9
7 : 3 : 0.2 upon which a broad band containing 9 and 10 eluted.
The solution was refluxed under N
the solvent was removed in vacuo. Toluene (20 cm ) was added
2
for 1 h and upon cooling
3
The fractions that contained the band were evaporated in vacuo to
yield 9 and 10 as a black residue (108 mg, 27%).
3
and evaporated in vacuo. A mixture of toluene (20 cm ) and water
3
(
0.5 cm ) was added and the two-phase mixture was stirred for
Separation of DPIX amide-linked mono-metronidazole mono-
3
0 min. The solvent was removed in vacuo to yield a mixture of
products as a black residue. The residue was purified by silica
gel chromatography with an initial eluting solvent of CH Cl
CH OH–CH NO (30 : 1 : 1). The polarity was increased to 20 :
: 1 upon which the first band eluted. The polarity was further
acid adducts 9 and 10. The residue of 9 and 10 from method
2
(108 mg) was re-dissolved in a small amount of CH
passed through a Zorbax Rx-SIL column eluting isocratically with
CH Cl –CH OH–(CH CH N (99.5 : 0.5 : 0.2) to resolve the two
2
Cl
2
and
2
2
–
3
3
2
2
2
3
3
)
2 3
1
isomers 9 and 10. The fractions were re-chromatographed 12 times
until sufficient purity was obtained as confirmed by analytical
HPLC.
increased to 10 : 1 : 1 upon which the second band eluted. The
fractions that contained the first band were evaporated in vacuo
to yield the di-metronidazole-substituted adduct 14 (12 mg, 4%)
3
-1
-1
as a black solid. lmax (CHCl
3.99), 530 (3.72), 566 (3.63) and 620 (3.40) nm; nmax (CHCl
w (NH), 2992 m (aromatic CH), 2900 m (aliphatic CH), 1737 s
C=O), 1661/1465 m (C=C), 1528/1364 w (N=O), 1220 m (C–
3
) 400 (log e dm mol cm 5.10), 497
DPIX amide-linked mono-metronidazole mono-acid adducts 11
(
3
) 3316
and 12. Method 1. Hydrolysis of the combined isomers 9 and 10
(
68 mg, 0.101 mmol) occurred with LiOH (222 mg, 9.30 mmol)
in CH OH–THF–H O (3 : 4 : 1) with stirring for 2 h to give 11
and 12 (67 mg, quant.). lmax (CHCl
(
3
2
-
1
1
3
-1
-1
O) cm ; H NMR (300 MHz; CDCl
inner NH), 1.54 (3H, s, N=C–CH ), 1.56 (3H, s, N=C–CH
.17 (4H, t, J 7.4, –CH ), 3.56/3.57 (6H, s, –CH ), 3.70/3.72
6H, s, –CH ), 3.77–3.86 (4H, m, –CH ), 4.07–4.14 (4H, m, –
), 4.32 (4H, t, J 7.4, –CH ), 7.42 (1H, s, NO
C=CH), 7.47
3
; SiMe
4
) d
H
-4.04 (2H, s,
3
) 399 (log e dm mol cm
3
3
),
4
.55), 496 (3.72), 531 (3.60), 565 (3.54), 621 (3.45) nm; nmax (KBr)
3
2
3
3
493 w (NH), 3068 m (OH), 2980 m (aromatic CH), 2855 m
(
3
2
(
aliphatic CH), 1710 s (C=O), 1650/1467 m (C=C), 1536/1364
-1 1
CH
2
2
2
w (N=O), 1150 m (C–O) cm ; H NMR (300 MHz; DMSO-d
-4.09 (2H, s, inner NH), 2.36 (3H, s, N=C–CH
), 2.95–2.97
4H, m, –CH ), 3.48–3.50 (2H, m, –CH ), 3.57–3.74 (12H, m,
), 4.14–4.17 (2H, br t, –CH ), 4.30–4.32 (4H, m, –CH ),
.98 (1H, s, NO
)
6
(
1H, s, NO
2
C=CH), 9.05 (2H, s, b-pyrrolic H), 9.91 (1H, s, meso
d
H
3
H), 9.96 (1H, s, meso H), 10.03 (1H, s, meso H), 10.15 (1H, s,
(
2
2
+
meso H); m/z (MALDI-TOF) 817.7 [(M + H) requires 817.9];
–CH
3
2
2
+
m/z (FTICR-MS) 817.3349; [(M + H) , calcd for [C42
H
44
N
10
8
O +
7
2
C=CH), 9.27 (2H, s, b-pyrrolic), 9.70 (1H, br s,
+
H ]: 817.3416]. The fractions that contained the second band were
OH), 10.21–10.27 (4H, m, meso H), 10.54 (1H, s, NH); m/z (ESI-
MS) 663.5 [(M + H) requires 663.7]; m/z (FTICR-MS) 663.3012
+
evaporated in vacuo to yield 13a and 13b (26 mg, 8%) as a black
3
-1
-1
+
+
solid. lmax (CHCl
5
3
) 399 (log e dm mol cm 5.11), 497 (3.99),
) 3694 m
[
(M + H) , calcd for [C36
H
38
N
8
O
5
+ H ]: 663.3038]. Method 2.
30 (3.70), 566 (3.59) and 620 (3.36) nm; nmax (CHCl
3
Separated isomer residues 9 (5 mg, 0.007 mmol) and 10 (5 mg,
3
(NH), 3581 m (OH), 3089 m (aromatic CH), 2927 m (aliphatic
0
.007 mmol) were re-dissolved in CH
2
Cl (2 cm ), then a solution
2
3
3
3
CH), 1730 s (C=O), 1602/1467m (C=C), 1520/1364 w (N=O),
of NaOH (2 M, 0.8 cm ), CH OH (4 cm ) and dioxane (2 cm ) was
added. The mixture was stirred for 9 h at room temperature. The
mixture was acidified to approximately pH 3 with a solution of
hydrochloric acid (2 M) and extracted into CH
3
-
1 1
1
220 m (C–O) cm ; H NMR (300 MHz; CDCl
3
; SiMe
4
) d -4.32
H
(
2H, s, inner NH), 0.78 (3H, s, N=C–CH
3
), 3.09–3.15 (2H, br t,
), 3.43–3.65 (12H, m, –CH ),
), 4.04–4.21 (4H, m, –CH ), 7.11 (1H, s,
3
–CH
2
), 3.23–3.27 (2H, br t, –CH
2
3
2
Cl
2
(3 ¥ 10 cm ).
3
.90–3.97 (4H, m, –CH
2
2
The combined organic extracts were washed with water (2 ¥
3
3
NO
2
C=CH), 8.92/8.96 (2H, s, b-pyrrolic H), 9.81–9.91 (4H, m,
10 cm ), brine (1 ¥ 10 cm ), dried (anhydrous sodium sulfate)
+
meso H); m/z (MALDI-TOF) 664.5 [(M + H) requires 664.7];
and the solvent removed in vacuo to yield 11 (5 mg, quant.) and
+
1
m/z (FTICR-MS) 664.2869 [(M + H) , calcd for [C36
H
37
N
7
6
O +
1
2 (5 mg, quant.) as black residues. 11/Fr 1: H NMR (200 MHz;
DMSO-d ) d ),
-3.97 (2H, s, inner NH), 2.17 (3H, s, N=C–CH
.05 (2H, t, J 4.8, –CH ), 3.28 (2H, t, J 4.8, –CH ), 3.44 (2H, t,
J 3.9, –CH ), 3.69 (3H, s, –CH ), 3.70 (3H, s, –CH
CH ), 3.83 (3H, s, –CH ), 4.24 (2H, t, J 3.9, –CH
J 4.9, –CH
+
H ]: 664.2879].
6
H
3
3
2
2
DPIX ester-linked mono-metronidazole mono-acid adducts 13a
2
3
3
2
), 3.79 (3H, s,
), 4.36 (2H, t,
and 13b. DPIX 3 (50 mg, 0.098 mmol) was dissolved in refluxing
–
3
3
3
thionyl chloride (10 cm ) under N
2
and reacted for 45 min. The
2
), 4.44 (2H, t, J 4.9, –CH
.29 (1H, br s, O=C–NH), 9.38/9.40 (2H, s, b-pyrrolic H), 10.35–
2
), 8.00 (1H, s, NO
2
C=CH),
mixture was cooled and excess thionyl chloride was removed
8
1
3
in vacuo. The residue was dissolved in CH
7 mg, 0.041 mmol) and (CH CH N (0.07 cm , 0.501 mmol)
were added. The solution was refluxed under N for 3 h and
allowed to cool. DMF (0.03 cm ), dimethyl-tert-butylsilyl chloride
18 mg, 0.119 mmol) and imidazole (16 mg, 0.235) were added
2
Cl
2
(50 cm ) and 4
0.39 (4H, m, meso H), 12.35 (1H, br s, O=C–OH). 12/Fr 2:
3
(
3
)
2 3
1
H NMR (200 MHz; DMSO-d
6
) d
H
-3.98 (2H, s, inner NH),
2
2
.14 (3H, s, N=C–CH
J 6.9, –CH ), 3.36 (2H, t, J 5.7, –CH
3H, s, –CH ), 3.78 (3H, s, –CH ), 3.82 (3H, s, –CH
J 5.7, –CH ), 4.35 (2H, t, J 6.8, –CH ), 4.45 (2H, t, J 6.8, –CH
.99 (1H, s, NO
C=CH), 8.26 (1H, br s, O=C–NH), 9.38/9.40
3
), 3.06 (2H, t, J 6.9, –CH
), 3.66 (3H, s, –CH
), 4.23 (2H, t,
),
2
), 3.29 (2H, t,
3
2
2
3
), 3.72
(
(
3
3
3
and the mixture was stirred for 14 h. The solvent was removed
in vacuo to yield a mixture of products as a black residue. The
residue was purified by silica gel chromatography with the eluting
2
2
2
7
2
(
2H, s, b-pyrrolic H), 10.34–10.38 (4H, m, meso H), 12.35 (1H,
solvent of 13a and 13b (15 mg, 23%) being CH
10 : 1).
2
Cl
2
–CH OH
3
br s, O=C–OH).
(
DPIX ester-linked mono-metronidazole mono-acid adducts 13a
General procedures for biological testing. The translated prod-
and 13b and DPIX ester-linked di-metronidazole adduct 14. DPIX
uct of a synthetic HA2 gene, derived from the sequence of
3
(200 mg, 0.392 mmol) was converted to 7 as above. The
RgpA, was expressed in E. coli with a C-terminal polyhistidine
tag, was functionally purified by Hb-agarose affinity chro-
matography as previously described and is referred to as rHA2.
3
18
product was dissolved in CH
2
2
Cl (50 cm ) and metronidazole 4
CH N (5 drops) were added.
(
27 mg, 0.157 mmol) and (CH
3
2
)
3
2
862 | Org. Biomol. Chem., 2009, 7, 2855–2863
This journal is © The Royal Society of Chemistry 2009