Inactivation of S-adenosyl-L-homocysteine hydrolase and antiviral activity with 5',5',6',6'-tetradehydro-6'-deoxy-6'-halohomoadenosine analogues (4'-haloacetylene analogues derived from adenosine)

Article abstract of DOI:10.1021/jm980163m

Treatment of a protected 9-(5,6-dideoxy-β-D-ribo-hex-5- ynofuranosyl)adenine derivative with silver nitrate and N-iodosuccinimide (NIS) and deprotection gave the 6'-iodo acetylenic nucleoside analogue 3c. Halogenation of 3-O-benzoyl-5,6-dideoxy-1,2-O-isopropylidene-α-D-ribo-hex- 5-enofuranose gave 6-halo acetylenic sugars that were converted to anomeric 1,2-di-O-acetyl derivatives and coupled with 6-N-benzoyladenine. These intermediates were deprotected to give the 6'-chloro 3a, 6'-bromo 3b, and 6'- iodo 3c acetylenic nucleoside analogues. Iodo compound 3c appears to inactivate S-adenosyl-L-homocysteine hydrolase by a type I ('cofactor depletion') mechanism since complete reduction of enzyme-bound NAD⁺ to NADH was observed and no release of adenine or iodide ion was detected. In contrast, incubation of the enzyme with the chloro 3a or bromo 3b analogues resulted in release of Cl^- or Br^- and Ade, as well as partial reduction of E-NAD⁺ to E-NADH. Compounds 3a, 3b, and 3c were inhibitory to replication of vaccinia virus, vesicular stomatitis virus, parainfluenza-3 virus, and reovirus-1 (3a < 3b < 3c, in order of increasing activity). The antiviral effects appear to correlate with type I mechanism-based inhibition of S- adenosyl-L-homocysteine hydrolase. Mechanistic considerations are discussed.

Full text of DOI:10.1021/jm980163m

J . Med. Chem. 1998, 41, 3857-3864
3857
In a ctiva tion of S-Ad en osyl-L-h om ocystein e Hyd r ola se a n d An tivir a l Activity
w ith 5′,5′,6′,6′-Tetr a d eh yd r o-6′-d eoxy-6′-h a loh om oa d en osin e An a logu es
(4′-Ha loa cetylen e An a logu es Der ived fr om Ad en osin e)
Morris J . Robins,*,† Stanislaw F. Wnuk,†,‡ Xiaoda Yang,§ Chong-Sheng Yuan,§,| Ronald T. Borchardt,§
J an Balzarini, and Erik De Clercq
Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602-5700, Department of Biochemistry,
The University of Kansas, Lawrence, Kansas 66045, and Rega Institute for Medical Research, Katholieke Universiteit of
Leuven, Leuven, Belgium
Received March 18, 1998
Treatment of a protected 9-(5,6-dideoxy-â-D-ribo-hex-5-ynofuranosyl)adenine derivative with
silver nitrate and N-iodosuccinimide (NIS) and deprotection gave the 6′-iodo acetylenic
nucleoside analogue 3c. Halogenation of 3-O-benzoyl-5,6-dideoxy-1,2-O-isopropylidene-R-D-
ribo-hex-5-enofuranose gave 6-halo acetylenic sugars that were converted to anomeric 1,2-di-
O-acetyl derivatives and coupled with 6-N-benzoyladenine. These intermediates were depro-
tected to give the 6′-chloro 3a , 6′-bromo 3b, and 6′-iodo 3c acetylenic nucleoside analogues.
Iodo compound 3c appears to inactivate S-adenosyl-L-homocysteine hydrolase by a type I
(“cofactor depletion”) mechanism since complete reduction of enzyme-bound NAD+ to NADH
was observed and no release of adenine or iodide ion was detected. In contrast, incubation of
the enzyme with the chloro 3a or bromo 3b analogues resulted in release of Cl- or Br- and
Ade, as well as partial reduction of E-NAD+ to E-NADH. Compounds 3a , 3b, and 3c were
inhibitory to replication of vaccinia virus, vesicular stomatitis virus, parainfluenza-3 virus,
and reovirus-1 (3a < 3b < 3c, in order of increasing activity). The antiviral effects appear to
correlate with type I mechanism-based inhibition of S-adenosyl-L-homocysteine hydrolase.
Mechanistic considerations are discussed.
In tr od u ction
dative (C3′) activities of AdoHcy hydrolase were differ-
entiated effectively with the 6′-fluoro analogue
(EDDFHA).7b Lys-426 was identified as an important
residue for the hydrolytic activity.8 We recently found
that geminal and vicinal (dihalohomovinyl)adenosine
analogues are new putative hydrolytic substrates for
mechanism-based inhibition of AdoHcy hydrolase.1,9
Electrophilic acyl halides and/or R-halomethyl ketones
might result from addition of enzyme-sequestered water
at C5′/C6′ followed by loss of hydrogen halide. Attack
by amino acid functionalities (e.g., an amino group on
Lys-426 or Arg-196) might cause type II (covalent
binding2b) inhibition of the enzyme.1,9
The cellular enzyme S-adenosyl-L-homocysteine
(AdoHcy) hydrolase (EC 3.3.1.1) effects cleavage of
AdoHcy to adenosine and L-homocysteine. AdoHcy is a
potent feedback inhibitor of crucial transmethylation
enzymes so the design of inhibitors of AdoHcy hydrolase
represents a rational approach for anticancer and
antiviral chemotherapy.2 ZDDFA3 (the Z-isomer of 4′,5′-
didehydro-5′-deoxy-5′-fluoroadenosine) and its chloro
analogue4 are mechanism-based inactivators of AdoHcy
hydrolase. Inactivation by ZDDFA involves addition of
water at C5′, elimination of hydrogen fluoride to give
adenosine 5′-carboxaldehydes5a (nonlethal event), and
their oxidation to 3′-keto derivatives (lethal event) with
concomitant reduction of E-NAD+ to E-NADH5b (“co-
factor depletion” or type I inhibition2b). This indicates
that AdoHcy hydrolase has hydrolytic activity (addition
of water at C5′) that functions independently of its C3′-
oxidation activity.5b
Homologated 5′,5′-dibromomethylene-5′-deoxyuridine10
and adenosine1,11 analogues had been synthesized from
nucleoside 5′-carboxaldehydes (with CBr4/PPh3/Zn12
)
and converted into 5′-deoxy-5′-methynylnucleosides (the
4′-acetylenic derivatives).10,11 We had prepared such 4′-
acetylenic derivatives by oxidative destannylation of
vinyl 6′-stannanes.6,13 The corresponding adenosine
analogue is a type II inhibitor of AdoHcy hydrolase6,14
with antiviral6,11 and cytostatic activity.6 Examples of
2′-15 and 3′-ethynyl16 nucleoside derivatives have been
synthesized, and 1-(3-C-ethynyl-â-D-ribo-pentofuranos-
yl)cytosine has antitumor activity.16b An acetylenic
linkage has been substituted for the phosphodiester
moiety in an oligonucleotide mimic,17 and a 2′,3′-
dideoxyuridine analogue with ethynyl groups at C3′ and
C4′ has been reported.18
Our homologated analogues6 [(E)-5′,6′-didehydro-6′-
deoxy-6′-halohomoadenosines, EDDHHAs] inhibit
AdoHcy hydrolase and are enzymatically hydrolyzed to
give “homoadenosine 6′-carboxaldehyde” which under-
goes decomposition.7 The hydrolytic (C5′/C6′) and oxi-
* To whom correspondence should be addressed at Brigham Young
University.
† Brigham Young University.
§ University of Kansas.
Katholieke Universite of Leuven.
‡ Current address: Department of Chemistry, Florida International
University, Miami, FL.
We now describe syntheses of the first (6′-halo)-
acetylenic adenine nucleosides (from adenosine or glu-
cose), their inhibitory effects on AdoHcy hydrolase, and
| Current address: Tanabe Research Laboratories, USA, Inc., San
Diego, CA.
S0022-2623(98)00163-0 CCC: $15.00 © 1998 American Chemical Society
Published on Web 09/03/1998

3858 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 20
Robins et al.
Sch em e 2a
F igu r e 1. Possible generation of active intermediates from
the 6′-halo acetylenic nucleoside analogues 3 by the “hydrolytic
activity” of AdoHcy hydrolase.
Sch em e 1a
a
(a) BuLi/THF/-78 °C. (b) AgNO3/NIS/Me2CO. (c) NH3/MeOH.
(d) TFA/H2O.
a
(a) H5IO6/EtOAc. (b) Ph3P/CBr4/CH2Cl2. (c) BuLi/THF/-78 °C.
(d) BzCl/pyridine. (e) NaOCl/H2O. (f) NBS/AgNO3/Me2CO. (g) i,
TFA/H2O; ii, Ac2O/pyridine/DMAP. (h) 6-N-Benzoyladenine/SnCl4/
CH3CN. (i) NH3/MeOH.
their antiviral activities. Addition of enzyme-seques-
tered water at C5′/C6′ followed by tautomerization of
hydroxyvinyl intermediates might generate acyl halides
and/or R-halomethyl ketones (Figure 1). Attack of
protein nucleophiles might cause type II (covalent)
inhibition of AdoHcy hydrolase.1,9
from 9b (TFA/H2O) and the product was acetylated to
give the anomeric acetates 10. Coupling (SnCl4/CH3-
CN25) of 10 and 6-N-benzoyladenine followed by deacy-
lation (NH3/MeOH) gave the crystalline bromoacetylene
homonucleoside 3b (20% from 9b).
Ch em istr y
Moffatt oxidation19 of 6-N-benzoyl-2′,3′-O-isoprop-
ylideneadenosine and treatment of the crude 5′-carbox-
aldehyde with “(dibromomethylene)triphenylphos-
phorane” (CBr4/Ph3P/Zn12) gave the dibromovinyl ana-
logue 11,11 (74%, Scheme 1). Treatment of 1 with excess
BuLi gave the acetylenic derivative 2 (53%) plus byprod-
As anticipated,20 treatment of 6 or 7 with NCS/AgNO3
failed to effect 6-chlorination. However, treatment of
6 with an aqueous sodium hypochlorite solution21a
(commercial laundry bleach) at ambient temperature
gave the chloroacetylene derivative 8a (87%, purifica-
tion by chromatography on silica gel). Benzoylation of
8a , hydrolysis of the acetal, and acetylation gave the
anomeric acetates 10a . Coupling of 10a with 6-N-
benzoyladenine, deacylation, and chromatography gave
the chloroacetylene nucleoside 3a (36% from 9a ). Com-
pound 3a was unstable upon heating and underwent
decomposition upon attempted removal of solvent mol-
ecules (1H NMR) for elemental analysis. The crystalline
solvate was stored at 0 °C and checked for chromato-
graphic homogeneity before biological testing. Terminal
chloroacetylenes are known to be somewhat unstable,
and testing results must be considered tentative (al-
though testing data were repeatable). All attempts to
make the fluoroacetylenic analogue failed.
In a ctiva tion of S-Ad en osyl-L-h om ocystein e Hy-
d r ola se. Recombinant human placental AdoHcy hy-
drolase was inactivated upon incubation with 3a , 3b,
or 3c by concentration-dependent (Table 1) and time-
dependent (data not shown) processes. The maximal
inactivation at the highest concentration used (100 µM)
was approximately 55% of the original enzyme activity
with the chloro 3a and bromo 3b analogues. In con-
trast, the 6′-iodo analogue 3c caused complete inactiva-
tion of AdoHcy hydrolase at a concentration of 100 µM.
Kinetic analysis of the inactivation processes by the Kitz
and Wilson method gave the k2 and Ki values in Table
2. The chloro 3a and bromo 3b analogues appear to
have similar kinetic properties. In contrast, the 6′-iodo
analogue 3c inactivated the enzyme at a substantially
uct(s), as noted by others.11 Treatment of 2 with
N-iodosuccinimide (NIS) and AgNO3
equimolar) resulted in efficient 6′-iodination. Sequen-
tial removal of the 6-N-benzoyl (NH3/MeOH) and iso-
propylidene [CF3CO2H (TFA)/H2O] groups gave the
iodoacetylene derivative 3c (42% from 2).
20,21
(catalytic or
The bromo-, 3b, and chloro-, 3a , acetylene analogues
were prepared from sugar precursors (Scheme 2). At-
tempted bromination (NBS) and especially chlorination
of 2 did not proceed cleanly, in contrast with iodination
(NIS). Attempted base-promoted dehydrobromination
of 1 [BuLi, tetrabutylammonium fluoride17a (TBAF), or
1,8-dizazbicyclo[5.4.0]undec-7-ene (DBU)] also was prob-
lematic. Oxidation of 1,2:5,6-di-O-isopropylidene-R-D-
glucofuranose and stereoselective reduction of the 3-keto
intermediate gave the R-D-allofuranose derivative 4 as
reported.22 Sequential (one-flask) selective hydrolysis
of the 5,6-O-isopropylidene acetal and oxidative cleavage
of the exposed glycol with periodic acid23 gave the
dehomologated 5-carboxaldehyde. Wittig-type olefina-
tion with the dibromomethylene reagent (generated in
situ24) gave the (dibromohomovinyl)ribose 5 [58% over-
all; analogous treatment of the 3-O-benzoyl derivative
of 422c gave 3-O-benzoyl-5 (71%)]. Treatment of 5 with
Mg/THF24 failed to produce the acetylenic derivative,
but BuLi12 effected its conversion to 6 (81%). Benzoy-
lation of 6 and treatment of the product 7 with NBS/
AgNO3 (catalytic20) gave the bromoacetylene sugar 9b
(79% from 6). The isopropylidene group was removed

Inactivation of S-Adenosyl-L-homocysteine Hydrolase
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 20 3859
Ta ble 1. Inhibition of AdoHcy Hydrolase with 3a -c
We presently cannot rule out the possibility of cova-
lent modification of AdoHcy hydrolase by 3c, but it could
not have occurred by an activation process involving the
5′/6′-hydrolytic activity (as illustrated in Figure 1) since
Ade or I- release was not detected. However, it is
possible that covalent modification of the enzyme oc-
curred via oxidation of 3c to its 3′-keto derivative
followed by isomerization to give allenic ketone(s) and
attack by protein nucleophile(s) on such electrophilic
species analogous to the mechanism proposed by Parry
et al. for the nonhalogenated analogue.14 Mechanisms
by which 3c inactivate AdoHcy hydrolase are currently
under investigation.
enzyme activity remaininga,b (%)
concn (µM)
3a
3b
3c
0.1
1.0
10
89.3 ( 2.8
64.0 ( 0.6
58.9 ( 1.3
55.0 ( 0.6
91 ( 1.5
80.0 ( 0.8
44.6 ( 1.0
8.8 ( 0.2
<2.0
73.2 ( 3.2
62.9 ( 0.7
56.0 ( 3.1
100
a
AdoHcy hydrolase (25 nM) was incubated with 3a -c in buffer
A at 37 °C for 20 min, and the remaining activity was assayed as
described in the Experimental Section. Data are the averages of
b
duplicate determinations.
Ta ble 2. Kinetic Constants for Inhibition of AdoHcy
Hydrolase with 3a -ca
Results observed with the chloro 3a and bromo 3b
analogues are different from those with 6′-iodo analogue
3c. Partial (∼50%) reduction of E-NAD+ to E-NADH
was observed with 3a or 3b with concomitant partial
loss (∼50%) of enzyme activity. Incubation of 3a or 3b
with AdoHcy hydrolase resulted in release of adenine
(3b > 3a ) and halide ions (3b > 3a ). These results
suggest that 3a and 3b are substrates for both the 3′-
oxidative activity (partial reduction of E-NAD+ to E-
NADH) and the 5′/6′-hydrolytic activity (release of Ade
and halide ions) of AdoHcy hydrolase. More specifically,
the release of Ade suggests 6′-hydrolytic activity, whereas
release of halide ions could result from either the 5′- or
6′-hydrolytic activity of the enzyme.7 Since halide ions
are released from the chloro 3a and bromo 3b analogues
upon incubation with AdoHcy hydrolase, it is plausible
that these inhibitors are converted into acyl halides and/
or R-halomethyl ketones (Figure 1) and subsequently
react with protein nucleophiles. However, 3a and/or 3b
also might inactive the enzyme by type I (cofactor
depletion) and/or type II (covalent binding via an allenic
ketone14) mechanisms. Studies to characterize the
molecular mechanism(s) involved in these inactivation
processes are in progress.
compd
k2 (min-1
)
Ki (µM)
k2/Ki (M-1 min-1
)
3a
3b
3c
0.48 ( 0.05
0.49 ( 0.03
0.095 ( 0.004
1.7 ( 0.2
3.8 ( 0.3
1.10 ( 0.04
2.82 × 105
1.29 × 105
8.64 × 104
a
Kinetic constants k2 and Ki were calculated from the pseudo-
first-order rate constants (kapp) for enzyme inactivation with
various concentrations of 3a -c as described in the Experimental
Section. See ref 2c (p 54) for kinetic data with some type I
inhibitors.
Ta ble 3. Effects of 3a -c on AdoHcy Hydrolase Activity and
NADH Content, and Enzyme-Mediated Release of Ade and
Halide Ions from 3a -ca
enzyme
activity
Ade
release
(%)
NADH
content (% of
halide ion
release (%) total coenzyme)
compd remaining (%)
3a
3b
3c
47 ( 3
18 ( 2
28 ( 2
28 ( 2
70 ( 15
0
53 ( 2
49 ( 2
96 ( 3
49 ( 3
1.4 ( 0.2
5.7 ( 0.5
a
AdoHcy hydrolase was incubated with 3a -c (100 µM) in buffer
A at 37 °C for 20 min. Enzyme activity remaining, amounts of
Ade and halide ions released, and NADH content were assayed
as described in the Experimental Section.
slower rate than 3a or 3b. Kinetic data for some “pure
type I” inhibitors are given in ref 2c (p 54).
We also determined the effects of 3a -c on the NADH
content of the enzyme as well as the enzyme-mediated
release of halide ions and/or Ade from the inhibitors
(Table 3). It can be determined whether the inactivation
process involves a type I (cofactor depletion) mechanism
of inactivation by monitoring the NADH content of the
enzyme2b and whether inhibitors are substrates for the
5′/6′-hydrolytic activity of AdoHcy hydrolase by monitor-
ing the release of Ade and/or halide ions.7 The data in
Table 3 show that the 6′-iodo analogue 3c causes
complete conversion of the enzyme from its E-NAD+ to
E-NADH form (with loss of enzyme activity). This
indicates that 3c is a substrate for the 3′-oxidative
activity of AdoHcy hydrolase. Since Ade or I- is not
released upon incubation of 3c with the enzyme, it
appears that this compound inactivates AdoHcy hydro-
lase by a type I process. The present results with 3c
are in marked contrast with those reported by Parry et
al. for the nonhalogenated acetylenic analogue.14 In
that case, complete inactivation of AdoHcy hydrolase
was observed but only partial (∼50%) conversion of
E-NAD+ to E-NADH occurred. The remaining 2 equiv
of NAD+ was “lost” from the tetramer. It also was
reported that 4 equiv of the inhibitor was bound to the
tetramer upon inactivation and that 2 equiv of the
inhibitor was “more tightly” bound.14 This suggests
partial covalent modification of the enzyme (type II
mechanism).
An tivir a l Activity. Haloacetylenes 3a , 3b, and 3c
were evaluated for antiviral activity in cell culture
(Table 4). They were inactive against HSV-1 and HSV-2
in E6SM cells, Coxsackie virus B4 in HeLa and Vero
cells, and Sindbis and Punta Toro viruses in Vero cells
at subtoxic concentrations. Chloro derivative 3a was
marginally effective against vaccinia virus (VV) and
vesicular stomatitis virus (VSV) in E6SM cells and
parainfluenza virus type 3 in Vero cells. However, the
bromo 3b and especially the iodo 3c derivatives were
markedly inhibitory to these viruses [EC50 values for
3c of 0.08 µM against VSV in E6SM cells, 0.26 µM
against VV in E6SM cells, 0.78 µM against reovirus-1,
and 2.6 µM against parainfluenza-3 virus in Vero cells
(Table 4)]. Surprisingly, 3c was much less inhibitory
to VSV in HeLa than in E6SM cells. Also, 3a and 3b
were less active against VSV in HeLa than in E6SM
cells. Since 3c was more toxic to E6SM than HeLa cells,
differences in the antiviral potencies against VSV in E6-
SM and HeLa cell cultures might result from differences
in metabolism of the compounds in these two cell lines.
The inhibitory effects of 3a , 3b, and 3c against
vaccinia virus, vesicular stomatitis virus, parainfluenza
virus, and reoviruses are in overall harmony with the
activity spectrum of AdoHcy hydrolase inhibitors.2c The
order of increasing antiviral activity was 3a < 3b < 3c,
irrespective of the virus (VV, VSV, parainfluenza, or

3860 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 20
Robins et al.
reoviruses). However, inactivation of purified human
placental AdoHcy hydrolase was approximately the
same with 3a and 3b, which both were considerably less
potent than 3c. The modes of action also appear to be
different with 3a and 3b (mixed type I and type II
inactivation) relative to that of 3c (type I inactivation).
Su m m a r y a n d Con clu sion s
The 6′-chloro 3a , 6′-bromo 3b, and 6′-iodo 3c acety-
lenic homonucleosides were prepared from adenosine or
hexofuranose precursors. Incubation of AdoHcy hydro-
lase with 3a or 3b resulted in partial (∼50%) reduction
of E-NAD+ to E-NADH (3′-oxidative activity) and paral-
lel loss of enzyme activity. In contrast, incubation with
3c resulted in complete reduction of E-NAD+ to
E-NADH and complete loss of enzyme activity. Release
of Ade plus Cl- or Br- (5′/6′-hydrolytic activity) was
observed with 3a or 3b, but no Ade or I- release was
detected with 3c. These results are consistent with type
I (cofactor depletion) inactivation for 3c and mixed type
I and type II (covalent binding) inactivation for 3a and
3b, although other mechanistic interpretations have not
been excluded. It is remarkable that such apparent
changes in mechanisms occur with the bromo 3b versus
iodo 3c congeners. Greater antiviral potency was
observed with 3c relative to 3b and 3a . These observa-
tions are compatible with results on our dihalohomo-
vinyl analogues1,9 which appear to be activated by the
5′/6′-hydrolytic activity of AdoHcy hydrolase to generate
electrophiles that undergo nucleophilic attack by protein
residues. Half of the site’s covalent binding was indi-
cated, and the resulting functional tetrameric-subunit
complexes retained both oxidative and hydrolytic activ-
ity. Treatment of such partially inactivated enzymes
with a type I inhibitor caused complete inactivation. The
dihalohomovinyl analogues had no significant antiviral
activity and were not cytotoxic to certain cell cultures.1,9
It is possible that antiviral activity was not observed
owing to insufficient perturbation of cellular AdoMet/
AdoHcy ratios. Reduction of E-NAD+ to NADH with
iodo analogue 3c caused loss of AdoHcy hydrolase
activity, and potent antiviral activity was observed. In
contrast, approximately 50% of the E-NAD+ is reduced
to E-NADH with 3a or 3b, and lower antiviral effects
are observed with these chloro and bromo congeners.
Exp er im en ta l Section
Uncorrected melting points were determined with a capil-
lary apparatus. UV spectra were measured with MeOH
solutions. 1H (200 or 500 MHz) and 13C (125 MHz) NMR
spectra were determined with CDCl3 solutions unless specified.
Mass spectra (MS and HRMS) were obtained by electron
impact (20 eV), chemical ionization (CI, isobutane), or fast
atom bombardment (FAB, thioglycerol matrix). Merck kie-
selgel 60-F254 sheets were used for TLC (detection at 254 nm,
or by color with I2 in a sealed chamber). Merck kieselgel 60
(230-400 mesh) was used for column chromatography. Pre-
parative reversed-phase (RP)-HPLC was performed with a
Dynamax C18 column and a Spectra Physics SP 8800 ternary
pump system (gradient solvent systems are noted). Elemental
analyses were determined by M-H-W Laboratories, Phoenix,
AZ. AdoHcy, Ado, Hcy, and calf intestinal Ado deaminase (EC
3.5.4.4) were obtained from Sigma. Standard Cl-, Br-, and
I- ions were obtained from P. J . Cobert Association, Inc. (St.
Louis, MO). Recombinant human placental AdoHcy hydrolase
was overexpressed and purified as described.7a Reagent grade
chemicals were used and solvents were dried by reflux over

Inactivation of S-Adenosyl-L-homocysteine Hydrolase
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 20 3861
and distillation from CaH2 (except THF/potassium) under
argon. Sonication was performed with a 300 Ultrasonik unit.
stirred at ∼0 °C for 2 h and then at ambient temperature
overnight. The solution was washed (NaHCO3/H2O, brine) and
dried (MgSO4), and volatiles were evaporated. Chromatogra-
phy (EtOAc/hexanes, 2:3) of the residue gave 5 (4.99 g, 58%)
as a solidified syrup: 1H NMR δ 1.38 and 1.60 (2 × s, 2 × 3,
2 × Me), 2.47 (d, J ) 11.0 Hz, 1, OH3), 3.75-3.88 (m, 1, H3),
4.45 (t, J ) 8.6 Hz, 1, H4), 4.56 (t, J ) 4.4 Hz, 1, H2), 5.79 (d,
J ) 3.6 Hz, 1, H1), 6.42 (d, J ) 8.4 Hz, 1, H5); HRMS (CI) m/z
346.9135 (3, MH+ [C9H1381Br2O4] ) 346.9140), 344.9153 (6,
6-N-Ben zoyl-9-(5,6-d id eoxy-2,3-O-isop r op ylid en e-â-D-
r ibo-h ex-5-yn ofu r a n osyl)a d en in e (2). BuLi/hexane (1.6 M;
1.25 mL, 2 mmol) was added dropwise to a solution of 6-N-
benzoyl-9-(6,6-dibromo-5,6-dideoxy-2,3-O-isopropylidene-â-D-
ribo-hex-5-enofuranosyl)adenine1,11 (1; 282 mg, 0.5 mmol) in
THF (20 mL) at -78 °C under Ar, and stirring was continued
for 2 h (TLC indicated conversion to a more polar product and
minor fluorescent byproducts). The mixture was neutralized
(HOAc, pH ∼6) and evaporated, and the residue was parti-
tioned (NaHCO3/H2O//CHCl3). The organic layer was washed
(brine), dried (MgSO4), and evaporated. Chromatography
(hexanes/EtOAc, 1:4) of the residue gave 214 (107 mg, 53%):
1H NMR δ 1.42 and 1.62 (2 × s, 2 × 3, 2 × Me), 2.48 (d, J )
2.0 Hz, 1, H6′), 5.10 (br s, 1, H4′), 5.16 (d, J ) 5.9 Hz, 1, H3′),
5.77 (d, J ) 5.9 Hz, 1, H2′), 6.31 (s, 1, H1′), 7.48-8.10 (m, 5,
Arom), 8.35 (s, 1, H2), 8.84 (s, 1, H8), 9.30 (s, 1, NH); HRMS
(CI) m/z 406.1519 (50, MH+ [C21H20N5O4] ) 406.1515).
MH+ 81/79Br2] ) 344.9160), 342.9162 (3, MH+ [79Br2] )
[
342.9181).
Analogous treatment of the 3-O-benzoyl derivative of 422c
gave the 3-O-benzoyl derivative of 5 (71%).
5,6-Did eoxy-1,2-O-isop r op ylid en e-r-D-r ibo-h ex-5-yn o-
fu r a n ose (6). BuLi/hexane (2.5 M; 28 mL, 70 mmol) was
added dropwise to a solution of 5 (3.44 g, 10 mmoL) in dried
THF (50 mL) at -78 °C, and stirring was continued for 3 h.
The mixture was neutralized (AcOH, pH ∼6.5), concentrated
(∼10 mL, <25 °C), and partitioned (NaHCO3/H2O//CHCl3). The
aqueous layer was extracted (CHCl3, 2×), the combined organic
phase was washed (brine) and dried (MgSO4), and volatiles
were evaporated. The residue was chromatographed (CHCl3
f 2% MeOH/CHCl3) to give 6 (1.49, 81%) as a white solid: 1H
NMR δ 1.36 and 1.57 (2 × s, 2 × 3, 2 × Me), 2.51 (d, J ) 2.0
Hz, 1, H6), 2.60 (d, J ) 9.2 Hz, 1, OH3), 3.98-4.10 (m, 1, H3),
4.36 (dd, J ) 1.6, 8.6 Hz, 1, H4), 4.59 (t, J ) 4.4 Hz, 1, H2),
5.84 (d, J ) 4.0 Hz, 1, H1); HRMS (CI) m/z 185.0815 (100,
MH+ [C9H13O4] ) 185.0814).
3-O-Ben zoyl-5,6-dideoxy-1,2-O-isopr opyliden e-r-D-r ibo-
h ex-5-yn ofu r a n ose (7). Treatment of 6 (276 mg, 1.5 mmol)
with BzCl (0.3 mL, 363 mg, 2.58 mmol) at ∼0 °C for 2 h and
30 min at ambient temperature, and chromatography (as
described for 9a ) gave 7 (380 mg, 88%) as an amorphous
solid: 1H NMR δ 1.30 and 1.57 (2 × s, 2 × 3, 2 × Me), 2.54 (d,
J ) 1.8 Hz, 1, H6), 4.88 (dd, J ) 1.6, 9.0 Hz, 1, H4), 4.94-5.07
(m, 2, H2,3), 5.92 (d, J ) 3.8 Hz, 1, H1), 7.40-8.10 (m, 5,
Arom); HRMS (CI) m/z 289.1069 (100, MH+ [C16H17O5] )
289.1076).
6-Ch lor o-5,6-d id eoxy-1,2-O-isop r op ylid en e-r-D-r ibo-
h ex-5-yn ofu r a n ose (8a ). A suspension of 6 (276 mg, 1.5
mmol) in NaOCl/H2O (5.25% commercial laundry bleach, 50
mL) was stirred at ambient temperature for 16 h (the reaction
mixture became homogeneous within 30 min). The solution
was partitioned (NaHCO3/H2O//CHCl3), the organic layer was
washed (H2O, brine) and dried (MgSO4), and volatiles were
evaporated. The residue was chromatographed (CHCl3 f 1.5%
MeOH/CHCl3) to give 8a (289 mg, 87%) as a syrup: 1H NMR
δ 1.37 and 1.58 (2 × s, 2 × 3, 2 × Me), 2.57 (d, J ) 10.6 Hz, 1,
OH3), 3.95-4.07 (m, 1, H3), 4.36 (d, J ) 8.6 Hz, 1, H4), 4.57
(t, J ) 4.4 Hz, 1, H2), 5.82 (d, J ) 4.0 Hz, 1, H1); HRMS (CI)
m/z 221.0390 (10, MH+ [C9H1237ClO4] ) 221.0395), 219.0421
(36, MH+ [35Cl] ) 219.0424).
3-O-Ben zoyl-6-ch lor o-5,6-dideoxy-1,2-O-isopr opyliden e-
r-D-r ibo-h ex-5-yn ofu r a n ose (9a ). BzCl (0.2 mL, 239 mg, 1.7
mmol) was added via syringe into a solution of 8a (219 mg, 1
mmol) in dried pyridine (5 mL) at ∼0 °C (ice bath) under N2,
and stirring was continued for 4 h. NaHCO3/H2O (2 mL) was
added, stirring was continued for 10 min, and volatiles were
evaporated. The residue was partitioned (H2O/CHCl3), the
organic layer was washed (HCl/H2O, NaHCO3/H2O, brine),
dried (MgSO4), and volatiles were evaporated. The residue
was chromatographed (EtOAc/hexane, 1:4) to give 9a (264 mg,
82%) as a solidified syrup: 1H NMR δ 1.31 and 1.53 (2 × s, 2
× 3, 2 × Me), 4.87-5.04 (m, 3, H2,3,4), 5.91 (d, J ) 3.6 Hz, 1,
H1), 7.42-8.10 (m, 5, Arom); HRMS (CI) m/z 325.0677 (40,
MH+ [C16H1637ClO5] ) 325.0657), 323.0703 (100, MH+ [35Cl]
) 323.0686).
9-(5,6-Did e oxy-6-iod o-â-D-r ibo-h e x-5-yn ofu r a n osyl)-
a d en in e (3c). A solution of 2 (102 mg, 0.25 mmol) in Me2CO
(7 mL) was added dropwise to a stirred solution of AgNO3 (65
mg, 0.38 mmol) in H2O (3 mL, to which “1 drop” of diluted
NH3/H2O had been added). Separation of the silver acetylide
salt began almost immediately, and after 5 min NIS (90 mg,
0.4 mmol) was added. Stirring was continued for 30 min (the
reaction mixture became homogeneous after ∼5 min and then
became turbid again), and volatiles were evaporated. The
residue was partitioned (NaHCO3/H2O//CHCl3), and the or-
ganic layer was washed (H2O) and dried (MgSO4). Volatiles
were evaporated to give the protected iodoalkyne (∼90%) [MS
(CI) m/z 532 (10, MH+), 406 (100), 240 (45), 135 (50)]. This
material was stirred overnight with NH3/MeOH (20 mL) at
ambient temperature, and volatiles were evaporated. Chro-
matography of the residue (EtOAc f 2% MeOH/EtOAc) gave
the 2′,3′-O-isopropylidene compound (76 mg, 71% from 2): 1H
NMR (CD3OD/CDCl3, ∼4:1) δ 1.32 and 1.48 (2 × s, 2 × 3, 2 ×
Me), 5.02 (s, 1, H4′), 5.08 (d, J ) 5.5 Hz, 1, H3′), 5.71 (d, J )
5.5 Hz, 1, H2′), 6.16 (s, 1, H1′), 8.08 (s, 1, H2), 8.24 (s, 1, H8);
MS (CI) m/z 428 (80, MH+), 302 (60), 242 (70), 213 (75), 135
(100). A solution of this material (68 mg, 0.16 mmol) in TFA/
H2O (9:1, 3 mL) was stirred at ∼0 °C (ice bath) for 1 h, and
volatiles were evaporated. EtOH was added and evaporated,
and the residue was chromatographed (EtOAc f 10% MeOH/
EtOAc) and crystallized (MeOH) to give 3c (27 mg, 44%; 31%
from 2). The mother liquor was purified by RP-HPLC (pre-
parative C18 column; program 15% CH3CN/H2O for 30 min
followed by a gradient of 15 f 40% for 30 min at 2.8 mL/min;
tR ) 53 min) to give additional 3c (10 mg, 16%; 42% total from
2): mp 180-183 °C dec; UV max 259 (ꢀ 14 200), min 228 nm
1
(ꢀ 2800); H NMR (Me2SO-d6) δ 4.32 (q, J ) 4.6 Hz, 1, H3′),
4.62 (d, J ) 4.6 Hz, 1, H4′), 4.77 (q, J ) 5.1 Hz, 1 H2′), 5.66
(d, J ) 6.2 Hz, 1, OH3′), 5.69 (d, J ) 6.0 Hz, 1, OH2′), 5.87 (d,
J ) 4.8 Hz, 1, H1′), 7.35 (br s, 2, NH2), 8.14 (s, 1, H2), 8.24 (s,
1, H8); 13C NMR (Me2SO-d6) δ 15.95, 73.36, 74.68, 75.30, 87.68,
90.45, 119.45, 139.75, 149.69, 153.09, 156.09; HRMS (CI) m/z
387.9915 (100, MH+ [C11H11IN5O3] ) 387.9907). Anal. [C11H10
-
IN5O3‚0.25H2O‚0.5 EtOAc (435.7)] C, H, N. The presence of
1
0.5 equiv of EtOAc was verified by integration of the H NMR
solvent peaks.
Treatment of 2 with catalytic AgNO3 (0.125 equiv; as
described for 9b with NIS instead of NBS), and deprotection
gave 3c in similar yield. The sugar/coupling route 7 f 9c f
10c f 3c (as described for 3b with NIS instead of NBS) also
gave 3c (28% overall).
6,6-Dibr om o-5,6-dideoxy-1,2-O-isopr opyliden e-r-D-r ibo-
h ex-5-en ofu r a n ose (5). H5IO6 (6.84 g, 30 mmol) was added
to a solution of 422b (6.5 g, 25 mmol) in dried EtOAc (200 mL)
at ambient temperature, and stirring was continued for 2 h.
The mixture was filtered, the filter cake was washed (EtOAc),
and the combined filtrate was evaporated. The residue was
dissolved in dried CH2Cl2 (200 mL), and the solution was
cooled to ∼0 °C (ice bath). CBr4 (16.6 g, 50 mmol) and Ph3P
(26 g, 100 mmol) were added, and the brown solution was
3-O-Ben zoyl-6-br om o-5,6-dideoxy-1,2-O-isopr opyliden e-
r-D-r ibo-h ex-5-yn ofu r a n ose (9b). AgNO3 (25 mg, 0.15
mmol) was added to a solution of 7 (288 mg, 1 mmol) and NBS
(205 mg, 1.15 mmol) in Me2CO (10 mL) at ambient tempera-
ture, and stirring was continued for 30 min. Volatiles were
evaporated, and the residue was partitioned (H2O/CHCl3). The
aqueous layer was extracted (CHCl3), the combined organic

3862 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 20
Robins et al.
phase was washed (NaHCO3/H2O, brine) and dried (MgSO4),
and volatiles were evaporated to give 9b (351 mg, 96%) as an
amorphous solid: 1H NMR δ 1.35 and 1.57 (2 × s, 2 × 3, 2 ×
Me), 4.89-5.06 (m, 3, H2,3,4), 5.92 (d, J ) 3.4 Hz, 1, H1), 7.42-
adenine (252 mg, 57%): HRMS (FAB) m/z 592.0668 (96, MH+
[C27H2181BrN5O6] ) 592.0655), 590.0671 (100, MH+ [79Br] )
590.0675). Deprotection (NH3/MeOH), chromatography, and
“diffusion crystallization”26 (as with 3a ) gave 3b (65 mg, 2
crops; 20% from 9b): mp 120-130 °C (transition), >160 °C
8.10 (m, 5, Arom); HRMS (CI) m/z 369.0154 (10, MH+ [C16H16
-
1
81BrO5] ) 369.0161), 367.0180 (10, MH+ [79Br] ) 367.0181).
dec; UV max 259 nm (ꢀ 13 800), min 228 (ꢀ 2900); H NMR
(Me2SO-d6) δ 4.36 (q, J ) 4.8 Hz, H3′), 4.56 (d, J ) 4.2 Hz,
H4′), 4.82 (q, J ) 5.2 Hz, H2′), 5.69 (d, J ) 5.8 Hz, 1, OH3′),
5.75 (d, J ) 5.6 Hz, 1, OH2′), 5.88 (d, J ) 4.8 Hz, 1, H1′), 7.32
(br s 2, NH2), 8.14 (s, 1, H2), 8.28 (s, 1, H8); 13C NMR (Me2-
SO-d6) δ 50.89, 73.45, 74.37, 75.41, 77.83, 88.09, 119.48,
140.20, 150.011, 153.40, 156.41; HRMS (FAB) m/z 342.0038
(25, MH+ [C11H1181BrN5O3] ) 342.0025), 340.0039 (25, MH+
[79Br] ) 340.0045). Anal. [C11H10BrN5O3‚0.5 H2O (349.2)] C,
H, N.
Compound 3b also was obtained (∼9% overall) by the
nucleoside route 1 f 2 f 3b, as described for 3c, with NBS
instead of NIS. Treatment of 1 with TBAF/THF,17a DBU/THF,
or BuLi/THF (1-2 equiv) did not give clean elimination of HBr.
Ad oHcy Hyd r ola se Activity. Activity was assayed in the
synthetic direction by measuring rates of formation of AdoHcy
from Ado and Hcy. The enzyme was incubated with Ado (1
mM) and Hcy (5 mM) in potassium phosphate buffer [500 µL;
50 mM, pH 7.2, containing EDTA (1 mM)] (buffer A) at 37 °C
for 5 min. Reaction was terminated by addition of HClO4/H2O
(5 M, 25 µL), the reaction mixture was cooled in ice-water
for 15 min, and the clear supernatant was collected and
analyzed for AdoHcy by HPLC using a C18 reverse-phase
column (Econosphere C18, 5 µm, 250 × 4.6 mm, Alltech,
Deerfield, IL) as described.5b
9-(6-Ch lor o-5,6-d id eoxy-â-D-r ibo-h ex-5-yn ofu r a n osyl)-
a d en in e (3a ). (a ) Hyd r olysis a n d Acetyla tion . A solution
of 9a (250 mg, 0.77 mmol) in TFA/H2O (9:1, 10 mL) was stirred
at ∼0 °C (ice bath) for 1 h. Volatiles were evaporated, and
traces were coevaporated from the residue (toluene 2 ×,
pyridine). Pyridine (8 mL), Ac2O (1 mL), and 4-(dimethylami-
no)pyridine (DMAP, 5 mg) were added, the mixture was stirred
at ∼0 °C for 2 h and at 10-15 °C for 2 h, and MeOH (5 mL)
was added. Stirring was continued for 20 min, volatiles were
evaporated, the residue was dissolved (EtOAc), and the
solution was washed (HCl/H2O, NaHCO3/H2O, brine) and dried
(Na2SO4). Volatiles were evaporated, and the residue was
chromatographed (20 f 30% EtOAc/hexanes) to give 1,2-di-
O-acetyl-3-O-benzoyl-6-chloro-5,6-dideoxy-R,â-D-ribo-hex-5-
ynofuranose (10a , R/â, ∼1:4; 245 mg, 87%): 1H NMR δ 1.99
and 2.14 (2 × s, 2 × 0.2, 2 × Ac), 2.02 and 2.15 (2 × s, 2 × 0.8,
2 × Ac), 4.96 (d, J ) 4.6 Hz, 0.8, H4), 5.01 (d, J ) 1.6 Hz, 0.2,
H4), 5.48 (t, J ) 5.1 Hz, 0.2, H2), 5.57 (dd, J ) 1.8, 4.6 Hz,
0.8, H2), 5.67 (dd, J ) 1.8, 5.8, 0.2, H3), 5.69 (t, J ) 4.6 Hz,
0.8, H3), 6.26 (d, J ) 2.0 Hz, 0.8 H1), 6.50 (d, J ) 4.4 Hz, 0.2,
H1), 7.40-8.10 (m, 5, Arom); HRMS (FAB) m/z 391.0374 (34,
MNa+ [C17H1537ClNaO7] ) 391.0375), 389.0396 (100, MNa+
[35Cl] ) 389.0404).
(b) Cou p lin g. SnCl4 (0.18 mL, 391 mg, 1.5 mmol) was
added dropwise to a suspension of 6-N-benzoyladenine (240
mg, 1 mmol) and the anomeric sugar [from step (a): 245 mg,
0.67 mmol] in dried CH3CN (15 mL), and stirring was
continued for 16 h at ambient temperature. Volatiles were
evaporated, the residue was partitioned (NaHCO3/H2O//
CHCl3), and the organic layer was washed (brine) and dried
(MgSO4). Volatiles were evaporated, and the residue was
chromatographed (CHCl3 f 2% MeOH/CHCl3) to give 9-(2-O-
acetyl-3-O-benzoyl-6-chloro-5,6-dideoxy-â-D-ribo-hex-5-yno-
furanosyl)-6-N-benzoyladenine (274 mg, 75%): HRMS (FAB)
m/z 548.1144 (7, MH+ [C27H2137ClN5O6] ) 548.1151), 546.1162
(23, MH+ [35Cl] ) 546.1180).
In activation of AdoHcy Hydr olase. Incubation of AdoHcy
hydrolase (25 nM) with various concentrations of 3a -c (0-
100 µM) in buffer A at 37 °C for 20 min gave data shown in
Table 1, and that with various concentrations of 3a -c for
different times (0-5 min) gave data shown in Table 2. The
remaining enzyme activity was assayed in the synthetic
direction as described in the previous experimental procedure.
Time-dependent inactivation data were used to calculate the
pseudo-first-order rate constants (kapp) by plotting the loga-
rithm of the remaining activity versus time. The kinetic
constants k2 and Ki were calculated from the kapp values and
inhibitor concentrations ([I]) by the method of Kitz and
Wilson27 with eq 1.
(c) Dep r otection . A solution of the product from step b
(274 mg, 0.5 mmol) in NH3/MeOH (20 mL) was stirred for 24
h at ∼5 °C. Volatiles were evaporated, and the residue was
flash chromatographed (EtOAc f 15% MeOH/EtOAc). “Dif-
fusion crystallization”26 (MeOH/EtOAc) of the residue gave 3a
(52 mg, 2 crops) as a white crystalline solvate (variable). RP-
HPLC purification (preparative C18 column, gradient 15 f 45%
CH3CN/H2O for 80 min at 2 mL/min; tR ) 64 min) of the
mother liquor gave additional 3a (29 mg; ∼36% total from
9a ): mp ∼160-185 °C dec; UV max 259 nm (ꢀ 13 800), min
228 nm (ꢀ 3100); 1H NMR (Me2SO-d6) δ 4.36-4.44 (m, 1, H3′),
4.59 (d, J ) 4.4 Hz, 1, H4′), 4.83-4.92 (m, 1, H2′), 5.72 (d, J
) 5.6 Hz, 1, OH3′), 5.79 (d, J ) 5.4 Hz, 1 OH2′), 5.91 (d, J )
5.0 Hz, 1, H1′), 7.30 (br s, 2, NH2), 8.16 (s, 1, H2), 8.31 (s, 1,
H8); 13C NMR (Me2SO-d6) δ 66.43, 67.32, 73.19, 73.68, 75.25,
87.94, 119.29, 140.15, 149.82, 153.23, 156.16; HRMS (FAB)
m/z 298.0503 (38, MH+ [C11H1137ClN5O3] ) 298.0521), 296.0539
(100, MH+ [35Cl] ) 296.0550).
9-(6-Br om o-5,6-d id eoxy-â-D-r ibo-h ex-5-yn ofu r a n osyl)-
a d en in e (3b). Hydrolysis and acetylation (as with 3a ) of 9b
(330 mg, 0.9 mmol) gave anomeric 10b (R/â, ∼1:3; 310 mg,
84%): 1H NMR δ 1.99 and 2.11 (2 × s, 2 × 0.25, 2 × Me), 2.02
and 2.12 (2 × s, 2 × 0.75, 2 × Me), 4.97 (d, J ) 4.4 Hz, 0.75,
H4), 5.03 (d, J ) 1.6 Hz, 0.25, H4), 5.49 (t, J ) 5.3 Hz, 0.25,
H2), 5.58 (dd, J ) 1.6, 4.6 Hz, 0.75, H2), 5.69 (dd, J ) 1.8, 6.0
Hz, 0.25, H3), 5.73 (t, J ) 4.7 Hz, 0.75, H3), 6.26 (d, J ) 1.6
Hz, 0.75, H1), 6.51 (d, J ) 4.6 Hz, 0.25, H1), 7.41-8.11 (m, 5,
Arom); HRMS (EI) m/z 411.9964 (15, M+ [C17H1581BrO7] )
411.9981), 409.9993 (15, M+ [79Br] ) 410.0001). Coupling of
10b (310 mg, 0.75 mmol) with 6-N-benzoyladenine and puri-
fication (as with 3a ) gave 9-(2-O-acetyl-3-O-benzoyl-6-bromo-
5,6-dideoxy-â-D-r ibo-h ex-5-yn ofu r a n osyl)-6-N -ben zoyl-
1/kapp ) 1/k2 + (Ki/k2)/[I]
(1)
An a lysis of Ha lid e Ion s Relea sed fr om 3a -c. The Cl-
or Br- ions released upon incubation of AdoHcy hydrolase with
3a or 3b were analyzed by ion-exchange chromatography. The
enzyme (10 µM) was incubated with 3a or 3b (100 µM) in
buffer A at 37 °C for 20 min. An aliquot of the reaction
mixture was applied to an Amicon Centricon-3 microconcen-
trator (3000 Mr cutoff). The filtrate was applied to an ion-
exchange column (Anion/R, 250 × 4.1 mm, 10 µm, Alltech,
Deerfield, IL) in a HPLC system equipped with a conductivity
detector (690 Ion Chromatograph, Omega, Metrohm, Ltd.,
Herisau, Switzerland) for halide ion analysis. The ions were
eluted isocratically [1.5 mM p-hydroxybenzoic acid/2% metha-
nol (pH 8.5, LiOH)] at a flow rate of 2 mL/min. Comparison
of ion peak areas with those from known quantities of standard
ions with standardized curves allowed quantitation of Cl- and
Br- ions.
Iodide ions released by incubation of AdoHcy hydrolase with
3c were detected as described28 [C18 reversed-phase column
(ODS Hypersil 250 × 4.6 mm, Alltech, Deerfield, IL), 0.5 mM
tetrabutylammonium hydroxide/5% CH3OH (pH 7.1 adjusted
with 0.1 M potassium hydrogen phthalate), flow rate of 1.5
mL/min, detection at 260 nm]. Quantitation of I- employed
comparison of peak areas with those of known quantities of
standard I- with a standardized curve.
An a lysis of Ad en in e. AdoHcy hydrolase and 3a , 3b, or
3c were incubated in buffer A under the conditions described
for the halide ion determinations except at each time point
the reaction was stopped by addition of 5 M HClO4/H2O (10

Inactivation of S-Adenosyl-L-homocysteine Hydrolase
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 20 3863
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µL). The precipitate was removed by centrifugation, and the
supernatant was analyzed for Ade (HPLC with a reverse-phase
C18 column as described7a). The HPLC peak assigned to Ade
was verified (co-injection with authentic Ade) and confirmed
by mass spectral analysis (CI/NH3).
An a lysis of E-NAD+ a n d E-NADH. The extent of conver-
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curve.
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Ack n ow led gm en t. We thank the American Cancer
Society (Grant DHP-34), Brigham Young University
development funds, the United States Public Health
Service (Grant GM-29332), the Belgian Fonds voor
Geneeskundig Wetenschappelijk Onderzoek (Project
3.0026.91), the Belgian Geconcerteerde Onderzoeksac-
ties (Conventie 95/5), and the Biomedical Research
Program of the European Commission for support. We
thank Anita Van Lierde and Frieda De Meyer for
excellent technical assistance and Mrs. J eanny K.
Gordon for assistance with the manuscript.
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