A new class of pentacoordinate ribonuclease inhibitors: Synthesis, characterization, and inhibition studies of ribonucleoside and anhydropentofuranose oxorhenium(V) complexes

Article abstract of DOI:10.1021/ja9611248

This article describes the synthesis, characterization, and kinetic studies of a new class of stable ribonuclease (RNase) inhibitors. Herein the first synthesis of oxorhenium(V) complexes of diaminonucleosides and diaminoaldopentofuranoses is reported. St

Full text of DOI:10.1021/ja9611248

J. Am. Chem. Soc. 1996, 118, 12521-12527
12521
A New Class of Pentacoordinate Ribonuclease Inhibitors:
Synthesis, Characterization, and Inhibition Studies of
Ribonucleoside and Anhydropentofuranose Oxorhenium(V)
Complexes
Paul Wentworth, Jr., Torsten Wiemann, and Kim D. Janda*
Contribution from the Departments of Chemistry and Molecular Biology, The Scripps Research
Institute, La Jolla, California 92037
ReceiVed April 5, 1996X
Abstract: This article describes the synthesis, characterization, and kinetic studies of a new class of stable ribonuclease
(RNase) inhibitors. Herein the first synthesis of oxorhenium(V) complexes of diaminonucleosides and diaminoal-
dopentofuranoses is reported. Stability studies have shown that the complexes are stable between pH 3 and 13 at
temperatures of 25-80 °C for 24 h with no occurrence of ligand exchange or epimerization at the RedO core.
Kinetic studies show that both the syn (1a) and anti (1b) diastereoisomers of oxorhenium(V) complexes of 9-(2′,3′-
diamino-2′,3′-dideoxy-â-D-ribofuranosyl)adenine (13) are excellent inhibitors of the purine specific ribonuclease,
RNase U2. Dixon and Lineweaver-Burke analyses suggest that the inhibition is competitive, with inhibitory constants
(Kis) of 15 and 93 µM, respectively. These complexes are of equivalent inhibitory potency to previously reported
nucleoside vanadium alkoxide inhibitors with the added advantage of being completely stable in aqueous solution.
The oxorhenium(V) complexes of 2,5-anhydro-3,4-diamino-3,4-dieoxy-D-ribitol (2a-Re) and (2b-Re) display much
poorer inhibition (Ki > 2 mM), highlighting the critical nature of the purine base for successful inhibition.
Introduction
Scheme 1
Ribonuclease U2 (EC 3.1.27.4) secreted by the smut fungus
Ustilago sphaerogena1,2 is an endoribonuclease which catalyzes
the hydrolysis of RNA, specifically at the 3′-phosphodiester
bond of adenylic acid, via a two-step mechanism very similar
to that of RNases A and T1.3-8 Usher9,10 and co-workers have
shown that both steps of the RNase catalyzed reaction follow
an in-line associative mechanism with phosphorus existing in
a theoretical pentacoordinate trigonal-bipyramidal geometry
through each of the transition states (TS) (Scheme 1). Although
Usher did not identify the amino acid residues in the binding
site responsible for catalysis, these have since been inferred from
a number of substrate modification experiments and published
structures of RNase-inhibitor complexes.11-16
* Author to whom correspondence should be addressed.
X Abstract published in AdVance ACS Abstracts, December 1, 1996.
(1) Arima, T.; Uchida, T.; Egami, F. Biochem. J. 1968, 106, 601-607.
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(3) Takahashi, K.; Moore, S. The Enzymes; Academic Press: New York,
1982; Vol. 15, pp 435-467.
Pentacoordinate oxyphosphoranes are generally unstable;
therefore, other elements that exist as five-coordinate species
have been developed as models of the postulated TS for
inhibition and mechanistic characterization of RNase activity.
From as early as 1973, complexes of uridine with oxovanadium-
(IV) and vanadium(V) ions have been shown to be “potent”
inhibitors of RNases (Ki 8-12 µM).17 At the time it was
suggested by the authors and later validated by X-ray crystal-
lography that the vanadate was mimicking the pentacoordinate
TS for phosphodiester hydrolysis.18,19 However, vanadium
alkoxides are very unstable and undergo rapid ligand exchange
(4) Takahashi, K. J. Biochem. (Tokyo) 1985, 98, 815-817.
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S0002-7863(96)01124-9 CCC: $12.00 © 1996 American Chemical Society

12522 J. Am. Chem. Soc., Vol. 118, No. 50, 1996
Wentworth et al.
in aqueous solution making a direct structural analysis of the
inhibitory species and its kinetic parameters very complex.20-24
In a recent strategy, we have shown that a diastereoisomeric
mixture of a ribonucleoside oxotechnetium(V) chelate inhibits
RNase U2, heralding the first water-stable pentacoordinate
inhibitor of a RNase.25 Modeling shows that the technetium
exists in a distorted TBP geometry, suggesting that the inhibitor
is indeed mimicking the putative transition state for RNA
hydrolysis. However, because of the radioactivity associated
with technetium and the inherent difficulties of both purifying
and analyzing its diastereomers on a preparative scale, we have
sought alternative stable, pentacoordinate metal chelates for
RNase inhibition.
Rhenium, in common with its congener technetium occupies
a central position in the d-block elements and exists in a wide
variety of oxidation states and coordination numbers. A number
of bifunctional ligands have been developed for Re coordination
and these metallocomplexes have found use as radioimaging
agents or for the labeling of monoclonal antibodies (radio-
immunoassay).26-32 A number of structural studies have shown
that in all cases the Re atom is coordinated to five atoms (N,
N, S, S, and O for Re-N2S2 complexes, or N, N, N, S, and O
for Re-N3S complexes) in a distorted square-pyramidal
geometry.33-36 This paper describes the synthesis, characteriza-
tion, solution properties, and RNase U2 inhibition of novel N2S2
(diamide dimercaptide) oxorhenium(V) complexes of ribo-
nucleosides and anydropentofuranoses (Figure 1).
Figure 1. (A) The complexes synthesized as a novel class of
pentacoordinate inhibitors for RNase U2, based on the ribonucleoside
adenosine (1a and 1b) and a modified anhydroribitol (2a and 2b). B
shows the DADT ligand with the four deprotonation sites which
generates the pentacoordinate, negatively charged oxorhenium(V)
complexes.
Scheme 2
Results and Discussion
Synthesis of Rhenium Inhibitors (2a and 2b). The
synthetic route comprised of three main parts. First, a de noVo
construction of the 1,4-anhydro-2,3-diamino-2,3-dideoxy-D-
ribitol ring; second, incorporation of the complete DADT ligand
system; and third, metal insertion. The initial steps from
L-xylose (3) via the L-arabino tosylate 4 led to the reported
lyxo D-epoxide 5 (Scheme 2).37
Opening of the epoxide 5 with lithium azide gives an isomeric
mixture of azido alcohols which were isolated as their respective
acetates (6a-arabino) and (6b-xylo) in good yields, 32% and
47%, respectively (Scheme 3).
(19) Wlodawer, A.; Miller, M.; Sjolin, L. Proc. Natl. Acad. Sci. U.S.A.
1983, 80, 3628-3631.
(20) Paulsen, K.; Rehder, D.; Thoenne, D. Z. Naturforsch. A 1978, 33A,
834-839.
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28.
(22) Crans, D. C.; Rithner, C. D.; Theisen, L. A. J. Am. Chem. Soc.
1990, 112, 2901-2908.
Progression to the bis azide 7, which has the nitrogen
heteroatoms of the DADT ligand inserted with the correct regio-
and stereochemistry is accomplished equally well with both
isomers 6a and 6b. Methanolysis of either 6a or 6b gives the
corresponding azido alcohol (8). Epimerization of the alcohol
8 is achieved by activation as its trifluoromethanesulfonate ester
followed by displacement with sodium azide and 18-crown-6.
The ribo bis azide 7, following catalytic hydrogenation, is
acylated immediately with chloroacetyl chloride to give the ribo
bis(chloroacetamido) derivative 9 in 90% overall yield for the
two steps. The intermediate diamine 7a was not isolated due
to its high polarity and tendency to oxidation. Treatment of 9
with sodium thiobenzoate in refluxing ethanol now inserts the
masked thiol components of the DADT ligand and gives the
bis thioester 10 in excellent yields (94%). Because of the well-
known propensity of thiols to oxidation, metal insertion involves
a prior deprotection of the thioesters under basic conditions
followed by an immediate treatment of the crude dithiol 11 with
trichlorobis(triphenylphosphine)rhenium(V) oxide38 in ethanol
by the method described by O’Neill39 and co-workers. The
metal insertion reaction was found to be very base sensitive, if
the thioesters of 10 were deprotected with a mild base such as
(23) Crans, D. C.; Felty, R. A.; Miller, M. M. J. Am. Chem. Soc. 1991,
113, 265-269.
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74, 38-48.
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1489.
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M.; Fritzberg, A. R. In Technetium and Rhenium in Chemistry and Nuclear
Medicine 3; Nicolini, M., Bandoli, G., Massi, U., Eds.; Verona, 1990.
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heyden, J.-L.; Fritzberg, A. R.; Morgan, A. C., Jr.; Foon, K. A. Cancer
Res. 1990, 50, 7973-7978.
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W.; Bjorn, M. J.; Fer, M. F.; Wolf, S. B.; Ratliff, B. A.; Seiler, C. A.;
Foisie, D. C.; Fisher, D. R.; Schroff, R. W.; Fritzberg, A. R.; Abrams, P.
G. J. Nucl. Med. 1992, 33, 1099-1109.
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B.; Van Dongen, G. J. Nucl. Med. 1993, 34, 1953-1963.
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H. J. J. Nucl. Biol. Med. 1994, 38, 444-445.
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Biol. Med. 1994, 38, 485-486.
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Dunitz, J. D., Hafner, K., Ito, S., Lehn, J.-M., Raymond, K. N., Rees, C.
W., Thiem, J., Vogtle, F., Eds.; Springer-Verlag: Berlin, 1996; pp 275-
291.
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Am. Chem. Soc. 1990, 112, 5798-5804.
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1994, 33, 319-323.
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1992, 31, 2801-2808.

Pentacoordinate Ribonuclease Inhibitors
J. Am. Chem. Soc., Vol. 118, No. 50, 1996 12523
Scheme 3
Scheme 4
sodium acetate, the metal insertion process yielded a higher ratio
of anti/syn than if aqueous sodium hydroxide was employed.
By deprotection with sodium acetate a mixture of syn (12a)/
anti (12b) in the ratio 1:2 was obtained. Acetal hydrolysis with
TFA and reduction of the resultant aldehyde with sodium
borohydride then gave the required Re-anhydroribitol com-
plexes 2a and 2b.
Assignment of the oxorhenium(V) diastereoisomers either as
syn or anti is primarily based on the relative infrared stretches
of the RedO bond. Both complexes exhibit a strong absorbance
in the characteristic stretching frequency of a RedO bond (900-
100 cm-1).40 It is known that for a number of different
oxorhenium(V) complexes, the two isomers show different
infrared stretching frequencies, the syn epimer resonating at a
lower frequency than the corresponding anti isomer.41,42 Thus
2a was assigned as the syn epimer with an RedO stretching
frequency of 943 cm-1 and 2b denoted as the anti isomer with
a RedO frequency of 960 cm-1
.
Synthesis of Rhenium Inhibitors (1a and 1b). The starting
point for construction of the DADS ligand is 2,3-diamino-2,3-
dideoxyadenosine which we synthesized in seven steps, using
a modification of our procedure from 9-(â-D-arabinofuranosyl)-
adenine (Scheme 4).43
Chloroacetylation of 13 and thiobenzylation of the resultant
bis(chloroacetamide) intermediate 14 using the same procedure
described for 9 and 10, respectively, gave the fully protected
DADT ligand 15. Incorporation of the rhenium core was much
less sensitive to base strength for 16 than for 11, Vide supra.
By incubating in sodium hydroxide, 100% of the syn isomer
was produced. Whereas following incubation in sodium acetate
(1 M) a 20:1 ratio of syn/anti was produced (ratios determined
by HPLC). This high preference for the syn isomer (1a) in
this case may well be due to steric hindrance during the RedO
(40) Davison, A.; Jones, A. G.; Orvig, C.; Sohn, M. Inorg. Chem. 1981,
20, 1629.
(41) Chi, D.-Y.; Katzenellenbogen, J. A. J. Am. Chem. Soc. 1993, 115,
7045-7046.
(42) Dizio, J. P.; Fiaschi, R.; Davison, A.; Jones, A. G.; Katzenellen-
bogen, J. A. Bioconjugate Chem. 1991, 2, 353.
(43) Chen, Y. C. J.; Hansske, F.; Janda, K. D.; Robbins, M. J. J. Org.
Chem. 1991, 56, 3410-3413.

12524 J. Am. Chem. Soc., Vol. 118, No. 50, 1996
Wentworth et al.
Table 1. Comparative Inhibition Constantsa (Ki) and Binding
Energiesb (∆G) of Oxorhenium(V) Complexes 1a, 1b, 2a, and 2b
with RNase U2
All the rhenium complexes assayed were inhibitors of RNase
U2-catalyzed depolymerization of RNA. Figure 2 shows
representative Dixon48 plots for the rhenium complexes 1a and
1b, suggesting that the inhibition of RNase by these complexes
is competitive in nature. This result was further supported by
the fact that Lineweaver-Burke analyses gave similar Ki values
and there was no effect on Vmax with increase in inhibitor
concentration.
inhibitor
Ki, µM
∆G, kcal mol-1
1a (syn)
1b (anti)
2a (syn)
2b (anti)
adenine
15.7 ( 2
90 ( 9
7.1
6.0
<4.0
<4.0
>2000c
>2000c
no inhibitiond
It is generally accepted that the geometry of phosphorus in
the transition state for both of the steps in RNA depolymeri-
zation is trigonal bipyramidal (TBP),9,10 with the negative charge
around phosphorus being stabilized by key histidine and lysine
residues in the binding site. However there is also much
evidence to support a geometry of a distorted trigonal bipyramid
tending toward square pyramidal (SP).49 Theoretical calcula-
tions by Holmes50,51 and co-workers in the presence or absence
of amino acid residues, to allow a detailed study of the
enzymatic mechanism and catalytic residues, indicate a penta-
coordinate transition state for the enzyme-catalyzed reaction,
with geometry approaching SP. The O(2′)-P-O(5′) bond angle
is 162.2° in the minimum-energy pentacoordinate transition
state, which represents a 50% distortion of TBP geometry along
the Berry pseudorotation coordinate toward a SP structure. These
theoretical analyses are supported by X-ray crystallography data
for RNase A complexed with uridine-vanadate which show
the core vanadium atom existing in a distorted trigonal-
bipyramidal geometry with the axial planes O2′-V-O7 bond
angle being 156° rather than the theoretical 180°.19,23
a Ki values were determined by averaging the results of at least
duplicate runs in citrate buffer (pH 3.5, 16.5 mM), with urea (3.5 M)
and EDTA (0.85 mM) at 323 K. b Binding energies were calculated
from ∆G ) -RT ln (Ki). c Problems of solubility meant that an accurate
determination of Ki for this inhibitor was unfeasible. d Poor solubility
beyond a 5 mM assay concentration precluded a Ki determination.
insertion. The adenine ring sits trans to the RedO core during
generation of the syn isomer, but syn for the anti isomer.
Assignment of the two isomers syn (1a) and anti (1b) was based,
primarily, on the relative infrared stretching frequencies of the
RedO core, Vide supra.
Stability Studies. An important component of the strategy
for use of the DADT-based rhenium chelates was their known
stability in aqueous solution. In accordance with previous
reports, the ribonucleoside rhenium complexes (1a, 1b, 2a, and
2b) were all stable between pH 3 and 13 at temperatures of
25-80 °C for 24 h.44-46 No epimerization occurred at the
rhenium cores (assayed by HPLC) which allowed for an accurate
comparison of the inhibitory effect of each of the epimers in
the RNase assay, Vide infra.
Re complexes of diamide-dimercaptide (DADT)-based ligands
exist either in a pure or distorted SP geometry.34,35,52 The RedO
bond is axial and the rhenium classically adopts a position
approximately 0.72 Å above the basal plane of the N,N,S,S
ligands. There is a formal negative charge associated with these
complexes which resides in a delocalized manner around the
ligand sphere. Therefore these complexes seemed a good
starting point for development of a new class of RNase
inhibitors. The inhibition studies support our concept, with Kis
of the complexes 1a and 1b being 15 and 93 µM respectively
(Table 1), which for comparison is the same order of magnitude
as the inhibition by vanadate complexes reported by Leinhard17
for RNase A. If we assume that the Km is a true dissociation
constant, then the purine-based Re complexes bind a factor of
50-300 times stronger to RNase U2 than does the substrate,
which is a critical aspect of a potential TS analog. While at
present, we do not have any X-ray diffraction analyses of these
Re complexes, there is little reason to doubt that they exist as
distorted SP complexes. Therefore, our results add further
weight to the theory of a distorted TBP/SP transition state in
the RNase mechanism. However, there are a number of
reservations that makes further extrapolation of these complexes’
mechanistic role limited. First, we have no direct structural
information about these complexes which makes their precise
orientation in the RNase U2 binding site only speculative. In
addition, the necessary DADT ligand structure around the Re
core, which is not a component of vanadate inhibitors, may be
too bulky to occupy other RNase binding sites meaning that
these complexes may not serve as general RNase inhibitors.
Work in our laboratory is underway to answer these questions.
In this study it was of interest to quantify the enzyme
interactions and hence binding contribution of the purine base,
The pH optimum of RNase U2 is around 4.0,7 and a concern
was that the purine complexes 1a and 1b by being linked to
the organometallic may be susceptible to acid-catalyzed depu-
rination during the kinetic assay. However, no decomposition
was detected either during the stability or inhibition studies.
Kinetic and Inhibition Studies. The enzyme-catalyzed and
background transphosphorylation of the dinucleotide ApA and
individual inhibition constants for the Re complexes (1a, 1b,
2a, 2b), adenosine, and adenine were followed at pH 3.5
(sodium citrate) and 323 K by monitoring the increase in
adenosine concentration by HPLC (Table 1). Data was analyzed
using initial rates (1-2% of the reaction) and each data point
is an average of at least two determinations. Due to the poor
solubility of 1a and 1b in water, the assay included 4% DMSO
as a cosolvent. The kinetic parameters, Km (Michaelis-Menten
constant) and kcat (catalytic rate constant) of RNase U2 with
ApA were redetermined using our assay conditions and found
to be 4.5 mM and 3 min-1 respectively. The value for Km is
an order of magnitude greater than previously reported while
the kcat is in general accord with literature precedence.7 The
apparent elevation of the Km may be a result of two factors.
First, the binding affinity of RNase U2 for its substrates is known
to be perturbed by increasing the percentage of certain organic
solvents in acetate buffer.47 Second, the dinucleotide ApA is
only poorly water soluble and the original Km (234 ( 35 µM)
was determined by fitting experimental data obtained from only
10-80 µM of substrate.7 The addition of DMSO confers
increased solubility on the dinucleotide, and our estimation of
Km was based on substrate concentrations up to 2 mM.
(44) Fritzberg, A. R.; Kuni, C. C.; Klingensmith, W. C.; Stevens, J.;
Whitney, W. P. J. Nucl. Med. 1982, 23, 592.
(45) Fritzberg, A. R.; Abrams, P. G.; Beaumier, P. L.; Kasina, S. A.;
Morgan, C.; Rao, T. N.; Reno, J. M.; Sanderson, J. A.; Srinivasan, A.;
Wilbur, D. S. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 4025-4029.
(46) Kasina, S.; Fritzberg, A. R.; Johnson, D. L.; Eshima, D. J. Med.
Chem. 1986, 29, 1933-1940.
(47) Hirato, S.; Hirai, A. J. Biochem. 1979, 85, 327-334.
(48) Dixon, M.; Webb, E. C. Enzymes; Academic Press: New York,
1964.
(49) Thatcher, G. R. J.; Kluger, R. AdV. Phys. Org. Chem. 1989, 25,
99-265.
(50) Holmes, R. R.; Dieters, J. A.; Galluci, J. C. J. Am. Chem. Soc. 1978,
100, 7393-7402.
(51) Holmes, R. R. Pentacoordinated Phosphorus; American Chemical
Society: Washington D.C., 1980; Vol. 2.
(52) Rao, H. S. P.; Siva, P. Synth. Commun. 1994, 24, 549-555.

Pentacoordinate Ribonuclease Inhibitors
J. Am. Chem. Soc., Vol. 118, No. 50, 1996 12525
Figure 2. Dixon plots of the reciprocal reaction velocity Vs inhibitor concentration showing the inhibitory effects of (A) 1a syn and (B) 1b anti
in the hydrolysis of ApA by RNase U2. Each point is the average of at least duplicate determinations.
the ribose ring, and the rhenium complex. A valid approach
for assessing the binding contributions of different nucleoside
moieties is to determine the ∆G0 values for binding of
appropriate ligand pairs.53 The values of ∆∆G° obtained from
the comparison do not only reflect the intrinsic binding free
energy change of the specific ligand sites, but also include other
coupled free energy and entropy terms.54 This necessarily
means that the energy of binding of the purine rhenium
complexes 1a and 1b will be greater than the sum of the binding
energies of the anhydropentofuranose inhibitors 2a and 2b and
the base adenine. The kinetic studies support this view as there
is a large potentiation in binding involving the purine base and
the rhenium complexed with the ribose ring (Table 1). The Re
complexes with a â-adenin-9-yl group (1a and 1b) have ∆∆G°
of >2-3 kcal mol-1 over the anhydropentofuranose inhibitors
2a and 2b. A more complete thermodynamic analysis is not
possible because the low solubility of adenine meant that an
accurate determination of its Ki was unfeasible. What is
apparent however, is the importance of the purine base to the
inhibition by these TS analogs. Quantification of the binding
energy contribution in this case is not possible, but a recent
study has shown for RNase A as much as 50% of the binding
energy in the transition state is supplied by pyrimidine recogni-
tion by threonine, valine, and phenylalanine active-site resi-
dues.24
The most exciting feature of these oxorhenium(V)-based
inhibitors is their potential generality. Our synthetic route is
applicable for all nucleoside bases, and therefore rhenium
complexes containing all four nucleoside bases can be now
readily synthesized and their inhibition of a range of RNases
and other phosphodiesterases assessed.
Finally, these complexes offer the first real strategy, as TS
analogs for phosphodiester hydrolysis, which would be useful
for catalytic antibody generation. Their distorted TBP geometry,
and anionic charge coupled with aqueous stability makes them
ideal candidates for haptens that may generate phosphodiesterase
catalytic antibodies.
Experimental Section
General Procedures. Unless otherwise stated, all reactions were
performed under an inert atmosphere, with dry reagents and solvents
and flame-dried glassware. Analytical thin-layer chromatography
(TLC) was performed using 0.25 mm silica gel coated Kieselgel 60
F254 plates. Visualization of the chromatogram was by UV absorbance,
methanolic sulfuric acid, aqueous potassium permanganate, iodine, and
p-anisaldehyde. Liquid chromatography was performed using com-
pressed air (flash chromatography) with the indicated solvent system
and silica gel 60 (230-400 mesh). Preparative TLC was performed
using Merck 1 mm coated silica gel Kieselgel 60 F254 plates. Infrared
spectra were recorded on a Nicolet FTIR spectrometer and are reported
as wavenumbers (cm-1). 1H NMR spectra were recorded on either a
Bruker AMX-300 or a Bruker AMX-500 spectrometer. Chemical shifts
are reported in parts per million (ppm) on the δ scale from an internal
standard. 13C NMR (proton decoupled) spectra were recorded on a
Bruker AMX-500 spectrometer at 125.77 MHz. High-resolution mass
spectra (HRMS) were recorded on a VG ZAB-VSE mass spectrometer.
High-performance liquid chromatography (HPLC) was performed either
with a Hitachi D7000 series machine equipped with an Adsorbosphere
HS RP-C18 analytical column, or a Rainin preparative HPLC instrument
with a VYDAC RP-C18 90 Å pharmaceutical semipreparative column.
2,5:3,4-Dianhydro-D-lyxose Diethyl Acetal (5). Sodium methoxide
(684 mg, 11.8 mmol) in methanol (50 mL) was added dropwise to a
stirring solution of 2,5-anhydro-3-O-benzoyl-1,2-O-methylethylidene-
4-O-(p-toluenesulfonyl)-L-xylose diethyl acetal (4) (5.0 g, 10.7 mmol)
in methanol (120 mL) which had been cooled on ice. The reaction
mixture was allowed to warm to room temperature and was stirred for
8 h. The volatiles were removed in Vacuo, and the residue, dissolved
in ethyl acetate (EtOAc) (100 mL), was washed with water (3 × 50
mL), 5% citric acid (2 × 50 mL), saturated aqueous sodium bicarbonate
(NaHCO3) (3 × 50 mL), and brine (2 × 50 mL). The combined organic
fractions were then dried over anhydrous Na2SO4 and evaporated in
Vacuo to give a tan oil which was purified by silica gel chromatography
Concluding Discussion
In this study we have developed a new class of stable
pentacoordinate oxometal complexes based on rhenium(V) with
diaminonucleoside or diaminoaldopentofuranose ligands. The
diaminonucleoside-based inhibitors 1a and 1b are excellent
competitive inhibitors of a purine specific RNase. The geometry
of these complexes is believed to be distorted TBP/SP which
supports the rational of a distorted TBP transition state for RNA
hydrolysis.
The ribonucleoside oxorhenium(V) are as potent as previously
described vanadate-nucleoside complexes and other metallo-
nucleoside complexes.55 However, their much greater aqueous
stability and decreased susceptibility to ligand exchange means
that structural characterization of the oxorhenium(V) complexes
is much more straightforward.
(53) Jencks, W. P. AdV. Enzymol. 1975, 43, 219-410.
(54) Jencks, W. P. Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 4046-4050.
(55) Georgalis, Y.; Zouni, A.; Hahn, U.; Saenger, W. Biochim. Biophys.
Acta (Protein Structure and Molecular Enzymology) 1991, 1118, 1-5.

12526 J. Am. Chem. Soc., Vol. 118, No. 50, 1996
Wentworth et al.
(25% EtOAc/hexane). This gave the title compound as a colorless oil
ether 1:10) to give the title compound as a colorless oil (532 mg,
80%): Rf ) 0.4 (EtOAc/petroleum ether 1:10); H NMR (300 MHz,
1
1
(1.5 g, 74.6%): Rf ) 0.3 (50% EtOAc/hexane); H NMR (300 MHz,
CDCl3) δ 4.53 (d, J 7.5 Hz, 1H, H1), 4.08 (d, J2 10.7 Hz, 1H, H5a),
3.83 (dd, J 2.0 Hz, 1H, H2), 3.80-2.60 (m, 7H, H3, H4, H5b, 2 ×
CH3CH2O), 1.26 (t, J 7.1 Hz, 3H, CH3CH2O), 1.25 (t, J 7.1 Hz, 3H,
CH3CH2O); 13C NMR (125.77 MHz, CDCl3) δ 101.52, 78.07, 67.91,
63.85, 62.32, 56.47, 55.95, 15.26, 15.11; LRFABMS (m/z)+ 211 (M
+ Na)+.
CDCl3) δ 4.54 (d, J 3.5 Hz, 1H, H1), 4.33 (dd, J 5.6 Hz, J 5.6 Hz, 1H,
H3), 4.11 (ddd, J 5.0 Hz, I 4.1 Hz, 1H, H4), 4.03 (dd, 1H, J2 9.5 Hz,
H5a), 4.00 (dd, 1H, H2), 3.81 (dd, 1H, H5b) 3.86-3.71 (m, 2H,
CH3CH2O), 3.66-3.53 (m, 2H, CH3CH2O), 1.26 (t, J 7.1 Hz, 3H, CH3-
CH2O), 1.24 (t, J 7.1 Hz, 3H, CH3CH2O); LRFABMS (m/z)+ 279 (M
+ Na)+, 211 (M - OEt)+.
4-O-Acetyl-2,5-anhydro-3-azido-3-deoxy-D-arabinose Diethyl Ac-
etal (6a) and 3-O-Acetyl-2,5-anhydro-4-azido-4-deoxy-D-xylose Di-
ethyl Acetal (6b). NaN3 (7.5 g, 115.00 mmol) was added to a stirred
solution of the epoxide 5 (4.74 g, 25.18 mmol) in DMF (100 mL), and
the mixture was heated under reflux for 8 h. The DMF was removed
in Vacuo, and the brown residue was dissolved in ethyl acetate and
washed sequentially with water, aqueous NaHCO3, and brine. The
combined organic fractions were then dried over Na2SO4. Following
filtration of the drying agent, evaporation of the EtOAc in Vacuo gave
a crude mixture of diastereomeric azido alcohols. This mixture was
stirred overnight in acetic anhydride (15 mL, 125 mmol) and pyridine
(15 mL). The pyridine was then removed in Vacuo to yield a brown
residue which was dissolved in DCM (200 mL) and washed with water,
aqueous NaHCO3 and brine, and the combined organic fractions were
dried over Na2SO4 and evaporated to dryness. The two title diaster-
eomeric acetates were isolated following silica gel purification (EtOAc/
petroleum ether 1:10) as pale yellow oils (6a, 2.20 g, 32%, 6b, 3.22 g,
47%) in a ratio of arabino/xylo (2:3), 6a-arabino: Rf ) 0.5 (EtOAc/
petroleum ether 1:10); 1H NMR (300 MHz, CDCl3) δ 4.99 (ddd, J 2.5
Hz, J 5.1 Hz, J 2.6 Hz, 1H, H4), 4.56 (d, J 5.3 Hz, 1H, H1), 4.1 (dd,
J 5.2 Hz, J 2.5 Hz, 1H, H2), 3.98 (dd, 1H, H5a), 3.92 (dd, 1H, 5b),
3.84-3.70 (m, 3H, H3, CH3CH2O), 3.68-3.58 (m, 2H, CH3CH2O),
2.10 (s, 3H, COCH3), 1.25 (t, J 7.1 Hz, 3H, CH3CH2O), 1.23 (t, J 7.1
Hz, 3H, CH3CH2O); LRFABMS (m/z)+ 296 (M + Na)+, 228 (M -
OEt)+, M - OEt - HOAc)+. 6b-xylo; Rf ) 0.3 (EtOAc/petroleum
2,5-Anhydro-3,4-bis(2-chloroacetamido)-3,4-dideoxy-D-ribose Di-
ethyl Acetal (9). Palladium on carbon (50 mg) was added to a solution
of the diazide 7 (479 mg, 1.87 mmol) in MeOH (5 mL). The reaction
mixture was then shaken on a Parr hydrogenator under 30 psi of H2.
After 2 h, the reaction mixture was filtered through Celite and the
solvent was removed in Vacuo to give the diamine 7a as a colorless oil
1
(332 mg, 82%): Rf ) 0.2 (isopropyl alcohol/ethanol/water 5:3:2); H
NMR (300 MHz, CDCl3) δ 4.48 (d, J 5.3 Hz, 1H, H1), 4.02 (dd, 1H,
J2 9.2 Hz, J 4.8 Hz, H5a), 3.86-3.69 (m, 2H, CH3CH2O), 3.66-3.55
(m, 4H, H5b, CH3CH2O), 3.47-3.42 (m, 1H), 3.35 (dd, 1H, J 6.0 Hz,
J 6.7 Hz, 1H), 1.23 (t, J 7.1 Hz, 3H, CH3CH2O), 1.24 (t, J 7.1 Hz, 3H,
CH3CH2O); LRFABMS (m/z)+ 205 (M + H)+, 113 (M - 2OEt)+. A
solution of the diamine 7a (317 mg, 1.55 mmol) in DCM (5 mL) was
added dropwise to a cold (0 °C), vigorously stirred, suspension of
NaHCO3 (1.3 g, 15.5 mmol) and chloroacetyl chloride (876 mg, 7.8
mmol) in DCM (10 mL). TLC analysis (EtOAc/petroleum ether 1:10)
showed the reaction was complete after 2 h. The reaction mixture was
diluted with DCM (20 mL), poured onto ice water, and partitioned.
The organic layer was repeatedly washed with water (3 × 50 mL),
aqueous NaHCO3 (2 × 50 mL), and brine (2 × 50 mL). The combined
organic fractions were then dried and evaporated as described above
for 4. The residue was purified by silica gel chromatography (EtOAc/
petroleum ether 1:7) to give the title compound as a white solid, which
crystallized from EtOAc/Ether to give needles (431 mg, 90%); Rf )
0.3 (EtOAc/petroleum ether 5:1); mp 194-198 °C; 1H NMR (300 MHz,
CDCl3) δ 6.94 (bs, 1H, NH), 6.86 (bs, 1H, NH), 4.69 (dddd, J 7.0 Hz,
J 5.8 Hz, J 4.7 Hz, J 6.5 Hz, 1H, H4), 4.55 (d, J 4.4 Hz, 1H, H1), 4.42
(ddd, J 7.0 Hz, J 7.0 Hz, H3), 4.25 (dd, J2 9.5 Hz, 1H, H5a), 4.05 (s,
4H, 2 × CH2Cl), 3.97 (dd, 1H, H2), 3.88 (dd, 1H, H5b), 3.84-3.72
(m, 2H, CH3CH2O), 3.66-3.55 (m, 2H, CH3CH2O), 1.24 (t, J 7.1 Hz,
3H, CH3CH2O), 1.22 (t, J 7.1 Hz, 3H, CH3CH2O); 13C NMR (125.77
MHz, DMSO-d6) δ 165.94, 165.61, 102.30, 81.07, 70.07, 63.44, 62.19,
51.79, 51.09, 42.63, 15.31, 15.26; LRFABMS (m/z)+ 357/359 (M +
H)+, 313/311 (M - OEt)+; HRFABMS calcd for C13H22N2O5Cl2
3570984, obsd 357.0977.
1
ether 1:10); H NMR (300 MHz, CDCl3) v 5.25 (dd, J 4.9 Hz, J 1.3
Hz, 1H, H3), 4.66 (d, J 7.5 Hz, 1H, H1), 4.21 (dd, J2 9.9 Hz, J 5.6 Hz,
1H, H5a), 4.09 (dd, J 4.9 Hz, 1H, H2), 4.08 (dd, J 4.2 Hz, 1H, H5b),
3.78-3.61 (m, 3H, H4, CH3CH2O), 3.55-3.45 (m, 2H, CH3CH2O),
2.11 (s, 3H, COCH3), 1.26 (t, J 7.1 Hz, 3H, CH3CH2O), 1.17 (t, J 7.1
Hz, 3H, CH3CH2O); LRFABMS (m/z)+ 296 (M + Na)+, 228 (M -
OEt)+.
2,5-Anhydro-4-azido-4-deoxy-D-xylose Diethyl Acetal (8). The
azidoacetate (3.25 g, 11.91 mmol) was dissolved in MeOH (70 mL)
and cooled on ice. To this stirred solution was added sodium methoxide
(90 mg, 0.60 mmol) in one portion. The reaction mixture was then
allowed to warm to room temperature and stirred for 3 h. The volatiles
were then removed in Vacuo, and the crude residue was purified by
silica gel chromatography (EtOAc/petroleum ether 1:10). This yielded
the title compound as a colorless oil (2.60 g, 94%): Rf ) 0.1 (EtOAc/
2,5-Anhydro-3,4-bis[(benzoylthio)acetamido]-3,4-dideoxy-D-ri-
bose Diethyl Acetal (10). A freshly prepared solution of sodium
thiobenzoate in ethanol [3.13 mL (0.84 M), 2.62 mmol)] was added
dropwise to a stirred solution of 9 (447 mg, 1.19 mmol) in ethanol (20
mL). The reaction mixture was heated under reflux for 2 h. After
cooling, the volatiles were removed in Vacuo, and the residue was
dissolved in DCM (50 mL), and partitioned with water (50 mL). The
organic layer was then washed with aqueous NaHCO3 (3 × 50 mL)
and brine (2 × 50 mL). The combined organic fractions were dried
and evaporated as described for 4. This gave a yellow solid which
was purified by silica gel chromatography to yield the title compound
as a white solid (491 mg, 74%): Rf ) 0.8 (EtOAc/methanol 20:1); mp
1
petroleum ether 1:10); H NMR (300 MHz, CDCl3) δ 4.77 (d, J 4.6
Hz, 1H, H1), 4.37 (ddd, J 3.5 Hz, J 3.5 Hz, J 1.5 Hz, 1H, H3), 4.24
(dd, J2 9.8 Hz, J 4.7 Hz, 1H, H5a), 4.05 (ddt, 1H, J 1.9 Hz, H4), 4.00
(dd, 1H, H2), 3.82 (dd, 1H, H5b), 3.85-3.74 (m, 2H, CH3CH2O), 3.72-
3.56 (m, 2H, CH3CH2O), 1.60 (s, 1H, OH), 1.26 (t, J 7.1 Hz, 3H, CH3-
CH2O), 1.24 (t, J 7.1 Hz, 3H, CH3CH2O); LRFABMS (m/z)+ 254 (M
+ Na)+, 232 (M + H)+.
1
208-210 °C; H NMR (300 MHz, CD3OD) δ 7.95 (m, 4H, Ar-H),
2,5-Anhydro-3,4-azido-3,4-dideoxy-D-ribose Diethyl Acetal (7).
Trifluoromethanesulfonic anhydride (733 mg, 2.6 mmol) was added
dropwise, over a 10 min period, to a cold (0 °C) stirred solution of the
alcohol 8 (0.5 g, 2.16 mmol) and (N,N-dimethylamino)pyridine (DMAP)
(792 mg, 6.48 mmol) in DCM (20 mL). After 15 min the reaction
mixture was poured into a cold 1% acetic acid solution (20 mL). The
mixture was partitioned and the organic layer washed with cold
NaHCO3 (3 × 20 mL) and brine (1 × 20 mL). The combined organic
fractions were then dried over Na2SO4, filtered, and dried in Vacuo, to
give the crude triflate (850 mg, 90%) which was used without further
purification. The triflate was dissolved in DMF (6 mL) and LiN3 (217
mg, 4.35 mmol) was added in one portion. The reaction mixture was
stirred at room temperature for 4 h and then diluted with EtOAc (50
mL). The mixture was then washed with water (3 × 100 mL), aqueous
NaHCO3 (2 × 50 mL), and brine (2 × 50 mL). The combined organic
fractions were then dried and evaporated as described for 4. The crude
residue was purified by silica gel chromatography (EtOAc/petroleum
7.62 (m, 2H, Ar-H), 7.48 (m, 4H, Ar-H), 4.61-4.56 (m, 2H, H3, H4),
4.50 (d, J 4.7 Hz, 1H, H1), 4.13 (dd, J2 9.0 Hz, J 5.7 Hz, H5a), 3.90
(dd, J 5.6 Hz, 1H, H2), 3.84 (s, 2H, COCH2S), 3.82 (s, 2H, COCH2S),
3.78-3.66 (m, 3H, H5b, CH3CH2O), 3.65-3.52 (m, 2H, CH3CH2O),
1.17 (t, J 7.1 Hz, 3H, CH3CH2O), 1.16 (t, J 7.1 Hz, 3H, CH3CH2O);
13C NMR (125.77 MHz, DMSO-d6) δ 192.82, 191.65, 166.42, 165.98,
153.21, 150.15, 140.21, 136.05, 135.95, 133.15, 133.02, 102.35, 86.21,
84.29, 81.01, 79.98, 51.82, 51.15, 51.09, 42.48, 15.30, 15.29; LR-
FABMS (m/z)+ 583 (M + Na)+, 561 (M + H)+, 515 (M - OEt)+;
HRFABMS calcd for C27H32N2O7S2 561.1729, obsd 561.1717.
[1,4-Anhydro-2,3-dideoxy-2,3-bis(mercaptoacetamido)-D-ribitol]-
oxorhenium(V) (2a-syn and 2b-anti). A. Rhenium Incorporation.
The bis(thiobenzoate) 10 (68 mg, 0.12 mmol) was dissolved in a
methanolic sodium acetate (1 M) solution (5 mL) and heated under
reflux for 1 h. Immediately after cooling of the reaction mixture
oxotrichlorobis(triphenylphosphine)rhenium (V) (111 mg, 0.15 mmol)

Pentacoordinate Ribonuclease Inhibitors
J. Am. Chem. Soc., Vol. 118, No. 50, 1996 12527
was added, and the mixture was reheated under reflux for a further 45
min. The solution turns black during the reaction. The solvent was
removed in Vacuo, and the residue was purified by silica gel chroma-
tography (EtOAc/MeOH 5:1), to give a 1:2 syn/anti mixture which
was used without further purification; LRESMS (m/z)- 551/549 B.
Acetal Hydrolysis and Aldehyde Reduction. The diastereomeric syn/
anti mixture from above was dissolved in a 50% aqueous TFA solution
(3 mL) and stirred overnight at room temperature. Following repeated
evaporation of the volatiles in Vacuo, the crude mixture of aldehydes
were dissolved in ethanol (4 mL) and 4 mL of an ethanolic sodium
borohydride solution (0.1 M) were added dropwise with stirring. The
reaction was stirred at room temperature for 6 h, and then the solvent
was removed in Vacuo. The solid residue was purified by silica gel
chromatography (EtOAc/MeOH 3:1) to give the title compound as a
1:2 syn/anti mixture (40 mg, 98%). The diastereoisomers were
separated by preparative HPLC [(65% acetonitrile/35% water (0.1%
TFA)]. 2a syn: tR ) 10.51 min; mp 288-292 °C; 1H NMR (300 MHz,
CD3OD) δ 5.00 (m, J 6.8 Hz, J 5.6 Hz, J 5.3 Hz, 1H, H2), 4.92 (dd,
J 8.4 Hz, 1H, H3), 4.54 (d, J2 17.9 Hz, 1H, COCHRS), 4.50 (dd, J2 9.3
Hz, 1H, H1R), 4.45 (d, J2 18.3 Hz, 1H, COCHRS), 4.09 (d, 1H,
COCHâS), 4.07 (d, 1H, COCHâS), 3.92 (m, J2 12.5 Hz, J 6.5 Hz, 2H,
H4, H5a), 3.82 (m, J2 12.5 Hz, 1H, H5b), 3.73 (dd, 1H, H1â); 13C
NMR (101 MHz, CD3OD) δ 192.01, 191.87, 87.10, 74.26, 72.4, 72.08,
62.81, 42.09, 41.92; UV λmax (H2O) 228 nm, ꢀ 8600; IR (KBr) 943
cm-1; LRESMS (m/z)- 477/479 (M)-. 2b anti: tR ) 10.09 min; mp
294-297 °C; 1H NMR (300 MHz, CD3COCD3) δ 4.92 (d, J2 18.2 Hz,
1H, COCHâS), 4.90 (m, J 6.3 Hz, J 6.3 Hz, 1H, H2), 4.73 (dd, J 9.1
Hz, J 3.9 Hz, 1H, H3), 4.72 (d, J2 18.0 Hz, 1H, COCHâS), 4.49 (dd,
J2 9.3 Hz, 1H, H1R), 3.99 (m, J 7.6 Hz, J 3.9 Hz, 1H, H4), 3.92 (m,
2H, 5a, 5b), 3.89 (d, 1H, COCHRS), 3.82 (d, 1H, COCHRS), 3.43 (dd,
1H, H1â); 13C NMR (125.77 MHz, CD3OD) δ 191.25, 190.51, 87.05,
74.27, 73.31, 71.05, 70.66, 44.82, 44.61; UV λmax (H2O) 230 nm ꢀ
8900; IR (KBr) 960 cm-1; LRESMS (m/z)- 479/477 (M)-.
9-[2′,3′-Bis(chloroacetamido)-2′,3′-dideoxy-â-D-ribofuranosyl]ad-
enine (14). Diaminoadenosine (13) (1.7 g, 6.6 mmol) was added in
one portion to a stirred suspension of sodium bicarbonate (5.5 g, 65.7
mmol) and chloroacetyl chloride (1.15 mL, 14.5 mmol) in DMF (100
mL) on ice. The reaction mixture was allowed to warm to room
temperature and stirred until reaction was complete as judged by TLC
(80% DCM/MeOH). The DMF was then evaporated in Vacuo. The
residue was dissolved in methanol (50 mL), silica gel (100 g) was added
and then evaporated to dryness in Vacuo. Silica gel chromatography
(95% DCM/MeOH) of the crude residue gave the title compound (1.9
g, 80%) as a white solid: Rf 0.4 (80% DCM/MeOH); mp 220 °C dec;
1H NMR (300 MHz, DMSO-d6) δ 8.55 (d, 1H, J 8.6 Hz, NH), 8.40 (d,
1H, J 9.0 Hz, NH), 8.35 (s, 1H, Ar-H), 8.05 (s, 1H, Ar-H), 7.35 (bs,
2H, NH2), 6.12 (d, 1H, J 7.5 Hz, 1H, H1′), 5.40 (t, 1H, J 6.4 Hz, OH),
5.05 (dd, 1H, J), 4.6; 13C NMR (125.77 MHz, DMSO-d6) δ 166.42,
166.27, 156.13, 152.67, 149.29, 139.18, 119.01, 85.40, 83.89, 61.91,
54.49, 50.73, 42.71, 42.36; HRFABMS calcd for C14H18N7O4Cl2
418.0755, obsd 418.0762.
[9-[2′,3′-Bis(mercaptoacetamido)-2′,3′-dideoxy-â-D-ribofuranosyl]-
adenine]oxorhenium(V) (1a-syn and 1b-anti). To a stirred solution
of the bis thioester (15) (200 mg, 0.32 mmol) in degassed ethanol/
water (1:1) (40 mL) was added trichlorobis(triphenylphosphine)-
rhenium(V) oxide (400 mg, 0.48 mmol) and aqueous 1 M sodium
hydroxide (3.2 mL, 3.2 mmol). The reaction mixture was heated at
80 °C for 20 min. On cooling, a white precipitate of sodium benzoate
formed and was filtered. The mother liquor was evaporated in Vacuo
to give a brown solid. Purification by reversed-phase preparative HPLC
[22% acetonitrile, 78% water (0.1% TFA)] gave the title compounds
as orange powders 1a syn (86 mg, 44%) and 1b anti (44 mg, 23%). 1a
1
(syn): tR 22.4 min; Rf 0.2 (EtOAC/MeOH 3:1); mp 285-289 °C; H
NMR (500 MHz, DMSO-d6) δ 8.80 (s, 1H, Ar-H), 8.45 (s, 1H, Ar-
H), 6.08 (d, 1H, J 3.5 Hz, H1′), 5.15 (m, 2H, H2′, H3′), 4.10 (m, 1H,
H4′), 4.04 (d, 1H, J2 17 Hz, CHâS), 3.94 (d, 1H, J2 18.3 Hz, CHâS),
3.87 (dd, 1H, J2 11.97 Hz, J 2.35 Hz, H5a′), 3.78 (d, 1H, CHRS), 3.75
(dd, 1H, J 5.93 Hz, H5b′), 3.65 (d, 1H, COCHRS); 13C NMR (125.77
MHz, DMSO-d6) δ 192.08, 191.74, 150.91, 148.29, 146.16, 142.86,
118.47, 90.17, 88.04, 75.61, 69.91, 62.81, 40.71, 40.18; UV λmax 236
nm, ꢀ 21 680; IR (KBr) 950 cm-1; LRESMS (m/z)- 612/610 (M)-,
477/475 (M - adenine)-. 1b anti: tR 26.8 min; Rf 0.25 (EtOAC/MeOH
3:1); mp 273-278 °C; 1H NMR (500 MHz, DMSO-d6) δ 8.55 (s, 1H,
J 8.6 Hz, Ar-H), 8.65 8.32 (s, 1H, Ar-H), 8.12 (s, 1H, Ar-H), 6.12
(d, 1H, J 4.3 Hz, H1′), 5.35 (m, 1H, H2′), 5.15 (m, 1H, H3′), 4.10 (dd,
1H, J 5.7 Hz, H4′), 4.07 (d, 1H, J2 16.3 Hz, COCHRS), 3.86 (d, 1H, J2
18.4 Hz, COCHRS), 3.85 (dd, 1H, J2 11.8 Hz, J 2.02 Hz, H5a′), 3.80
(m, 2H, H5b′, COCHâS), 3.60 (d, 1H, J2 18.4 Hz, COCHâS); 13C NMR
(125.77 MHz, DMSO-d6) δ 192.08, 191.95, 154.01, 149.82, 146.72,
128.93, 90.24, 89.52, 72.93, 69.82, 62.83, 40.91, 40.68; UV λmax 238
nm, ꢀ 19 670; IR (KBr) 975 cm-1; LRESMS (m/z)- 612/610 (M)-.
General Kinetic Method. Rigorous precautions were taken to
minimize the risk of nonspecific ribonuclease contamination. All
glassware was heated at 180 °C for 5 h prior to use. All buffers were
prepared daily and were autoclaved prior to use. The urea was
purchased as RNase free from Sigma Chemical Co. and used as
received.
We used a modification of our previously reported procedure to
follow the formation of adenosine and 3′,5′-cyclic adenosine mono-
phosphate from ApA, catalyzed by RNase U2.25 The assay mixture
comprised of 10 units of sequencing grade RNase U2, 16.6 mM sodium
citrate (pH 4.5), 0.85 mM EDTA, 3.5 M urea, and the desired
concentration of substrate and inhibitor (a stock solution prepared in
DMSO) to a final volume of 50 µL (4% DMSO). After addition of
the substrate the reaction vessels were vortexed and heated in a water
bath at 50 ( 0.1 °C.
Data Collection. Sealed tubes, compatible with use in a D-7000
series Hitachi autosampler, were used as reaction vessels. Each tube
was taken out of the incubator every 1-2 h for HPLC analysis.
Aliquots were removed, and the components were separated on an
Alltech Adsorbosphere HS C-18 reversed-phase column at 260 nm,
using an isocratic elution system with a flow rate of 1 mL min-1. The
mobile phase was 7% acetonitrile/93% ammonium acetate (50 mM,
pH 4.5). The observed rates were linear over the time period followed.
To obtain each rate, 5 or 6 time points were taken. The substrate (ApA)
concentrations ranged from 60 µM to 4 mM for the Km and kcat
determinations. Inhibitor concentrations ranged from 6 µM to 2 mM.
Every rate plot had a correlation coefficient of at least 0.985 and was
repeated either two or three times. The data was analyzed by using
standard nonlinear regression (enzyme kinetics) analysis using the
GraFit computer program on a Compaq PC and linear regression
analysis for the Dixon plots on a Macintosh computer.
9-[2′,3′-Bis[(benzoylthio)cetamido]-2′,3′-dideoxy-â-D-ribofurano-
syl]adenine (15). Freshly prepared sodium thiobenzoate solution (10.14
mL, 0.25 M, 2.6 mmol) was added dropwise to a stirred solution of
the bis(chloroacetamide) 14 (370 mg, 0.88 mmol) in ethanol (50 mL).
The reaction mixture was then heated under reflux for 2 h. During
cooling, a white solid precipitated from the reaction mixture. This
precipitate was filtered and recrystallized (ethanol/ether) to give the
title compound as white needles (421 mg, 78%): Rf 0.3 (80% DCM/
MeOH); mp 202-204 °C; 1H NMR (500 MHz, DMSO-d6) δ 8.80 (d,
1H, J 8.6 Hz, NH), 8.65 (d, 1H, J 9.0 Hz, NH), 8.35 (s, 1H, Ar-H),
8.12 (s, 1H, Ar-H), 7.94 (d, 2H, Ar-H), 7.85 (d, 2H, Ar-H), 7.78
(m, 2H, Ar-H), 7.55 (m, 4H, Ar-H), 7.42 (bs, 2H, NH2), 6.12 (d,
1H, J 7.5 Hz, H1′), 5.45 (bs, 1H, OH), 5.05 (dd, 1H, J 3.5 Hz, H2′),
4.58 (ddd 1H, J 5.6 Hz, H3′), 4.12 (m, 1H, H4′), 3.92 (s, 2H, CH2Cl),
3.82 (s, 2H, CH2Cl), 3.66 (dd, 1H, J2 9.8 Hz, H5a′), 3.55 (dd, 1H, J
3.7 Hz, H5b′); 13C NMR (125.77 MHz, DMSO-d6) δ 190.47, 190.12,
167.28, 167.10, 155.82, 152.26, 149.17, 139.51, 135.96, 135.85, 134.09,
134.04, 129.17, 129.12, 126.88, 118.96, 86.21, 84.27, 61.83, 54.96,
54.51, 50.68, 50.57; HRFABMS calcd for C28H27N7O6S2 622.1543, obsd
622.1557.
Acknowledgment. This work was supported in part by the
National Institutes of Health (NIH-GM48351), the Alfred P.
Sloan foundation and the Alexander von Humboldt foundation
(Feodor Lynen programme). T.W. also acknowledges the
support of Dr. Richard Jacob.
JA9611248