2′-Fluorosugar analogues of the highly potent anti-varicella-zoster virus bicyclic nucleoside analogue (BCNA) Cf 1743

Article abstract of DOI:10.1016/j.bmcl.2009.09.116

We report the preparation of 2′-α-F, 2′-β-F and 2′,2′-difluoro analogues of the leading anti-varicella zoster virus (VZV) pentylphenyl BCNA Cf 1743. VZV thymidine kinase showed the highest phosphorylating capacity for the β-fluoro derivative, that retaine

Full text of DOI:10.1016/j.bmcl.2009.09.116

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6264–6267
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
20-Fluorosugar analogues of the highly potent anti-varicella-zoster virus
bicyclic nucleoside analogue (BCNA) Cf 1743
a
a
b
b
Christopher McGuigan a, , Marco Derudas , Maurizio Quintiliani , Graciela Andrei , Robert Snoeck ,
*
Geoffrey Henson c, Jan Balzarini b
a Welsh School of Pharmacy, Cardiff University, King Edward VII Avenue, Cardiff CF10 3XF, UK
b Rega Institute for Medical Research, Minderbroedersstraat 10, Leuven B-3000, Belgium
c Inhibitex, 9005 Westside Parkway, Alpharetta, GA 30004, USA
a r t i c l e i n f o
a b s t r a c t
We report the preparation of 20- -F, 20-b-F and 20,20-difluoro analogues of the leading anti-varicella zoster
a
virus (VZV) pentylphenyl BCNA Cf 1743. VZV thymidine kinase showed the highest phosphorylating
Article history:
Received 4 September 2009
Revised 29 September 2009
Accepted 29 September 2009
Available online 3 October 2009
capacity for the b-fluoro derivative, that retained equal antiviral potency as the parent compound. In con-
trast, the
a
-fluoro- and 20,20-difluoro BCNA derivatives were markedly less (ꢀ100-fold) antivirally active.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
VZV
BCNAs
Cf 1743
Thymidine kinase
Zoster
Shingles
Nucleosides
Herpes
Fluorosugars
In 1999 we first reported the anti-VZV activity of the bicyclic
nucleoside analogue family now known as the BCNAs.1 We subse-
quently reported nanomolar activity and exclusive anti-VZV selec-
tivity for the pentylphenyl BCNA (Fig. 1, 1).2
The desired 20-fluoro-substituted BCNAs were prepared via su-
gar base coupling of protected 5-iodouracil to the appropriate flu-
oro sugar followed by construction of the BCNA base.
Thus, as shown in Scheme 1, 5-iodouracil (3) was silylated to
give (4) and 1,3,5-tri-O-benzoyl-2-deoxy-2-b-fluororibose (5) was
converted to its 1-bromo analogue (6) using HBr/AcOH,9 and these
reagents were allowed to couple using HMDS and ammonium sul-
fate to give mixed anomers of the protected nucleoside (7).9
The two isomers were separated by filtration to give the pure
b-anomer as a white solid 7 in a yield of 51%. The deprotection
of the benzoyl groups was performed using sodium methoxide
and the corresponding unprotected compound 8 converted to its
p-pentylphenyl BCNA (9) by standard methods1,2 in a yield of
45% from (7). Compound (9) showed spectroscopic (1H NMR and
13C NMR) and analytical data entirely as expected including a 19F
NMR peak at ꢁ198 ppm and other data (high resolution mass spec-
tra and HPLC) confirmed its structure and purity.10 Similarly pre-
As its 50-valyl Pro-Drug FV100 (Fig. 1, 2) human phase 2 clinical
trials for VZV shingles have recently commenced.3
We have reported extensively on the structure–activity rela-
tionships surrounding this family of potent antivirals.4 In general,
there is little tolerance for structural modifications; indeed we re-
cently reported that the corresponding carbocycle, a modification
often tolerated amongst antiviral nucleosides, is very poorly active
in this case.5 Also, the arabinosyl BCNA was considerably less
inhibitory than its parental 20-deoxyribosyl BCNA.6
There have been a number of cases where 20-modification, in
particular 20-fluorination of bioactive nucleosides leads to
enhancements in the biological activity profile, notably, the
anti-cancer agent gemcitabine with a 20,20-difluoro pattern7 and
Pharmasset0s anti-HCV agent PSI6130 (20-deoxy-20-
a
-fluoro-20-b-
pared by analogous methods were the
and the 20,20-difluoro BCNA derivative (11). Compound (10) was
prepared starting from the commercial available 20- -F-20-deoxy-
a-fluoro analogue (10)
C-methyl cytidine).8 Therefore, we were interested to prepare var-
ious 20-fluorinated analogues of the parent BCNA (1) (Fig. 2).
a
uridine which was iodinated at the 5-position and then coupled
with the phenyl acetylene under standard method. Compound
(11) was prepared starting from commercially available 2-deoxy-
* Corresponding author. Tel./fax: +44 2920874537.
E-mail address: mcguigan@cf.ac.uk (C. McGuigan).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.09.116

C. McGuigan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6264–6267
6265
O
N
O
N
N
O
N
O
O
C
O
O
(1)
(2)
HO
HC
O
NH2.HCl
OH
OH
Figure 1.
CH3
(H2C)4
CH3
(H2C)4
CH3
(H2C)4
Table 1
Compound
20-Fluoro position
VZVTK IC50/lM
1
9
10
11
—
3.3
38
2.7
ꢀ13
b-Fluoro
O
O
O
a
-Fluoro
Difluoro
N
N
N
N
O
N
O
N
O
O
O
O
the phosphorylation of 1
binant VZV TK.
l
M [CH3-3H]thymidine by purified recom-
HO
HO
HO
F
It is notable from Table 1 that the
a-fluoro analogue (10) retains
F
F
F
HO
HO
HO
low M potency as an inhibitor of VZV TK-catalysed dThd phos-
l
9
10
11
phorylation. The b-fluoro derivative 9 was at least 10-fold less
inhibitory. The 20,20-difluoro analogue 11 showed an IC50 value in
between 9 and 10 (Table 1).
Figure 2.
We then measured substrate activity of the BCNA derivative for
VZV TK at different compound concentrations by determining both
Km and Vmax values for each compound (Table 2, SI).
2,2-difluoro-D-erythro-pentafuranos-1-ulose-3,5-dibenzoate which
was reduced to benzoylated lactol using LiAl(O-tBu)3H and then
mesylated. The crude mesylate was coupled with silyl-protected
5-iodouracil in dichloroethane and the b-anomer was obtained
by precipitation from the organic solvent after the work-up. Depro-
tection of the benzoyl groups by sodium methoxide in methanol
provided the desired nucleoside.
Compared with the parent compound 1 (Cf 1743), the three flu-
oro derivatives were endowed with Km values that were somewhat
(up to threefold for compound 10) lower than for compound 1.
Whereas the Vmax value for 9 was ꢀ2.5-fold higher and for 11
was 1.5-fold lower than noticed for 1, the Vmax for the
a-fluoro
Given the crucial requirement for the BCNAs to be 50-phosphor-
ylated by VZV thymidine kinase (TK) for their antiviral activity11 we
first probed their interaction with VZV TK.12 Thus, in Table 1 we
show the 50% inhibitory concentrations (IC50) of (1) and (9–11) for
derivative 10 was markedly lower than observed for the parent
compound (6–7-fold). As a result, the phosphorylating capacity
(Vmax/Km) of the enzyme proved highest for the b-fluoro derivative
9 and lowest for the
a
-fluoro derivative 10 (Table 2).
OSi(CH3)3
N
O
O
O
I
I
OBz
Br
NH
BzO
BzO
i
ii
+
N
OSi(CH3)3
95%
N
H
O
BzO
BzO
F
F
5
4
6
3
CH3
(H2C)4
51%
iii
O
O
N
O
I
I
N
NH
NH
N
O
O iv
v
N
O
O
O
O
BzO
HO
HO
Overall yield
45%
BzO
F
HO
F
HO
F
7
8
9
Scheme 1. Reagents and conditions: (i) HBr in acetic acid, DCM, rt, 22 h; (ii) hexamethyldisilazane, ammonium sulfate, acetonitrile, 70 °C, 5 h; (iii) NaI, DCM, acetonitrile, rt,
1 week; (iv) MeONa, MeOH, 1 h, rt; (v) 4-n-pentylphenylacetylene, tetrakis (triphenylphosphine)Pd(0), CuI, DIPEA, DMF, rt, overnight, then CuI, TEA, 85 °C, 8 h.

6266
C. McGuigan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6264–6267
2. McGuigan, C.; Barucki, H.; Blewett, S.; Carangio, A.; Erichsen, J. Y.; Andrei, G.;
Table 2
Kinetic values of the 20-fluoro-substituted test compounds
Snoeck, R.; De Clercq, E.; Balzarini, J. J. Med. Chem. 2000, 43, 4993.
3. Hutchins, J.; Chamberlain, S.; Chang, C.; Ganguly, B.; Gorovits, E.; Hall, A.;
Henson, G.; Kolykhalov, A.; Liu, Y.; Muhammad, J.; Perrone, P.; Gilles A.; Holl, S.;
Madela, K.; McGuigan, C.; Patti, J. 22nd International Conference on Antiviral
Research, Miami, Antiviral Res. 2009, 82, A81.
Compound
Km
(lM)
Vmax
(lmol/
l
g protein/h)
Vmax/Km
1
9
10
11
1.58
1.11
0.47
0.95
22
50
3.4
14
14
45
7.2
14
4. McGuigan, C.; Balzarini, J. Antiviral Res. 2006, 71, 149.
5. Migliore, M. D.; Zonta, N.; McGuigan, C.; Henson, G.; Andrei, G.; Snoeck, R.;
Balzarini, J. J. Med. Chem. 2007, 50, 6485.
6. McGuigan, C.; Migliore, M.; Henson, G.; Patti, J.; Andrei, G.; Snoeck, R.;
Balzarini, J. 21st International Conference on Antiviral Research, Montreal,
Antiviral Res. 2008, 78, A1–A76, 33.
The kinetic values shown in Table 2 were derived from the Lineweaver–Burk dia-
grams based on the data shown in Supplementary data.
7. Parker, W. B. Chem. Rev. 2009, 109, 2880.
8. Stuyver, L. J.; McBrayer, T. R.; Thranish, P. M.; Clark, J.; Hollecker, L.; Lostia, S.;
Nachman, T.; Grier, J.; Bennett, M. A.; Xie, M.-Y.; Schinazi, R. F.; Morrey, J. D.;
Julander, J. L.; Furman, P. A.; Otto, M. J. Antiviral Chem. Chemother. 2006, 17, 79.
9. Tann, C. H.; Brodfuehrer, P. R.; Brundidge, S. P.; Sapino, C., Jr.; Howell, H. G. J.
Org. Chem. 1985, 50, 3644.
Table 3
MCCb
(lM)
a
10. Procedure for the preparation of (9). Synthesis of 5-iodo-20-b-fluoro-
Compound
VZV EC50
YS
(lM)
20deoxyuridine (8). To
a stirring solution of (7) (2.40 g, 4.14 mmol) in
OKA
TKꢁ 07-1
anhydrous methanol (60 mL) was added NaOMe (0.49 g, 9.70 mmol) and the
reaction mixture was stirred at room temperature for 1 h. After this period, the
reaction was neutralized with Amberlite, filtered and concentrated to give the
desired product, which was used in the following step without further
1
9
10
11
0.0097
0.007
0.75
—
>20
>50
>20
>50
P50
>50
>50
0.011
—
purification. Synthesis of 3-(20-b-fluoro-20-deoxy-b-
D-ribofuranosyl)-6-(4-n-
1.5
6.7
P50
pentylphenyl)-2,3-dihydrofuro [2,3-d]pyrimidin-2-one (9). To a solution of
(8) (1.56 g, 4.14 mmol) in anhydrous DMF (20 mL) were added: 4-n-
pentylphenylacetylene (2.40 mL, 12.41 mmol), tetrakis triphenylphosphine
palladium(0) (0.48 g, 0.41 mmol), copper(I) iodide (0.16 g, 0.83 mmol) and
DIPEA (1.44 mL, 8.27 mmol) and the reaction mixture was stirred at room
temperature, under an Argon atmosphere overnight. After this period were
added copper(I) iodide (0.16 g, 0.83 mmol) and anhydrous TEA (20 mL) and the
reaction mixture was stirred at 85 °C for 8 h. The solvent was then removed in
vacuo and the residue was triturated with DCM and stirred at room
temperature for 2 h. The solid was filtered and washed with DCM to give the
a
50% Effective concentration, or compound concentration required to reduce
viral plaque formation by 50% in the VZV-infected human embryonic (HEL) cell
cultures.
b
Minimal cytotoxic concentration, or compound concentration that results in a
microscopical alteration of HEL cell morphology.
When tested in vitro against two strains of TK-competent VZV13
(Table 3) we found that (10) was moderately antivirally active, being
ca. 100 times less active than (1). The 20,20-difluoro BCNA (11) was
also markedly less active than the parent drug 1. By contrast, the b
fluoro derivative (9) was highly active, being comparable to (1) in
its anti-VZV activity. As previously shown,1,2 all compounds lost
activity versus VZV TK- deficient virus strains, confirming their need
for VZV TK-mediated activation (phosphorylation).
desired compound as light brown solid (0.78 g, 45%).
A sample of this
compound was further purified by filtration trough silica gel for testing. 19F
NMR (DMSO-d6, 471 MHz): d ꢁ197.85. 1H NMR (DMSO-d6, 500 MHz): d 8.73
(1H, s, H-4), 7.74 (2H, d, J = 8.25 Hz, Ph), 7.33 (2H, d, 8.25 Hz, Ph), 7.22 (1H, s, H-
5), 6.25, 6.22 (1H, 2d, J = 3.70 Hz, JH–F = 17.10 Hz, H-10), 6.07 (1H, d, J = 4.60 Hz,
30-OH), 5.28 (1H, t, J = 5.80 Hz, 50-OH), 5.24, 5.14 (1H, 2dd, J = 2.35 Hz, J = 3.65,
JH–F = 52 Hz, H-20), 4.29, 4.26 (1H, 2dd, J = 4.30 Hz, J = 6.10, JH–F = 18.16 Hz, H-
30), 3.99 (1H, q, H-40), 3.71–3.63 (2H, m, H-50), 2.62 (2H, t,
a-CH2), 1.59 (2H, qn,
b-CH2), 1.35–1.24 (4H, m, 2 ꢂ CH2), 0.86 (3H, t, CH3). 13C NMR (DMSO-d6,
As noticed before for other BCNA derivatives,11 phosphorylation
by VZV TK proved necessary, but not sufficient to display potent
antiviral efficacy. In fact, the parent compound 1 showed an equal
capacity for phosphorylation to that of compound 11 (difluoro-
derivative) but proved P100-fold more antivirally active than 11.
Instead, the b-fluoro derivative 9 could be ꢀ3.5-fold better phos-
phorylated than 1, but was found equally antivirally active as 1.
Thus, there is no correlation between antiviral potency and VZV
TK affinity (substrate) properties indicating that other factors are
important for eventual antiviral action.11
126 MHz): d 13.85 (CH3), 21.87 (CH2), 30.32 (b-CH2), 30.78 (CH2), 34.87 (a-
CH2), 60.13 (C-50), 73.08 (d, JC–F = 24.49 Hz, C-30), 85.22 (C-40), 86.15 (d, JC–
F = 16.64 Hz, C-10), 94.66 (d, JC–F = 191.53 Hz, C-20) 98.60 (C-5), 107.14 (C-4a),
124.61 (Ph), 125.70 (ipso-C), 128.99 (Ph), 138.70 (C-4), 144.20 (para-C), 153.47
(C-6), 154.20 (C-2), 171.30 (C-7a). EI MS = 416.1749 (M+). HPLC = H2O/CH3CN
from 100/0 to 0/100 in 30 min = retention time 23.81 min. 3-(20-
a
-Fluoro-20-
[2,3-
deoxy-b-D-ribofuranosyl)-6-(4-n-pentylphenyl)-2,3-dihydrofuro
d]pyrimidin-2-one (10). 19F NMR (DMSO-d6, 471 MHz): d ꢁ201.19. 1H NMR
(DMSO-d6, 500 MHz):
d
8.94 (1H, s, H-4), 7.75 (2H, Ha) ꢁ7.32 (2H, Hb)
(3J = 8.10), 7.22 (1H, s, H-5), 6.02 (1H, d, J = 17.0, H-10), 5.60 (1H, d, J = 6.60, 30-
OH), 5.42 (1H, t, J = 4.90, 50-OH), 5.00 (1H, dd, J = 3.70, JF = 52.65, H-20), 4.26–
4.20 (1H, m, H-30), 3.95 (1H, dd, J = 12.4, 2.9, H-40), 3.77–3.67 (2H, m, H-50),
2.61 (2H, t, J = 7.6,
a-CH2), 1.65–1.61 (2H, m, b-CH2), 1.42–1.18 (4H, m, g/d-
Thus, in conclusion, we report the synthesis of the
a- and b-
CH2), 0.86 (3H, t, J = 6.90, CH3). 13C NMR (DMSO-d6, 126 MHz): d 13.84 (CH3),
21.88, 30.33, 30.79, 34.88 (C4H8), 58.42 (C-50), 66.31 (d, JC–F = 16.3, C-30), 83.04
(C-40), 89.54 (d, JC–F = 34.0, C-10), 94.16 (d, JC–F = 185.68, C-20), 98.56 (C-5),
107.19 (C-4a), 124.58 (C-Hb), 125.77 (ipso-C), 128.96 (C-Ha), 137.77 (C-4),
144.14 (para-C), 153.69 (C-6), 154.12 (C-2), 171.27 (C-7a). EI MS = 416.1738
(M+). Anal. Calcd for C22H25FN2O5ꢃ0.5H2O: C, 62.11; H, 6.16; N, 6.58. Found: C,
mono-20-fluoro analogues 10 and 9 of the potent anti-VZV BCNA
(1) and also the 20,20-difluoro BCNA 11. Only the 20-b-fluoro ana-
logue retains full low-nanomolar potency versus VZV in cell
culture.
61.73; H, 6.15; N, 6.41. 3-(20-Difluoro-20-deoxy-b-
D-ribofuranosyl)-6-(4-n-
pentylphenyl)-2,3-dihydrofuro [2,3-d]pyrimidin-2-one (11). 19F NMR (DMSO-
d6, 471 MHz): d ꢁ116.84. 1H NMR (DMSO-d6, 500 MHz): d 8.75 (1H, s, H-4),
7.76 (2H, Ha) ꢁ7.34 (2H, Hb) (3J = 8.20), 7.23 (1H, s, H-5), 6.35 (1H, d, J = 6.50, H-
10), 6.33–6.30 (1H, m, 30-OH), 5.43 (1H, t, J = 5.30, 50-OH), 4.36–4.18 (1H, m, H-
30), 3.99–3.95 (1H, m, H-40), 3.91–3.85 (1H, m, H-50), 3.75–3.69 (1H, m, H-50),
Acknowledgements
The authors would like to thank Inhibitex for their support. This
work was also supported by the Geconcerteerde Onderzoeksacties
(GOA) Grant No. 05/19. We are grateful to Ms. H. Murphy for sec-
retarial services, and L. van Berckelaer, R. Van Berwaer, A. Camps, L.
Van den Heurck and S. Carmans for technical assistance.
2.63 (2H, t, J = 7.6
a-CH2), 1.68–1.52 (2H, m, b-CH2), 1.40–1.21 (4H, m, g/d-
CH2), 0.87 (3H, t, J = 7.00, CH3). 13C NMR (DMSO-d6, 126 MHz): d 13.85 (CH3),
21.88, 30.32, 30.79, 34.90 (C4H8), 58.62 (C-50), 68.14 (t, JC–F = 22.40, C-30), 81.11
(C-40), 85.14 (t, JC–F = 31.2, C-10), 98.37 (C-5), 107.94 (C-4a), 120.84, 122.91 (C-
20), 124.75 (C–Hb), 125.56 (ipso-C), 129.01 (C–Ha), 137.26 (C-4), 144.44 (para-
C), 153.68 (C-6), 154.87 (C-2), 171.53 (C-7a). EI MS = 435.1731 (M+H). Anal.
Calcd for C22H24F2N2O5ꢃ0.5H2O: C, 59.59; H, 5.68; N, 6.32. Found: C, 59.38; H,
5.59; N, 6.25.
Supplementary data
11. Sienaert, R.; Andrei, G.; Snoeck, R.; De Clercq, E.; McGuigan, C.; Balzarini, J.
Biochem. Biophys. Res. Commun. 2004, 315, 877.
12. Procedure of the VZV thymidine kinase experiments. The IC50 of the test
compounds against phosphorylation of [CH3-3H] dThd as the natural substrate
by VZV TK was determined under the following reaction conditions: the
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.09.116.
References and notes
standard reaction mixture (50
MgCl2, 10 mM dithiothreitol, 2.5 mM ATP, 10 mM NaF, 1.0 mg/mL bovine
serum albumin, 1 Ci), an appropriate amount of test
M [CH3-3H] dThd (0.1
compound and 5 L milli Q water. The reaction was started by the addition of
lL) contained 50 mM Tris–HCl, pH8.0, 2.5 mM
1. McGuigan, C.; Yarnold, C. J.; Jones, G.; Velázquez, S.; Barucki, H.; Brancale, A.;
l
l
Andrei, G.; Snoeck, R.; De Clercq, E.; Balzarini, J. J. Med. Chem. 1999, 42, 4479.
l

C. McGuigan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6264–6267
6267
enzyme, and incubated at 37 °C for 30 min, and the reaction was terminated by
spotting an aliquot of 45 L onto DE-81 discs (Whatman, Maidstone, England).
After 15 min, the discs were washed for three times 5 min in 1 mM HCOONH4
while shaking, followed by 5 min in ethanol (70%). Finally, the filters were
dried and assayed for radioactivity in a toluene-based scintillant. The IC50 was
gradient to 75% buffer and 25% ACN; 10 min linear gradient to 65% buffer and
35% ACN; 10 min linear gradient to 50% buffer and 50% ACN; 10 min isocratic
flow; 5 min linear gradient to 98% buffer and 2% ACN; 5 min equilibration at
the same conditions. Metabolites of the BCNAs were determined by
fluorescence detection (excitation at 340 nm and emission at 415 nm).
Retention times of BCNA nucleoside and 50-monophosphate derivatives were
as follows: 1 (Cf 1743): 30.2 and 22.0 min; 9 (Cf 2852): 31.8 and 24.0 min; 10
(Cf 2792): 31.3 and 22.7 min; 11 (Cf 2819): 32.8 and 24.5 min, respectively.
13. Procedure of the anti-VZV experiments in HEL cell cultures. The laboratory
wild-type VZV strain OKA and the thymidine kinase-deficient VZV strain 07/1
were used. The OKA strain was supplied by Dr. M. Takahashi, Osaka University,
Osaka, Japan. The YS strain was isolated from vesicular fluid of a patient with
varicella and the TK-deficient 07/1 strain was isolated alter exposure of BVaraU
to VZV (YS)—infected cell cultures (Sakuma, Antimicrob. Agents Chemother.
1984, 25, 742). Confluent HEL cell cultures grown in 96-well microtiter plates
were inoculated with VZV at an input of 20 PFU per well. After a 2-h incubation
period, residual virus was removed and varying concentrations of the test
compounds were added (in duplicate). Antiviral activity was expressed as the
50%-effective concentration required to reduce viral plaque formation after
5 days by 50% as compared with untreated controls. Cytotoxicity was
expressed as the minimum cytotoxic concentration (MCC) or the compound
l
defined as the drug concentration required to inhibit
1 lM thymidine
phosphorylation by 50%. Thymidine kinase assays to evaluate the test
compounds as a substrate for the enzyme were performed as follows: the
standard reaction mixture contained 50 mM Tris HCl pH 8, 2.5 mM MgCl2,
10 mM dithiothreitol, 2.5 mM ATP, 10 mM NaF, 10
compound at various concentrations (0.5, 2, 5 and 12.5
l
l
L milli Q water, test
M) dissolved in DMSO
and 5
mixture of 50
the reaction was terminated by transferring the contents into 150
methanol, followed after 10 min by centrifugation at 12,000g. The resulting
samples were injected on Waters HPLC to separate and quantify the
nucleoside and 50-monophosphates of the BCNAs. HPLC analysis to separate
and quantitate the lipophilic reaction products was done on Merck
(Darmstadt, Germany) LiChroCART 125-4 RP column (5 m) using the
l
L of an appropriate amount (1.5 pg) of protein VZV TK in a total reaction
L. The reaction mixture was incubated at 37 °C for 60 min, and
L ice-cold
l
l
a
a
l
following gradient (flow 1 mL/min); 2 min at 98% NaH2PO4 (Acros, New
Yersey, USA) 50 mM + heptanesulfonic acid 5 mM pH 3.2 (buffer) (Sigma, St.
Louis, MO) and 2% acetonitrile (ACN) (Biosolve, Valkenswaard, The
Netherlands); 6 min linear gradient to 80% buffer and 20% ACN; 2 min linear
concentration that causes
morphology.
a microscopically detectable alteration of cell