Synthesis of 8-aminoadenosine 5′-(aminoalkyl phosphates), analogues of aminoacyl adenylates

Article abstract of DOI:10.1016/j.bmc.2005.11.049

A short and efficient route for the synthesis of aminoalkyl 8-aminoadenylates, potential aminoacyl-tRNA synthetase inhibitors, is presented. Aminoalkyl 8-aminoadenylates were synthesized using a 5′-H-phosphonate strategy involving minimal protecting group manipulations and a single final deprotection step.

Full text of DOI:10.1016/j.bmc.2005.11.049

Bioorganic & Medicinal Chemistry 14 (2006) 2653–2659
Synthesis of 8-aminoadenosine 50-(aminoalkyl phosphates),
analogues of aminoacyl adenylates
Esmail Yousefi-Salakdeh,a Merita Murtola,b Anders Zetterberg,c
Esther Yeheskielya and Roger Stro¨mberga,b,
*
aDivision of Organic and Bioorganic Chemistry, MBB, Scheele Laboratory, Karolinska Institutet, S-17177 Stockholm, Sweden
bDepartment of Biosciences, Novum, Karolinska Institutet, S-14157 Huddinge, Sweden
cDepartment of Oncology–Pathology, Cancer Center Karolinska, CCK, R:804, Karolinska Hospital, S-17176 Stockholm, Sweden
Received 20 September 2005; revised 23 November 2005; accepted 23 November 2005
Available online 20 December 2005
Abstract—A short and efficient route for the synthesis of aminoalkyl 8-aminoadenylates, potential aminoacyl-tRNA synthetase
inhibitors, is presented. Aminoalkyl 8-aminoadenylates were synthesized using a 50-H-phosphonate strategy involving minimal
protecting group manipulations and a single final deprotection step.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
Translation of the genetic code and protein synthesis
from amino acids takes place in the ribosomes where
aminoacyl-tRNAs act as key intermediates. The tRNAs
are loaded with the specific amino acids by the action of
aminoacyl-tRNA synthetases. There are specific amino-
acyl-tRNA synthetases for each amino acid, which
means 20 different enzymes for the common 20 amino
acids.1 The aminoacylation reaction, that is, ÔchargingÕ
of tRNA is carried out in two steps. The first step is
the formation of aminoacyl adenylates (I, Fig. 1) by
a specific reaction involving amino acids, adenosine
triphosphate, and an aminoacyl-tRNA synthetase. The
amino acids are thus activated and subsequently trans-
ferred to their corresponding tRNA through a highly
specific reaction catalyzed by the same enzymes.
Figure 1. Aminoacyl adenylate I and analogues II and III.
its methionyl-tRNA synthetase in Escherichia coli which
results in the blocking of acylation of tRNAMet, whereas
acylation of other tRNAs remains unaffected.3
Aminoalkyl adenylates (II) are inhibitors of these en-
zymes and are thought to occupy the binding site for
the aminoacyl adenylate.2 This class of compounds is
valuable as inhibitors of amino acid activation and as
stable substrates for studies of enzyme–substrate inter-
action. For example, the L-methioninyl adenylate inhib-
It has also been reported that adenosine has an inhibito-
ry effect on ATP-PPi exchange and the aminoacylation
of tRNA catalyzed by methionyl-tRNA synthetase from
E. coli.4 This inhibition is greatly influenced by substitu-
tion in the 8-position, as shown by studies with modified
adenosines. 8-Aminoadenosine displays a Ki 300 times
lower than that of adenosine.4 It has been suggested that
with the 8-aminoadenosine, the 8-position of the imidaz-
ole ring comes in close proximity to the functional group
Keywords: Aminoacyl-tRNA synthetase inhibitors; Aminoacyl aden-
ylate analogues; Aminoalkyl adenylates; H-Phosphonates.
*
Corresponding author. Tel.: +46 8 608 9115; fax: +46 8 608 9127;
e-mail: Roger.Stromberg@biosci.ki.se
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.11.049

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E. Yousefi-Salakdeh et al. / Bioorg. Med. Chem. 14 (2006) 2653–2659
of an amino acid residue in the enzyme, which can form
a strong hydrogen bond with the 8-amino function.4
can be converted to the azido intermediates 6b–d, which
upon reduction should afford the desired 8-amino ad-
ducts. For the introduction of the phosphodiester bond
between the 50-hydroxy of the nucleoside and the amino
alcohol we selected the H-phosphonate approach be-
cause of its experimental simplicity, efficiency of genera-
tion of H-phosphonate mono- and diesters and stability
of the monoester intermediate during prolonged storage.
Moreover, the reagents used in this approach do not af-
fect the nucleobase and hence, masking of the exocyclic
amino group on the purine is not mandatory. Apart
from this, a key element in the route towards the prep-
aration of compounds (III) is the single step removal
of the protecting groups, which will be accompanied
by simultaneous reduction of the 8-azido to the 8-amino
function. We selected the benzyloxycarbonyl (Z) group
for the amino function of the amino alcohol component
(and the imidazole moiety of histidinol) and the benzyl-
idene group for the cis-diol function of the adenosine.
Both groups can be removed by hydrogenolysis, which
is also a suitable method for conversion of the azido
function into an amine.
The inhibitory effects found with 8-aminoadenosine and
aminoalkyl adenylates suggest that it may be fruitful to
combine these modifications in order to obtain more
efficient inhibitors of tRNA acylation. Thus, we decided
to develop a route for synthesis of the nucleopeptide
analogues, aminoalkyl 8-aminoadenylates (III).
The synthesis of nucleopeptide analogues is still a chal-
lenge due to the variety of functional groups that can be
involved. A chemical synthesis of some aminoalkyl
adenylates containing unmodified adenosine has been
published2 as well as a method that involves enzyme
catalyzed condensation of adenosine-50-pyrophosphate
with the amino alcohol.5 However, there are no reports
on the preparation of aminoalkyl 8-aminoadenylates
(III). In the present study, we report an efficient and
short route for synthesis of aminoalkyl 8-aminoadeny-
lates involving minimal protecting group manipulations
and a single final deprotection step.
As a reference compound, an aminoalkyl adenylate con-
taining unmodified adenosine (II) was also synthesized
following the same general route, but without the
manipulations involving the 8-position. The synthesis
of this compound will be described first. In the first step,
masking of 20- and 30-OH of adenosine was done in
order to selectively phosphonylate the 50-OH function.
A benzylidene acetal is a commonly used protecting
2. Results and discussion
Aminoalkyl 8-aminoadenylates (III) unlike aminoalkyl
adenylates (II) contain an additional amino function
on the nucleobase. We chose to start from adenosine
(1) and to introduce the amino group at the 8-position
via the 8-bromo derivatives 2 (Scheme 1). The latter
Scheme 1. Reagents: (i) benzaldehyde, ZnCl2; (ii) 1. phosphonic acid, Piv-Cl, pyridine; 2. Et3N–H2O; (iii) protected amino alcohol, bis(2-oxo-3-
oxazolidinyl)phosphinic chloride (OXP); (iv) I2/pyridine/H2O; (v) NaN3, DMSO; (vi) Pd–C, 80% acetic acid; (vii) Pd-black, 80% acetic acid,
(viii) Na2S2O4. X = CH(CH3)CH2CH3, Y = CH2(imidazole), and Z = CH2CH2SCH3.

E. Yousefi-Salakdeh et al. / Bioorg. Med. Chem. 14 (2006) 2653–2659
2655
group for 1,2- and 1,3-diols in carbohydrate chemistry
and there are different routes towards the benzylidene
protection but a,a-dimethoxytoluene or benzaldehyde
are the most used reagents for this purpose.6 In the first
attempt to protect the cis-diol system, a,a-dimethoxytol-
uene was used. An isomeric mixture of products, for
example, 20,30- and 30,50-protected adenosine, was ob-
tained, and due to the low yield of the desired 20–30 iso-
mer, alternative conditions, that is, benzaldehyde in the
7,8
presence of ZnCl2 were implemented. After stirring
compound 1 for three days, in a mixture of benzalde-
hyde and ZnCl2 at ambient temperature and purification
by silica gel chromatography, the protected compound
3a was obtained in good yield.
Figure 2. Protected amino alcohols.
The next step was to establish the proper phosphonyla-
tion conditions. The free amino group of adenosine can
react with a variety of electrophiles and therefore it is of-
ten necessary to mask it during synthesis, for example,
during phosphoryl-, phosphityl- and phosphonylation
processes.9 However, a few examples of the O-selective
phosphonylation of N-unprotected nucleosides have
been reported. Phosphonylation reagents containing
the P–H function such as pyrophosphonate10 and
diphenyl H-phosphonate11–13 were completely inert to-
wards the exocyclic amino groups of the naturally
occurring nucleosides.9 The reaction of 3a with diphenyl
H-phosphonate (7 equiv) in pyridine, proceeded fast and
efficiently (complete in less than 20 min), to give after
addition of triethylamine in water compound 4a. How-
ever, when following this procedure, difficulties during
work-up and purification were encountered, resulting
in a low yield of purified 4a. These purification problems
were probably caused by the similar solubilities and
chromatographic mobility of 4a and the side product
from the phosphonylation reagent (i.e., phenyl H-phos-
phonate monoester). This experience led us to adopt an
alternative phosphonylation reagent, that is, pyro-
phosphonate. Pyrophosphonate can be generated practi-
cally as a single species from phosphonic acid in pyridine
in the presence of 0.5 equiv of pivaloyl chloride.10 The
reaction of equimolar amounts of the pyrophosphonate
and the nucleoside proved to be slow, but using a higher
concentration of the reagent gave faster and more efficient
conversion. Thus, when 3a was phosphonylated using
0.4 M (2.5 equiv) of pyrophosphonate reagent the reac-
tion was complete within 4 h giving compound 4a in high
yield after purification by silica gel chromatography. No
reaction on the unprotected amino group was detected.
ride,16 which are commonly used in the H-phosphonate
approach15,17–21 can react with unprotected amino
groups of the nucleoside.22 Although no reaction was
found with the adenine base, we thought that for gener-
ality of the method, another condensing agent would be
a better alternative. It has been reported that bis (2-oxo-
oxazolin-3-yl) phosphonic chloride (OXP), first used in
peptide chemistry for peptide bond formation23 and lat-
er in the H-phosphonate method for introduction of a
phosphodiester linkage,24,25 does not react readily with
the exocyclic amino groups of all common nucleosides.9
Condensation of amino alcohol 9 with compound 4a un-
der the agency of OXP in pyridine proceeded smoothly
giving, after oxidation with iodine in aqueous pyridine,
compound 5a. After purification, deprotection of the Z
and the benzylidene groups was affected by catalytic
hydrogenolysis. Thus, treatment of the isoleucinol con-
taining 5a with palladium–carbon in 80% acetic acid
solution for 24 h afforded completely deprotected aden-
osine 50-(L-isoleucinyl phosphate) (6a) in 82% yield after
purification.
Synthesis of L-isoleucinol, L-histidinol and L-methioni-
nol containing III also started from adenosine. In the
first step, adenosine was converted into 8-bromoadeno-
sine (2) with aqueous bromine in acetate buffer (pH 4).26
Introduction of the benzylidene group for protection of
the cis-diol function was performed as described above
for adenosine (1). Phosphonylation of protected 8-
bromoadenosine was affected using phosphorous acid/
pivaloyl chloride in pyridine under conditions identical
to those for the preparation of 4a. The Z-protected
amino alcohols were used together with OXP in the
condensation step affording after oxidation and purifica-
tion the diesters 5b–d in high yields.
Introduction of the Z group for protection of the amino
moiety of the amino alcohol was achieved by following a
standard peptide chemistry protocol for building block
synthesis14 giving (Na-benzyloxycarbonyl-L-methioninol
(10) and Na-benzyloxycarbonyl-L-isoleucinol (9)) in
nearly quantitative yields (Fig. 2). For L-histidinol both
the a-amino and the s-amino functions were protected
in one step to give the Na,Ns-dibenzyloxycarbonyl-L-his-
tidinol (11). With the properly protected amino alcohols
(9–11) and phosphonylated adenosine (4a) in hand, we
then focused our attention on the condensation step. It
has been established that condensing reagents such as
pivaloyl chloride15 and 1-adamantanecarbonyl chlo-
Conversion of 8-bromopurines directly to the 8-amino
counterparts has been reported27 but is usually unsuccess-
ful with adenosine derivatives. Reaction with ammonia
was unsuccessful below 125 ꢁC and considerable decom-
position was observed at higher temperatures. Reaction
with hydrazine, which with 8-bromoguanosine gives good
yield of 8-aminoguanosine, was unsuccessful with 8-
bromoadenosine and produced mainly 8-hydrazinoade-
nosine. Instead, the most efficient method to the 8-amino-
adenosine was via reduction of8-azidoadenosine.27 In our
synthesis strategy towards aminoalkyl adenylates this

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E. Yousefi-Salakdeh et al. / Bioorg. Med. Chem. 14 (2006) 2653–2659
reduction can be accomplished simultaneously to remov-
al of the Z and benzylidene protection, thus obviating the
need for an additional step.
the preparation of other aminoalkyl 8-aminoadenylates,
since most side chains can be protected with Z (Lys and
Trp), as benzyl esters (Asp and Glu) or benzyl ethers
(Ser, Thr and Tyr), all of which can be readily removed
by hydrogenolysis. At the moment, evaluation of the
biological activity of L-isoleucinyl, L-methioninyl and
L-histidinyl 8-aminoadenylates is in progress.
Compounds 5b–d were treated with sodium azide in
dimethylsulfoxide at 65 ꢁC during 16 h to give, after
work-up and purification, homogeneous compounds
6b–d. The final stage of the preparation of aminoalkyl-
8-aminoadenylates was catalytic hydrogenolysis on pal-
ladium. Thus, treatment of the L-isoleucinol containing
6b using palladium–carbon in 80% acetic acid solution
for 24 h afforded completely deprotected L-isoleucinyl
8-aminoadenylate (7a) in a yield of 76% after purifica-
tion. Using palladium–carbon for deprotection/reduc-
tion of compounds containing methionine or histidine
is often troublesome and the procedure applied for 6b
was unsuccessful when adopted to 6c and d. Prolonged
exposure (three days) to the conditions described above
for 5a and 6b did not result in the conversion of signif-
icant amounts of 6c and d into the corresponding 7b and
8. To circumvent this problem, the more reactive cata-
lyst palladium black28 was selected, which has been used
successfully for the hydrogenolysis of the Z group from
the amino acids L-methionine and L-histidine.29 Conse-
quently, L-histidinol and L-methioninol derivatives (6c
and d) were dissolved in 80% acetic acid and subjected
to treatment with palladium black in a H2-saturated
atmosphere. As a result of this, compound 7b was isolat-
ed after work-up and purification in a yield of 75%.
Although palladium black catalyzed hydrogenation
has been reported to be successful with methionine
derivatives we faced problems with the L-methioninol
derivative. It is not unlikely that this is due to poisoning
of the catalyst and difficulties with washing out of the
product from the catalyst due to adsorbance of the
thioether to the palladium. However, we noticed that
addition of thiophenol helped in washing out of the
product and additional treatment with 80% acetic acid
ensured more complete removal of the benzylidine
group. In addition, we always observed some accompa-
nying oxidation of the thioether, although thorough
flushing of all solutions with nitrogen was performed,
and we could also isolate the corresponding sulfoxide
(7c) in minor quantities. To simplify the isolation of
the pure non-oxidized methioninol derivative 8, we also
introduced a sodium hydrosulfite (dithionite) treatment
in the work-up scheme. This additional reduction step
can conveniently be executed without intermediate
purification of 7c. By taking the above findings into
account we could modify the procedure so that hydrog-
enolysis of 6d and subsequent treatment with dithionite
gave a reasonably acceptable isolated yield (35%) of
L-methioninyl-8-aminoadenylate (8).
3. Experimental
3.1. Materials and methods
All commercial reagents were of synthesis grade and
used without further purification. 8-Bromoadenosine26
(2) and 20,30-O,O-benzylidene-adenosine7,8 (3a) were
synthesized using published procedures. Pyridine (Lab-
˚
scan) and DMSO were dried and stored over 4 A molec-
ular sieves. THF was distilled at atmospheric pressure
from CaH2. Dichloromethane, chloroform and metha-
nol were of pro analysis commercial quality. The reac-
tions were monitored by thin-layer chromatography
(TLC) analysis using silica gel plates (Kieselgel 60
F254, E. Merck). Compounds were visualized by
UV-light. Silica gel chromatography was performed on
˚
Matrix silica (60 A, 35–70 lm, Amicron). General mass
spectrometry analysis was performed using electrospray
quadropole time-of-flight mass spectrometry (Q-TOF)
and accurate mass was determined using electrospray
time-of-flight mass spectrometry (Micromass LCT,
ESI-TOF). NMR-spectra were obtained on a Bruker
AVANCE DRX-400 instrument (400 MHz in 1H,
100 MHz in 13C and 162 MHz in 31P). Chemical shifts
(d) are reported in parts per million downfield from
tetramethylsilane, with the residual nondeuterated sol-
vent signal as an internal standard. 31P chemical shifts
are calibrated with respect to H3PO4 (1% v/v) in D2O.
3.2. 20,30-O,O-Benzylidene-8-bromoadenosine (3b)
20, 30-O,O-Benzylidene-8-bromoadenosine (3b) was syn-
thesized using a similar procedure as for compound
3a.7,8 Compound 2 (1.82 g, 5.27 mmol) was dissolved in
benzaldehyde (8.61 g, 81.16 mmol) and ZnCl2 (2.82 g,
20.70 mmol) was added to the solution. The reaction mix-
ture was stirred at room temperature for three days. The
mixture was then diluted with ethyl acetate (200 ml)
and washed with water (2· 300 ml). The combined water
layers were washed with ethyl acetate (100 ml). The
organic layers were pooled, dried with MgSO4 and
evaporated to dryness. The crude product was purified
by silica gel column chromatography (stepwise gradient
of 0–10% v/v MeOH in CHCl3). The product was
obtained as a mixture of endo/exo stereoisomers.
Yield (1.70 g, 3.91 mmol) 74%. Rf = 0.3 (MeOH/CHCl3,
1:19, v/v).
In conclusion, aminoalkyl (L-isoleucinyl and L-histidi-
nyl) 8-aminoadenylates and aminoalkyl (L-isoleucinyl)
adenylate were synthesized in high yields. L-Methioni-
nyl-8-aminoadenylate could also be produced although
the yield is here limited by problems in final deprotec-
tion. The general procedure involves few steps without
protection of the nucleobase and with a single deprotec-
tion step which simultaneously produces the 8-amino
function. The above method should be applicable for
3.3. Synthesis of 20,30-O,O-benzylideneadenosine and
20,30-O,O-benzylidene-8-bromoadenosine 50-hydrogen-
phosphonate triethylammonium salts (4a and b)
For this reaction the in situ pyrophosphonate method
was used.10 To a solution of 3a or b (0.69 mmol) in

E. Yousefi-Salakdeh et al. / Bioorg. Med. Chem. 14 (2006) 2653–2659
2657
dry pyridine (2 ml) a prepared solution of phosphonic
3.7. Synthesis of protected 8-bromoadenosine and aden-
osine 50-(aminoalkyl phosphate) triethylammonium salts
(5a–d)
acid (282.9 mg, 3.45 mmol) and pivaloyl chloride
(208 mg, 1.72 mmol) in pyridine (2 ml) was added. The
reaction was followed by TLC (CHCl3/MeOH, 8:2,
v/v). The reaction was quenched after 4 h by addition
of a mixture of triethylamine (1.92 ml, 13.8 mmol) in
water (1 ml). The reaction mixture was evaporated to
dryness and the residue was dissolved in THF and was
passed through a filter. The THF solution was evaporat-
ed to dryness and purification was performed by silica
gel chromatography (flash mode) using MeOH/CHCl3
1:19 v/v, containing 0.1% triethylamine. Products were
obtained as a mixture of endo/exo stereoisomers. 20,
30-O,O-Benzylideneadenosine 50-hydrogenphosphonate
triethylammonium salt (4a): yield 55%. 20,30-O,O-Ben-
Compounds 4a or b (0.25 mmol) and Z-amino alcohol
(1.2 equiv, 0.30 mmol) were dried by evaporation of
added pyridine (2· 3 ml) and then dissolved in dry
pyridine (10 ml). The stirred reaction mixture was then
treated with bis (2-oxo-oxazolin-3-yl) phosphonic chlo-
ride (OXP) (2 equiv, 0.5 mmol). The reaction was fol-
lowed by TLC (CHCl3/MeOH, 8:2, v/v) and after
30 min, iodine in a mixture of pyridine and water
(2% w/v, 4.9 ml pyridine in 0.1 ml water) was added.
The oxidation mixture was kept for 15 min, diluted
with dichloromethane (10 ml) and evaporated to dry-
ness. The residue was dissolved in dichloromethane
(15 ml) and washed with a solution of Na2S2O3
(10% aq) and triethylammonium bicarbonate (aq,
2 M) (1:1, v/v) (10 ml). The organic layer was dried
with MgSO4 and evaporated to dryness. The residue
was purified by silica gel column chromatography
(flash mode) using CHCl3/MeOH (19:1) containing
0.1% triethylamine. Products were obtained as a
mixture of endo/exo stereoisomers. 20,30-O,O-Benzylid-
zylidene-8-bromoadenosine
triethylammonium salt (4b): yield (335 mg, 0.56 mmol)
81%.
50-hydrogenphosphonate
3.4. Na-Benzyloxycarbonyl-L-isoleucinol (9)
Na-Benzyloxycarbonyl-L-isoleucinol (9) was synthe-
sized using published procedure.14 To a solution of
L-isoleucinol (200 mg, 1.71 mmol) in dried dioxane
(1.70 ml),
a
solution of Na2CO3 (580.12 mg,
eneadenosine
50-(Na-benzyloxycarbonyl-L-isoleucinyl
5.47 mmol) in water (2 ml) was added. Benzyl chloro-
formate (337.61 mg, 1.98 mmol) was added in five por-
tions, while the mixture was stirred in an ice bath.
The reaction was monitored by TLC (CHCl3/MeOH,
19:1, v/v). After 1 h, the mixture was neutralised to
pH 7 by KH2PO4 (pH 3) and extracted with H2O
(20 ml) and ethyl acetate (2· 30 ml). The organic layer
was dried with MgSO4 and evaporated to dryness.
The residue was purified by silica gel chromatography
using methanol in chloroform (1:19, v/v) as eluent.
Yield (430 mg, 1.71 mmol) P 99%. Rf = 0.7 (MeOH/
CHCl3, 1:19, v/v).
phosphate) triethylammonium salt (5a): yield 88%,
Rf = 0.2 (MeOH/CHCl3, 2:8, v/v). 20,30-O,O-Benzyli-
dene-8-bromoadenosine 50-(Na-benzoxycarbonyl-L-isol-
eucinyl phosphate) triethylammonium salt (5b): yield
93%, Rf = 0.5 (MeOH/CHCl3, 2:8, v/v). 20,30-O,O-Ben-
zylidene-8-bromoadenosine 50-(Na,Ns-Dibenzyloxycar-
bonyl-L-histidinyl phosphate) triethylammonium salt
(5c): yield 93%, Rf = 0.4 (MeOH/CHCl3, 2:8, v/v).
20,30-O,O-Benzylidene-8-bromoadenosine 50-(Na-ben-
zyloxycarbonyl-L-methioninyl phosphate) triethylam-
monium salt (5d): yield 86%, Rf = 0.3 (MeOH/
CHCl3, 2:8, v/v).
3.5. Na-Benzyloxycarbonyl-L-methioninol (10)
3.8. Synthesis of protected 8-azidoadenosine (aminoalkyl
phosphate) triethylammonium salts (6b–d)
Compound 10 was prepared using L-methioninol as start-
ing material following the above-described procedure for
the synthesis of compound 9. Yield (297 mg, 1.17 mmol)
94%. Rf = 0.5 (MeOH/CHCl3, 1:19, v/v).
Compounds 5b,c or d (0.23 mmol) was dissolved in
DMSO (1 ml) and NaN3 (4 equiv, 0.99 mmol) was
added. The resulting solution was heated at 75 ꢁC
for 16 h, cooled to room temperature and diluted with
THF (10 ml) whereupon excess NaN3 largely precipi-
tated. The mixture was passed through a filter and
the solution was evaporated to dryness. The residue
was purified by silica gel chromatography using
CHCl3/MeOH (19:1) containing 0.1% triethylamine.
Products were obtained as a mixture of endo/exo ster-
eoisomers. 20,30-O,O-Benzylidene-8-azidoadenosine 50-
(Na-carbobenzoxy-L-isoleucinyl phosphate) triethylam-
monium salt (6b): yield 70%, Rf = 0.7 (THF/pyridine/
H2O, 8:1:1, v/v/v). 20,30-O,O-Benzylidene-8-azido-
adenosine 50-(Na,Ns-dibenzyloxycarbonyl-L-histidinyl
phosphate) triethylammonium salt (6c): yield 77%,
Rf = 0.7 (THF/pyridine/H2O, 8:1:1, v/v/v). 20,30-O,O-
Benzylidene-8-azidoadenosine 50-(Na-benzyloxycarbon-
yl-L-methioninyl phosphate) triethylammonium salt
(6d): yield 76%, Rf = 0.8 (THF/pyridine/H2O, 8:1:1,
v/v/v).
3.6. Na,Ns-Dibenzyloxycarbonyl-L-histidinol (11)
Na,Ns-Dibenzyloxycarbonyl-L-histidinol (11) was syn-
thesized using published procedure.14 To a solution of
L-histidinol dihydrochloride (104 mg, 0.486 mmol) in
dried dioxane (3 ml), a solution of Na2CO3 (483 mg,
4.55 mmol) in water (1.42 ml) was added. Benzyl chloro-
formate (182 mg, 1.07 mmol) was added in five portions,
while the mixture was stirred in an ice bath. The reaction
was followed by TLC (CHCl3/MeOH, 19:1, v/v). After
1 h, the mixture was neutralised to pH 7 by KH2PO4
(pH 3) and extracted with H2O (20 ml) and ethyl acetate
(2· 30 ml). The organic layer was dried with MgSO4 and
evaporated to dryness. The residue was purified by silica
gel chromatography using methanol in chloroform
(1:19, v/v) as eluent. Yield (82 mg, 0.20 mmol) 41%.
Rf = 0.8 (MeOH/CHCl3, 1:19, v/v).

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3.9. 8-Aminoadenosine and adenosine 50-(L-isoleucinyl
phosphate) triethylammonium salts (7a and 6a)
3.12. 8-Aminoadenosine 50-(L-histidinyl phosphate) trie-
thylammonium salt (7b)
To palladium on carbon (100 mg), under a hydrogen
atmosphere in 80% acetic acid (1 ml), was added 5a
or 6b (27 mg, 0.033 mmol) in 80% acetic acid (2 ml).
The reaction was stirred followed by TLC (2-propa-
nol/ammonia/water, 7:1:2, v/v/v). After 24 h under
hydrogen atmosphere, the mixture was diluted with
water and methanol (50:50, v/v) (5 ml). The solution
was filtered through a column of Celite, which was
washed repeatedly with methanol, methanol–water
and water. The combined solutions were evaporated
to dryness under reduced pressure and the residue
was redissolved in water and lyophilized. Reverse-
phase HPLC purification was carried out using
Jones (Genesis C18, 4l, 10 · 250 mm) chromatogra-
phy column employing a flow rate of 4 ml/min with
a linear gradient 0–12.5% acetonitrile in 0.1 M trie-
thylammonium acetate buffer (pH 6.5) at 30 ꢁC for
90 min. The retention time of 6a was 32 min and
of 7a 35 min. The purified product was lyophilized,
redissolved in water and lyophilized again three
times.
A solution of 6c (33 mg, 0.034 mmol), in 80% acetic acid
(2 ml) was added to a round-bottomed flask containing
approximately 100 mg palladium black catalyst and 80%
acetic acid (2 ml) with stirring under a hydrogen atmo-
sphere. The progress of hydrogenation was followed
by TLC (2-propanol/ammonia/water, 7:1:2, v/v/v) which
indicated that the reaction was complete after 24 h. The
crude compound was isolated by filtering off the catalyst
through Celite and washing with methanol-water (50:50,
v/v) (30 ml), followed by methanol (40 ml) and water
(40 ml). The combined filtrates were then concentrated
by evaporation under reduced pressure. The product
was redissolved in water and lyophilized. Reverse-phase
HPLC purification was carried out using Jones (Genesis
C18, 4l, 10 · 250 mm) chromatography column
employing a flow rate of 4 ml/min with a linear gradient
0–12.5% acetonitrile in 0.1 M triethylammonium acetate
buffer (pH 6.5) at 30 ꢁC for 90 min. Retention time was
19.2 min. The purified product was lyophilized, redis-
solved in water and lyophilized again three times. Yield
(15 mg, 0.025 mmol) 75%. Electrospray-MS: m/z, calcd:
[M+H]+ = 486.1615,
found
[M+H]+ = 486.1611,
[MꢀHis] = 363.0818. Rf = 0.7 in 2-propanol/ammonia/
3.10. Adenosine 50-(L-isoleucinyl phosphate) triethy-
lammonium salt (6a)
1
water, 7:1:2, v/v/v. H NMR (400 MHz, D2O) d 1.18
(t, N(CH2CH3)3), 2.85 (d, 2H, bCH2), 3.08 (q,
N(CH2CH3)3), 3.60 (m, 1H, aCH), 3.81 (m, 1H,
CH2O), 3.93 (m, 1H, CH2O), 4.03 (m, 2H, 50), 4.15
(m, 1H, 40), 4.26 (dd, 1H, 30), 4.64 (1H, 20) 5.88 (d,
1H, 10), 6.96 (s, 1H, CH(im)), 7.67 (s, 1H, CH(im)),
7.93 (s, 1H, 2) ppm. 13C NMR (100 MHz, D2O) d 8.9,
26.7, 47.3, 51.8, 51.9, 65.0, 65.9, 70.3, 71.3, 83.9, 84.0,
87.6, 116.8, 117.3, 131.6, 136.4, 149.8, 150.1, 152.4,
152.9 ppm. 31P NMR, (162 MHz, D2O/CD3OD/
DMSO-d6) d ꢀ0.7 ppm.
Yield 82%. MS, (Q-TOF, m/z) calcd: [M+H]+ = 447.17,
found: [M+H]+ = 447.07. Rf = 0.6 (2-propanol/ammo-
nia/water, 7:1:2, v/v/v). 1H NMR (400 MHz, D2O) d
0.74 (m, 6H, 2CH3), 0.82 (m, 1H, CH), 1.17 (m,
1H, CH2A), 1.34 (t, N(CH2CH3)3), 1.60 (m, 1H,
CH2B), 3.27 (q, N(CH2CH3)3), 3.27 (1H, aCH under
N(CH2CH3)3), 3.81 (t, 2H, CH2OP), 4.16 (m, 2H,
50A/50B), 4.42 (m, 1H, 40), 4.62 (t, 1H, 30), 4.92 (t,
1H, 20), 6.16 (d, 1H, 10), 8.27 (s, 1H, 2), 8.51 (s,
1H, 8) ppm. 13C NMR (100 MHz, D2O) d 8.9,
10.9, 13.6, 25.4, 34.8, 47.3, 55.9, 56.0, 63.6, 63.6,
65.4, 65.4, 70.9, 74.1, 84.1, 84.2, 87.5, 119.1, 140.3,
149.8, 153.6, 156.2 ppm. 31P NMR (162 MHz, D2O)
d ꢀ0.3 ppm.
3.13. 8-Aminoadenosine 50-(L-methioninyl phosphate)
triethylammonium salt (8)
A solution of 6d (20 mg, 0.024 mmol), in 80% acetic acid
(1 ml) was added to a round-bottomed flask containing
approximately 50 mg palladium black catalyst and 80%
acetic acid (2 ml) with stirring under a hydrogen atmo-
sphere. After 36 h, 40 ll thiophenol was added and the
mixture was stirred for 1 h. The crude compound was
isolated by filtering off the catalyst through Celite and
washing with methanol/water (50:50, v/v, 20 ml), fol-
lowed by methanol (20 ml) and water (20 ml). The com-
bined filtrates were then concentrated by evaporation
under reduced pressure. The crude product was dis-
solved in 80% acetic acid (3 ml) and the reaction solu-
tion was stirred for 100 h. The crude product was
evaporated to dryness, the residue was dissolved in
water (25 ml) and washed with CHCl3 (2· 10 ml). The
aqueous layer was evaporated to dryness, redissolved
in water and lyophilized.
3.11. 8-Aminoadenosine 50-(L-isoleucinyl phosphate)
triethylammonium salt (7a)
Yield (14 mg, 0.024 mmol), 76%. Electrospray-MS:
m/z, calcd: [M+H]+ = 462.1866, found: [M+H]+ =
462.1862, [MꢀIle] = 363.0817. Rf = 0.5 (2-propanol/am-
monia/water, 7:1:2, v/v/v). 1H NMR, (400 MHz,
D2O/CD3OD) d 0.71 (m, 6H, 2CH3), 0.93 (m, 1H,
CH), 1.21 (t, N(CH2CH3)3), 1.21 (1H, CH2A under
N(CH2CH3)3), 1.59 (m, 1H, CH2B), 3.14 (q,
N(CH2CH3)3), 3.29 (m, 1H, aCH), 3.82 (m, 1H,
CH2OP), 3.93 (m, 1H, CH2OP), 4.12 (m, 2H, 50A/
50B), 4.23 (s, 1H, 40), 4.44 (t, 1H, 30), 4.86 (m, 1H,
20), 5.91 (d, 1H, 10), 7.97 (s, 1H, 2) ppm. 13C NMR
(100 MHz, D2O/CD3OD)
34.9, 47.3, 56.0, 56.0, 63.9, 64.0, 65.5, 65.5, 70.0,
d 8.9, 10.9, 13.8, 25.5,
The crude product was dissolved in water (1 ml) and an
excess of sodium hydrosulfite (dithionite) (10 mg,
0.06 mmol) was added. The solution was heated up to
60 ꢁC and the reaction was followed by TLC (2-propa-
71.2, 83.7, 83.8, 87.6, 116.9, 149.8, 150.2, 152.6,
d
153.0 ppm. 31P NMR (162 MHz, D2O/CD3OD)
ꢀ0.4 ppm.

E. Yousefi-Salakdeh et al. / Bioorg. Med. Chem. 14 (2006) 2653–2659
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Acknowledgment
We thank the Swedish Research Council for financial
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Supplementary material
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmc.2005.11.049.
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