = 0.011 (p = 0.01).
The activity of BAP in the presence of compounds 3b
–
6b is particularly relevant. Compounds
3b
–6b (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 (3b–6b) 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 [37–39].

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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 3b–6b, 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
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 [ ]. The proposed Y3H system
,39]). The proposed Y3H system is very unlikely to
8,9
has the potential to identify and clone these soluble proteins and thus define new regulatory CBPs
in plants.
3. Experimental Section
3.1. Thin-Layer Chromatography (TLC)
Merck silica gel 60 PF254 was used for normal phase TLC and layers were developed in
tanks lined with filter paper saturated with solvent. Solvents used were (proportions are by vol.):
(A) chloroform/methanol (9:1); (B) chloroform/methanol (9:1) with 14 M NH4OH added to solvent
in tank (0.2 mL/100 mL) and to paper lining tank (several drops); (C) chloroform/methanol (4:1);
(D) chloroform/methanol (4:1) plus NH4OH as for solvent B; (E) n-butanol/H2O/14 M NH4OH
(6:2:1) upper phase; (F) methyl acetate/H2O/acetic acid (500:125:40). Products were eluted with the
minimum volume of ethanol (unless stated otherwise) after the silica gel had been packed into a
small column.
3.2. UV and Mass Spectra
UV spectra were recorded in ethanol–H2O (95:5 v/v) containing 0.1 M HCI (acid spectra) or 0.2 M
NH4OH (base spectra) using a Shimadzu UV-240 spectrophotometer. Electron-impact mass spectra
(EI-MS) were determined at 70 eV and samples were introduced via the direct probe inlet. A VG
ZAB/2F mass spectrometer was used to determine fast atom bombardment mass spectra (FAB-MS) at
8 kV using a glycerol matrix. For electrospray ionization spectra (ESI-MS), the samples were introduced
by direct infusion in a methanol solution (0.3–0.6 mM) for positive ion spectra. Molecular formulae
were determined by high resolution (HR) ESI-MS, with a Waters Xevo G1 QTof mass spectrometer
using the following parameters: capillary v˝oltage, 2.5 kV; source temperature, 80 ˝C; sampling cone
voltage, 20 V; desolvation temperature, 350 C.
3.3. Synthesis of Aminoalkylaminopuries
3.3.1. 2-(10-Aminodecylamino)-6-benzylaminopurine (3a)
2,6-Dichloropurine was reacted with benzylamine to yield 6-benzylamino-2-chloropurine.
This compound (21 mg) and 1,10-diaminodecane (46 mg) were suspended in isoamyl alcohol (0.75 mL)
and the mixture was heated at 156 ˝C for 8 h. The reaction solution was diluted with water, acidified
with acetic acid and extracted with n-butanol/ethylacetate (1:1 by vol). The extracts were concentrated
and subjected to preparative TLC on silica gel (solvent D) yielding compound 3a. UV (acid):
λ
max
(log ) 237 (4.30), 252 (4.32), 291 (4.20) nm; λmin (log ) 242 (4.29), 271 (3.94) nm. UV (base): (log ε)
ε
ε
λ
max

Molecules 2016, 21, 576
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232 (4.36), 255 (sh) (3.96), 291 (4.03) nm; λmin (log
ε
) 268 (3.72) nm. EI-MS m/z (rel. int.) main ions:
395 (M+, 57%), 267 (21), 253 (54), 240 (37), 106 (18), 91 (100).
3.3.2. 3-(6-Benzylaminopurin-9-yl)propionamido-decylamine (4a)
BAP was reacted with ethyl acrylate to yield ethyl 3-(6-benzylaminopurin-9-yl)propionate [39].
This ester with MS, NMR and other properties identical to those previously reported [36] (102 mg),
1,10-diaminodecane (163 mg) and toluene (200 µ
L) were heated in a sealed vial at 110 ˝C for 2.5 h.
The reaction mixture was dissolved in ethanol (2 mL) and water (4 mL) added. The solution was
acidified with HCl and extracted with ethyl acetate (extracts discarded). The aqueous phase was then
adjusted to pH 11–12 with KOH and extracted again with ethyl acetate. The extracts were washed with
water, concentrated and subjected to preparative TLC on silica gel (solvent D) yielding compound 4a
.
UV (acid):
λ
(log
ε
) 268 (4.23) nm; λmin (log
ε
) 237 (3.47) nm. UV (base):
λ
271 nm; λmin 233 nm.
max
max
EI-MS m/z main ions: 451 (M+, 70%), 253 (24), 252 (19), 225 (100), 224 (49), 106 (82), 91 (90).
3.3.3. 8-(10-Aminodecylamino)-6-benzylamino-9-β-D-ribofuranosylpurine (5a)
6-Benzylamino-8-bromopurine 9-riboside was first prepared as an intermediate in the synthesis
of 5a. In an initial reaction, benzylbromide (130
dissolved in dry DMF (250
3 days. After addition of further triethylamine (10
µ
mol) was added to 8-bromoadenosine (22 ˝
µmol)
µ
L) containing triethylamine (7 µL) and the mixture was kept at 37 C for
µL), the solvent was evaporated and the residue
dissolved in 50% ethanol (0.5 mL) to which 0.5 M aqueous NH4OH (0.5 mL) was added. The solution
˝
was kept at 37 C for 24 h to induce rearrangement of the N1-benzyl product (identified by characteristic
UV spectrum [33]) to the N6 compound. Preparative TLC (solvent E) yielded two main products:
A (RF 0.45) and B (RF 0.83) together with some unreacted bromoadenosine (RF 0.40). Compound A
(identified as 6-benzylamino-8-bromopurine 9-riboside): UV (100% ethanol)
λ
273 nm, λmin 238 nm;
max
EI-MS m/z 435/437 (M+, 1.2%), 405/407 (1.4), 346/348 (11), 332/334 (8), 303/305 (74), 224 (94),
198/200 (10), 119 (9), 106 (100); CI-MS (CH4) m/z 464/466 ([M + C2H5]+, 3%), 436/438 ([M + H]+,
32), 304/306 (80), 226 (100). HR ESI-MS: found m/z 436.0629, calc. for C17H19N5O4Br 436.0620.
Compound B (identified as 6-benzylamino-9-benzyl-8-bromopurine): UV (100% ethanol),
λmax 274 nm,
λmin 243 nm; EI-MS m/z 393/395 (M+, 100%), 392/394 ([M H]+, 66), 314 (33), 302/304 (90),
´
197/199 (10), 106 (96). HR ESI-MS: found m/z 394.0659, calc. for C19H17N5Br 394.0667.
Based on the above and the observation that addition of CaCO3 to the benzylation reaction
reduced the amount of compound B formed, a large scale preparation with simplified purification
was devised. Thus, 8-bromo-adenosine (3.2 g), dry DMF (110 mL), benzylbromide (12 mL) and dried
˝
CaCO3 (4.4 g) were stirred at 25 C for 7 days. After evaporation of the DMF (rotary evaporator, under
vacuum, bath temperature 40 ˝C) and the rearrangement step, the centrifuged (13,000
temperature for 30 s) product was dissolved in 50% methanol and extracted first with equal volumes
of benzene (2 times) and then ethyl acetate. The latter extract yielded compound A (2.5 g) free of B and
in sufficient purity to use in further synthesis.
ˆ
g at room
This bromoriboside A (160 mg), n-propanol (4 mL), triethylamine (50 µL) and 1,10-diaminodecane
˝
(190 mg) were mixed and heated at 100 C for 10 h. The resulting solution was subjected to prep.
TLC on silica gel (solvent E) and the main zone was eluted with ethanol–H2O–acetic acid (70:30:3
by vol). The evaporated eluate was dissolved in water (pH to 11) and extracted with ethyl acetate.
The extracts were washed with water and evaporated to yield compound 5a. ESI-MS: m/z 528
([M + H]+, 18%), 396 ([M + H
m/z 528.3294, calc. for C27H42N7O4 528.3298. UV (acid):
´
ribose]+, 45), 379 ([396-NH3]+, 25), 91 (100); HR ESI-MS: found
218, 279 nm; λmin 244 nm. UV (base):
λ
max
λmax 220, 286 nm; λmin 245 nm.
3.3.4. 8-(10-Aminodecylamino)-6-benzylaminopurine (6a)
6a was prepared directly from 5a by methanolysis to cleave the ribosyl moiety. Crude 5a (160 mg)
was dissolved in methanol (10 mL) containing 2,2-dimethoxypropane (2 mL) and conc. HCI (0.4 mL).

Molecules 2016, 21, 576
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˝
˝
The solution was kept at 37 C for 24 h and then at 25 C for 24 h. Ethanol was added to the evaporated
solution and also evaporated under vacuum. After this step was repeated, the residue was dissolved
in 2 M NH4OH and the solution evaporated under vacuum. An aqueous solution (pH 6–7) of the
residue was chromatographed on a column of C18-silica (40 µm, 7.5 g of packing) which was developed
sequentially with water, 15% methanol, 25% methanol and 80% ethanol all of which contained acetic
acid (1%). 6a was eluted as a sharp fraction by the ethanol. Ethyl acetate extracts of an aqueous
solution (pH to 11) of the evaporated eluate yielded compound 6a. UV (acid): λmax (log ε) 238 (4.11),
307 (4.25) nm; λmin (log ε) 255 (3.56) nm. UV (base): λmax (log ε) 221 (4.41), 286 (4.31) nm; λmin (log ε)
248 (3.45) nm. ESI-MS: m/z 396 ([M + H]+, 100%), 379 (10). HR ESI-MS: found m/z 396.2880, calc. for
C22H34N7 396.2876.
3.3.5. 3-(6-Aminopurin-9-yl)propionamido-decylamine (7a) and
8-(10-Aminodecylamino)-6-amino-9-β-D-ribofuranosylpurine (8a)
Analogues of 4a and 5a termed compounds 7a and 8a respectively with a 6-aminopurine moiety
instead of a 6-benzylaminopurine were synthesised by methods analogous to those used for 4a and
5a. All the above amines (3a–8a) were obtained as white powders and analytical TLC of each using
additional solvents revealed only a single ninhydrin-positive UV absorbing component.
3.4. Preparation of Dexamethasone-Cytokinin Conjugates
The hydroxymethyl ketone moiety of dexamethasone was oxidised by periodate according to
Govindan and Manz [40] to yield the corresponding carboxylic acid, 9-fluoro-16
dihydroxy-3-oxo-1,4-androstadiene-17 -carboxylic acid (henceforth termed “dexamethasone acid”).
This acid was converted in small batches to the N-hydroxysuccinimide ester ( ) immediately before
reaction with a purine derivative having a free amino group (3a to 8a). To form the active ester ( ),
ethyl acetate solutions (64 nM) of 1,3-dicyclohexylcarbodiimide (DCHC) (0.95 mL, 61 mol) and of
N-hydroxysuccinimide (0.95 mL, 61 mol) were added to dexamethasone acid (22.5 mg, 60 mol) and
the mixture was gently shaken to suspend all solids at 20 ˝C for 18 h. The solution was centrifuged
(13,000 g at room temperature for 30 s) to remove dicyclohexylurea, which was washed with a small
vol of ethyl acetate. The combined ethyl acetate solutions containing were washed twice with a one
α-methyl-11β,17-
β
1
1
µ
µ
µ
ˆ
1
˝
third vol of water and kept at 4 C until required. TLC of the ethyl acetate solution (solvent A) revealed
only one prominent UV absorbing component, the N-hydroxysuccinimide ester of dexamethasone
acid (1
). HREI-MS found m/z 475.2013 (M+), calc. for C25H30NO7F 475.2006; found m/z 455.1950
(M+-HF), calc. for C25H29NO7 455.1944.
To conjugate the amino-alkyl purines (3a to 8a) to dexamethasone, the former were dissolved
in purified dimethylformamide (20
µL/mg) and the ethyl acetate solution of 1 prepared above was
added to give a 50%–75% excess. The mixture was shaken sufficiently to maintain a suspension of any
˝
solids that separated during 12 h at 23 C. TLC then indicated that the aminopurines were completely
conjugated. The conjugates 3b 8b were purified by preparative TLC of the reaction mixture using
solvents as follows: 3b, B; 4b 5b and 6b, A; 7b and 8b, C. The yield of all purified products was >75%.
–
,
Mass and UV spectral data are in Table 1 and analysis of UV spectra confirming structure has been
discussed in Results. The purity of all conjugates was confirmed by the TLC in additional solvents.
3.5. Preparation of Cytokinin-Biotin Conjugates
To a solution of d-biotin (14.5 mg, 60
µmol in dimethylformamide (0.6 mL), 61 µmol of both
DCHC and N-hydroxysuccinimide (each in ethyl acetate 1.0 mL) were added. After 2 days at 25 ˝C,
the solution was centrifuged (13,000
ˆ
g˝at room temperature for 30 s) and this solution of biotin
hydroxysuccinimide ester was stored at 4 C. The amino-alkyl cytokinins 5a and 6a were reacted with
a 50% excess of the ethyl acetate solution of this ester, as for the preparation of the dexamethasone
conjugates, yielding the biotin conjugates 5c and 6c purified by TLC (solvent D).

Molecules 2016, 21, 576
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Conjugate (5c): UV (acid):
λ
212, 278 nm; λmin 243 nm. ESI-MS: m/z 754 ([M + H]+, 100%),
´
max
776 ([M + Na]+, 8), 622 ([M + H
Ri]+, 13). HR ESI-MS: found m/z 754.4074 and 776.3889, calc. for
C37H56N9O6S and C37H55N9O6SNa, 754.4074 and 776.3894 respectively. ESI-MS-MS: MS2 (622) m/z
396, 379.
Conjugate (6c): UV (acid):
λ
(log ε) 207, 237 (4.00), 308 (4.23) nm; λmin 256 nm. ESI-MS:
max
m/z 622 ([M + H]+), 644 ([M + Na]+). HR ESI-MS: found m/z 622.3659 and 644.3469, calc. for
C32H48N9O2S and C32H47N9O2SNa, 622.3652 and 644.3471 respectively. ESI-MS-MS: MS2 (622) m/z
396 ([M + H ´ biotinyl]+), 379.
By the above methods, biotin succinimidyl ester was also reacted with 6-aminohexanoic acid and
the product was esterified to yield succinimidyl 6-(biotinamido)hexanoate (now available from Sigma).
Amino alkylamino adenines with short alkyl moieties can be conjugated to this biotin derivative to
yield further compounds for FACS. Thus, after TLC purification (solvent F) of the reaction mixture,
the readily synthesised amine, 9-(3-aminopropyl)-6-benzylaminopurine [41] yielded the BAP-biotin
conjugate
9
. UV (acid):
λ
(log
ε
) 205, 268 (4.23) nm; UV (base):
λ
(log ε) 211, 271 (4.25) nm.
max
max
ESI-MS: m/z 622 ([M + H]+), 644 ([M + Na]+). HR ESI-MS: found m/z 622.3284, 644.3103; calc. for
C31H44N9O3S and C31H43N9O3SNa, 622.3288 and 644.3107 respectively.
3.6. Cytokinin Bioassay
This assay was similar to the improved method of Elliott [36]. The cotyledons and the attached
upper hypocotyl were excised from dark-grown Amaranthus caudatus (cv. Tricolor) seedlings and
incubated in petri dishes (8.5 cm diam.) on Whatman No. 1 filter paper wetted with phosphate buffer
˝
(3 mL) containing L-tyrosine and the compound being tested. After 18 h in darkness at 26 C, replicate
samples of 15 cotyledons were extracted with 3 mM acetic acid (1 mL) and the supernatants (centrifuged
at 13,000ˆ g at room temperature for 30 s) were used to determine absorbance (A542nm–A620nm).
4. Conclusions
After periodate oxidation to a carboxylic acid and conversion of this to an activated ester,
dexamethasone can be conjugated by amide formation to a C10 spacer linked to positions 2, 8 or
9 of BAP. The methods described yielded mg amounts of conjugates, which were purified directly from
the reaction mixtures by preparative TLC using carefully selected solvents. The simple purification
methods developed will facilitate preparation of the compounds by molecular biologists with limited
chemical ability and facilities. The conjugate involving the 8 position of the purine ring (i.e., 6b
)
appeared to exhibit the strongest binding to the BAP receptor in the bioassay. This trend also applied
to alkyl (C6–C8) derivatives of BAP (compounds A, B and C) without an attached steroid moiety.
Hence position 8 of BAP appears to be the preferred site of linkage attachment for retention of plant
receptor binding affinity.
The dexamethasone conjugates especially 6b would enable a Y3H system to be developed
to characterise CBPs that are not membrane bound. The synthesised biotin-BAP conjugates, also
involving linkage at purine C-8, would provide a complementary system using FACS as it would
recognise receptors on cell membranes. The systems proposed herein are also potentially applicable to
characterisation of cytokinin-binding proteins in animal cells, especially human cancer cells that are
inhibited by cytokinin.
Acknowledgments: The authors wish to thank Jan Elliot for typing assistance, and also Charles Hocart (ANU),
Jennifer Saville (University of Wollongong) and Research School of Chemistry (ANU), for mass spectra.
Author Contributions: D.S.L., P.C.L.J. and R.Z. conceived and designed the experiments. Y.W. and D.S.L.
performed the chemical synthesis and bioassay. D.S.L. wrote the draft and all contributed to the revision.
Conflicts of Interest: The authors declare no conflict of interest.

Molecules 2016, 21, 576
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References
1.
2.
Kaminek, M. Tracking the story of cytokinin research. J. Plant Growth Regul. 2015, 34, 723–739. [CrossRef]
Stolz, A.; Riefler, M.; Lomin, S.N.; Achazi, K.; Romanov, G.A.; Schmülling, T. The specificity of cytokinin
signalling in Arabidopsis thaliana is mediated by differing ligand affinities and expression profiles of the
receptors. Plant J. 2011, 67, 157–168. [CrossRef] [PubMed]
3.
4.
5.
Wulfetange, K.; Lomin, S.N.; Romanov, G.A.; Stolz, A.; Heyl, A.; Schmülling, T. The cytokinin receptors of
Arabidopsis are located mainly to the endoplasmic reticulum. Plant Physiol. 2011, 156, 1808–1018. [CrossRef]
[PubMed]
Murray, J.D.; Karas, B.J.; Sato, S.; Tabata, S.; Amyot, L.; Szczyglowski, K.A. Cytokinin perception mutant
colonized by Rhizobium in the absence of nodule organogenesis. Science 2007, 315, 101–104. [CrossRef]
[PubMed]
Dolezal, K.; Popa, I.; Krystof, V.; Spichal, L.; Fojtikova, M.; Holub, J.; Lenobel, R.; Schmülling, T.; Strnad, M.
Preparation and biological activity of 6-benzylaminopurine derivatives in plants and human cancer cells.
Bioorg. Med. Chem. 2006, 14, 875–884. [CrossRef] [PubMed]
6.
7.
8.
Brinegar, C. Cytokinin binding proteins and receptors. In Cytokinins—Chemistry, Activity, and Function;
Mok, D.W.S., Mok, M.C., Eds.; CRC Press: Boca Raton, FL, USA, 1994; pp. 217–232.
Brault, M.; Maldiney, R.; Miginiac, E. Cytokinin-binding proteins. Physiol. Plant. 1997, 100, 520–527.
[CrossRef]
Kulaeva, O.N.; Zagranichnaya, T.K.; Brovko, F.A.; Karavaiko, N.N.; Selivankina, S.Y.; Zemlyachenko, Y.V.;
Hall, M.; Lipkin, V.M.; Boziev, K.M. A new family of cytokinin receptors from Cereales. FEBS Lett. 1998, 423,
239–242. [CrossRef]
9.
Selivankina, S.Y.; Karavaiko, N.N.; Maslova, G.G.; Zubkova, N.K.; Prokoptseva, O.S.; Smith, A.R.; Hall, M.A.;
Kulaeva, O.N. Cytokinin-binding protein from Arabidopsis thaliana leaves participating in transcription
regulation. Plant Growth Regul. 2004, 43, 15–26. [CrossRef]
10. Brovko, F.A.; Vasil’eva, V.S.; Lushnikova, A.L.; Selivankina, S.Y.; Karavaiko, N.N.; Boziev, K.M.;
Shepelyakovskaya, A.O.; Mashkov, D.A.; Pavlik, L.L.; Kusnetsov, V.V.; et al. Cytokinin-binding protein
(70‘kDa) from etioplasts and amyloplasts of etiolated maize seedlings and chloroplasts of green plants and
its putative function. J. Exp. Bot. 2010, 61, 3461–3475. [CrossRef] [PubMed]
11. Godge, M.R.; Kumar, D.; Kumar, P.P. Arabidopsis HOG1 gene and its petunia homolog PETCBP act as key
regulators of yield parameters. Plant Cell Rep. 2008, 27, 1497–1507. [CrossRef] [PubMed]
12. Rattan, S.I.S.; Sodagam, L. Gerontomodulatory and youth-preserving effects of zeatin on human skin
fibroblasts undergoing aging in vitro. Rejuvenation Res. 2005, 8, 46–57. [CrossRef] [PubMed]
13. Johnson, G.S.; D’Armiento, M.; Carchman, R.A. N6-Substituted adenines induce cell elongation irrespective
of the intracellular cyclic AMP levels. Exp. Cell Res. 1974, 85, 47–56. [CrossRef]
14. Olsen, A.; Siboska, G.E.; Clark, B.F.; Rattan, S.I. N6-Furfuryladenine, kinetin, protects against Fenton
reaction-mediated oxidative damage to DNA. Biochem. Biophys. Res. Commun. 1999, 265, 499–502. [CrossRef]
[PubMed]
15. Verbeke, P.; Siboska, G.E.; Clark, B.F.C.; Rattan, S.I.S. Kinetin inhibits protein oxidation and glycoxidation
in vitro. Biochem. Biophys. Res. Commun. 2000, 276, 1265–1270. [CrossRef] [PubMed]
16. Hsiao, G.; Shen, M.Y.; Lin, K.H.; Chou, C.Y.; Tzu, N.H.; Lin, C.H.; Chou, D.S.; Chen, T.F.; Sheu, J.R.
Inhibitory activity of kinetin on free radical formation of activated platelets in vitro and on thrombus
formation in vivo. Eur. J. Pharmacol. 2003, 465, 281–287. [CrossRef]
17. Fleysher, M.H.; Hakala, M.T.; Bloch, A.; Hall, R.H. Synthesis and biological activity of some
N6-alkyladenosines. J. Med. Chem. 1968, 11, 717–720. [CrossRef] [PubMed]
18. Fleysher, M.H. N6-Substituted adenosines: Synthesis, biological activity and some structure-activity
relationships. J. Med. Chem. 1972, 15, 187–191. [CrossRef] [PubMed]
19. Ishii, Y.; Sakai, S.; Homma, Y. Cytokinin-induced differentiation of human myeloid leukemia HL-60
cells is associated with the formation of nucleosides but not with incorporation into DNA or RNA.
Biochim. Biophys. Acta 2003, 1643, 11–24. [CrossRef] [PubMed]

Molecules 2016, 21, 576
13 of 14
20. Mlejnek, P.; Dolezel, P. Apoptosis induced by N6-substituted derivatives of adenosine is related to
intracellular accumulation of corresponding mononucleotides in HL-60 cells. Toxicol. In Vitro 2005, 19,
985–990. [CrossRef] [PubMed]
21. Choi, B.H.; Kim, W.; Wang, Q.C.; Kim, D.C.; Tan, S.N.; Yong, J.W.H.; Kim, K.T.; Yoon, H.S. Kinetin riboside
preferentially induces apoptosis by modulating Bcl-2 family proteins and caspase-3 in cancer cells. Cancer Lett.
2008, 261, 37–45. [CrossRef] [PubMed]
22. Cheong, J.; Goh, D.; Yong, J.W.; Tan, S.N.; Ong, E.S. Inhibitory effect of kinetin riboside in human heptamoa,
HepG2. Mol. Biosyst. 2009, 5, 91–98. [CrossRef] [PubMed]
23. Tiedemann, R.E.; Mao, X.; Shi, C.X.; Zhu, Y.X.; Palmer, S.E.; Sebag, M.; Marler, R.; Chesi, M.; Fonseca, R.;
Bergsagel, P.L.; et al. Identification of kinetin riboside as a repressor of CCND1 and CCND2 with preclinical
antimyeloma activity. J. Clin. Investig. 2008, 118, 1750–1764. [CrossRef] [PubMed]
24. Voller, J.; Zatloukai, M.; Lenobei, R.; Dolczal, K.; Béres, T.; Krystof, V.; Spíchai, L.; Niemann, P.; Dzubák, P.;
Hajdúch, M.; et al. Anticancer activity of natural cytokinins: A structure-activity relationship study.
Phytochemistry 2010, 71, 1350–1359. [CrossRef] [PubMed]
25. Casati, S.; Ottria, R.; Baldoli, E.; Lopez, E.; Maier, J.A.; Ciuffreda, P. Effects of cytokinins, cytokinin ribosides
and their analogs on the viability of normal and neoplastic human cells. Anticancer Res. 2011, 31, 3401–3406.
[PubMed]
26. Stirk, W.A.; van Staden, J. Flow of cytokinins through the environment. Plant Growth Regul. 2010, 62, 101–116.
[CrossRef]
27. Tsoupras, G.; Luu, B.; Hoffmann, J.A. A cytokinin (isopentenyl-adenosyl-mono-nucleotide) linked to
ecdysone in newly laid eggs of Locusta migratoria. Science 1983, 220, 507–509. [CrossRef] [PubMed]
28. Becker, J.L.; Roussaux, J. 6-Benzylaminopurine as a growth factor for Drosophila melanogaster cells grown
in vitro. In Metabolism and Molecular Activities of Cytokinins; Guern, J., Peaud-Lenoël, C., Eds.; Springer-Verlag:
Berlin, Germany, 1981; pp. 319–328.
29. Sharma, S.P.; Kaur, P.; Rattan, S.I.S. Plant-growth hormone kinetin delays ageing, prolongs the life span
and slows development of the fruit fly Zaprionus paravittiger. Biochem. Biophys. Res. Commun. 1995, 216,
1067–1071. [CrossRef] [PubMed]
30. Cottier, S.; Monig, T.; Wang, Z.; Svoboda, J.; Boland, W.; Kaiser, M.; Kombrink, E. The yeast three-hybrid
system as an experimental platform to identify proteins interacting with small signalling molecules in plant
cells: potential and limitations. Front. Plant Sci. 2011. [CrossRef] [PubMed]
31. Galbraith, D.W.; Bartos, J.; Dolezel, J. Flow cytometry and cell sorting in plant biotechnology. In Flow
Cytometry in Biotechnology; Sklar, L.A., Ed.; Oxford University Press: New York, NY, USA, 2005; pp. 291–322.
32. Mattanovich, D.; Borth, N. Applications of cell sorting in biotechnology. Microb. Cell Fact. 2006. [CrossRef]
[PubMed]
33. Leonard, N.J.; Fujii, T. The use of a blocking group at the 9-position of adenine for the synthesis of
1-substituted adenines. Proc. Natl. Acad. Sci. USA 1964, 51, 73–75. [CrossRef] [PubMed]
34. Macon, J.B.; Wolfenden, R. 1-Methyladenosine. Dimroth rearrangement and reversible reduction.
Biochemistry 1968, 7, 3453–3458. [CrossRef] [PubMed]
35. Biddington, N.L.; Thomas, T.H. A modified Amaranthus betacyanin bioassay for the rapid determination of
cytokinins in plant extracts. Planta 1973, 111, 183–186. [CrossRef] [PubMed]
36. Elliott, D.C. Analysis of variability in the Amaranthus bioassay for cytokinins. Plant Physiol. 1979, 63, 269–273.
[CrossRef] [PubMed]
37. Nisler, J.; Zatloukal, M.; Popa, I.; Dolezal, K.; Strnad, M.; Spíchal, L. Cytokinin receptor antagonists derived
from 6-benzylaminopurine. Phytochemistry 2010, 71, 823–830. [CrossRef] [PubMed]
38. Krivosheev, D.; Kolyachkina, S.; Mikhailov, S.; Tararov, V.; Vanyushin, B.; Romanov, G. N6-(benzyloxymethyl)
adenosine is a novel anticytokinin, an antagonist of cytokinin receptor CRE1/AHK4 of Arabidopsis.
Dokl. Biochem. Biophys. 2012, 444, 178–181. [CrossRef] [PubMed]
39. Spíchal, L.; Werner, T.; Popa, I.; Riefler, M.; Schmülling, T.; Strnad, M. The purine derivative PI-55 blocks
cytokinin action via receptor inhibition. FEBS J. 2009, 276, 244–253. [CrossRef] [PubMed]

Molecules 2016, 21, 576
14 of 14
40. Govindan, M.V.; Manz, B. Three step purification of glucocorticoid receptors from rat liver. Eur. J. Biochem.
1980, 108, 47–53. [CrossRef] [PubMed]
41. Letham, D.S.; Palni, L.M.S.; Tao, G.Q.; Gollnow, B.I.; Bates, C.M. Regulators of cell division in plant
tissues XXIX. The activities of cytokinin glucosides and alanine conjugates in cytokinin bioassays. J. Plant
Growth Regul. 1983, 2, 103–115. [CrossRef]
Sample Availability: Samples of the compounds are no longer available from the authors.
©
2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).