Template-competitive inhibitors of HIV-1 reverse transcriptase: design, synthesis and inhibitory activity.

Article abstract of DOI:10.1016/S0968-0896(01)00297-8

We report the design, synthesis and activity studies on a novel class of template-competitive reverse transcriptase inhibitors (TCRTIs). The TCRTIs are 1,N(6)-etheno analogues of a series of dATP-based template-competitive DNA polymerase inhibitors synthesized in our laboratory (Moore, B. M.; Jalluri, R.; Doughty, M.B. Biochemistry 1996, 35, 11634). Thus, nucleotides 2-(4-azidophenacyl)thio-1,N(6)-etheno-2'-deoxyadenosine 5'-triphosphate 1, the tetrafluoro analogue 2-(4-azido-2,3,5,6-tetrafluorophenacyl)thio-1,N(6)-etheno-2'-deoxyadenosine 5'-triphosphate 2 and its analogues were synthesized by alkylation of 2-thio-1,N(6)-etheno-2'-deoxyadenosine 5'-monophosphate with the corresponding chloro- or bromo-alkyl halides and converted to the triphosphate. Kinetically, nucleotides 1 and 2 are both competitive inhibitors of reverse transcriptase versus template/primer with K(i)'s of 8.0 and 7.4 microM, respectively, and non-competitive inhibitors versus TTP with K(i)'s of 15 and 10 microM, respectively. Nucleotide 3, which differs from 1 only in that it lacks the etheno group, non-complementary nucleotide triphosphates, and related monophosphates and nucleosides, are completely inactive as inhibitors of reverse transcriptase at concentrations up to 1 mM. Photoinactivation of RT by 1 was both time- and concentration-dependent, and protected by template/primer but not by dNTPs. The concentration-dependent inactivation data gave a K(D,app) of 17.2 microM and maximum inactivation of 90%, and radiolabeled [beta, gamma-32P]-1 photoincorporated specifically and covalently into the p66 subunit of RT. Thus the photoinactivation data support our main conclusion from the kinetic data that this class of RT inhibitors are non-substrate and template-competitive.

Full text of DOI:10.1016/S0968-0896(01)00297-8

Bioorganic & Medicinal Chemistry 10 (2002) 507–515
Template-Competitive Inhibitors of HIV-1 Reverse Transcriptase:
Design, Synthesis and Inhibitory Activityy
Ke Li, Weiying Lin, Kar Hua Chong, Bob M. Moore and Michael B. Doughty*
Department of Medicinal Chemistry, The University of Kansas, 1250 Wescoe Hall Drive, Lawrence, KS 66045, USA
Received 8 March 2001; accepted 10 August 2001
Abstract—We report the design, synthesis and activity studies on a novel class of template-competitive reverse transcriptase inhi-
bitors (TCRTIs). The TCRTIs are 1,N6-etheno analogues of a series of dATP-based template-competitive DNA polymerase inhi-
bitors synthesized in our laboratory (Moore, B. M.; Jalluri, R.; Doughty, M.B. Biochemistry 1996, 35, 11634). Thus, nucleotides 2-
(4-azidophenacyl)thio-1,N6-etheno-20-deoxyadenosine 50-triphosphate 1, the tetrafluoro analogue 2-(4-azido-2,3,5,6-tetra-
fluorophenacyl)thio-1,N6-etheno-20-deoxyadenosine 50-triphosphate 2 and its analogues were synthesized by alkylation of 2-thio-
1,N6-etheno-20-deoxyadenosine 50-monophosphate with the corresponding chloro- or bromo-alkyl halides and converted to the
triphosphate. Kinetically, nucleotides 1 and 2 are both competitive inhibitors of reverse transcriptase versus template/primer with
Ki’s of 8.0 and 7.4 mM, respectively, and non-competitive inhibitors versus TTP with Ki’s of 15 and 10 mM, respectively. Nucleotide
3, which differs from 1 only in that it lacks the etheno group, non-complementary nucleotide triphosphates, and related mono-
phosphates and nucleosides, are completely inactive as inhibitors of reverse transcriptase at concentrations up to 1mM. Photo-
inactivation of RT by 1 was both time- and concentration-dependent, and protected by template/primer but not by dNTPs. The
concentration-dependent inactivation data gave a KD,app of 17.2 mM and maximum inactivation of 90%, and radiolabeled [b,g-32P]-
1 photoincorporated specifically and covalently into the p66 subunit of RT. Thus the photoinactivation data support our main
conclusion from the kinetic data that this class of RT inhibitors are non-substrate and template-competitive. # 2002 Elsevier
Science Ltd. All rights reserved.
Introduction
Two classes of RT inhibitors, including nucleoside
(NRTIs) and non-nucleoside (NNRTIs) inhibitors, have
been described. NRTIs, including 30-azido-30-deoxy-
thymidine (zidovudine, AZT),7,8 20,30-dideoxyinosine
(didanosine, ddI),9 20,30-dideoxycytidine (ddC),10 and
20,30-dideoxy-30-thiacytidine (3TC)11,12 are an important
class of drugs clinically available for AIDS treatment.
These nucleoside RT inhibitors are classic anti-metabo-
lites, needing activation by normal cellular kinases to
the active 50-triphosphates. The triphosphates are alter-
nate substrates and are readily incorporated into the
growing DNA chain. Kinetic studies have shown that
AZT 50-triphosphate is an alternate substrate of RT
with a Km,app of 3.0 mM, which is similar to the Km of
2.5 mM for TTP.13 The binding site for the activated
NRTIs is thought to be identical to that for normal
dNTP substrates. In the crystal structure of an inactive
ternary complex, TTP is oriented in a position where the
a-phosphate is adjacent to the 20-position on the primer
by triphosphate ligation to two magnesium ions bound
to the active site carboxylates of Asp110, Asp185, and
Asp186 and by hydrogen bonding to a complementary
base on the template.6 AZT-50-triphosphate as an alter-
nate substrate also forms a dead-end complex which is a
potent inhibitor of RT.13
HIV-1reverse transcriptase (RT) is the enzyme neces-
sary for the transformation of HIV genomic RNA into
double-stranded proviral DNA and has been the object
of extensive biochemical, structural, and mutagenesis
studies. The p66/p51heterodimer of RT is the mature
form purified from virions and infected cells.1 Crystal
structures have been reported for heterodimer RT com-
plexed with either a non-competitive inhibitor2ꢀ4 or an
18-base pair double-stranded DNA and Fab antibody,5
and RT complexed with covalently modified template/
primer and dNTP.6 The RT heterodimer is highly
asymmetric, but the p66 subunit contains the three sub-
domains named the fingers, palm, and thumb that
together constitute the polymerase domain and form a
large template/primer binding cleft.
§Taken in part from the PhD dissertation of Ke Li, The University of
Kansas, 1996 and the PhD dissertation of Weiying Lin, The University
of Kansas, 2000.
*Corresponding author at present address: Department of Chemistry
and Physics, Southeastern Louisiana University, SLU Box 10878,
Hammond, LA 70402, USA. Tel.: +1-985-549-5123; fax: +1-
985+549-5126; e-mail: mdoughty@selu.edu
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00297-8

508
K. Li et al. / Bioorg. Med. Chem. 10 (2002) 507–515
Many HIV-1-specific non-nucleoside RT inhibitors have
also been described.14 These compounds belong to
structurally distinct classes, and are non-competitive
inhibitors with respect to the deoxynucleoside tripho-
sphate substrate and show non-competitive or mixed
type inhibition with respect to the template/primer. X-
ray structure elucidation,2ꢀ4 inhibitor photoaffinity
labeling,15,16 and mutagenesis studies17 indicate that
despite their structural diversity, non-nucleoside inhibi-
tors share a common binding site in the RT p66 subunit
involving amino acid residues surrounding Tyr181 and
method.21 The products were >95% pure based on
elution as a single peak from a C18 reverse-phase HPLC
column, elution as a single peak on mono-Q FPLC ion-
1
exchange chromatography, and 31P and H NMR spec-
troscopy
The reversible inhibition of HIV-1RT and the Klenow
fragment by nucleotides 1 and 2 was investigated in order
to ascertain if the nucleotides specifically interact with
the substrate binding sites and to provide information on
the binding modes of these inhibitors. Table 1sum-
marizes the kinetic analysis of the inhibition of RT and
KF by nucleotide 1, and on the inhibition of only RT by
2. Both KF and RT were inhibited competitively by 1
relative to template/primer as the varying substrate with
Ki’s of 8.0 and 96 mM, respectively, and non-competitive
relative to TTP with Ki’s of 15 mM for RT and 78 mM
for KF. TCRTI 2 was evaluated only as a reverse tran-
scriptase inhibitor; this analogue had activity similar to
that of 1 with competitive inhibition relative to varying
template/primer and non-competitive inhibition relative
to varying TTP with Ki’s of 7.4 and 10 mM, respectively.
Tyr188 16
This allosteric, non-substrate binding site is
.
near, but not coincident with, the primer binding site,
and is distal from the template and dNTP binding sites.
Our laboratory has synthesized a series of nucleoside 50-
triphosphate photoprobes to explore the structural
regions of DNA polymerases important in substrate
binding and catalysis. One such photoprobe, 2-(4-azi-
dophenacyl)thio-20-deoxyadenosine 50-triphosphate (3),
binds to the DNA polymerase Klenow fragment (KF)
with a Kd in the low mM range and is an efficient pho-
toprobe of this enzyme.18,19 In the present communica-
tion we demonstrate that its 1,N6-etheno bridged
analogue 1 and its etheno and tetrafluoro analogue 2 are
poor inhibitors of the Klenow fragment but show sur-
prisingly good activity as inhibitors of HIV-1reverse
transcriptase. In addition, their inhibition is competitive
with respect to template-primer and non-competitive
with respect to nucleoside 50-triphosphate. The kinetic,
photochemical, and structural data provided evidence
to differentiate the TCRTIs from both the nucleoside
and non-nucleoside inhibitors.
To further define the interaction of this inhibitor class
with reverse transcriptase, we studied the photoinactiva-
tion and photoincorporation of 1 into RT in the absence
and presence of substrates. RT was rapidly inactivated
by 100 mM 1 with 83 and 87% inactivation after 6 and
12 min, respectively (Table 2). This inactivation was
protected (11% inactivation relative to control) by tem-
plate/primer, but not by TTP, a result consistent with the
poor binding of dNTPs to RT in the absence of template/
primer (see Discussion). The concentration-dependent
photoinactivation data displayed saturation, reaching
90% maximum inactivation with IC50 of 17 mM when fit
to a binding model where photoinactivation occurs from
an enzyme-inhibitor binary complex (Fig. 1). As sug-
gested in the time-dependent inactivation data, the con-
centration-dependent inactivation was protected by
template-primer; at lower concentrations of template/
primer the dose-response curve was shifted to the right
without changes in total photoinactivation (not shown).
Additionally, [32P]-1 incorporated specifically into the
p66 subunit (>98%) at 100 mM (Fig. 1).
For structure–activity studies, a series of nucleotide tri-
phosphates and their monophosphate and nucleoside
analogues were tested for RT inhibition based on their
IC50 at substrate concentrations equal to the Km, and the
data were analyzed for Ki using the method of Chou.22
The results given in Table 3 first demonstrate that both
the etheno group and the triphosphate are necessary
requirements for RT inhibition. Second, the series in
Table 3 demonstrates that the 2-position side chain is
sensitive to steric interaction at the para position (either
electron donating or releasing), but the side chain can be
lengthened by addition of a carbon to the side chain.
Results
The monophosphates 1m, 2m, and 5m–8m were synthe-
sized by alkylation of 2-thio-1,N6-etheno-20-deox-
yadenosine 50-monophosphate20 4 with substituted
phenyl or benzyl acylhalides at pH 7–8 in aqueous
DMF (Scheme 1). The 50-monophosphates 1m, 2m, and
5m–8m were then converted to the 50-triphosphates 1, 2,
and 5–8 using the diphenylphosphoryl anhydride
Discussion
Our approach to the design of nucleotide inhibitors of
DNA polymerases was based on a thermodynamic

K. Li et al. / Bioorg. Med. Chem. 10 (2002) 507–515
509
Scheme 1. (1) alkyl halides, pH 7–8; (2) diphenyl chlorophosphate, pyridine; (3) tributylammonium pyrophosphate, pyridine.
model of Klenow fragment-substrate interactions
developed by Doronin and Kolocheva.23,24 In this
model, the enzyme interacts with about seven nucleo-
sides in the primer strand and about 12 nucleosides in
the template strand. Numerous interactions contribute
to formation of the enzyme-template/primer complex,
interactions which include: (1) hydrogen bonds with
phosphoryl oxygens (ꢀ4.7 kcal/mol), (2) ionic interac-
tions with phosphate anions (ꢀ1.2 kcal/mol), (3) hydro-
phobic interactions with the deoxyribose sugar and
bases (ꢀ0.32 kcal/mol) and (4) interactions with the
primer 30 terminus (ꢀ5.8 kcal/mol). The incoming
dNTP binds to the active site of the binary E-template/
primer complex near the 30 end of the primer and base-
pairs with the complimentary nucleoside in the template
strand. In contrast to the extensive binding interactions
between the enzyme and the template/primer in which
no single interaction dominates, the binding energy for
the dNTP substrate is mainly contributed from the inter-
action of the enzyme with the magnesium-complexed tri-
phosphate moiety (about ꢀ5.9 to ꢀ6.7 kcal/mol) and by
hydrogen bonding (about ꢀ0.35 kcal/mol) to the com-
plementary template.
interactions with the template and primer. To overcome
this inherent instability of the binary enzyme-inhibitor
complex, we appended 2-alkyl substituents in order to
stabilize binding interactions. We reasoned that a minor
groove substituent would be in position to stabilize the
modified dNTP by interaction with a lipophilic site in
the template binding domain (as predicted from the
thermodynamic model; Figure 2). Although stabilizing
this dead-end complex in DNA polymerase has to date
no clinical relevance, the stabilized complex provides a
method for exploring polymerase structure and func-
tion. Indeed, nucleotide 3 is an efficient inhibitor and
photolabel of KF. With photolysis at 3000 A, probe 3
inactivates >91% of KF polymerase activity, and
[8-3H]-3 covalently incorporates into the Klenow frag-
ment with an IC50 of 0.75 mM, an incorporation which is
protected competitively by template/primer, at residue
Asp732 in the template-binding finger subdomain.18,19
Thus, the azidophenacyl side chain in 3 does stabilize a
binary complex between the Klenow fragment and
modified dNTPs as proposed in the model.
Nucleotides in this series are inactive as reverse tran-
scriptase inhibitors. For example, nucleotide 3 shows no
inhibition of reverse transcriptase at concentrations up
to 1mM (the highest concentration tested), indicating
that its Ki must be >5 mM (Table 2). This lack of
binding of nucleotide is consistent with other studies
Nucleotide-based photoaffinity probes of DNA poly-
merases which rely only on the triphosphate for binding
interaction generally show low incorporation and weak
affinity. Examples include TTP,25,26 8-azido-dATP,25,27,28
and the 2-azido-dATP and 2-azido-1,N6-etheno-dATP
synthesized in our laboratory.20 We concluded that
these photoprobes are poor polymerase labels since in
binding to the free enzyme they lack the stabilizing
Table 2. Time-dependent photoinactivation of RT by 1 and substrate
protectiona
Time (min)b
Additions
mM 1
100 mM 1
100 mM 1
% Actc
Table 1. Reverse transcriptase and Klenow fragment steady-state
inhibition patterns and constants for TCRTIs 1 and 2 as reversible
inhibitors
0
1
10000
17
13
76.3
87.5
17
6.0
12.0
6.0
6.0
6.0
.
100 mM 1+ 500 nM poly(dA) (dT)10
Inhibitor Enzyme Variable substrate Inhibition Pattern
Ki
(none; controld)
(mM)
100 mM 1+5 mM TTPe
a
.
1
1
1
1
2
2
RT
RT
KF
KF
RT
RT
poly(dA) (dT)10
Competitive
Non-competitive
Competitive
Non-competitive
Competitive
Non-competitive
8
15
92
78
7.4
10
TTPb
aStandard reaction conditions: 2 mM RT, 3 mM MgCl2, and 2 mM 2-
mercaptoethanol as scavenger.
bPhotolysis time at 25 ꢁC.
c
.
poly(rA) (dT)10
TTPb
a
.
poly(dA) (dT)10
cPercent activity remaining after photolysis as measured by initial rate
data after dilution of the photolysis mixture.
dPhotolysis control used standard reaction conditions with pre-photo-
lyzed 100 mM 1.
TTPb
aTTP was maintained at 50 mM.
bTemplate/primer was maintained at 20 nM.
eMagnesium chloride was increased to 50 mM to account for the high
triphosphate concentration.
cTPP was maintained at 100 mM.

510
K. Li et al. / Bioorg. Med. Chem. 10 (2002) 507–515
showing very weak binding of dNTPs to free RT.29,30
Thus our model of modified dNTP interaction to KF in
a dead-end complex in the dATP series was not directly
applicable to reverse transcriptase.
the binding site occupied by dNTP’s in the kinetic
mechanism.36 Thus, the TCRTIs, unlike the tripho-
sphate activated NRTIs, are not RT substrates. The Ki’s
from the two sets of kinetic experiments show a good
correlation, indicating similar binding modes to the free
enzyme in the two sets of experiments.
We subsequently added the 1,N6-etheno group into 3
with the expectation that increased lipophilicity might
increase binding to the free forms of both DNA poly-
merases and reverse transcriptases. But in the Klenow
fragment the etheno analogue is significantly less active
than the corresponding adenosine analogue (by a factor
of 50–100), a result consistent with the general activity
of other 1,N6-ethenoadenosine analogues.31,32 In addi-
tion to its poor inhibition, nucleotide 1 is also not an
effective photoprobe of DNA polymerase I Klenow
fragment since it photoincorporates by only 35%.33
Nevertheless, the etheno modification had a dramatic
and positive effect on the inhibition of HIV-1reverse
transcriptase. Thus both nucleotides 1 and 2 are com-
petitive with template/primer with Ki’s in the low
micromolar range, which is a >500-fold increase over
the adenosine congener, suggesting the etheno group is
a key modification in this series. The exact role of the
etheno group is not clear, but preliminary molecular
modeling studies suggest that the etheno group shifts
the conformational equilibrium of the 2-position side
chain due to etheno-H to side chain CH2 steric interac-
tions.33 Whether the etheno group shifts the conforma-
tional equilibrium or makes direct contact with the
enzyme in the binary E-I complex remains to be deter-
mined.
Reverse transcriptase is irreversibly photoinactivated by
1 at 3500 A photolyis in a time- and concentration-
dependent manner. The photoincorporation of [32P]-1 is
>98% into the catalytically active p66 subunit, a result
consistent with an active site dependent binding. Addi-
tionally, the photoinactivation of 1 into RT saturates
with a Kd nearly identical to that determined from
kinetic studies, and the photoinactivation is protected
competitively by template/primer. Thus the photo-
Table 3. Reverse transcriptase inhibition by 20-deoxynucleoside 50-
triphosphates and analogues
Compd
R1
R2
Ki (mM)a
8
Triphosphate
6.0
1
Triphosphate
Triphosphate
Triphosphate
Triphosphate
Triphosphate
Monophosphate
10
10
In addition to being competitive relative to template-
primer, the inhibitors also show non-competitive inhi-
bition relative to the nucleotide triphosphate substrate
(TTP in this case). Since dNTP binds to the E-template/
2
primer binary complex in the kinetic mechanism,34,35
a
noncompetitive inhibition is fully consistent with bind-
ing of the inhibitor to the free enzyme form and not to
5
10
7
55
6
75
8m
>5000
Figure 1. Evaluation of nucleotide 1 as an RT photoprobe. Left:
Photoinactivation of RT by 1. Standard reaction conditions included
2 mM RT, 3 mM MgCl2, 2 mM 2-mercaptoethanol as scavenger, and
varying concentrations of 1 (0–80 mM), in the absence (diamonds) or
presence (triangles) of 500 mM template primer. The photolysis mix-
ture was photolyzed for 6 min at 3500 A, and residual polymerase
activity was measured after dilution. The solid line is the best fit to eq
(1). Right: Photoincorporation of 1 into RT. Standard reaction con-
ditions included 2 mM RT, 3 mM MgCl2, 2 mM 2-mercaptoethanol as
scavenger, and 100 mM 1. The photolysis mixture was subjected to
SDS–PAGE, and analyzed by autoradiography (lane 1) or coomassie
stain (lane 2). Lane 3 contains coomassie stain of MW standards of
(top to bottom) 200, 116, 97, 66, and 45 kD.
2nb
H
>5000
>5000
3b
—
—
aKi’s are calculated from concentration-dependent inhibition initial
rate data as described in the Experimental.
bAssays performed in 10% DMSO.

K. Li et al. / Bioorg. Med. Chem. 10 (2002) 507–515
511
inactivation and photoincorporation data indicate that
this class of inhibitors occupy a site on the free enzyme
which is competitive to template/primer binding.
response.37 In any case, given that TCRTIs 1 and 2 do
not support the polymerization reaction, and that the
inhibition patterns and structural requirements are dis-
tinct from that observed for the nucleoside and non-
nucleoside inhibitors of RT, the data support our con-
clusion that nucleotides 1 and 2 are unique template-
competitive inhibitors of HIV-1reverse transcriptase.
We are currently mapping the binding site for inhibitors
1 and 2 in order to gain further insights into this unique
class of RT inhibitors.
Our limited SAR studies shed some light on the nature
of the binding interactions between RT and the
TCRTIs. First, the monophosphates of all analogues
and the nucleoside analogue of 8 are inactive, clearly
establishing that the triphosphate is a required sub-
stituent. This structural prerequisite distinguishes this
group of RT inhibitors from the lipophilic NNRTIs,14
and is our only current evidence that the triphosphate
binding site of dNTP substrates, which in the crystal
structure of an inactive ternary complex is composed of
three active site carboxylates in the palm subdomain of
the p66 subunit,6 is an important recognition site for
TCRTIs. Second, the combination of both the side
chain and the 1,N6-etheno group are required for RT
inhibition. Analogue 3 lacking the etheno group is
inactive, as are 1,N6-etheno nucleotide triphosphates
lacking a 2-position side chain.33 This requirement
structurally distinguishes this class of RT inhibitors
from NRTIs, which all have heterocyclic bases which
can form complementary hydrogen bonds to template.
The side chain shows some preference for small sub-
stituents (e.g., -F or -H) at the para position, and does
not tolerate larger substituents, either electron donating
or withdrawing. Finally, the most active analogue in
this series is the 3-phenyl-2-propanone analogue 8,
indicating that some lengthening of the side chain is
permissible or preferred.
Experimental
Chemicals and solvents of analytical or reagent grade
were purchased from commercial suppliers and used
without purification. Recombinant HIV-1RT was
kindly provided by Professor John V. Schloss or pur-
chased from Worthington Biochemical Corporation
(Freehold, NJ, USA). The Klenow fragment of DNA
polymerase I was purchased from United States Bio-
chemical Corporation (Cleveland, OH, USA) and dia-
lyzed against phosphate buffer (50 mM, pH 7.4)
containing 1mM 2-mercaptoethanol and 50% glycerol
to remove dithiothreitol. Polynucleotides, oligonucleo-
tides, and nonradioactive nucleotides were purchased
from Pharmacia (Piscataway, NJ, USA). Tritiated thy-
midine ([3H]TTP) was purchased form DuPont New
England Nuclear (Boston, MA, USA).
Inhibitor precursor 4 was purified as described earlier,20
a-bromo-4-azido-2,3,5,6-tetrafluoroacetophenone was
prepared by a standard literature procedure,38 a-bro-
moacetophenones were prepared by bromination of the
corresponding ketone with bromine and toluenesulfonic
acid catalyst, and 3-chloro-1-phenyl-2-propanone was
prepared by acidification of the corresponding a-diazo-
ketone.39 All manipulations of phenylazides were con-
ducted in the dark or under a red light. Proton (1H)
nuclear magnetic resonance (NMR) spectra were recor-
ded on General Electric QE-300 and Varian VXR-500S
500 MHz instruments. Chemical shifts are reported as
parts per million (ppm) relative to HOD peak (d=4.80)
in D2O. Negative fast atom bombardment mass spectra
(ꢀFABMS) were obtained on a VG Analytical ZAB
spectrophotometer, and ultraviolet spectra were recor-
ded on a Hewlett-Packard 8450A diode array spectro-
photometer. FPLC chromatography was performed
using a Pharmacia FPLC system. Reverse-phase high
pressure liquid chromatography (HPLC) was conducted
on a Beckman dual pump gradient HPLC system with
System Gold software on a Vydac C-18 analytical or
semi-preparative column. Solvent flow rates were 2.5
and 1mL/min for semi-preparative and analytical col-
umns, respectively. The purity of the nucleotides was
>95% as determined by analytical HPLC using a
30 min linear gradient from 0 to 50% acetonitrile in
10 mM triethylammonium acetate, analytical FPLC on
a Mono-Q column using a linear gradient from 0.2 to
The thermodynamic model used for design of these
inhibitors (see Fig. 2) cannot fully explain the increases
in inhibitory activity observed for the binding of 1 and 2
to reverse transcriptase relative to related dNTPs. The
steric/electronic interactions of the side chain of 3 sta-
bilized the dead-end complex with KF by about 150
fold relative to dNTP binding, so it is unlikely that the
steric/electronic effects of the side chain/etheno group
combination of 1 and 2 alone can account for a >500-
fold increase in binding affinity at RT. One possible
explanation is that the combined substrate (or bisub-
strate) nature of the TCRTIs, with binding interactions
in both the dNTP and template binding sites (Fig. 2),
causes a more than additive, or synergistic, inhibitory
1
2 M NH4OAc, pH 4, and by H NMR analysis.
Figure 2. Binding model for the interaction of template-competitive
inhibitors with DNA polymerase. Potential sites of hydrogen bonding
to template which are not available in the binary enzyme–inhibitor
complex are marked with arrows.
Photolysis was conducted in a Rayonet Photochemical
Mini-Reactor chamber Model RMR-600 purchased

512
K. Li et al. / Bioorg. Med. Chem. 10 (2002) 507–515
from the Southern New England Ultraviolet Company
(Branford, CT, USA). Samples were photolyzed in
1.0 mL water jacketed quartz cells. Liquid scintillation
counting was conducted in a Packard 1900 TR liquid
scintillation counter (Meridian, CT, USA). SDS gel
electrophoresis was performed on a Protean II unit and
gels were dried on a Model 543 gel drier, both from
BioRad (Richmond, CA, USA). X-ray films were
developed using an X-Omat developer (AFP Imaging,
Elmsford, NY, USA).
2-(4-Fluorophenylacyl)-thio-1,N6-etheno-20-deoxyadeno-
sine 50-monophosphate (5m). This was prepared from
ammonium 2-thio-1,N6-etheno-20-deoxyadenosine 50-
monophosphate (4, 120 mg, 0.29 mmol) and a-bromo-4-
fluoroacetophenone (69 mg, 0.32 mmol) as described
above for the synthesis of 2, yielding 140 mg (92%) of
pure 5m. H NMR (DMSO) d 1.95 (m, 1H, 20H1), 2.52
1
(m, 1H, C20H), 3.80 (t, 2H, C50H), 4.10 (m, 1H, C40H),
5.10 (m, 1H, C30H), 6.00 (t, 1H, C110H), 6.90 (m, 2H, Ph
H), 7.30 (m, 2H, PhH), 7.60 (d, 1H, C10H), 7.92 (s, 1H,
C11H), 8.30 (s, 1H, C8H). High resolution MS (+FAB,
glycerol) m/e 524.0804; calcd for C20H20N5O7SFP
(M+H+), 524.0805.
Ammonium 2-(4-azidophenacyl)thio-1,N6-etheno-20-deoxy-
adenosine 50-monophosphate (1m). The tetrabutyl-
ammonium salt of 2-thio-1,N6-etheno-dAMP (120 mg,
0.29 mmol) in water (2 mL) was added to a solution of
para-azidophenacyl bromide (78 mg, 1.1 equivalent) in
acetonitrile (2 mL) in a brown reacti-vial. The pH of the
solution was adjusted to 8 using KOH (6 N); the result-
ing clear yellow solution was purged with nitrogen and
stirred at room temperature for 3 h. The reaction mix-
ture was then diluted with water (10 mL), acidified to
pH 4 with HCl (1N), and extracted with three volumes
of ethyl ether. The aqueous phase was neutralized with
1N NaOH, and the product was purified by preparative
HPLC on a C18 column using a 30 min linear gradient
from 0 to 50% acetonitrile in 10 mM ammonium ace-
tate. Collection and lyopholization of the major peak
with a retention time of 29 min afforded 52.3 mg (50%)
of 1m: IR (KBr) 2116 (N3); UV lmax (eꢂ10ꢀ4): H2O, 297
2-(4-N-acetylamino-phenylacyl)-thio-1,N6 -etheno-20 -
deoxyadenosine 50-monophosphate (6m). This was pre-
pared from triethylammonium 2-thio-1,N6-etheno-20-
deoxyadenosine
50-monophosphate
(4,
100 mg,
0.26 mmol) and a-bromo-4-N-acetylamino-acetophen-
one (73.2 mg, 0.28 mmol) using the method described
above for the synthesis of 2, yielding 95 mg (66%) of
pure 6m. 1H NMR (DMSO) d 2.02 (s, 3H, CH of
amide), 2.12 (m, 1H, 20H1), 2.52 (m, 1H, C20H), 3.85 (t,
2H, C50H), 4.15 (m, 1H, C40H), 4.78 (s, 2H, CH of
ketone), 5.10 (m, 1H, C30H), 6.10 (t, 1H, C10H), 7.60 (d,
1H, C10H), 7.80 (m, 2H, Ph H), 7.92 (s, 1H, C11H),
8.10 (m, 2H, PhH), 8.40 (s, 1H, C8H). High-resolution
MS (+FAB, glycerol) m/e 563.1103; calcd for
C22H24N6O8SP (M+H+), 563.1114.
(1.61); H NMR (D2O) d 2.08 (m, 1H, C20H1), 2.62 (m,
1
1H, C20H2), 3.86 (br s, 2H, C50H), 4.33 (br s, 1H, C40H),
4.78 (br s, 1H, C30H), 5.70 (t, J=6.3 Hz, 1H, C10H), 7.10
(d, J=8.4 Hz, 2H, PhH), 7.71 (s, 1H, C10H), 7.53 (s, 1H,
C11H), 7.98 (d, J=8.4 Hz, 2H, PhH), 8.25 (s, 1H, C8H).
High-resolution FABMS (glycerol) m/e 545.0772, calcd
for C20H18N8O7PS (MꢀH+), 545.0757.
2-(4-Trifluoromethylphenylacyl)-thio-1,N6-etheno-20-deoxy-
adenosine 50-monophosphate (7m). This was prepared
from triethylammonium 2-thio-1,N6-etheno-20-deoxy-
adenosine 50-monophosphate (4, 100 mg, 0.26 mmol)
and a-bromo-4-trifluoromethylacetophenone (86.1mg,
0.32 mmol) using the method described above for the
synthesis of 2, yielding 53 mg (36%) of pure 7m. 1H
NMR (DMSO) d 1.92 (m, 1H, 20H1), 2.50 (m, 1H,
C20H), 3.45 (t, 2H, C50H), 4.10 (m, 1H, C40H), 5.10 (m,
1H, C30H), 5.25 (s, 2H, CH2), 6.00 (t, 1H, C10H), 7.60
(m, 2H, Ph H), 7.95 (m, 2H, PhH), 8.12 (d, 1H, C10H),
8.33 (s, 1H, C11H), 8.85 (s, 1H, C8H). High resolution
MS (+FAB, glycerol) m/e 574.0776; calcd for
C21H20N5O7SF3P (M+H+), 574.0773.
2-(2,3,5,6-Tetrafluoro-4-azidophenacyl)-thio-1,N6-etheno-
20 deoxyadenosine 50-monophosphate (2m). Triethyl-
ammonium 2-thio-1,N6-etheno-20-deoxyadenosine 50-
monophosphate (4, 276 mg, 0.4 mmol) in water (10 mL)
was added to a solution of a-bromo-4-azido-2,3,5,6 tet-
rafluoroacetophenone (136 mg, 0.44 mmol) in acetoni-
trile (2 mL). The pH of the solution was adjusted to 7.0
using 0.2 N HCl. The reaction mixture was stirred at
room temperature for 1h. The solvents were removed
under vacuum, and the resulting residue was dissolved
in water (10 mL). The aqueous solution was washed
with methylene chloride. The aqueous layer was sepa-
rated and lyophilized to dryness to give the crude pro-
duct. The crude product was purified on a reverse-phase
C18 HPLC column using a 30 min gradient from 0 to
50% acetonitrile in 0.1% TFA. Collection and lyopho-
lization of appropriate fractions afforded 50.3 mg (50%)
of the product as a off-white solid: IR (cmꢀ1, KBr) 2112
(N3); UV lmax (eꢂ10ꢀ4): H2O, 310 (2.01); 1H NMR
(DMSO) d 2.35 (m, 1H, 20H1), 2.56 (m, 1H, C20H), 3.94
(t, 2H, C50H), 4.35 (m, 1H, C40H), 4.89 (m, 1H, C30H),
6.15 (t, 1H, C10H), 7.70 (d, 1H, C10H), 8.02 (d, 1H,
C11H), 8.42 (s, 1H, C8H); 19FNMR (DMSO) d
ꢀ141.38 (dd, 2F), ꢀ151.75 (dd, 2F); High-resolution
MS (+FAB, glycerol) m/e 619.0519; calcd. for
C20H16N5O7SF4P (M+H+), 619.0536.
2-(3-phenyl-2-propanonyl)-thio-1,N6-etheno-20-deoxyade-
nosine 50-monophosphate (8m). This was prepared from
triethylammonium 2-thio-1,N6-etheno-20-deoxyadeno-
sine 50-monophosphate (4, 138 mg, 0.2 mmol) and ben-
zyl chloromethyl ketone (36.9 mg, 0.22 mmol) as
described above for the synthesis of 2, yielding 41.8 mg
1
(41%) of pure 8m. H NMR (DMSO) d 2.35 (m, 1H,
20H1), 2.68 (m, 1H, C20H), 3.88 (t, 2H, C50H), 3.98 (m,
1H, C40H), 4.06 (s, 2H, CH of benzyl), 4.42 (m, 1H,
C30H), 6.12 (t, 1H, C10H), 7.18 (d, 3H, PhH), 7.62 (d,
1H, C10H), 7.95 (s, 1H, C11H), 8.40 (s, 1H, C8H); MS
(-FAB, glycerol) m/e 517 (MꢀH+).
2-(2,3,5,6-Tetrafluoro-4-azidophenacyl)-thio-1,N6-etheno-
20-deoxyadenosine 50-triphosphate (2). The triphosphate 2
was prepared from 2-(2,3,5,6-tetrafluoro-4-azidophena-
cyl)-thio-1,N6-etheno-20-deoxyadenosine 50-monopho-
sphate (2m) by a previously published procedure.18

K. Li et al. / Bioorg. Med. Chem. 10 (2002) 507–515
513
Briefly, the trioctylammonium salt of 2m (10 mg,
16.2 mmol) was activated with diphenyl chlorophosphate
(3.7 mL, 1.1 equiv) and tributylamine (3.8 mL, 1equiva-
lent) in anhydrous dioxane, and after removal of excess
activating reagent, the mixed anhydride (in pyridine)
was treated with a solution of tributylammonium pyr-
ophosphate (8.5 mg, 1.1 equiv) in pyridine (1 mL). The
crude product was precipitated with ether and purified
by FPLC ion exchange chromatography on a Mono-Q
column using a gradient of 2 M ammonium acetate
containing 30% acetic acid (pH 4). The appropriate
fractions were collected, pooled, and lyophilized. The
resulting residue was further purified on a reverse phase
C18 HPLC column using a 30 min gradient from 0 to
50% acetonitrile in 5 mM triethylammonium bicarbo-
nate. After HPLC purification, 1.2 mg (9.6%) of highly
pure product as a light yellow solid was obtained: Rf
0.13 (20:12:1 propanol/NH4OH/H2O); UV lmax
(eꢂ10ꢀ4): H2 O, 309 (2.00); 31P NMR (D2O) dꢀ6.83 (d,
50-monophosphate (5m, 20 mg, 0.038 mmol) as descri-
bed above for the synthesis of 2, yielding 7.8 mg (30%)
of pure 5 as a light-yellow solid: Rf 0.13 (20:12:1 pro-
panol/NH4OH/H2O); MS (ꢀFAB, glycerol) m/e 684
(MꢀH+).
2-(4-N-Acetylamino-phenylacyl)-thio-1,N6 -etheno-20 -
deoxyadenosine 50-triphosphate (6). This was prepared
from 2-(4-N-acetylamino-phenylacyl)-thio-1,N6-etheno-
20-deoxyadenosine 50-monophosphate (6m, 20 mg,
0.035 mmol) as described above for the synthesis of 2,
yielding 7.8 mg (31%) of pure 6 as a light-yellow solid:
Rf 0.10 (20:12:1 propanol/NH4OH/H2O); 1H NMR
(DMSO) d 1.98 (m, 1H, 20H1), 2.14 (s, 3H, CH of
amide), 2.55 (m, 1H, C20H), 3.75 (t, 2H, C50H), 4.05 (m,
1H, C40H), 4.68 (s, 2H, CH of ketone), 4.95 (m, 1H,
C30H), 5.85 (t, 1H, C10H), 7.58 (d, 1H, C10H), 7.85 (m,
2H, Ph H), 7.90 (s, 1H, C11H), 8.11 (m, 2H, PhH), 8.25
(s, 1H, C8H); MS (ꢀFAB, glycerol) m/e 721(M ꢀH+).
2 - (4 - Trifluoromethylphenylacyl) - thio - 1,N6 - etheno -20 -
deoxyadenosine 50-triphosphate (7). This was prepared
from 2-(4-trifluoromethylphenylacyl)-thio-1,N6-etheno-
20-deoxyadenosine 50-monophosphate (7m, 20 mg,
0.038 mmol) as described above for the synthesis of 2,
yielding 5.6 mg (20%) of pure 7 as a light-yellow solid:
Rf 0.11 (20:12:1 propanol/NH4OH/H2O); MS (ꢀFAB,
glycerol) m/e 723 (MꢀH+).
2-(3-phenyl-2-propanon-1-yl)-thio-1,N6-etheno-20-deoxy-
adenosine 50-triphosphate (8). This was prepared from
2-(benzylacyl)-thio-1,N6-etheno-20-deoxyadenosine 50-
monophosphate (8m, 19 mg, 0.036 mmol) as described
above for the synthesis of 2, yielding 13.8 mg (57%) of
pure 8 as a light yellow solid: Rf 0.11 (20:12:1 propanol/
NH4OH/H2O); MS (ꢀFAB, glycerol) m/e 677
(MꢀH+).
1
gP), ꢀ10.41 (d, aP), ꢀ21.71 (t, bP); H NMR (D2O) d
2.23 (m, 20H1), 2.47 (m, 1H, C2H), 4.04 (t, 2H, C50H),
4.41(m, 1H, C4 0H), 4.91(m, 1H, C3 0H), 6.21(t, 1H,
C10H), 7.73 (d, 1H, C10H), 8.10 (d, 1H, C11H), 8.45 (s,
1H, C8H).
Ammonium 2-(4-azidophenacyl)thio-1,N6-etheno-20-deoxy-
adenosine 50-triphosphate (1). Compound 1m (30 mg,
42.5 mmol) was converted to the free acid by HPLC
purification using a semi-preparative C18 column, elut-
ing with a 30 min linear gradient from 0 to 50% aceto-
nitrile in 0.1% trifluoroacetic acid. The appropriate
fractions were pooled and lyophilized to dryness. The
resulting solid (20 mg, 37 mmol) was converted to the
triphosphate according to the method used for the pre-
paration of 2: MS (ꢀFAB, glycerol) m/e 705 (MꢀH+).
Ammonium
[ꢀ,ꢁ-32P]-2-(4-azidophenacyl)thio-1,N6-
etheno-dATP. [b,g-32P]-1 was prepared from 1m and
32P-tributylammonium pyrophosphate as described
above for the synthesis of 2. Tributylammonium [32P]-
pyrophosphate was prepared immediately before use by
addition of tetrasodium [32P]-pyrophosphate (103 ml,
0.365 mCi) to a solution of tributylammonium pyr-
ophosphate (7.4 mg, 16 mmol, 200 equiv) in 50% aqu-
eous ethanol (2 mL). The mixture was stirred at room
temperature for 10 min, and after removing the solvent
by roto-evaporation, the residue was resuspended in
ethanol (2 mL) and dried by evaporation under vacuum.
Anhydrous pyridine (2 mL) was then added and evapo-
rated to remove traces of water. The residue was dis-
solved in anhydrous pyridine (0.5 mL) and added to the
P1-nucleotide-50 P2-diphosphate. The final product was
then purified on an analytical HPLC C18 column as
described for 1. The fractions were collected every 1min
and the radioactivity of each fraction was measured by
scintillation counting. The appropriate radioactive frac-
tions were pooled and lyophilized, and the residue was
dissolved in water (86 mL), yielding a 1mM solution of
[b,g-32P]-1.
KF and RT inhibition assays
The reverse transcriptase activity of HIV-1reverse
transcriptase was assayed using freshly prepared poly(-
.
rA) (dT)10 as template/primer and TTP as the nucleo-
side triphosphate substrate. The assays were performed
using 0.5 units of RT in 50 mM Tris–HCl buffer (pH
7.4) containing 3 mM of MgCl2 and 1mg/mL of BSA.
For kinetic analysis, the enzyme was assayed at 12.5–
100 nM of the template/primer and 50 mM of TTP or at
5–25 mM of TTP and 10 nM of the template/primer; for
IC50 determinations, both substrates were held at their
Km concentrations (10 nM for template/primer and
3.3 mM for TTP) and inhibitor concentration was var-
ied. The enzyme and substrates (with or without the
inhibitor) were diluted separately in 50 mM Tris–HCl
buffer (pH 7.4) containing 3 mM magnesium chloride,
and the reaction was initiated by addition of 20 mL of
the enzyme solution to 30 mL of substrate solution. The
reaction was incubated at 37 ꢁC for 30 min and termi-
nated by addition of an aliquot (40 mL) of the reaction
mixture to an equal volume of 30 mM EDTA, and the
quenched solution was immediately mixed by vortexing.
Aliquots (20 mL) were spotted on DEAE cellulose filter
paper (swatches of 2ꢂ2 cm2) in triplicate. The filter
2-(4-Fluorophenylacyl)-thio-1,N6-etheno-20 deoxyadeno-
sine 50 triphosphate (5). This was prepared from 2-(4-
fluorophenylacyl)-thio-1,N6-etheno-20 -deoxyadenosine

514
K. Li et al. / Bioorg. Med. Chem. 10 (2002) 507–515
papers were washed in ammonium formate solution
(0.3 M, pH 8, 3ꢂ200 mL) with gentle stirring, dehy-
drated in ethanol (150 mL, 95%) and ethyl ether
(100 mL) and air-dried. The radioactivity on each filter
paper was then measured by scintillation counting in
7 mL of Scintiverse II. The initial rate of the poly-
merization was determined as the picomoles of [3H]TTP
incorporated into DNA per min. The initial velocity
versus substrate data were fit to competitive, non-
competitive and uncompetitive models using non-linear
regression analysis using the program KINETICS as
previously described.18
the photolysis mixture. The photolysis and enzyme
assays were performed using the same procedures as
described for enzyme photoinactivation in the absence
of substrate except that the enzyme and the substrate
were preincubated for 4 min prior to addition of the
photoprobe.
Photoincorporation of [ꢀ,ꢁ-32P]-1 into reverse
transcriptase
HIV-1reverse transcriptase (158 pmol) was incubated in
the presence of various concentrations of [b,g-32P]-1(5–
100 mM) in 50 mM Tris–HCl buffer (pH 7.4) (0.78 mL)
containing MgCl2 (3.0 mM) and 2-mercaptoethanol
(2 mM or 10 times the concentration of the photoprobe)
at 37 ꢁC in the dark for 10 min. The solutions were
irradiated at 3500 A at 25 ꢁC for 5 min. For the control
experiment, the photoprobe was preirradiated before
addition to the enzyme solution. The photolysis mix-
tures were diluted with 2-times treatment buffer (50 mM
Tris–HCl, pH 6.8, containing 100 mM dithiothreitol,
2% SDS, 10% glycerol, and 1% bromophenol blue)
and denatured on a boiling water bath for 3 min. The
samples were subjected to denaturing gel electrophoresis
using the methodology of Laemmli.40 For auto-
radiography, gels were fixed in destaining solution (10%
acetic acid, 5% methanol, and 85% water) for 10 min
and dried at 80 ꢁC under vacuum for 2 h. The dried gel
was exposed to Kodak XRA-5 X-ray film in a cassette
with an intensifying screen. After 5 days at ꢀ70 ꢁC, the
film was developed, and band intensities were measured
by densitometry scanning.
The Klenow fragment polymerase assays were per-
formed at 1unit/assay of the Klenow fragment, varying
concentrations of TTP (10–100 mM) and a fixed con-
.
centration of the poly(dA) (dT)10 (20 nM) or at varying
.
concentrations of poly(dA) (dT)10 (5–50 nM) and a
fixed concentration of TTP (100 mM) in the absence or
presence of varying concentrations of inhibitor 2 (5–
100 mM). The enzyme and substrates (with or without
the inhibitor) were diluted separately in 50 mM Tris–
HCl buffer (pH 7.4) containing 3 mM magnesium chlo-
ride, and the reaction was initiated by addition of 20 mL
of the enzyme solution to 30 mL of substrate solution.
The assay procedure and kinetic analyses were per-
formed as described above for the polymerase assay of
RT.
Irreversible photoinactivation of reverse transcriptase
A standard irradiation mixture contained 2 mM HIV-1
reverse transcriptase, 2 mM 2-mercaptoethanol, 3 mM
magnesium chloride and various concentrations of the
photoprobe (5–100 mM) in 100 mM Tris–HCl buffer
(pH 7.4) in a total volume of 40 mL. The photolysis
mixture was incubated at 37 ꢁC for 10 min in the dark
and irradiated at 3500 A at 25 ꢁC. Aliquots of 5 mL were
removed at indicated times and diluted 10-fold in assay
buffer. An aliquot (5 mL) of the diluted sample was
assayed for polymerase activity as described above. The
% photoinactivation at each concentration of the pho-
toprobe was calculated as the percentage loss of enzyme
activity relative to the enzyme activity of the control in
which the enzyme was photolyzed in the absence of the
photoprobe. The concentration-dependent photo-
inactivation data were fit to eq (1):
Acknowledgements
This work was supported in part by grants from the
U.S. Public Health Service (GM38722) and The Uni-
versity of Kansas General Research Fund.
References and Notes
1. Di Marzo Veronese, F.; Copeland, T. D.; DeVico, A. L.;
Rahman, R.; Oroszlan, S.; Gallo, R. C.; Sarngadharan, M. G.
Science 1986, 231, 1289.
2. Kohlstaedt, L. A.; Wang, J.; Friedman, J. M.; Rice, P. A.;
Steitz, T. A. Science 1992, 256, 1783.
3. Ding, J.; Das, K.; Moereels, H.; Koymans, L.; Andries, K.;
Janssen, P. A. J.; Hughes, S. H.; Arnold, E. Struct. Biol. 1995,
2, 407.
½IiŠ
½IŠÃImax
¼
ð1Þ
½ItŠ ½IŠ þ KD;app
4. Ding, J.; Das, K.; Tantillo, C.; Zhang, A.; Clark, A. D.;
Jessen, S.; Lu, X.; Hsiou, Y.; Jacobo-Molina, A.; Andries, K.;
Pauwels, R.; Moereels, H.; Koymans, L.; Janssen, P. A. J.;
Smith, R. H., Jr.; Kroeger Koepke, M.; Michejda, C. J.;
Hughes, S. J.; Hughes, S. H.; Arnold, E. Structure 1995, 3, 365.
5. Jacobo-Molina, A.; Ding, J.; Nanni, R. G.; Clark, A. D.;
Lu, X.; Tantillo, C.; Williams, R. L.; Kamer, G.; Ferris, A. L.;
Clark, P.; Hizi, A.; Hughes, S. H.; Arnold, E. Proc. Natl.
Acad. Sci. U.S.A. 1993, 90, 6320.
where [Ii]/[IT] is the fraction of photoinactivation of the
enzyme by photoprobe I, Imax is the maximum inacti-
vation at saturation, [I] is the photoprobe concentra-
tion, and KD,app is the concentration of the photoprobe
which causes 50% of maximum photoinactivation of
the enzyme.18 KD,app values were calculated by non-
linear regression using the software MINSQ.
For substrate protection experiments, 5 mM TTP or
.
500 nM template-primer poly(rA) (dT)10 was added to
6. Huang, H.; Chopra, R.; Verdine, G. L.; Harrison, S. C.
Science 1998, 282, 1669.

K. Li et al. / Bioorg. Med. Chem. 10 (2002) 507–515
515
7. Chen, M. S.; Oshana, S. C. Biochem. Pharmacol. 1987, 36,
4361.
8. Mitsuya, H.; Jarrett, R. F.; Matsukura, M.; Di Marzo
Veronese, F.; DeVico, A. L.; Sarngadharan, M. G.; Johns,
D. G.; Reitz, M. S.; Broder, S. Proc. Natl. Acad. Sci. U.S.A.
1987, 84, 2033.
9. Yarchoan, R.; Mitsuya, H.; Thomas, R. V.; Pluda, J. M.;
Hartman, N. R.; Perno, C.-F.; Marczyk, K. S.; Allain, J.-P.;
Johns, D. G.; Broder, S. Science 1989, 245, 412.
10. Mitsuya, H.; Broder, S. Proc. Natl. Acad. Sci. U.S.A.
1986, 83, 1911.
11. Coates, J. A. V.; Cammack, N.; Jenkinson, H. J.; Jowett,
A. J.; Jowett, M. I.; Pearson, B. A.; Penn, C. R.; Rouse, P. L.;
Viner, K. C.; Cameron, J. M. Antimicrob. Agents Chemother.
1992, 36, 733.
20. Flaherty, D.; Balse, P.; Li, K.; Moore, B. M.; Doughty,
M. B. Nucleosides Nucleotides 1995, 14, 65.
21. Michelson, A. M. Biochim. Biophys. Acta 1964, 91, 1 .
22. Chou, T.-C. Mol. Pharmacol. 1974, 10, 235.
23. Kolocheva, T. I.; Nevinsky, G. A.; Levina, A. S.; Khomov,
V. V.; Lavrik, O. I. J. Biomol. Struc. Dynam. 1991, 9, 169.
24. Doronin, S. V.; Lavrik, O. I.; Nevinsky, G. A.; Podust,
V. N. FEBS Lett. 1987, 216, 221.
25. Abraham, K. I.; Modak, M. J. Biochemistry 1984, 23,
1176.
26. Pandey, V. N.; Williams, K. R.; Stone, K. L.; Modak,
M. J. Biochemistry 1987, 26, 7744.
27. Evans, R. K.; Coleman, M. S. Biochemistry 1989, 28, 707.
28. Rush, J.; Konigsberg, W. H. J. Biol. Chem. 1990, 265,
4821.
12. Van Leeuwen, R.; Lange, J. M. A.; Hussey, E. K.; Donn,
K. H.; Hall, S. T.; Harker, A. J.; Jonker, P.; Danner, S. A.
AIDS 1992, 6, 1471.
13. Heidenreich, O.; Kruhoffer, M.; Grosse, F.; Eckstein, F.
Eur. J. Biochem. 1990, 192, 621.
14. De Clercq, E. Il Farmaco 1999, 54, 26.
15. Cohen, K. A.; Hopkins, J.; Ingraham, R. H.; Pargellis, C.;
Wu, J. C.; Palladino, D. E. H.; Kinkade, P.; Warren, T. C.;
Rogers, S.; Adams, J.; Farina, P. R.; Grob, P. M. J. Biol.
Chem. 1991, 266, 14670.
16. Wu, J. C.; Warren, T. C.; Adams, J.; Proudfoot, J.; Skiles,
J.; Raghavan, P.; Perry, C.; Potocki, I.; Farina, P. R.; Grob,
P. M. Biochemistry 1991, 30, 2022.
29. Reddy, G.; Nanduri, V. B.; Basu, A.; Modak, M. J. Bio-
chemistry 1991, 30, 8195.
30. Delahunty, M. D.; Wilson, S. H.; Karpel, R. L. J. Mol.
Biol. 1994, 236, 469.
31. Jones, G. H.; Murthy, D. V. K.; Tegg, D.; Golling, R.;
Moffatt, J. G. Biochem. Biophys. Res. Commun. 1973, 53,
1338.
32. Secrist, J. A. I.; Barrio, J. R.; Leonard, N. J.; Weber, G.
Biochemistry 1972, 11, 3499.
33. Li, K. PhD Dissertation, University of Kansas, 1996, p.
226.
34. Jaju, M.; Beard, W.; Wilson, S. J. Biol. Chem. 1995, 270,
9740.
17. Pauwels, R.; Andries, K.; Debyser, Z.; Van Daele, P.;
Schols, D.; Stoffels, P.; Vreese, K. D.; Woestenborghs, R.;
Vandamme, A.-M.; Janssen, C. G. M.; Cauwenbergh, G.;
Desmyter, J.; Heykants, J.; Janssen, M. A. C.; Clercq, E. D.;
Janssen, P. A. J. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 1711.
18. Moore, B. M.; Li, K.; Doughty, M. B. Biochemistry 1996,
35, 11634.
35. Pop, M.; Biebricher, C. Biochemistry 1996, 35, 5054.
36. Cleland, W. W. Biochim. Biophys. Acta 1963, 67, 173.
37. Page, M. I. In Enzyme Inhibition; Page, M. I., Ed.; Perga-
mon: New York, 1990; p 61–80.
38. Keana, J. F. W.; Cai, S. X. J. Org. Chem. 1990, 55,
3640.
39. McPhee, W.; Klingsberg, E. Org. Synth. Collec 1971, 3,
119.
40. Laemmli, U. K. Biochem, J. 1958, 68, 244.
19. Moore, B. M.; Jalluri, R.; Doughty, M. B. Biochemistry
1996, 36, 11642.