The synthesis of 3H-labelled 8-azido-N6-benzyladenine and related compounds for photoaffinity labelling of cytokinin-binding proteins

Article abstract of DOI:10.3390/molecules24020349

The biology of the group of plant hormones termed cytokinins is reviewed to reveal areas where further studies of cytokinin-binding proteins could be significant. Such areas include: inhibition of human tumour cell growth by cytokinin ribosides, the role of cytokinins in the development of diverse micro-organisms including the cyanobacteria and Mycobacterium tuberculosis, the very rapid responses of plant cells to exogenous cytokinins, and other aspects of cytokinin plant biology. Photoaffinity labelling (PAL) coupled to the recent advances in HPLC of proteins and mass spectral analysis and sequencing of proteins, may have relevance to these areas. To facilitate PAL, we present experimental details for two methods for synthesis of 8-azido-N⁶-benzyladenine, which has the azido affinity group in the preferred position of the purine ring. Synthesis from [2-³H]adenosine yielded the above-mentioned PAL reagent with ³H in the purine ring and also gave labelled 9-riboside and 8-azido-N⁶,9-dibenzyladenine. 8-Azido-N⁶-benzyladenine was also prepared from 6,8-dichloropurine by a facile synthesis, which would allow a label to be sited in the benzyl group where substituents can also be introduced to vary cytokinin activity. The use of inactive cytokinin analogues in assessing the significance of PAL is discussed.

Full text of DOI:10.3390/molecules24020349

molecules
Article
The Synthesis of 3H-Labelled
8-Azido-N6-Benzyladenine and Related
Compounds for Photoaffinity Labelling
of Cytokinin-Binding Proteins
David. S. Letham 1,
*
, Xue-Dong Zhang 1 and Charles H. Hocart 1,2
1
Research School of Biology, Australian National University, Canberra ACT 0200, Australia;
donxdzhang@yahoo.com.au (X.-D.Z.); Charles.Hocart@anu.edu.au (C.H.H.)
School of Chemistry and Chemical Engineering, Shaanxi University of Science and Technology,
Long Shuo Rd, Wei Yang District, Shaanxi 710021, China
2
*
Correspondence: david.letham@bigpond.com; Tel.: +61-2-4977-2008
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 19 November 2018; Accepted: 17 January 2019; Published: 18 January 2019
Abstract: The biology of the group of plant hormones termed cytokinins is reviewed to reveal areas
where further studies of cytokinin-binding proteins could be significant. Such areas include: inhibition
of human tumour cell growth by cytokinin ribosides, the role of cytokinins in the development
of diverse micro-organisms including the cyanobacteria and Mycobacterium tuberculosis, the very
rapid responses of plant cells to exogenous cytokinins, and other aspects of cytokinin plant biology.
Photoaffinity labelling (PAL) coupled to the recent advances in HPLC of proteins and mass spectral
analysis and sequencing of proteins, may have relevance to these areas. To facilitate PAL, we present
experimental details for two methods for synthesis of 8-azido-N6-benzyladenine, which has the
azido affinity group in the preferred position of the purine ring. Synthesis from [2-3H]adenosine
3
yielded the above-mentioned PAL reagent with H in the purine ring and also gave labelled
9-riboside and 8-azido-N6,9-dibenzyladenine. 8-Azido-N6-benzyladenine was also prepared from
6,8-dichloropurine by a facile synthesis, which would allow a label to be sited in the benzyl group
where substituents can also be introduced to vary cytokinin activity. The use of inactive cytokinin
analogues in assessing the significance of PAL is discussed.
Keywords: 8-azido-N6-benzyladenine; cytokinins; cytokinin-binding proteins; receptors;
photoaffinity labelling; synthesis of cytokinin analogues
1. Introduction
1.1. Binding of Cytokinins as Plant Regulators
The cytokinins, N6-substituted adenines, are a group of plant hormones that regulate many
features of plant development including cell division, leaf senescence, lateral shoot outgrowth
and nutrient translocation [1,2]. The natural cytokinins are of two types [3]: those with
an isoprenoid N6 substituent (e.g., zeatin, E-6-(4-hydroxy-3-methylbut-2-enylamino)-purine and
N6-isopent-2-enyladenine termed iP), and those with a benzyl or substituted-benzyl N6-substituent
(e.g., N6-benzyladenine, BA; 6-(o-hydroxybenzylaminopurine, termed o-topolin). The cytokinins occur
as free bases, 9-ribosides, riboside-50-phosphates, and as glucosides (sugar at position 3, 7 or 9 of
purine ring and at O of zeatin). However, the free bases appear to be the active form in plants and
bind to receptors [4].
Three histidine kinase cytokinin receptors (AHK2, AHK3, and AHK4 or CRE) have been
characterized in Arabidopsis [5] and three similar histidine kinase receptors have been defined in
Molecules 2019, 24, 349; doi:10.3390/molecules24020349
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Molecules 2019, 24, 349
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maize [
the former are active in signal transduction [
higher plants [ ] in which several binding proteins of uncertain significance have also been detected.
6
]. Both Arabidopsis and maize receptors appear to be predominantly localized in the ER and
7]. Other similar receptors remain to be characterized in
8,9
However, the rapid responses to exogenous cytokinin by isolated mitochondria suggest significant
binding that does not involve known receptors that regulate transcription [10]. The effects on oxygen
consumption, for example, are almost immediate. Rapid cytokinin effects on protein synthesis in
mitochondria and plastid preparations have also been recorded, as well as very rapid opening of
stomata [11].
In some situations, cytokinins may act at the site of biosynthesis, but these regulators can also
move from a site of synthesis to one of action and thus conform to the traditional definition of a
phytohormone. The view that root-produced cytokinin moves in the xylem to control numerous
phases of shoot development has been confirmed by recent evidence including: (1) root nodules that
overproduce cytokinins [12]; (2) supply of endogenous xylem cytokinins to excised monocot leaves at
natural flux rate [13]; and (3) grafting of normal roots to mutant shoots deficient in cytokinins [14,15].
Zeatin-type cytokinins predominate in xylem, but the isopentenyl type are dominant in the phloem
sap moving to control pattern development in the root [16]. This selective loading into xylem and
phloem must involve binding of cytokinins to specific trans-membrane transporter proteins and
details of this control are now emerging [17]. In contrast to this long-distance translocation, cytokinin
translocation within seeds is important in germination. Isoprenoid cytokinins in dry lupin seed,
for example, are completely degraded during imbibition [18], but cytokinins subsequently synthesized
in the embryonic axis [19] move to the cotyledons [20] to induce expansion, enzyme activities and
chloroplast formation [21,22]. The role of cytokinin binding proteins and receptors in seed germination
is not known. Proteins analogous to the cereal embryo proteins that bind and possibly stabilize
cytokinins, could be involved.
1.2. Cytokinins as Inhibitors of Tumour Cell Growth
Considerable interest now centres on the ability of cytokinin ribosides to inhibit the growth
of human cancer cells in culture and cause apoptosis [
occurring cytokinins (over 40 compounds), the ribosides of iP, 6-furfurylaminopurine, BA and o-topolin
were the most effective inhibitors, showing cytotoxicity at 0.5–5 M in 7 of 9 tumour cell lines [25].
9,23,24]. In a study of nearly all naturally
µ
The 2,6,9-trisubstituted purines are the other group of compounds related to cytokinins that inhibit
tumour cell growth. The prototype was 2-(2-hydroxyethylamino)-6-benzylamino-9-methylpurine,
which was originally designed and synthesized [26] as an inhibitor of a radish cotyledon enzyme
that glucosylates cytokinin bases at positions 7 and 9 [27
inhibitors of cyclin-dependent kinases (enzymes involved in regulation of the cell division cycle),
this trisubstituted purine (now termed olomoucine) was found to be the most effective compound [29
,28]. In a comparison of 81 purines as
]
and was also a potent inhibitor of tumour cell cultures. Determination of the mechanism of action
(competitive inhibitor of ATP, non-competitive for histone H1) [29], resulted in the design of closely
related compounds with enhanced inhibitory activity, one of which (roscovitine) is now in phase II
CLINICAL trials as an anti-cancer drug.
The mechanism of action of cytokinin ribosides as tumour cell inhibitors is not clear. Elucidation
may similarly lead to new cancer chemotherapies. Identification of cytokinin, and especially cytokinin
riboside binding proteins in tumour cells, is relevant in this regard.
1.3. Cytokinins and Microorganisms
A growing number of microorganisms have been found to produce cytokinins that are recognized
by the AHK-type receptors in intact plants. It is well established that plant microbial pathogens
use this signal to the plant to modify plant organ development and to increase host susceptibility,
thus facilitating infection [30]. However, recent studies indicate that this signal direction can be
reversed. Thus, partially characterized receptors resembling the plant AHK proteins have been

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detected in phytopathogenic bacteria and certain fungi, and also in cyanobacteria [31]. These bacteria
and fungi appear able to recognise cytokinin derived from plants.
In a recent advance, cytokinins were found to be produced by certain human microbial pathogens,
which also responded to applied cytokinin. Exogenous zeatin promoted cell cycle progression in an
obligate protozoan pathogen, which also produced zeatin and iP in nucleotide form [32]. However,
a receptor was not identified and a plant-type AHK receptor gene was absent suggesting the presence
of a new receptor type. A further observation of great interest is the secretion of several isoprenoid
cytokinins by Mycobacterium tuberculosis [33] which occurs naturally only in humans. The biosynthesis
and function of these cytokinins are unknown, but by analogy with plant systems, it has been suggested
that the cytokinins signal transcriptional changes in human cells to favour growth of the bacteria.
Exogenous iP induced transcriptional changes that altered the bacterial cell envelope showing that
M. tuberculosis responds to change in cytokinin level [34]. M. tuberculosis also contains a homologue of
the plant cytokinin activating enzyme LOG recently detected in other human pathogens including
Staphylococcus aureus (also termed “golden staph”). Thus, cytokinins appear to be recognized as
regulatory molecules in all the above microorganisms, opening possible strategies for pathogen
control and a new field for studies of cytokinin-binding proteins. Cytokinins have roles beyond plant
development that are yet to be characterized.
1.4. Cytokinins and Photoaffinity Labelling
Photoaffinity labelling (PAL, also denotes photoaffinity label) is a technique which could facilitate
the identification of cytokinin receptors and binding proteins in the diverse biologies already identified.
It has been used very successfully in drug research to identify receptors and depends on the presence of
a photoactivated group attached to the ligand [35]. The method can be applied to cytokinin analogues
with an azido group inserted on an aromatic ring. When such an analogue binds to a receptor or
binding protein, irradiation with UV light converts the N3 group into a nitrene that inserts into
any adjacent C–H, O–H or N–H bond, forming a covalent linkage with the binding protein [36].
The identification of PAL protein has been a major problem, which has now been greatly simplified
by developments in the HPLC of proteins coupled with mass spectral methods for protein analysis
and sequencing.
Certain synthetic phenylureas exhibit cytokinin activity. Tritium-labelled azido derivatives of
these ureas [37,38], as well as labelled 2-azido-BA [39,40] have been used in PAL to identify some
cytokinin-binding proteins in higher plants. However, these proteins are not functional receptors
and their significance is obscure. For PAL, 2-azido-purine cytokinins have an unsatisfactory feature.
These compounds are in equilibrium with isomers formed by ring closure of the azido substituent to
N-1 and N-3 (the azidoazomethine-tetrazole equilibrium in 2-azido-purines [41]). This appears to be
the cause of the undesirably long period required for photolysis, during which, secondary photolytic
reactions and unspecific labelling would occur.
Properties of 8-azido-N6-substituted adenines differ from those of the corresponding 2-azido
compounds in two important respects relevant to PAL. Firstly, the 8-azido compounds are markedly
more effective as cytokinins. Thus, in the tobacco tissue bioassay while 2-azido-BA has activity similar
to that of BA, 8-azido-BA is “over 25 times as active” as BA [42] (the least detectable concentrations are
−5
−3
µ
about 3
×
10 and 10
M respectively). Secondly, the 8-azido compounds do not require extended
irradiation for photolysis. These two properties of 8-azido cytokinins would be expected to minimize
unspecific labelling.
Detection of PAL-proteins requires a label in the probe. Biotin is often used in this way and it
also facilitates purification of the labelled protein. However, linkage to biotin would greatly reduce
or eliminate the activity of a purine cytokinin. Labelling with radioactivity appears to be necessary,
preferably tritium to give high specific activity.
Accordingly, in this paper, we discuss the biological areas to which PAL with cytokinins would
3
be relevant. The synthesis of H-labelled 8-azido-BA and also that of the corresponding riboside

Molecules 2019, 24, 349
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are described, providing the first syntheses of these tritium-labelled compounds. The role of azido
derivatives of inactive cytokinin analogues in the assessment of PAL binding is also discussed.
2. Results and Discussion
2.1. Possible Synthetic Routes to [3H]8-Azido-BA
Unlabelled 8-azido-BA has been synthesized by three methods:
1.
From 8-bromo-adenosine by conversion to 8-azido-adenosine, followed by benzylation and ribose
cleavage [43].
2.
3.
From 6,8-dichloropurine by four steps including blocking and deblocking N-9 [42].
By direct bromination of BA and reaction with sodium azido under novel conditions [44].
These methods involve macro-synthesis using gram amounts of compounds and purification by
classical methods. When using 3H-labelled starting compounds, micro synthesis from a few mg or less
is necessitated by the cost of 3H-labelled precursor. The purification methods given are therefore not
directly applicable to the synthesis of [3H]8-azido-BA. Furthermore, the yield of unlabeled compound
by method (1) was only 6% from 8-bromo-adenosine [43]. [3H]Adenosine is available commercially
at high specific activity to prepare 8-bromo-adenosine, but benzylamine and BA at this activity for
methods (2) and (3) respectively are not available. Provided adenosine can be brominated to yield the
8-bromo-derivative in high yield by micro-synthesis, and the low reported yield in its conversion to
8-azido-BA could be elevated, synthesis of this photoaffinity label from [3H]adenosine becomes the
preferable route, since it also yields the riboside needed for studies of tumour cells. This was achieved
in the optimized micro-synthesis discussed below and outlined in Figure 1. For the synthesis, an excess
of unlabeled adenosine was added to the high specific activity [3H]adenosine.
2.2. Synthesis of 8-Azido-N6-benzyl-[2-3H]adenine from [2-3H]Adenosine
The first step was bromination of adenosine (
C-8 of some nucleotides has been successful using water as solvent at 23 ◦C. However, TLC studies
indicated that the yield of [2-3H}8-bromo-adenosine ( ) by bromination at 4 ◦C in 50% methanol with
1) at C-8. Selective bromination at the purinyl
2
suspended CaCO3 markedly exceeded that obtained under the above conditions and in other solvents
(cf. Figure 2).
◦
When
2
was benzylated with benzyl bromide in dimethylformamide (DMF) at 37 C, the reaction
was slow and a significant amount of product was identified as a dibenzyl derivative. At
◦
higher temperature (60–85 C), the ratio of di- to mono-benzyl products was enhanced, but much
reduced at 23 ◦C with a slow reaction rate. However, the reaction can be catalyzed by the
addition of CaCO3. Under these conditions,
benzylbromide in 3 days, resulting in a mixture of 1-benzyl-8-bromo-adenosine (
2
was almost completely benzylated in DMF by
, the expected
) in a ratio of 3:1. The mixture was dissolved
3
product [45]) and 1, 9-dibenzyl-8-bromo-adenine (
4
◦
in 0.5 M NH4OH at 37 C and kept for 36 h to induce a Dimroth rearrangement (conversion of
1-substituted adenine to the N6 isomer [46]) yielding N6-benzyl-8-bromo-[2-3H]adenosine (
5) and
N6,9-dibenzyl-8-bromo-[2-3H]adenine (6).

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Figure 1. The sequence of reactions used to prepare photoaffinity compounds from [2-3H]adenosine.
The compounds and intermediates are numbered to as in the text. The 1-substituted adenosine
derivatives, and 4, were not preparatively separated but the mixture was subjected to conditions
to induce a Dimroth rearrangement yielding the N6-derivatives
and The reactions were
1
9
3
5
6.
(i) bromination; (ii) reaction with benzylbromide in DMF; (iii) rearrangement in 0.25 M NH4OH;
(iv) NaN3 at 70 oC; and (v) 0.5 M HCl at 80 oC.
Figure 2. The distribution of radioactivity over the TLC chromatograms show the effects of temperature
and solvent on bromination of [2-3H]adenosine. ( ) water at 26 oC (conditions often used to brominate
A
nucleotides); (B
) 50% methanol at 4 oC (conditions used herein). The quantities used in the reactions are
those given in Section 3.3.1. The letters a and b denote adenosine and 8-bromoadenosine, respectively.
For both graphs, the reaction time was 0.75 h and solvent A was used for TLC.

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For direct displacement of leaving groups (e.g., Br) from the C-8 position of purines, the acidic 7(9)
proton usually requires masking [42]. In the present situation, this was achieved by the ribosyl moiety
in the case of
NaN3 in DMF at 70 ◦C yielding the corresponding azides
from any unreacted bromide by HPLC (separation not achievable by TLC). Cleavage of the ribose
moiety from ).
by acid hydrolysis yielded [3H]8-azido-BA, i.e., 8-azido-N6-benzyl-[2-3H]adenine (
The overall yield from [2-3H] adenosine was 22%; the specific radioactivity of
was 21.8 MBq/ mol;
and the radiochemical purity (>98%) was established by normal TLC, reversed phase TLC and HPLC.
5
and benzyl in
6. Hence, these 8-bromo compounds were reacted with an excess of
7
and , respectively, which were separated
8
7
9
9
µ
The MS of 9
confirmed the assigned structure with prominent ions at m/z: 226 (M+), 238 (–N2 from
azido), 237 (further loss of H), 161 (loss of C6H5-CH=NH from M+ with H transfer to purine fragment
ion). Because of the molar excess (30-fold) of the unlabeled adenosine added to the [3H]adenosine for
the synthesis, the plotted MS for the above compounds fail to reveal the [3H]-labelled ions.
2.3. Synthesis of 8-Azido-N6-Benzyladenine from 6,8-Dichloropurine
Synthesis of the unlabeled compound is considered first. In an analogous situation,
2,6-dichloropurine was found to react with NaN3 to yield the 2,6-diazo compound, which was then
reacted with benzylamine to yield 2-azido-BA [42]. However, we found that this reaction sequence did
not convert 6,8-dichloropurine into 8-azido-BA. The first step in this synthesis from 6,8-dichloropurine
requires formation of N6-benzyl-8-chloro-adenine (10). In previous studies, replacement of the chlorine
in 10 by the azido group required prior blocking of the N-9 position followed by reaction with
triethylammonium azide. Deblocking then yielded 8-azido-BA [42]. A simple direct method for
this conversion is desirable, especially when 3H-labelled compounds are involved, and is detailed
in Section 3.4. It involves the reaction of 10 directly with excess NaN3 in propan-1-ol containing
acetic acid at 100 ◦C. The acid promoted the reaction probably by suppression of the ionisation of the
7/9 proton and formation of the 9-N anion [42]. This reaction provided a direct synthesis of unlabelled
8-azido-BA (Figure 3). However, it also allows the introduction of a label into the benzyl group if
labelled benzylamine was available for the synthesis of the precursor 10. The amine is available
commercially labelled with 14C, while catalytic reduction of iodobenzylamine with 3H gas should give
high-specific-activity labelling in the benzene ring. Reduction of benzonitrile with AlCl3-activated
[3H]NaBH4 [47] gives methylene labelling of low specific activity (unpublished data). However,
3
the optimum H-labelling of 8-azido-BA, based on synthesis from 6,8-dichloropurine, probably
involves formation of 8-chloro-N6-(3-iodobenzyl)adenine (11) for catalytic reduction with tritium
gas at room temperature giving deiodination and yielding N6-[3-3H]benzyl-8-chloro-adenine (12
)
(Figure 3). Based on the results of an analogous reaction [48]; note, this paper describes the synthesis
of N6-[3-3H]benzyl-2-chloro-adenine. In unlabeled form, this compound was previously converted
to 2-azido-BA through formation of the 2-hydrazino derivative], a specific radioactivity of about
5-6 Ci/mmol would be expected for 12 and the resulting 8-azido-BA (13).
As an alternative to the above methods, catalytic exchange with 3H gas at elevated temperatures
has been applied to label cytokinins [49] and may be applicable to 10, which may exhibit sufficient
stability because it lacks an azido group.
Some compounds that are potentially useful in the synthesis of di-azido derivatives of BA were
also prepared (see Experimental), namely: N6-benzyl-2,8-dichloro-9-(tetrahydropyran-2-yl)adenine
(A), 2,6,8-triazido-purine (B), and 2,6,8-trichloro-9-tetrahydropyran-2-yl)purine which was converted
to the 9-(tetrahydropyan-2-yl) derivative of B (termed C) by reaction with NaN3. A (by reaction
with NaN3), while B and possibly C (by reaction with benzylamine) could provide direct routes to
2,8-diazdio-N6-benzyladenine. The presence of two azido groups may facilitate the PAL reaction.

Molecules 2019, 24, 349
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Figure 3. Reaction sequences for synthesis of 8-azido-N6-benzyladenine from 2,6-dichloropurine (DCP).
A. Synthesis of the unlabelled compound (AZ) as described herein. B. Proposed synthesis of 3H-labelled
compound of high specific activity, i.e., 8-azido-N6-[3-3H]benzyladenine (13). Intermediates
shown in the figure are: N6-benzyl-8-chloro-adenine (10), 8-chloro-N6-(3-iodobenzyl)adenine (11),
N6-[3-3H]benzyl-8-chloro-adenine (12), 8-azido-N6-[3-3H]benzyladenine (13). The reactions were with:
(i) benzylamine at 100 oC; (ii) NaN3 in propan-1-ol containing acetic acid; (iii) 3-iodobenzylamine;
(iv) tritium gas over palladium catalyst.
2.4. Assessment of PAL
It is often suggested that when an active cytokinin competes with a cytokinin carrying a PAL at a
binding site, the site is involved in cytokinin action and could contain a functional receptor. In this
connection, it would be relevant to study the PAL in the presence of an inactive cytokinin analogue
that does not suppress the bioassay activity of active cytokinins. This analogue presumably does not
bind to the relevant receptor. If PAL is suppressed, the binding site cannot be a functional receptor,
but may be, for example, a metabolizing enzyme, or may have no relevance at all.
N6-(3,4-Dimethoxybenzyl)adenine (DMA) is a cytokinin analogue of the above type that was
noted in our previous studies [50]. While BA showed activity at 10 nM in the radish cotyledon bioassay,
DMA was inactive at 100 µM and did not suppress the activity of BA. DMA was also inactive in
the Amaranthus betacyanin bioassay and very weakly effective in a wheat leaf senescence assay [51].
This BA derivative appears to have relevance to PAL in relation to radish cotyledon expansion and
probably to other cytokinin-induced responses. It is also relevant that DMA did not activate the AHK3
and AHK4 receptors [51].
Very detailed compilations [9,51] of the activities of BA derivatives, including many newly
synthesized compounds, show that several are nearly inactive in some cytokinin bioassays and also in
activation of the AHK3 receptor. Thus, these compounds may resemble DMA in activity. Addition
of such BA analogues prior to PAL with 9, could yield significant conclusions, but synthesis of 2- or
8-azido derivatives of such inactive compounds for comparative PAL with 2- or 8-azido BA may be
more significant as all compounds then have an azido group, which can directly promote cytokinin
activity/binding [42]. A correlation between PAL labelling and cytokinin activity would be consistent
with the binding site having a functional significance in terms of growth or physiological activity.
The 2-azido cytokinins mentioned above can be synthesized readily from 2,6-diazidopurine
(the synthesis of 2-N3-DMA is detailed in the Experimental Section, while 2-N3-BA is a known
compound [52]). By heating with tritiated water (readily available at 5 Ci/mL), these 2-azido

Molecules 2019, 24, 349
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compounds can be labelled by exchange of the C-8 proton [53] to give a low but still useful specific
activity (maximum from above 3H-water, 45 mCi/mmol).
8-Azido-N6,9-dibenzyladenine (
in the synthesis of
PAL with . The benzyl substituent, like many other groups at N-9, markedly lowers cytokinin activity
8), the tritium-labelled compound formed as a secondary product
9
from adenosine, provides a further but structurally different compound to assess
9
and would be expected to greatly reduce PAL at a receptor site.
3. Experimental Section
3.1. Chromatographic Methods
Unless stated otherwise, Merck silica gel PF254 was used for normal phase TLC and layers were
developed in tanks lined with filter paper saturated with solvent. The solvents used were (proportions
are by volume): (A) butan-1-ol/H2O/14 M NH4OH (6:2:1) upper phase; (B) chloroform/methanol
(9:1); (C) chloroform/methanol (9:1) with 14 M NH4OH added to solvent in tank (0.2 mL/100 mL)
and to paper linking tank (several drops); (D) chloroform/methanol/acetic acid (95:5:0.5); and
(E) toluene/ethyl acetate/acetic acid (500:180:36). For reverse phase (RP) TLC, silica layers were
impregnated with DMPS-5X fluid [54] and the solvent was methanol/H2O (20:80). Compounds
separated on TLC plates were detected by short wavelength UV light. Azido compounds were
streaked across the origin of TLC plates for preparative separation. After TLC, only the edges of the
plates were exposed to UV light and these areas were discarded.
HPLC utilized a Waters system with effluent absorbance monitored at 265 and 280 nm.
The column was an RCM 100 Nova-Pak C-18 cartridge; solvent 35% ethanol with 1% acetic acid,
flow rate 2.4 mL/min.
3.2. UV and Mass Spectra
UV spectra in neutral solution were recorded in ethanol or aqueous ethanol. Spectra in acidic or
basic solution were recorded in aqueous ethanol containing 0.05 M HCl or 0.1 M NH4OH, respectively.
All UV spectra were determined with a Shimadzu-240 spectrophotometer (Shimadzu, Kyoto, Japan).
Mass spectra (MS) were obtained with a Finnigan 4500 instrument (Finnigan, San Jose, CA, USA)
◦
with samples introduced via the direct inlet probe. Electron impact (EI): 70 eV; source 150 C. Chemical
ionization (CI): methane reagent gas (1 torr); ionization energy 140 eV; source 100 ◦C.
3.3. Synthesis of 8-Azido-N6-benzyl-[2-3H]adenine from [2-3H]adenosine
3.3.1. 8-Bromo-[2-3H]adenosine (2)
A solution in 50% methanol (1 mL) was prepared containing unlabelled adenosine (3.0 mg,
11.2
µ
mol) and [2-3H]adenosine (296 MBq, 766 GBq/mmol). Bromine (38.2
µ
mol, 195
µ
L of a 1% v/v
◦
aqueous solution) and CaCO3 (2 mg) were added to this solution and the mixture was stirred at 4 C for
2 h, the optimal time determined in preliminary studies involving TLC (solvent A). The above reaction
mixture was evaporated in vacuo and the residue was dissolved in 50% ethanol for preparative TLC on
silica gel (solvent A), while the product eluting solvent was 50% ethanol. Small amounts of unreacted
[3H]adenosine were recycled through bromination and TLC giving a combined yield for
(10.0 moles, 23.9 MBq/
mol). EI-MS m/z (rel. int.): 345/347 (M+, 1%), 315/317 (3), 256/258 (20),
2 of 86%
µ
µ
242/244 (13), 213/215 (100) (loss of ribose), 186/188 (18), 135 (26). This spectrum and TLC comparisons
with authentic compound established the identity of the product as 8-bromo-adenosine (2).
3.3.2. N6-Benzyl-8-bromo-[2-3H]adenosine (5) and N,9-dibenzyl-8-bromo-[2-3H]adenine (6).
Benzylbromide (16
DMF (0.5 mL) and the mixture was stirred with CaCO3 (3.0 mg) at 23 ◦C for 3 days. After addition
of 10 L of triethylamine, the reaction was evaporated in vacuo. TLC combined with UV spectra
µL, 130 µmol) was added to the above dried 8-bromo-adenosine in dry
µ

Molecules 2019, 24, 349
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indicated a predominance of 1-substituted adenines (
3,
4
) and to induce rearrangement to the N6-benzyl
derivatives, the◦residue was dissolved in ethanol (1 mL) to which NH4OH (0.5 M, 1 mL) was added.
After 20 h at 37 C, rearrangement was complete as evidenced by TLC (solvent A) and the centrifuged
product was subjected to preparative TLC in this solvent, yielding N6-benzyl-8-bromo-[2-3H]adenosine
(5, 4.8
µmol) at Rf 0.46: UV (100% ethanol)
λ
max
273 nm, λmin 238 nm; 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 (MH+, 32), 304/306 (80), 226 (100). Yield of
5: 48% for
this step.
The above preparative TLC also gave a lesser component at Rf 0.83 identified as
N6,9-dibenzyl-8-bromo-[2-3H] adenine (
6
, 1.4
µ
mol): UV(100% ethanol)
λ
274 nm, λmin 243 nm;
max
EI-MS m/z: 393/395 (M+, 100%), 392/394 ([M
Yield of 6: 14% for this step.
−
H]+, 66), 314 (33), 302/304 (90), 197/199 (10), 106 (96).
3.3.3. 8-Azido-N6-benzyl-[2-3H]adenosine (7) and 8-azido-N6, 9-dibenzyl-[2-3H]adenine (8)
Compound above, was dissolved in DMF (2 mL) and all further procedures were conducted
under red light or in darkness. After the addition of NaN3 (200 mol), the solution of 5 was heated at
5
µ
70 ◦C for 80 h to achieve a complete conversion to the azido derivative. Since TLC did not achieve
resolution of the bromo and azido compounds, this conversion was monitored by HPLC (see 3.1).
The retention times were:
5 (5.88 min), azido 7 (6.50 min). When the reaction was complete, the residue
obtained by vacuum evaporation was dissolved in water (4 mL) and extracted 5 times with equal
volumes of ethyl acetate. Evaporation of the extracts yielded 8-azido-N6-benzyl-[2-3H] adenosine
(
7
). UV (50% ethanol):
λ
289 nm, λmin 255 nm. EI-MS m/z: 398 (M+, 22%), 368 (14), 309 (28),
max
295 (16), 266 (91), 238 (67), 237 (100), 224 (16), 161 (58). CI-MS (CH4) m/z: 439 ([M + C3H5]+, 3%),
427 ([M + C2H5] +, 5), 399 (MH+, 49), 267 (84), 239 (71). Yield of 7: 81% for this step.
Similarly, 6 was reacted with NaN3 yielding 8-azido-N6, 9-dibenzyl-[2-3H]adenine (8).
3.3.4. 8-Azido-N6-benzyl-[2-3H] adenine (9)
The foregoing riboside (7
) was hydrolyzed in 0.5 M HCl (1.5 mL) at 80 ◦C for 2 h. Hydrolysis
was monitored by TLC (solvent B) and the hydrolysate was then adjusted to pH 8-9 by addition of
solid KHCO3. Four extractions with ethylacetate (1.5 mL) yielded a product for preparative TLC
(solvent B) on HF254 silica gel. The dominant zone (Rf 0.76) was eluted with 50% ethanol to give
8-azido-N6-benzyl-[2-3H]adenine (
9
) 2.57
µmol). UV in 50% ethanol, the neutral, acidic and basic
spectra:
λ
max
290, 298 and 294 nm respectively; λmin 254, 251 and 258 nm, respectively. EI-MS m/z:
266 (M+, 100%), 238 (M+
this step; overall 22%.
−
N2, 58), 237 (89), 161 (45), 147 (21), 135 (34), 119 (14). Yield of 9: 66% for
3.4. N6-Benzyl-8-Chloro-Adenine (10), Ddirect Azido Insertion
6,8-Dichloropurine (3 mg, 15.8 mol) and benzylamine (8.5 mg, 79
were heated in a sealed vial at 100 ◦C for 6 h. After TLC (solvent C), elution of the main UV zone
µ
µmol) in butan-1-ol (0.15 mL)
yielded N6-benzyl-8-chloro-adenine (10), 2.7 mg (66% yield). UV acid:
λ
279 nm; UV base: λ
max max
276 nm. EI-MS m/z: 259/261 (M+, 30%), 224 (4), 223 (4), 154 (-C6H5-CH=NH, 6), 106 (66), 91 (100).
In synthesis designed to use all the supplied benzylamine (e.g., when the amine is 3H labelled),
a large excess of 6,8-dichloropurine is required as exemplified below. Benzylamine hydrochloride
(1.6 mg, 11.2
triethylamine (15
µ
mol), 6,8-dichloropurine (6.9 mg, 36.3 µm◦ ol), purified butan-1-ol (0.14 mL) and
µ
L) were heated in a sealed vial at 110 C for 10 h. TLC analysis of the reaction
product, involving ninhydrin application and UV characterization, indicated the benzylamine
was completely reacted and that 10 was the main reaction product. The excess of unreacted
6,8-dichloropurine remained at the origin during preparative TLC (solvent C), and was thus readily
separated from 10 which exhibited a low Rf.

Molecules 2019, 24, 349
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N6-Benzyl-8-chloro-adenine (10) (1.0 mg, 3.9
mol), propan-1-ol (540 L), and acetic acid (30
µ
mol), NaN3 aqueous solution (60
µ
L containing
◦
140
µ
µ
µL) were heated at 100 C in a sealed vial for
24 h. After addition of water (1.5 mL), the reaction mixture was extracted with three 1.5-mL volumes
of ethyl acetate. Preparative TLC (solvent C, eluent ethanol) of the evaporated extracts yielded a
product (0.81 mg, yield 79%; separable from 10 by TLC solvent E) identical to 8-azido-BA prepared
from adenosine. Both compounds turned yellow on TLC plates under UV light, co-chromatographed
and exhibited identical UV and mass spectra. UV in 95% ethanol, acidic spectrum:
λ
max
298 nm, λmin
252 nm, shoulder 225–234 nm; basic spectrum: λmax 294 nm, λmin 257 nm, shoulder 227–237 nm. EI-MS
m/z: 266 (M+), 238, 237, 161, 147, 135, 119 as for 9.
3.5. Additional Compounds
3.5.1. 2,6,8-Triazidopurine
Triethylammonium azide (TAA) was prepared by ion exchange from sodium azide and the
triethylammonium form of Dowex 50. 2,6,8-Trichloropurine (TCP, 7 mg) dissolved in dry DMF
(0.25 mL) was heated at 80 ◦C with dried TAA (100 mg, 7-fold excess) for 5 h. The reaction mixture
was partitioned between ethyl acetate and water and the aqueous phase was extracted with ethyl
acetate. TLC of the combined ethyl acetate solutions (solvent D) revealed only one UV absorbing
component (Rf rel. TCP = 1.93) and this gave a brown colour under UV light whereas TCP did not.
The new compound was further purified by retention from 25% (v/v) methanol on a C18 column
(40 µm, 0.5 g) and elution as a sharp fraction by 80% (v/v) ethanol containing acetic acid (1%) to yield
2,6,8-triazidopurine. EI-MS m/z: 243 (M+, 100%), 215 (48), 201 (29), 173 (17), 145 (68).
3.5.2. 2,6,8-Triazido-9-(tetrahydropyran-2-yl)purine
2,6,8-Trichloropurine (30 mg) was dissolved in dioxane (0.4 mL). After the addition of
3,4-dihydropyran (80 µL) and formic acid (40 µ
L), the solution was heated at 85 ◦C for 60 min before
all solvent was evaporated and the residue dissolved in chloroform. This was extracted with 0.5 M
aqueous ammonia (3 double volumes, discarded) to remove traces of the chloropurine. Evaporation
of the dried chloroform phase yielded 2,6,8-trichloro-9-(tetrahydropyran-2-yl)purine (yield 36 mg).
EI-MS m/z: 306/308 (M+, 6%), 222/224 (15), 187/189 (two Cl present, 9%), 85 (100), 84 (28), 67 (16).
The for◦egoing tetrahydropyranyl derivative (16 mg) was dissolved in ethanol (0.80 mL) and
heated at 75 C in a stirred sealed vial with NaN3 (20 mg) for 80 min. The evaporated reaction solution
was partitioned between chloroform and water, and the former phase was re-extracted twice with water.
Evaporation of the dried chloroform solution yielded 2,6,8-triazido-9-(tetrahydropyran-2-yl)purine.
EI-MS m/z: 327 (M+, 4%), 243 (40), 215 (25), 201(5) 85 (100), 84 (37), 67 (27).
3.5.3. 6-Benzylamino-2,8-Dichloro-9-(tetrahydropyran-2-yl)purine
A propan-1-ol solution (10 mL) of trichloropurine (240 mg) and benzylamine (400 mg) was
refluxed for 3 h. The evaporated solution was partitioned between ethyl acetate and water neutralized
with HCl. The ethyl acetate phase was washed with water and subjected to preparative TLC (solvent
D) yielding 6-benzylamino-2,8-dichloropurine. EI-MS m/z: 295 (M + 2, 64%), 293 (M+, 100%), 292 (38),
258 (19), 216 (8) 187 (12), 153 (11), 106 (84). The foregoing product was dissolved in dioxane and
converted to the 9-(tetrahydropyran-2-yl) derivative by the procedure described in Section 3.5.2.
3.5.4. 2-Azido-N6-(3,4-dimethoxybenzyl)adenine
For this synthesis, 2,6-diazidopurine
[42]
(85 mg, 421
µmol) was added to
3,4-dimethoxybenzylamine hydrochloride (1.2 mmol) dispersed in water (3 mL). After addition
◦
of triethylamine (0.25 mL), the mixture was heated at 100 C for 5 h and then neutralized (pH 7)
◦
with 0.5 M HCl. The product precipitated at 4 C over 3 days and was filtered off and washed
with water (yield 70 mg). UV in 95% ethanol, the neutral, acidic and basic spectra were:
λ
max
276

Molecules 2019, 24, 349
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and 236, 280 and 236, 277 and 238 nm, respectively (two maxima for each spectrum); λmin 254, 257
and 255 nm, respectively. EI-MS m/z: 326 (M+, 52%), 297 (27), 283 (56), 267 (28), 166 (29), 151 (100,
dimethoxy-benzyl or tropylium ion), 135 (46), 119 (27).
Author Contributions: Investigation, D.S.L., X.-D.Z. and C.H.H.; Writing—original draft, D.S.L. and X.-D.Z.;
Writing—review and editing, C.H.H.
Funding: This research received no external funding.
Acknowledgments: The authors thank the Research School of Chemistry (ANU) for some of the mass
spectra reported.
Conflicts of Interest: The authors declare no conflicts of interest.
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2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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(CC BY) license (http://creativecommons.org/licenses/by/4.0/).