Discovery of potent HIV integrase inhibitors active against raltegravir resistant viruses

Article abstract of DOI:10.1016/j.bmcl.2010.07.041

A series of novel HIV integrase inhibitors active against rategravir resistant strains are reported. Initial SAR studies revealed that activities against wild-type virus were successfully maintained at single digit nanomolar level with a wide range of substitutions. However, inclusion of nitrogen-based cyclic substitutions was crucial for achieving potency against mutant viruses. Several compounds with excellent activities against wild-type virus as well as against the viruses with the mutations Q148H/G140S or N155H/E92Q were reported.

Full text of DOI:10.1016/j.bmcl.2010.07.041

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5013–5018
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of potent HIV integrase inhibitors active against raltegravir
resistant viruses
a
a
a
a
b
Giang Le a, , Nick Vandegraaff , David I. Rhodes , Eric D. Jones , Jonathan A. V. Coates , Long Lu ,
*
Xinming Li b, Changjiang Yu b, Xiao Feng b, John J. Deadman a,
*
a Avexa Ltd, 576 Swan Street, Richmond, Victoria 3121, Australia
b Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Lu, Shanghai 200032, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel HIV integrase inhibitors active against rategravir resistant strains are reported. Initial
SAR studies revealed that activities against wild-type virus were successfully maintained at single digit
nanomolar level with a wide range of substitutions. However, inclusion of nitrogen-based cyclic substi-
tutions was crucial for achieving potency against mutant viruses. Several compounds with excellent
activities against wild-type virus as well as against the viruses with the mutations Q148H/G140S or
N155H/E92Q were reported.
Received 20 June 2010
Revised 9 July 2010
Accepted 12 July 2010
Available online 16 July 2010
Keywords:
Integrase
Inhibitor
Synthesis
HIV
Ó 2010 Elsevier Ltd. All rights reserved.
QHGS
NHEQ
Mutant virus
Recently, a new inhibitor of the human immuno-deficiency
virus (HIV) targeting the viral integrase enzyme was approved
for clinical use by the FDA. Raltegravir (RAL, marketed as
Isentress™ by Merck and Co.) disrupts the critical viral process of
integration in which newly made viral DNA is inserted into the
host cell chromosomal DNA.1 RAL inhibits integrase by binding
within the active site of a pre-assembled integrase–viral DNA
complex, and is able to co-ordinate two divalent metal (Mg) ions
known to be indispensable for enzyme catalysis.2 The core scaffold
of RAL comprises a phenolic hydroxyl group and one exocyclic and
one ring amide functionality arranged in such a way as to present
as a hydrogen bond acceptor–donor–acceptor motif responsible for
metal co-ordination.3
As with other classes of HIV drugs, under the highly selective
evolutionary pressures associated with exposure to integration
inhibitors, viral resistance emerges. To date, around 40 mutations
within the viral integrase gene have been associated with resis-
tance to either RAL and/or another integration inhibitor currently
in clinical trials, elvitegravir (EVR, Gilead Sciences).4 However,
two main pathways bestowing viral resistance to RAL have been
identified in the clinic and these involve mutations at either posi-
tions Gln-148 (Q148K/R/H) or Asn-155 (N155S/H) in the IN central
core domain.5 Mutations of these key amino acids have also been
associated with significant resistance to EVR. A third less common
pathway involving mutation at Tyr-143 (Y143C/R) has also been
associated with resistance to RAL.6 Additionally, specific secondary
mutations are also commonly associated with each primary muta-
tion and serve to significantly enhance resistance and/or the repli-
cative capacity of these viruses. A recent Letter indicates that the
Q148R natural polymorphism in HIV IN is present in a significant
proportion of therapy naïve as well as treatment experienced
patients studied as a minor quasi-species.7 Thus, there is a clear
need to identify scaffolds that allow the development of inte-
gration inhibitors that are active against RAL-resistant (and EVR-
resistant) HIV strains.
We have previously reported a series of bicyclic pyrimidinone
integrase inhibitors displaying excellent antiviral activity against
wild-type (WT) HIV in cell culture.8 Here we present evidence that
compounds active against viruses resistant to RAL, as well as wild-
type (WT) virus, can be derived from scaffolds containing a thiazole
replacement of the exocyclic amide. Furthermore, modification at
position 7 and/or 9 of the bicyclic pyrimidinone core can also sig-
nificantly influence the activity of compounds against drug resis-
tant viruses.
We have developed a convenient route for the formation of thi-
azole ring.8 The starting acid 1 was activated with 1,10-carbonyldi-
imidazole in the presence of NaSH, followed by substitution with
* Corresponding authors. Tel.: +61 408538488 (J.J.D.).
E-mail addresses: jjdeadman@hotmail.com, jdeadman@avexa.com.au (J.J. Dead-
man).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.07.041

5014
G. Le et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5013–5018
alpha bromoaldehyde to furnish thioester 2. Upon treating with an
ammonia source, such as ammonium acetate at elevated tempera-
ture, the aldehyde–thioester 2 (Scheme 1) cyclized to result in
thiazole 3 in reasonable overall yield. Intermediate 3 was further
derivatized in position 9 via Buchwald coupling of the versatile
bromo handle.9 Deprotection of the O-Bn group with TFA cleanly
gave the desired final products, of which the numerous examples
are exemplified by the general structure 5. Bromo precursor 3
was also utilized for other metal mediated reactions, such as car-
bamoylation to give the amide derivative 6.10
O
O
OH
S
N
N
N
N
O
N
F
S
13
O
To provide a key intermediate to facilitate analog generation,
the synthetic strategy shown in Scheme 2 was developed further
to allow diversification of both R7 and R9 substitutions of the fused
bicyclic core at a late stage. The versatile 7-Br-9-I thiazole precur-
sor 9 was prepared from acid 7 in two steps as described in
Scheme 2. The more active 9-I substituted carbon underwent cou-
pling with various nitrogen-based substitutions under Buchwald9
conditions using toluene as the solvent. The less active 7-Br group
was then coerced to undergo the second coupling reaction at high-
er temperatures in dioxane. These two coupling steps could be exe-
cuted sequentially in one-pot fashion or as separate reactions. The
final deprotection step of the O-Bn group was achieved by heating
in TFA as before, and all products were purified on preparative
HPLC to purity of greater than 95%.
From preliminary SAR studies we identified thiazole derivative
13 as a potent HIV integrase inhibitor, displaying an EC50 of 6.4 nM
in a cell-based assay.11 As this compound represents a novel
scaffold, we were interested to investigate its inhibitory potency
against RAL-resistant HIV mutants. To this end we have adapted
the single round infectivity assay for a large panel of mutant
viruses, including the clinically most important Q148H/G140S
and N155H/E92Q double mutations.12 Upon assaying we found
that compound 13 exhibited a reasonable level of potency against
QHGS and NHEQ mutants, with an EC50 of 280 nM and 49.5 nM,
respectively. The encouraging activity levels prompted us to inves-
tigate the SAR of compound 13. Initially we focused on the effect of
substitutions of the position C-9 of the bicyclic system. As reported
O
O
O
O
OBn
OBn
N
N
7
N
N
a
OH
S
N
N
9
Br
O
Br
O
O
F
2
1
b
O
O
N
O
O
N
N
OBn
N
OBn
N
N
c
S
S
N
N
Br
N
R1
R2
d
4
3
F
F
e, d
O
O
N
N
OH
O
O
N
N
N
O
S
N
O
S
N
N
R1
R2
N
5
N
F
6
F
Scheme 1. Reagents and conditions: (a) (i) CDI, NaSH and (ii) 2-bromo-3-(4-fluorophenyl)propanal, proton sponge, CH2Cl2, reflux; (b) ammonium acetate, AcOH, reflux, 20–
27% overall yield for (a) and (b); (c) NHR1R2, xantphos, Pd2(dba)3, Cs2CO3, toluene, 80 °C, 40–60%; (d) TFA, 80 °C, 3 h, 15–80%; (e) (i) Mo(CO)6, pyrrolidine, Pd(OAc)2, DBU,
dioxane, 80 °C, 5%.

G. Le et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5013–5018
5015
O
O
OBn
OBn
Br
Br
7
N
N
a
OH
S
N
N
9
I
O
I
O
O
F
8
7
b
O
N
O
N
Br
OBn
Br
OBn
N
N
c
S
S
N
N
I
N
R1
R2
10
9
F
F
d
R3
O
N
N
R3
O
N
OBn
N
R4
e
N
N
O
R4
N
S
S
N
R1
R2
N
N
11
R1
R2
F
12
F
Scheme 2. Reagents and conditions: (a) (i) CDI, NaSH and (ii) 2-bromo-3-(4-fluorophenyl)propanal, proton sponge, CH2Cl2, reflux; (b) ammonium acetate, AcOH, reflux, 20–
27% overall yield for (a) and (b); (c) NHR1R2, xantphos, Pd2(dba)3, Cs2CO3, toluene, 80 °C, 40–60%; d) NHR3R4, xantphos, Pd2(dba)3, Cs2CO3, dioxane, 100 °C, 30–70%; TFA, 80 °C,
3 h, 15–80%.
previously, unsubstituted derivative 14 or halogenated 15 exhib-
NHEQ double mutant viruses, respectively. This represents the
most potent integrase inhibitor for QHGS reported in the peer-
reviewed literature to date. We also examined cyclic ureas as
potential substitutions for position 9. The five-membered ring urea
24 had comparable activity to carbamate 22, however, its six-
membered congener 25 improved the QHGS activity by only two-
fold, resulting in an EC50 of 34 nM. Substitution on the second
nitrogen of the urea proved more beneficial. Replacement of the
methyl group with isopropyl furnished compound 26 which is four
times more active than 24 against the QHGS mutant virus. Interest-
ingly, opening up the cyclic urea ring (27) led to a significant loss of
QHGS activity, even though compound 27 was only slightly less
active than 25 with regard to WT virus. Removal of the N-Me substi-
tution caused an even more profound effect. Derivative 28 was not
capable of inhibiting QHGS infectivity up to a concentration of
ited comparable WT activity,8 however, these compounds inhib-
ited QHGS infectivity with only modest potency at 2–3 lM.
Likewise, methoxy (16) or carbon-based functionalities such as
acetyl (17), 2-hydroxyethane (18), tertiaryalkylamine (19), or
amide (6) in this position resulted in compounds with micromolar
or high nanomolar activities against QHGS mutant. The acetyl
group of compound 17 was introduced via Heck coupling into
the ester derivative of acid 1,8 from which the thiazole ring was
prepared as described in Scheme 1. Hydroxyethane congener 18
was obtained from 17 via a simple reduction step with NaBH4,
while 19 was formed through reductive amination (Table 1).
Introduction of a cyclic nitrogen-based substitution in this
position restored the mutant activities in contrast to compounds
14–19. For example, sultam derivative 20 was shown to be twice
as active against the QHGS or NHEQ mutants as the lead compound
13, while 21 with the lactam substitution maintained the same
level potency as 13 with an EC50 of 335 nM. The five-membered
cyclic carbamate (22) was beneficial, exhibiting an EC50 of
108 nM against QHGS virus. Enlarging the carbamate moiety to a
six-membered-ring resulted in the most significant improvement
of potency. Compound 23 displayed a WT EC50 of 4.3 nM, a value
which is about twice as active as RAL in our assays, and 23 had
an excellent activity of 16.5 nM and 10.5 nM against QHGS or
10
l
M, despite having an EC50 of 22 nM against WT virus (Table 2).
Having identified that cyclic amide analogs are essential for
good level of QHGS potency we set to investigate the effect of sub-
stitutions in position 7. For this purpose we chose cyclic urea 24 for
comparison. It was found that the bulky morpholine group could
be replaced with a smaller dimethyl amine group with negligible
loss of WT and QHGS potency. The NHEQ activity decreased by
threefold, however. Insertion of a methylene linker between the
morpholine and the bicyclic ring furnished compound 30 which

5016
G. Le et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5013–5018
Table 1
Table 2
SAR of R9 substitutions
SAR of R7 substitutions
F
O
O
O
N
OH
S
N
7
R7
N
OH
N
3
N
9
N
N
S
1
R9
F
N
O
R9
IC50
WT
EC50 (nM)
QHGS EC50
NHEQ EC50
Luc12 (nM)
13
N
Luc12 (nM)
11
(nM)
R7
IC50
WT EC50
QHGS EC50
Luc12 (nM)
NHEQ EC50
Luc12 (nM)
13
O
O
AVX
S
Luc11 (nM)
13
48
6.4
280
49.5
(nM)
N
14
15
16
H
Br
MeO-
20
42
na
32
14
100
3000
2500
5900
74.5
19
na
O
N
24
29
47
24
71.0
92.5
10
N
90
25
19.5
32.5
17
18
15
na
9.5
885
800
na
na
O
O
N
30
31
3.5
243
268
14.5
na
10.5
O
na
11.5
N
N
H
19
6
na
70
110
81
5500
1175
na
N
N
32
33
na
19
27
1100
21
na
O
O
N
6.8
8.9
N
200
N
O
O
S
20
21
38
na
8.5
21
112
335
18.5
na
N
N
Amide moieties, as exemplified by 32 retained the WT potency
but caused significant decrease of QHGS inhibitory effect, having
an EC50 of 1.1 lM. In contrast, cyclic amine derivative 33 exhibited
O
N
excellent potency against both WT and resistant viruses, with EC50
in the range of 6.8–21 nM. The results suggested that unlike posi-
tion 9, cyclic amines are preferable as R7 substitutions. However, in
both cases the steric effects are not the dominant factors, instead
electronic properties proved to be far more important in increasing
QHGS potency. With the SAR knowledge in hand, we prepared a
large number of compounds following the synthetic route depicted
in Scheme 2. Our investigations have resulted in the identification
of numerous potent inhibitors of HIV integrase (Table 3). Several
examples displayed excellent inhibitory activity against WT virus
in a high serum environment. For example, compound 33 shows
less than a twofold shift in EC50 from 6.8 nM to 12.5 nM in the
presence of 50% normal human serum (NHS). In addition, these
compounds also demonstrated outstanding potency against all
major RAL-resistant strains, such as QHGS and NHEQ. The specific-
ity of the inhibitors for the inhibition of integration during virus
infection of a cell was monitored during the development of this
series of compounds by quantitative PCR techniques,14,15 using
RAL as positive control. Both earlier compounds of the series and
examples of the most recent compounds containing the thiazole
moiety as reported here were demonstrated to specifically inhibit
integration events in the cell and had no effect on entry or reverse
transcription events occurring prior to integration. These results
taken together with the potency of the compounds against the iso-
lated enzyme demonstrate these compounds to be bona fide
integrase inhibitors. Initial PK studies indicated that these com-
pounds had relatively short half life in both in vitro microsomal
metabolism and in vivo pharmacokinetic experiments. The results
suggested that oxidative metabolism was the major pathway.
Compound 33 was treated with microsomes at high protein con-
centration in presence of NADPH and UDPGA co-factors. Analysis
O
22
23
24
26
na
47
7.1
4.3
24
108
16.5
71.0
25
N
O
N
10.5
10
O
O
O
N
N
N
O
25
na
11.5
34
21.5
N
N
O
26
27
28
19
na
na
7.5
16.5
22
18
120
9.7
na
na
N
O
O
N
N
N
>10000
N
is seven times more potent than 24 against WT virus, but surpris-
ingly its QHGS activity dropped threefold. Other aminomethylene
substitutions, for example, 31 displayed the same trend of SAR,
exhibiting a similar level to 30 of QHGS potency of 200–300 nM.

G. Le et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5013–5018
5017
Table 3
Activities and PK characteristics of selected examples
F
O
R7
OH
N
N
S
N
R9
R7
R9
WT EC50 (nM)
10% NHS11
WT EC50 (nM)
50% NHS
QHGS EC50 (nM)
10% FBS12
NHEQ EC50 (nM)
10% FBS12
In vitro
t1/2 (min)
In vivo
t1/2 (min)
%F
N
N
N
33
6.8
12.5
21
8.9
17.7
18
3
N
O
N
N
34
35
4
6.1
6.6
34.5
29.5
6.7
4.9
52
21.6
39
0.5
5
N
O
O
N
N
N
3.5
67.8
N
N
O
O
O
N
36
4.2
5.5
40.5
5.2
18
44
5.7
O
Monteagudo, E.; Muraglia, E.; Nizi, E.; Orvieto, F.; Pace, P.; Pescatore, G.; Scarpelli,
R.; Stillmock, K.; Witmer, V. M.; Rowley, M. J. Med. Chem. 2008, 51, 5843.
2. (a) Sunderland, J.; Botta, M.; Aime, S.; Raymond, K. N. Inorg. Chem. 2001, 40,
6746; (b) Hare, S.; Gupta, S. S.; Valkov, E.; Engelman, A.; Cherepanov, P. Nature
2010, 464, 232.
3. Vandegraaff, N.; Engelman, A. Expert Rev. Mol. Med. 2007, 9, 1.
4. Ceccherini-Silberstein, F.; Malet, I.; D’Arrigo, R.; Antinori, A.; Marcelin, A.;
Perno, C. AIDS Rev. 2009, 11, 17.
of the reaction mixture showed that the substituted piperazine in
position was metabolized extensively. The benzyl-thiazole
7
moiety also underwent oxidative metabolism, although the analy-
sis did not allow elucidation of the exact metabolic sites. In
addition, glucuronidation of the phenolic 3-OH was also identified
as a potential clearance pathway. Glucuronidation of a similar
hydroxyl group was also reported to be the major mechanism of
clearance for RAL.16
5. Hatano, H.; Lampiris, H.; Fransen, S.; Gupta, S.; Huang, W.; Hoh, R.; Martin, J. N.;
Lalezari, J.; Bangsberg, D.; Petropoulos, C.; Deeks, S. G. J. Acquir. Immune Defic.
Syndr. 2010, 54, 389.
In summary we have disclosed a series of potent HIV integrase
inhibitors based on a novel scaffold consisting of a hydroxypyridi-
none and a thiazole ring. The reported compounds are the first
inhibitors that exhibit low nanomolar potency against all major
clinically relevant RAL-resistant HIV strains, including double
mutants Q148HG140S and N155HE92Q. Initial SAR studies sug-
gested that electronic effects were dominant and important for
resistant activities, with nitrogen-based substitutions preferred in
both positions of the bicyclic ring system. We also demonstrated
that cyclic substitutions were crucial for maintaining mutant
activities while a range of diverse functionalities exhibited simi-
larly potent level of inhibition against WT virus. The data
6. Reigadas, S.; Anies, G.; Masquelier, B.; Calmels, C.; Stuyver, L. J.; Parissi, V.;
Fleury, H.; Andreola, M. L. PLoS One 2010, 5, 10311.
7. Charpentier, C.; Laureillard, D.; Piketty, C.; Tisserand, P.; Batisse, D.;
Karmochkine, M.; Si-Mohamed, A.; Weiss, L. AIDS 2010, 24, 867.
8. (Part 1) Jones, E.; Vandegraff, N.; Le, G.; Choi, N.; Issa, W.; Macfarlane, K.;
Thienthong, N.; Winfield, L.; Coates, J.; Lu, L.; Li, X.; Feng, X.; Yu, C.; Rhodes, D.;
Deadman, J. Bioorg. Med. Chem. Lett., accepted for publication; (part 2) Le, G.;
Vandegraff, N.; Rhodes, D.; Jones, E.; Thienthong, N.; Winfield, L.; Coates, J.; Lu,
L.; Li, X.; Feng, X.; Yu, C.; Deadman, J. Bioorg. Med. Chem. Lett., accepted for
publication.
9. Buchwald, S. L.; Yin, J. J. Am. Chem. Soc. 2002, 124, 6043.
10. (a) Ren, W.; Yamane, M. J. Org. Chem. 2010, 75, 3017; (b) Wu, X.; Rönn, R.;
Gossas, T.; Larhed, M. J. Org. Chem. 2005, 70, 3094.
11. Inhibition of HIV replication: Cells are seeded into 96 well microtitre plates at
50,000 cells per 50
2). Compounds are prepared to 4ꢀ final concentration in RF-10/2, and 30
added to cells. Virus (40 l in RF-10/2 containing 1600 pfu) is added to each
well or 40 RF-10/2 for negative controls and for assaying compound
cytotoxicity. After 24 h, an additional 90
l of media or media containing 1ꢀ
compound is added to each well. At 4 days post-infection, 100 l of media is
removed from each well and replaced with 100 l of fresh media with or
ll per well in RF-10 containing 2 lg/ml polybrene (RF-10/
ll
presented herein validated thiazole-pyridinone systems as
a
l
potential platform for developing second-generation integrase
inhibitors to treat RAL-resistant viruses. A comprehensive lead
optimization of these promising leads to optimize PK characteris-
tics is underway and the results will be reported in due course.
l
l
l
l
l
without compound. Forty eight hours later supernatants are harvested and
levels of extracellular p24 determined. Supernatants are diluted 1 in 10,000
and p24 levels assayed using the Vironostika p24 assay kit. EC50 is calculated as
the concentration required to inhibit HIV p24 production to 50%.
Acknowledgments
12. Chang, T. L.-Y.; Francois, F.; Mosiam, M.; Klotman, M. E. J. Virol. 2003, 77, 6777.
We are grateful to the Centre for drug candidate optimization,
Faculty of Pharmacy and Pharmaceutical Sciences, Monash
University, Melbourne, Australia, 3052 for the in-vitro metabolism
studies.
Infectivity assays used virus stocks generated by transfecting
a plasmid
encoding a full-length HIV-1 genome which had the envelope gene deleted and
contained a reporter luciferase gene cloned into the nef region (pHIV env-Luc)
together with an expression plasmid producing the vesicular stomatitis virus
envelope glycoprotein (VSV-G) into 293T cells. Culture supernatants containing
VSV-G pseudotyped virions were harvested 64 h post-transfection, clarified by
centrifugation to remove cell debris, and stocks stored at ꢁ70 °C. HIV integrase
mutations known to confer resistance to published integrase inhibitors were
prepared by site directed mutagenesis of the integrase gene in a shuttle vector
which contained the majority of the HIV-1 gag and pol sequence. The mutated
integrase coding region was then sequence verified and exchanged for the
References and notes
1. Summa, V.; Petrocchi, A.; Bonelli, F.; Crescenzi, B.; Donghi, M.; Ferrara, M.; Fiore,
F.; Gardelli, C.; Gonzalez Paz, O.; Hazuda, J. D.; Jones, P.; Kinzel, O.; Laufer, R.;

5018
G. Le et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5013–5018
wild-type coding sequence in the pHIV env-Luc reporter virus plasmid. Assay
ATTTTCCACACTGACTAA-AAGGGTC-DIG-30) or biotin (50-Bio-GACCCTTTTAGT
CAGTGT-GGAAAATCTCTAGCA-30) so that each substrate has either a DIG or Bio
tag on opposite strands. Reactions are carried out for 2 h at 37 °C, products
generated as a result of 30 processing and strand transfer activity are bound to
streptavidin plates and detected using anti-DIG-alkaline phosphatase
conjugate and p-nitro phenyl phosphate substrate.
method 293T cells were seeded in 96 well plates 16 h prior to compound
addition. Compounds were preincubated with cells for 4 h at 37 °C followed by
the addition of sufficient virus to generate approximately 10,000 Luciferase
light units in the absence of compound. Luciferase readout was measured 48 h
post-infection using the Bright-Glo™ reagent (Promega) according to the
manufacturer’s instructions.
14. Engelman, A.; Oztop, I.; Vandegraaff, N.; Raghavendra, N. K. Methods 2009, 47,
283.
15. Butler, S. L.; Hnasen, M. S.; Bushman, F. D. Nat. Med. 2001, 7, 631.
16. Kassahun, K.; McIntosh, I.; Cui, D.; Hreniuk, D.; Merschman, S.; Lasseter, K.;
Azrolan, N.; Iwamoto, M.; Wagner, J. A.; Wenning, L. A. Drug Metab. Dispos.
2007, 35, 1657.
13. (a) Ovenden, S. P.; Yu, J.; Wan, S. S.; Sberna, G.; Tait, R. M.; Rhodes, D.; Cox, S.;
Coates, J.; Walsh, N. G.; Meurer-Grimes, B. M. Phytochemistry 2004, 65, 3255;
(b) Combined strand transfer assay: Briefly, for a 96 well format, 400 ng IN is
incubated with 30 nM pre-processed substrate DNA, consisting of annealed U5
LTR sequence oligonucleotides tagged with Digoxigenin (DIG; 50-ACTGCTAGAG