Synthesis and biological activity of novel S-Adenosyl-L-homocysteine hydrolase inhibitors

Article abstract of DOI:10.1016/S0968-0896(03)00301-8

Four potential S-adenosyl-L-homocysteine hydrolase inhibitors were prepared and tested against purified recombinant rat liver enzyme. Preliminary studies indicate that three of these compounds, 1, 2, and 4, caused time-dependent inactivation of S-adenosyl-L-homocysteine hydrolase but showed a biphasic nature. Compound 3 was found to be a rapid equilibrium inhibitor of this enzyme.

Full text of DOI:10.1016/S0968-0896(03)00301-8

Bioorganic & Medicinal Chemistry 11 (2003) 3229–3236
Synthesis and Biological Activity of Novel
S-Adenosyl-L-homocysteine Hydrolase Inhibitors
Jennifer A. Steere and John F. Honek*
Department of Chemistry, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1
Received 21 January 2003; revised 21 April 2003; accepted 3 May 2003
Abstract—Four potential S-adenosyl-l-homocysteine hydrolase inhibitors were prepared and tested against purified recombinant
rat liver enzyme. Preliminary studies indicate that three of these compounds, 1, 2, and 4, caused time-dependent inactivation of
S-adenosyl-l-homocysteine hydrolase but showed a biphasic nature. Compound 3 was found to be a rapid equilibrium inhibitor of
this enzyme.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
effects.7a,b The antiviral activity associated with inhibit-
The cellular enzyme S-adenosyl-l-homocysteine
(AdoHcy) hydrolase (EC 3.3.1.1) plays an important
regulatory role in S-adenosyl-l-methionine (AdoMet)-
dependent methylation reactions.1a,b,c,d,e In AdoMet-
dependent transmethylation reactions, the methyl group
from AdoMet is transferred to various acceptor mole-
cules such as proteins, nucleic acids, and small mole-
cules by specific methyltransferase enzymes.2a,b,c
S-Adenosyl-l-homocysteine is a product of all AdoMet-
dependent transmethylation reactions. In eukaryotes,
the only known pathway for the catabolism of AdoHcy
is its hydrolysis to homocysteine (Hcy) and adenosine
(Ado) by AdoHcy hydrolase (Fig. 1).3a,b AdoHcy is a
potent competitive inhibitor of all AdoMet-dependent
methylation reactions. Inhibition of cellular AdoHcy
hydrolase results in an intracellular accumulation of
AdoHcy, causing a significant increase in the intracel-
lular AdoHcy/AdoMet ratio and the subsequent inhibi-
tion of AdoMet-dependent methylations. Failure to
regulate the intracellular levels of AdoHcy can therefore
lead to cellular toxicity.
ing AdoHcy hydrolase arises due to the observation that
most plant and animal viruses require a methylated cap
structure at the 50-terminus of their mRNA for viral
replication.8 Thus, by inhibiting AdoHcy hydrolase, the
virus-encoded methyltransferases that are involved in
the formation of this methylated cap structure can be
inhibited and viral replication can be slowed. AdoHcy
hydrolase inhibitors have been shown to be broad-
spectrum antiviral agents interfering with the replication
of (À) RNA viruses (e.g., Ebola, Marburg, measles,
influenza A and B and rabies), (+) RNA viruses (e.g.,
reo and rota), and some DNA viruses (e.g., vaccinia and
cytomegalovirus).4a,9a,b,c
The recent determination of the crystal structure of the
rat liver10a,b and human11 AdoHcy hydrolases have
greatly contributed to our understanding of this enzyme.
Even more recently the crystal structure of rat liver
AdoHcy hydrolase with the potent acyclic inhibitor
d-eritadenine bound has led to a greater understanding
of the interactions between inhibitor and enzyme.12 In
this paper, we describe the biological activity of four
nucleosides as potential inhibitors of AdoHcy hydrolase.
In recent years, AdoHcy hydrolase has become an
attractive target for drug design. Inhibitors of this
enzyme have been shown to exhibit antiviral,4a,b,c anti-
parasitic,5a,b,c anti-arthritic6 and immunosuppressive
The compound, 30-amino-30-deoxyadenosine (1) (Fig.
2), was first discovered to be a potent antibiotic in
1952;13a,b however, this compound, to our knowledge,
has never been studied as an inhibitor of AdoHcy
hydrolase. The study of this compound should allow for
the examination of the interactions an amino group at
*Corresponding author. Tel.:+1-519-888-4567; fax:+1-519-746-0435;
e-mail: jhonek@uwaterloo.ca
0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00301-8

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J. A. Steere, J. F. Honek / Bioorg. Med. Chem. 11 (2003) 3229–3236
The key intermediate in the preparation of compounds
2 and 3 was 4-(adenin-9-yl)-2,3-O-isopropylidenedioxy
butanal (5) which was obtained by the procedure
reported by Hirota et al. (Scheme 1).18 The first step
involves the selective cleavage of the C-10–O bond of
20,30-O-isopropylidene adenosine (6) with diisobutylalu-
minum hydride (DIBAL-H) to produce 9-(2,3-O-iso-
propylidene-d-ribityl)adenine (7) in 48% yield.19a,b
Oxidative cleavage of the 20,30-diol portion of 7 with
sodium periodate gave the key intermediate 5 in quanti-
tative yield as the gem diol.18 Compound 5 could then be
reacted with hydroxylamine hydrochloride and sodium
methoxide for 6 h to produce 4-(adenin-9-yl)-2,3-O-iso-
propylidenedioxybutanal oxime 8 in 86% yield as descri-
bed by Hirota et al.18 The isopropylidene protecting
group was then removed with trifluoroacetic acid (TFA)
to give compound 2 as a mixture of E:Z (15:85) isomers in
98% yield. The amine analogue 3 was also prepared from
compound 5 by reductive amination with N-a-Boc-l-a,g-
diaminobutyric acid, followed by reduction with sodium
cyanoborohydride to give [4-(adenin-9-yl)-2,3-O-iso-
propylidenedioxybutanal]-a-N-Boc-butanoic acid (9) in
36% yield. The protecting groups were then removed
with TFA to produce compound 3 in 96% yield.
Figure 1. Reaction catalyzed by S-adenosyl-l-homocysteine hydrolase.
Figure 2. Compounds prepared for this study.
Compound 4 was prepared by protecting the 20- and
30-hydroxyl groups of 50-deoxy-50-methylthioadenosine
(10) using 2,2-dimethoxypropane and p-toluenesulfonic
acid to give 9-(50-deoxy-20,30-O-isopropylidene-50-
methylthio) adenosine (11) in 99% yield (Scheme 2).20
The C-10–O bond of the now protected derivative 11
was cleaved using DIBAL-H to give 9-(50-deoxy-20,30-O-
isopropylidene-50-methylthio-d-ribityl)adenine (12) in
17% yield. Compound 12 was then reacted with TFA to
remove the protecting group to produce the desired
compound 4 in 97% yield.
the 30-position might have with the enzyme and what
effect this moiety has on inhibitory activity.
Most of the work previously reported on inhibitors of this
enzyme has focused on systematically altering the struc-
ture of adenosine and evaluating the derivatives. There
have been very few studies on acyclic analogues of ade-
nosine or AdoHcy as inhibitors of AdoHcy hydrolase. A
notable exception to this has been the work of Holy et al.
which describes a set of o-carboxyalkyl derivatives and
their corresponding MIC50 values.14a Their work indi-
cated that some of these analogues exhibit rapid irrever-
sible inactivation of SAH hydrolase. In addition,
compounds such as (S)-9-2,3-dihydroxypropyl)adenine
[(S)-DHPA], (R,S)-3-adenin-9-yl-2-hydroxypropanoic
acid [(R,S)-AHPA], and d-eritadenine exhibit Ki values
of 900 nM,14b 40 nM15 and 3 nM,16 respectively, against
murine L1210 leukemia AdoHcy hydrolase.
Kinetic studies
The recombinant rat liver AdoHcy hydrolase (MV
1304/pUCSAH) was purified as previously determined
by Gomi and coworkers with minor modifications.21a,b
Enzyme activity was measured in the hydrolytic direc-
tion, spectrophotometrically by the method of Mehdi
and coworkers.22 The Km for S-adenosyl-l-homo-
cysteine was determined to be 9.7 mMand the Vmax
was 1.1 mmol/min/mg which compares favourably with
previously reported values.22 The compound, 50-deoxy-
50-isobutylthioadenosine (SIBA), is a well studied
time-dependent inhibitor of AdoHcy hydrolase.23
When tested, we found it to have a kinact of 0.0488
We have prepared three acyclonucleosides: 4-(adenin-9-
yl)-2,3-dihydroxybutanal oxime (2), [4-(adenin-9-yl)-2,3-
dihydroxybutyl-l-a-amino]butanoic acid (3), and
9-(50-deoxy-50-methylthio-d-ribityl)adenine (4). Each of
these compounds should provide information about the
key components of AdoHcy hydrolase interactions with
substrates and inhibitors as their cyclic counterparts
have been investigated previously (see Kinetic studies).
21b
minÀ1 and a Ki of 105 mMin our system.
All compounds synthesized were tested as inhibitors
against the purified recombinant rat liver AdoHcy
hydrolase. Compound 1, 2, and 4 were found to inacti-
vate the enzyme in a time-dependent manner. Where
possible the kinetic constants for each compound were
determined according to the method of Kitz and Wil-
son.24 Compound 3 was not found to be a time-depen-
dent inhibitor of this enzyme and was therefore tested as
a rapid-equilibrium inhibitor. The kinetic results for all
compounds are listed in Table 1.
Results and Discussion
Chemistry
The compound, 30-amino-30-deoxyadenosine (1) was
prepared from adenosine in nine steps utilizing the
method by Samano and Robins.17 We achieved an
overall yield of 20% utilizing this method.

J. A. Steere, J. F. Honek / Bioorg. Med. Chem. 11 (2003) 3229–3236
3231
.
Scheme 1. The synthesis of compounds 2 and 3: (i) DIBAL-H (5 equiv), THF (ii) NaIO4, H2O (iii) NH2OH HCl, NaOMe, MeOH (iv) 8:1 TFA/H2O
(v) N-Boc-l-,-diaminobutyric acid, MeOH (vi) NaCNBH4.
Scheme 2. Synthesis of compound 4: (i) 2,2-dimethoxypropane, TsOH
(ii) DIBAL-H (5 equiv), THF (iii) 8/1 TFA/H2O.
Figure 3. Time-dependent inactivation of AdoHcy hydrolase with
compound 1. The enzyme was preincubated for the indicated times at
37 ꢀC in the presence of 25 mM (*), 50 mM (~), 75 mM (^), 100 mM
Table 1. Summary of the inhibition studies of AdoHcy hydrolase
(&) or 150 mM (!) of the compound. At the indicated time points,
Compd
AdoHcy hydrolase inhibition
residual enzyme activity was detÀer1mined in the hydrolytic direction as
stated in the text. Inset: plot of kobs versus [inhibitor]À1 from which the
Ki and kinact values were calculated. The values for compound 1 were
determined for time points ꢁ6 min. Data were the average of tripli-
cate measurements. This was reproducible throughout a number of
repeated trials.
1
Time-dependent kineticsa
Ki=48 mM, kinact=0.097 minÀ1
Very rapid biphasic time-dependent kinetics
Rapid-equilibrium kineticsb
Ki=66 uM
Very rapid biphasic time-dependent kinetics
2
3
4
30-position confers the ability of the nucleoside to act as a
time-dependent inhibitor of the enzyme.
aThe inhibition parameters were calculated from the Kitz and Wilson
plot shown in Figure 2.
bThe inhibition parameters were calculated from the plot of the slope
of the lines as a function of [I] shown in Figure 5.
Compound 2 proved to inactivate the rat liver AdoHcy
hydrolase in a time-dependent manner, also with non-
pseudo-first-order kinetics (Fig. 4a). There was a very
rapid decrease in enzymatic activity from 0 to 2 min
followed by a much slower decrease throughout the rest
of the experiment. Unfortunately, the initial decrease in
enzymatic activity was far too rapid for the extrapola-
tion of data to prepare a Kitz and Wilson plot. Com-
pound 4 also exhibited very similar kinetic results as
compound 2 but was not as active (Fig. 4b).
Compound 1 proved to inactivate the rat liver AdoHcy
hydrolase in a time-dependent manner, but was biphasic
in nature, showing pseudo-first-order kinetics only in the
first period of inactivation (Fig. 3). This biphasic process
has been observed quite frequently for other AdoHcy
hydrolase inhibitors25a,b and could be due to a decrease in
the concentration of the inhibitor during the experiment
or could occur if a generated product was a good inhibitor
of the enzyme.26 In both cases, the earlier time points are
more dependable. Further studies will be required in order
to determine the cause of this biphasic process; however, it
is clear that the presence of an amino function at the
Compound 3 was found not to inhibit the enzyme in a
time-dependent manner and was therefore tested as a
rapid-equilibrium inhibitor. Preliminary studies have

3232
J. A. Steere, J. F. Honek / Bioorg. Med. Chem. 11 (2003) 3229–3236
Figure 5. Reversible inactivation of AdoHcy hydrolase with com-
pound 3. The enzyme was tested in the presence of 0 mM (*), 5 mM
(~), 10 mM (^), 25 mM (&) or 50 mM (!) of the compound at 37 ꢀC.
Inset: Plot of the slope of the lines as a function of [I]. Data were the
average of triplicate measurements. This was reproducible throughout
a number of repeated trials.
0.14 mM),23 and 50-deoxy-50-methylthioadenosine
(MTA) inhibits rat liver AdoHcy hydrolase (Ki of 47
mM).28 For each of these compounds, except for Ng-
adenosyl-a,g-diaminobutyric acid, the enzymatic activ-
ity was measured in the synthetic direction.
Conclusions
Four potential S-adenosyl-l-homocysteine hydrolase
inhibitors were prepared and tested against purified
recombinant rat liver enzyme. One of the more potent
compounds tested proved to be compound 1 with Ki
and kinact values of 48 mMand 0.097 min À1, respec-
tively. To our knowledge this is the first evaluation of a
compound with an amine function at the 30-position
and strongly suggests that this replacement of function-
ality could lead to more potent and selective inhibitors.
Figure 4. Time-dependent inactivation of AdoHcy hydrolase with (a)
compound 2 and (b) compound 4. The enzyme was preincubated for
the indicated times at 37 ꢀC in the presence of 5 mM (*), 10 mM (~),
25 mM (^), 50 mM (&), 75 mM (!), 100 mM (*), or 150 mM (&) of
the appropriate compound. At the indicated time points, residual
enzyme activity was determined in the hydrolytic direction as stated in
the text. Data were the average of triplicate measurements. This was
reproducible throughout a number of repeated trials.
indicated that this compound is a mixed inhibitor of
AdoHcy hydrolase, where both the Vmax and Km values
are affected by the inhibitor. The Ki, as stated in Table
1, was determined by re-plotting the slope of each line
from the Lineweaver–Burk plot as a function of the
inhibitor concentration (Fig. 5).27
Compounds 2 and 4 showed very similar kinetic results
which could be due to a similar mechanism of inactiva-
tion. Further detailed biochemical studies would be
required to completely elucidate the chemical mechanism
of inhibition of AdoHcy hydrolase by these analogues.
Compound 3 is one of the few inhibitors of AdoHcy
hydrolase that contains an amino acid moiety. A crystal
stucture with this analogue bound to the enzyme should
give insight into the binding interactions experienced by
the amino acid portion of the natural substrate and may
provide additional information about the mechansim of
this target enzyme.
All compounds were tested as possible inhibitors of ade-
nosine deaminase since this protein is present in the assay
mixture for AdoHcy hydrolase. None of the compounds
showed significant inhibition against adenosine deami-
nase at the levels measured during this study.
In comparison with previously reported AdoHcy
hydrolase inhibitors similar to 1, 2, 3, and 4, respec-
tively, adenosine weakly inhibits rat liver AdoHcy
hydrolase (5% inhibition after 20 min at 20 mM;28 35%
inhibition after 3 h at 200 mM),29 adenosine 50-carbox-
aldehyde oxime inhibits human placental AdoHcy
hydrolase (Ki of 670 nM),30 Ng-adenosyl-a,g-diamino-
butyric acid inhibits beef liver AdoHcy hydrolase (I50 of
Experimental
Thin-layer chromatography was performed on Merck
silica gel plates (aluminum backed, 0.2 mm layer of
Kieselgel 60F254). Column chromatography was per-
formed using 70–230 mesh silica gel. Preparative scale

J. A. Steere, J. F. Honek / Bioorg. Med. Chem. 11 (2003) 3229–3236
3233
reverse phase separations were performed on a LOBAR
apparatus using Merck LiChroprep RP-18 gel. Eluent
was delivered through a metering pump (Fluid Metering
Inc., USA). Melting points were obtained on a Mel-
Temp melting point apparatus and are uncorrected.
Fourier transform infrared spectra were recorded on a
Perkin–Elmer 1600 FT-IR in chloroform or nujol. Pro-
ton (1H) and carbon (13C) magnetic resonance spectra
were obtained on Bruker AC-200, AM-250, AC-300 or
AC-500 spectrometers. Chemical shifts are reported
mmol) in 10 mL of H2O was added NaIO4 (0.407 g,
1.901 mmol) and the mixture was stirred for 2 h. The
mixture was quenched with ethylene glycol (0.071 mL,
1.267 mmol) and was further stirred at 0 ꢀC for 1 h. The
solvent was evaporated under reduced pressure and the
residue was dissolved in anhydrous methanol (25 mL).
The precipitate was filtered over a Celite pad and then
the filtrate was concentrated to dryness. The residue was
purified by column chromatography on silica gel (2%
MeOH/CHCl3) to give 0.349 g (99%) of the compound
as the gem diol structure as a yellow oil. Rf 0.46 (silica
1
downfield from TMS (d=0) for H NMR in CDCl3.
For DMSO-d6 the solvent shift of 2.54 ppm was used.
1
gel, 15% MeOH/CHCl3); H NMR (300 MHz, DMSO)
For MeOH-d4 dCD OD=3.34 and for D2O dHOD=4.67
d 8.33 (s, 1H, H8), 8.14 (s, 1H, H2), 7.34 (brs, 2H,
NH2), 6.11 (d, 2H, 40-OH), 5.33 (dd, 1H, H10), 5.23 (t,
1H, H40), 4.95 (dd, 1H, H10), 4.17–4.23 (m, 1H, H20),
3.52–3.59 (m, 1H, H30), 1.54 (s, 3H, isopropylidene),
1.31 (s, 3H, isopropylidene); 13C NMR (75 MHz,
MeOH) d 157.3 (C6), 153.8 (C2), 150.8 (C4), 143.5 (C8),
111.0 (C5), 109.1 (C(CH3)2), 97.1 (C40), 79.8 (C30), 76.5
(C20), 45.6 (C10), 28.4 (CH3), 25.7 (CH3); IR (neat) 3339
cmÀ1 (OH); Low Res FAB [m/z 300 (M+Na+); {calcd
for C12H15N5O3+Na+} 300.261].
3
ppm was used. For 13C NMR spectra, chemical shifts
are reported relative to the central CDCl3 (d=77.0),
CD3OD-d4 (d=49.5), or DMSO-d6 (d=39.5). Mass
spectra were recorded using FAB or EI on a VG 7070E
or a Concept 1S double focussing mass spectrometer.
30-Amino-30-deoxyadenosine (1). This compound was
prepared as previously described by Samano and
Robins17 in nine steps from adenosine. Mp 259–261 ꢀC
(decomp.) (lit. 259–261 ꢀC)78; Rf 0.05 (silica gel, 4/1/1
BuOH/H2O/AcOH); 1H NMR (500 MHz, DMSO) d
8.36 (s, 1H, H8), 8.12 (s, 1H, H2), 7.28 (s, 2H, NH2),
5.91 (d, 1H, H10), 4.36 (dd, 1H, H20), 3.80 (m, 2H, H40,
H30), 3.70 (m, 1H, H50), 3.55 (m, 1H, H50); 13C NM R
(125 MHz, DMSO) d 156.0 (C6), 152.4 (C2), 148.8 (C4),
139.3 (C8), 119.1 (C5), 89.0 (C10), 84.9 (C40), 74.3 (C20),
60.9 (C50), 52.2 (C30); IR (CHCl3) 3339 cmÀ1 (OH);
HRMS FAB [m/z 267.11673 (M+H+); {calcd for
C10H14N6O3+H+} 267.1127].
4-(Adenin-9-yl)-2,3-O-isopropylidenedioxybutanal oxime
(8). To a stirred solution of hydroxylamine hydrochlo-
ride (0.077 g, 1.107 mmol) in anhydrous MeOH (10 mL)
was added NaOMe (0.059 g, 1.107 mmol) at room tem-
perature. The mixture was stirred for 3 h and then the
aldehyde 5 (0.229 g, 0.775 mmol) was added and the
mixture was stirred at room temperature for 16 h. The
mixture was concentrated in vacuo and the residue was
purified by silica gel chromatography (2% MeOH/
CHCl3 to 15% MeOH/CHCl3) to give 0.195 g (86%, E/
Z=15/85) of a white solid. Mp 234–236 ꢀC (decomp.)
(lit. 234–237 ꢀC);18 Rf 0.32 (silica gel, 15% MeOH/
CHCl3); 1H NMR (300 MHz, DMSO) d (Z isomer) 11.28
(s, 1H, OH), 8.10 (s, 1H, H8), 8.03 (s, 1H, H2), 7.43 (d,
1H, H40), 7.21 (brs, 2H, NH2), 4.66–4.78 (m, 2H, H20 and
H30), 4.18 (d, 2H, H10), 1.43 (s, 3H, isopropylidene), 1.22
(s, 3H, isopropylidene); (E isomer) 11.65 (s, 1H, OH),
8.09 (s, 1H, H8), 8.01 (s, 1H, H2), 7.18 (brs, 2H, NH2),
6.94 (d, 1H, H40), 5.23 (dd, 1H, H30), 4.43 (ddd, 1H, H20),
4.14 (dd, 1H, H10), 3.96 (dd, 1H, H10), 1.45 (s, 3H, iso-
propylidene), 1.26 (s, 3H, isopropylidene); 13C NM R
(75 MHz, DMSO) d 156.5 (C6), 153.0 (C2), 150.0 (C4),
146.0 (C10), 141.4 (C8), 119.0 (C5), 109.9 (C(CH3)2), 75.8
(C30), 75.1 (C20), 44.1 (C40), 28.2 (CH3), 25.5 (CH3), (the
E isomer is difficult to distinguish); IR (CHCl3) 3232
cmÀ1 (OH), 1682 (C¼N); Low Res FAB [m/z 293
(M+H+); {calcd for C12H16N6O3} 292.296].
9-(2,3-O-Isopropylidene-D-ribityl)adenine (7). To a stir-
red solution of 20,30-isopropylidene adenosine (6) (1.60 g,
5.234 mmol) in anhydrous THF (100 mL) at 0 ꢀC under
argon, was added DIBAL-H (27 mL, 27.23 mmol, 1 M
solution in toluene) dropwise under argon. The solution
was stirred at room temperature for 24 h, the reaction
mixture was quenched at 0 ꢀC with saturated aqueous
potassium sodium tartrate (100 mL) and the solution was
stirred at room temperature overnight. The mixture was
concentrated in vacuo to remove the THF and the aqu-
eous layer was extracted with BuOH (3Â150 mL). The
combined organic layers were dried (Na2SO4) and con-
centrated in vacuo. The residue was purified by column
chromatography on silica gel (5% MeOH/CHCl3 to 15%
MeOH/CHCl3) to give 0.777 g (48%) of a white foam.
Mp 154–156 ꢀC (lit. 154–156 ꢀC);19a Rf 0.18 (silica gel,
1
15% MeOH/CHCl3); H NMR (300 MHz, DMSO) d
8.09 (s, 1H, H8), 8.03 (s, 1H, H2), 7.16 (brs, 2H, NH2),
5.11 (d, 1H, 40-OH), 4.62 (t, 1H, 50-OH), 4.48 (m, 2H,
H10, H20), 4.16 (dd, 1H, H10), 4.08 (dd, 1H, H30), 3.59–
3.62 (m, 2H, H40, H50), 3.38 (m, 1H, H50), 1.40 (s, 3H,
isopropylidene), 1.15 (s, 3H, isopropylidene); 13C NM R
(75 MHz, DMSO) d 156.5 (C6), 152.8 (C2), 150.1 (C4),
141.9 (C8), 119.3 (C5), 108.9 (C(CH3)2), 76.4 (C40), 75.4
(C30), 69.9 (C20), 64.4 (C50), 44.4 (C10), 28.5 (CH3), 26.1
(CH3); IR (nujol) 3331 cmÀ1 (OH); Low Res FAB [m/z
310 (M+H+); {calcd for C13H19N5O4} 309.3223].
4-(Adenin-9-yl)-2,3-dihydroxybutanal oxime (2). Com-
pound 8 (0.072 g, 0.246 mmol) was placed in a round
bottomed flask and dissolved in 5 mL of 8/1 TFA/H2O.
This was stirred for 1 h. The solvent was evaporated
under reduced pressure and redissolved in MeOH and
concentrated. This procedure was repeated three times
and the residue was dissolved in H2O and lyophilized to
give 0.060 g (98%) of a white powder. Mp 65–67 ꢀC; Rf
0.1 (silica gel, 15% MeOH/CHCl3); 1H NM
(300 MHz, DMSO) d (Z isomer) 11.06 (s, 1H, OH), 8.42
(s, 1H, H8), 8.32 (s, 1H, H2), 7.21 (d, 1H, H40),
4.33–4.46 (m, 2H, H20 and H30), 4.10 (d, 2H, H10); (the
R
4-(Adenin-9-yl)-2,3-O-isopropylidenedioxybutanal (5).
To a cold (0 ꢀC) stirring solution of 7 (0.392 g, 1.267

3234
J. A. Steere, J. F. Honek / Bioorg. Med. Chem. 11 (2003) 3229–3236
E isomer is difficult to distinguish); 13C NMR (75 MHz,
DMSO) d 151.5 (C6), 150.5 (C2), 149.4 (C4), 146.3
(C10), 145.0 (C8), 119.4 (C5), 71.6 (C30), 63.3 (C20), 47.3
(C40); (the E isomer is difficult to distinguish); IR
(CHCl3) 3257 cmÀ1 (OH), 1674 cmÀ1 (C¼N); HRMS
FAB [m/z 253.10811 (M+H+); {calcd for
C9H12N6O3+H+} 253.10487].
(0.128 g, 0.673 mmol) and 2,2-dimethyoxypropane
(0.332 mL, 2.688 mmol) under argon. This was dis-
solved in dry acetone (15 mL) and stirred at room tem-
perature for 2 h. The mixture was then neutralized with
10% NH4OH in H2O and then evaporated under
reduced pressure. The residue was purified by silica gel
chromatography (5% MeOH/CHCl3) to give 0.147 g
(99%) of a colourless oil. Rf 0.78 (silica gel, 15%
1
[4-(Adenin-9-yl)-2,3-O-isopropylidenedioxybutanal]-L-ꢀ-
N-t-Boc butanoic acid (9). Aldehyde 5 (0.331 g, 1.194
mmol) was placed in a round bottomed flask and dis-
solved in 20 mL of toluene. This mixture was refluxed
with a Dean-Stark trap for 1 h and then concentrated in
vacuo. The residue was placed under argon and the
N-l-a-Boc-a,g-diaminobutyric acid (0.287 g, 1.313
mmol) was added. Methanol (20 mL) was added and
this was stirred at room temperature for 2 h. Sodium
cyanoborohydride (0.225 g, 3.582 mmol) was added and
the mixture was stirred at room temperature overnight.
The reaction was concentrated under reduced pressure
and then purified on a reverse phase LOBAR column
C-18 (0–10% CH3CN/H2O in 2% increments of 100 mL
each) to give 0.206 g (36%) of a yellow crystal. Mp
232–234 ꢀC; Rf 0.16 (silica gel, 4/1/1 BuOH/H2O/
AcOH); 1H NMR (300 MHz, MeOH) d 8.21 (s, 1H,
H8), 8.13 (s, 1H, H2), 4.43 (dd, 2H, H10), 4.11 (dd, 1H,
H20), 3.98 (t, 1H, Ha), 3.82 (dd, 1H, H30), 2.83 (d, 2H,
H40), 2.71 (dd, 2H, Hg), 2.01 (m, 1H, Hb), 1.79 (m, 1H,
Hb), 1.39 (s, 9H, Boc, C(CH3)3), 1.33 (s, 3H, iso-
propylidene), 1.21 (s, 3H, isopropylidene); 13C NM R
(75 MHz, MeOH) d 177.9 (acid, C¼O), 156.3 (Boc,
C¼O), 155.9 (C6), 152.6 (C2), 149.6 (C4), 142.3 (C8),
118.2 (C5), 109.6 (C(CH3)2), 78.7 (Boc, C(CH3)3), 77.5
(C30), 77.1 (C20), 54.2 (Ca), 50.7 (C40), 46.2 (Cg), 44.3
(C10), 32.6 (Cb), 27.4 (Boc, C(CH3)3), 26.2 (CH3), 25.8
(CH3); IR (CHCl3) 3423 cmÀ1 (OH), 1636 cmÀ1 (C¼O);
HRMS FAB [m/z 502.20628 (M+Na+); {calcd for
C21H33N7O6+Na+} 502.20897].
MeOH/CHCl3); H NMR (300 MHz, MeOH) d 8.24 (s,
1H, H8), 8.19 (s, 1H, H2), 6.15 (d, 1H, H10), 5.48 (dd,
1H, H20), 5.02 (dd, 1H, H30), 4.32 (m, 1H, H40), 2.72 (m,
2H, H50), 2.02 (s, 3H, SCH3), 1.55 (s, 3H, iso-
propylidene), 1.35 (s, 3H, isopropylidene); 13C NM R
(75 MHz, MeOH) d 156.1 (C6), 152.7 (C2), 148.9 (C4),
140.5 (C8), 119.2 (C5), 114.1 (C(CH3)2), 90.3 (C10), 86.4
(C40), 83.8 (C30), 78.2 (C20), 35.9 (C50), 26.1 (CH3), 24.2
(CH3), 14.6 (SCH3); Low Res FAB [m/z 338 (M+H+);
{calcd for C14H19N5O3S} 337.395].
9-(50-Deoxy-20,30-O-isopropylidene-50-thiomethyl-D-ribi-
tyl)adenine (12). To a stirred suspension of 11 (0.048 g,
0.142 mmol) in anhydrous THF (10 mL) at room tem-
perature was added DIBAL-H (0.711 mL, 0.711 mmol)
dropwise, under argon. The solution was stirred at
room temperature for 24 h and the reaction was quen-
ched with saturated potassium sodium tartrate (10 mL).
The solvent was evaporated and the aqueous residue
was extracted with n-BuOH (30 mLÂ2). The organic
layers were combined, dried (Na2SO4) and concentrated
in vacuo. This was purified by silica gel chromatography
(2% MeOH/CHCl3 to 18% MeOH/CHCl3) to give 0.008
g (17%) of a yellow oil. Rf 0.52 (silica gel, 15% MeOH/
CHCl3); 1H NMR (300 MHz, MeOH) d 8.19 (s, 1H, H8),
8.11 (s, 1H, H2), 4.68 (dd, 1H, H40), 4.58 (m, 1H, H20),
4.30 (m, 1H, H30), 4.17 (m, 1H, H10), 3.93 (m, 1H, H10),
2.92 (dd, 1H, H50), 2.60 (dd, 1H, H50), 2.16 (s, 3H,
SCH3), 1.47 (s, 3H, isopropylidene), 1.26 (s, 3H, iso-
propylidene); 13C NMR (75 MHz, MeOH) d 156.2 (C6),
152.3 (C2), 148.8 (C4), 142.0 (C8), 119.6 (C5), 109.2
(C(CH3)2), 78.3 (C30), 75.6 (C40), 68.2 (C20), 44.2 (C10),
39.4 (C50), 27.0 (CH3), 24.3 (CH3), 15.2 (SCH3); IR
(CHCl3) 3330 cmÀ1 (OH); HRMS FAB [m/z 340.14297
(M+H+); {calcd for C14H21N5O3S+H+} 340.14430].
4-[(Adenin-9-yl)-2,3-dihydroxybutylL–ꢀ-amino] butanoic
acid (3). Compound 9 (0.029 g, 0.061 mmol) was
placed in a round bottomed flask and dissolved in 8/1
TFA/H2O (5 mL) and stirred overnight at room tem-
perature. The solvent was evaporated under reduced
pressure and redissolved in MeOH and concentrated.
This procedure was repeated three times and the residue
was dissolved in H2O and lyophilized to give 0.02 g
(96%) of a white powder. Mp 158–160 ꢀC; Rf 0.1 (silica
gel, 4/1/1 BuOH/H2O/AcOH); 1H NMR (300 MHz,
D2O) d 8.31 (s, 1H, H8), 8.27 (s, 1H, H2), 4.28 (dd, 2H,
H10), 3.94 (m, 1H, H20), 3.76 (m, 2H, H40), 2.71 (m, 4H,
H30, Ha, Hg), 2.14 (m, 2H, Hb); 13C NMR (75 MHz,
MeOH) d 163.0 (C¼O), 149.9 (C6), 149.5 (C2), 145.4
(C4), 144.4 (C8), 112.0 (C5), 76.3 (C30), 69.9 (C20), 66.9
(Ca), 49.9 (C40), 47.1 (Cg), 44.8 (C10), 26.6 (Cb); IR
(CHCl3) 3301 cmÀ1 (OH), 1676 cmÀ1 (C¼O); HRMS
FAB [m/z 340.17447 (M+H+); {calcd for
C13H21N7O4+H+} 340.17328].
9-(50 -Deoxy-50 -thiomethyl-D-ribityl)adenine (4). Com-
pound 12 (10 mg, 0.025 mmol) was dissolved in 8/1
TFA/H2O (0.5 mL) in a round bottom flask was stirred
for 1.5 h at room temperature. The solvent was removed
in vacuo. Methanol (5 mL) was added to the mixture
and subsequently evaporated under reduced pressure.
The treatment with methanol was repeated three times.
The residue was dissolved in H2O and lyophilized over-
night to give 0.007 g (97%) of a yellow solid. Mp
73–75 ꢀC; Rf 0.15 (silica gel, 15% MeOH/CHCl3); H
1
NMR (300 MHz, MeOH) d 8.34 (s, 1H, H8), 8.29 (s,
1H, H2), 4.68 (dd, 1H, H40), 4.56 (m, 1H, H20), 4.36 (m,
1H, H30), 4.06 (m, 1H, H10), 3.84 (m, 1H, H10), 2.90 (dd,
1H, H50), 2.60 (dd, 1H, H50), 2.11 (s, 3H, SCH3); 13C
NMR (75 MHz, MeOH) d 156.4 (C6), 151.4 (C2), 148.6
(C4), 145.1 (C8), 119.3 (C5), 74.4 (C30), 71.4 (C40), 70.5
(C20), 44.5 (C10), 37.5 (C50), 14.8 (SCH3); IR (CHCl3)
3342 cmÀ1 (OH); HRMS FAB [m/z 300.11535
(M+H+); {calcd for C11H17N5O3S+H+} 300.15115].
9-(50-Deoxy-20,30-O-isopropylidene-50-thiomethyl) adeno-
sine (11). In a round bottomed flask under argon was
placed 50-deoxy-50-methylthioadenosine (10) (0.1 g,
0.336 mmol), p-toluenesulfonic acid monohydrate

J. A. Steere, J. F. Honek / Bioorg. Med. Chem. 11 (2003) 3229–3236
3235
Protein purification. The recombinant rat liver AdoHcy
hydrolase was purified from the cell-free extract of
E. coli transformed with plasmid pUCSAH and grown
in the presence of isopropyl b-d-thiogalactopyransoide
according to the procedure described by Gomi et al. for
the purification, including DEAE Sepharose and Sepha-
cryl S-300 chromatography.21 About 12 mg of enzyme
was obtained from a 1 L culture. The homogeneity of the
AdoHcy hydrolase preparation was checked by ESI-MS,
the major molecular species being detected at 47,407 Da
for each enzyme subunit. The protein concentration was
determined by the Lowry method using bovine serum
albumin (BSA) as a standard.31
added and the resulting mixture was mixed rapidly and
the absorbance was measured at 265 nm, 25 ꢀC.
Acknowledgements
We gratefully acknowledge Drs. R. Aksamit and G.
Cantoni for the Escherichia coli MV1304/pUCSAH
used in these studies and the Natural Sciences and
Engineering Research Council of Canada for financial
assistance. Graduate student funding in the form of an
OGSST to J.A.S. is also gratefully appreciated. We
would also like to thank Tim Jones and Jan Venne for
conducting MS and NMR studies, respectively and to
A. Litchfield for preliminary synthetic studies.
Enzyme assay. AdoHcy hydrolase activity was mea-
sured spectrophotometrically22 by following the forma-
tion of homocysteine, which was detected by reaction
with 5,50-dithiobis(2-nitrobenzoic acid) (DTNB) to give
the chromophoric thiolate (e412nm=13.6 mMÀ1 cmÀ1).
The reaction mixture (1 mL total) had the following
composition: 100 mMDTNB, 62.5 mM S-adenosyl-l-
homocysteine, 2 U adenosine deaminase (calf intestine)
and AdoHcy hydrolase in 10 mMpotassium phosphate
buffer, 1 mMEDTA, pH 7.5. The assay buffer con-
tained 10 mMpotassium phosphate buffer, 0.1 mM
DTNB, 1 mMEDTA, pH 7.5 in which the adenosine
deaminase, AdoHcy and AdoHcy hydrolase are added.
The resulting mixture was mixed rapidly and the
absorbance was measured at 412 nm with respect to
time.22
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