S. Neelakantan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5722–5726
5725
LysLysLys-isoluminol probe from all bacteria equally. However,
sufficient probe remained after the second or third washings to al-
low bacterial estimates to be observed. A second isoluminol probe
containing GluGlu as the active binding moiety appeared to be
slightly more specific than the LysLysLys-isoluminol probe, in that
it had a greater affinity for L. lactis ssp. lactis C2 than for either S.
typhimurium, S. aureus, or E. coli. This specificity was most notice-
able after the second and third washings, which would be the ex-
pected clean-up procedure for a commercial test. These data
suggest that reporter probes derived from peptides which mimic
bacteriophage attachment protein binding regions can be synthe-
sized using the chemistry described. As more complex peptide se-
quences are utilized in the synthesis and design of second
generation probes, the specificity is expected to improve. Thus,
specific probes suitable for a rapid assay procedure might be pro-
duced using these procedures, and these could be utilized to iden-
tify a particular genus of bacteria, such as Listeria and E. coli, which
are known to be problematic pathogens in foods.
2 h at 0 °C. The reaction mixture was acidified to pH 2.0 with concentrated
hydrochloric acid and then concentrated under vacuum. The solid obtained
was refluxed with 20 ml of acetic anhydride for 15 min and allowed to stand
overnight. The crystals obtained were washed with acetic acid, then water, and
dried under vacuum. The mother liquor, which contained additional product,
was evaporated and the slurry was subjected to silica gel (240–300 mesh) flash
column chromatography with ethyl acetate and petroleum ether as eluant to
afford additional amounts of 7. The total yield of 2-methyl-5-nitro-isoindole-
1
1,3-dione (7) was 4.9 g (48%), mp 177 °C. H NMR (CDCl
3
300 MHz) d 8.74(lH,
13
s), 8.62(1H, d, J = 7.0 Hz), 8.10 (1H, d, J = 7.0 Hz), 3.21 (3H, s) ppm. C NMR
CDCl , 75 MHz): d 165.9, 151.4, 136.3, 133.3, 129.0, 124.1, 118.4, 24.5 ppm.
MS: m/z 206 (M ). 5-Amino-2-methyl-isoindole-1,3-dione (8): to a solution of
SnCl dihydrate (19.2 g, 85 mmol) in 8 ml water and 24 ml of concentrated
hydrochloric acid was added 2-methyl-5-nitro-isoindole-1,3-dione (7) (4.5 g,
1.8 mmol). The mixture was stirred vigorously for 2 h, during which time the
(
3
+
2
2
cloudy solution turned clear. The reaction was then allowed to stand overnight,
and the mixture concentrated to three fourths its volume on a rotovac. The
solid obtained was filtered and washed with water several times (Note: on
washing with water the solid becomes yellow in color). The resulting solid was
then dried under vacuum to afford 5-amino-2-methyl-isoindole-1,3-dione (8);
1
yield: 2.1 g (54.7%), mp 247–248 °C. H NMR (DMSO-d , 300 MHz): d 7.50 (1H,
6
1
3
m), 6.90 (1H, m), 6.76 (1H, m), 6.44 (2H, s), 2.95 (3H, s) ppm. C NMR (DMSO-
, 75 MHz): d 168.1, 154.6, 134.4, 124.5, 116.7, 106.8, 23.3 ppm. MS: m/z 176
M ). N-Methyl-4-N-[6-(N-phthalimido)hexyl]aminophthal- imide (9): to 2-(6-
d
6
+
(
bromohexyl)isoindole-1, 3-dione (4) (7.04 g, 22.7 mmol) in 20 ml of anhydrous
Acknowledgement
DMF under a nitrogen atmosphere was added 5-amino-2-methyl-isoindole-
1
1
,3-dione (8) (4 g, 22.7 mmol). The mixture was stirred under nitrogen at
20 °C for 30 h, and then cooled to room temperature and poured into ice-cold
This work was supported by a grant from the Kentucky Science
water with stirring. A yellow precipitate was obtained, which was filtered and
&
Engineering Foundation KSEF-311-RDE-003.
recrystallized from 70% aqueous acetic acid to afford N-methyl-4-N-[6-(N-
phthalimido)hexyl]amino phthalimide (9); yield: 4.5 g (49%), mp 169–170 °C.
1
6
H NMR (DMSO-d , 300 MHz): d 7.82 (4H, m), 7.46 (1H, m), 6.96 (1H, m), 6.86
References and notes
(
1
1H, d, J = 7.0 Hz), 6.74 (1H, m), 3.55 (2H, t, J = 9.0 Hz), 3.20 (2H, m), 2.90 (3H, s),
.60 (4H, m), 1.30 (4H, m) ppm. 13C NMR (DMSO-d
6
, 75 MHz): d 168.1, 167.6,
1
.
Doyle, M. P.; Beuchat, L. R. Food Microbiology, 3rd ed.; ASM Press: Washington
DC, 2007. 757–759;775–778. ISBN-13:978-1-55581-407-6.
Kraus, J.; Geller, B. L. J. Dairy Sci. 1998, 81, 2329.
Garbutt, K. C.; Kraus, J.; Geller, B. L. J. Dairy Sci. 1997, 80, 1512.
Lubbers, M. W.; Waterfield, N. R.; Beresford, T. P. J.; LePage, R. W. F.; Jarvis, A.
W. Appl. Environ. Microbiol. 1995, 61, 4348.
153.8, 134.4, 134.1, 124.4, 122.7, 116.2, 114.3, 105.6, 42.3, 37.3, 28.6, 27.9,
+
26.09, 26.04, 23.4 ppm. MS: m/z 405 (M ). 4-[N-Ethyl-N-(6-N-phthal-
2
3
4
.
.
.
imido)hexyl]amino-N-methylphthalimide (10): to N-methyl-4-N-[6-(N-phtha-
limido)hexyl]aminophthal- imide (9) (3.7 g, 9.2 mmol) in a flask under
nitrogen was added diethyl sulfate (11 ml, 70.84 mmol). The mixture was
gradually heated with stirring at 110 °C for 2 h, and then at 160 °C for a further
20 min, during which time the reaction turned brown in color. The mixture
was then cooled to room temperature and poured into ice-cold water with
stirring. The yellow precipitate obtained was filtered and recrystallized from
5
6
7
8
.
.
.
.
Ustunol, Z.; Hicks, C. L. J. Dairy Sci. 1994, 77, 1479.
Onourah, C. E.; Hicks, C. L.; Oleary, J. J. Dairy Sci. 2000, 78, 110. D48.
Hicks, C. L.; Onuorah, C. E.; Surjawan, I. J. Dairy Sci. 2000, 83, 1196.
Hicks, C. L.; Clark-Safko, P. A.; Surjawan, I.; O’Leary, J. J. Food Res. Int. 2004, 37,
70% aqueous acetic acid to yield 4-[N-ethyl-N-(6-N-phthalimido)hexyl]-
1
1
15.
amino-N-methylphthalimide (10); yield: 2.3 g (58%), mp 195–196 °C.
H
9
.
Robbins, J. C.; Lam, M. H.; Tripp, C. S.; Bugianesi, R. L.; Ponpipom, M. M.; Shen, T.
NMR (DMSO-d , 300 MHz): d 7.80 (4H, m), 7.45 (1H, m), 6.90 (1H, s), 6.80
6
Y. Proc. Nat. Acad. Sci. 1981, 78, 7294.
(1H, m), 3.54 (2H, t, J = 9.0 Hz), 3.44 (2H, m), 3.34 (2H, t, J = 7.0 Hz), 2.94 (3H, s),
1.56 (4H, m), 1.32 (4H, m), 1.10 (3H, t, J = 12.0 Hz) ppm. 13C NMR (DMSO-d6,
75 MHz): d 168.2, 167.7, 134.5, 134.2, 131.4, 124.5, 122.8, 115.7, 114.0, 104.5,
1
0. Representative characterization data for conjugates and intermediates. 2-(6-
Hydroxyhexyl)-isoindole-1,3-dione (3): to mixture of phthalic anhydride
14.8 g, 100 mmol) and 6-aminohexan-1-ol (11.7 g, 100 mmol) in a round-
a
+
(
49.8, 44.7, 37.3, 27.9, 26.6, 26.1, 25.9, 23.4, 11.9 ppm. MS: m/z 434(M ). 6-[N-
bottomed flask was added 120 ml of anhydrous toluene. The flask was then
fitted with a Dean-Stark apparatus and the mixture refluxed for 12 h at 120 °C.
The water formed during the reaction was removed as an azeotrope. On
completion of the reaction, toluene was removed on a rotovac. Little unreacted
amine was detected is the resulting residue when monitored by silica gel thin
layer chromatography. The residue was subjected to flash column
chromatographic separation over silica gel (230–400 mesh) with ethyl
(6-Aminohexyl)-N-ethylamino]-2,3-dihydrophthalazine-l,4-dione (11): to 4-[N-
ethyl-N-(6-N-phthalimido)- hexyl]amino-N-methylphthalimide (10) (1.75 g,
4.04 mmol) in 20 ml absolute ethanol under nitrogen was added hydrazine
(5 ml, 147.1 mmol). The reaction mixture was refluxed for 3 h under nitrogen.
The solvent was removed under vacuum and the resulting solid dried at 110 °C
under vacuum. The residue was then dissolved in 10% HCl and filtered to
remove insoluble impurities. The filtrate was then adjusted to pH 8 with
potassium hydroxide solution. The precipitate obtained was filtered and
crystallized from aqueous DMF to yield 6-[N-(6-aminohexyl)-N-ethylamino]-
2,3-dihydro- phthal-azine-l,4-dione (11); yield: 220 mg (18%) mp 151–152 °C.
acetate and petroleum ether as eluant, to afford 2-(6-hydroxy-hexyl)-
1
isoindole-1,3-dione (3) as a white solid; yield: 22.7 g (92%), mp 49–50 °C.
NMR (CDC1
.65 (2H, t, J = 7.0 Hz), 1.70 (2H, m), 1.55 (4H, m), 1.40 (4H, m) ppm. C NMR
CDC1 , 75 MHz): d 168.2, 133.6, 131.8, 122.9, 62.5, 37.7, 32.4, 28.4, 26.4,
H
3
, 300 MHz): d7.85 (2H, m), 7.65 (2H, m), 3.70 (2H, t, J = 7.0 Hz),
13
1
3
H NMR (DMSO-d , 300 MHz): d 7.80 (1H, m), 7.1 (2H, m), 3.45 (2H, m), 3.34
6
(
2
3
(2H, t, J = 9.0 Hz), 2.73 (2H, t, J = 7.0 Hz), 1.54 (4H, m), 1.34 (4H, m), 1.14 (3H, t,
+
13
5.1 ppm. MS: m/z 247 (M ). 2-(6-Bromohexyl)-isoindole-1,3-dione (4): to 6-
J = 12.0 Hz) ppm. C NMR (DMSO-d , 75 MHz): d 156.3, 155.5, 149.8, 129.8,
6
hydroxy hexylphthalimide (22.5 g, 0.09 mol) in 100 ml of anhydrous toluene at
126.9, 116.0, 103.6, 49.4, 44.3, 29.9, 26.3, 25.9, 25.8, 12.0 ppm. MS: m/z 304
+
1
0
10 °C was slowly added phosphorous tribromide in anhydrous toluene (17 g,
.063 mol in 20 mL of toluene) through a pressure-equalizing funnel. The
(M ). ({6-[(1,4-Dioxo-1,2,3,4-tetrahydrophthalazin-6-yl)-ethylamino]-hexylcarb-
amoyl}-methoxy)-acetic acid (12): to 6-[N-(6-aminohexyl)-N-ethylamino]-2,3
dihydrophthalazine-l,4-dione (11) (608 mg, 2 mmol) in 10 ml of anhydrous
DMF under nitrogen was added diglycolic anhydride (232 mg, 2 mmol) and
triethylamine (205 mg, 2 mmol). The reaction mixture was stirred under
nitrogen for 18 h. The solvent was removed under vacuum and the resulting oil
was dissolved in anhydrous methanol and kept at 4 °C. The product crystallized
from the solution after 18 hours and was filtered and dried under vacuum;
mixture was refluxed for 3 h and allowed to cool to room temperature. The
orange precipitate that formed was removed by filtering the reaction mixture
through Celite. The filtrate was then concentrated on a rotovac to afford an oil.
The resulting oil was crystallized from absolute ethanol to afford 2-(6-bromo-
hexyl)-isoindole-1, 3-dione (4) of analytical purity; yield: 25.1 g (90%), mp 59–
1
6
0 °C. H NMR (CDC1
3
, 300 MHz): d 7.82 (2H, m), 7.65 (2H, m), 3.65 (2H, t,
1
J = 7.0 Hz), 3.40 (2H, t, J = 7.0 Hz), 1.85 (2H, m), 1.65 (2H, m), 1.45 (2H, m), 1.34
2H, m) ppm. 13C NMR (CDC1
, 75 MHz): d 168.1, 133.6, 131.8, 122.9, 37.7,
3.6, 32.5, 28.3, 27.6, 25.9 ppm. MS: m/z 310 (M ). 5-Nitro-isobenzofuran-1,3-
yield: 420 mg (50%) mp 170–171 °C. H NMR (DMSO-d , 300 MHz): d 7.82 (1H,
6
(
3
3
m), 7.16 (1H, m), 7.10 (l H, s), 4.04 (2H, s), 3.94 (2H, s), 3.46 (2H, m), 3.36 (2H, t,
+
J = 9.0 Hz), 3.14 (2H, t, J = 7.0 Hz), 1.56 (2H, m), 1.44 (2H, m), 1.32 (4H, m) ppm.
13
dione (6): to 4-nitrophthalic acid (21.1 g, 0.1 mols) in a round-bottomed flask
was added acetic anhydride (40 ml) and the mixture refluxed for 1 h. The
mixture was brought to room temperature and the acetic anhydride was
removed under vacuum. The concentrated mixture was allowed to stand
6
C NMR (DMSO-d , 75 MHz): d 176.0, 170.9, 156.3, 155.5, 149.8, 129.8, 126.9,
116.0, 103.6, 74.7, 73.6, 49.4, 44.3, 29.9, 26.3, 25.9, 25.8, 12.0 ppm. MS: m/z 420
+
(M ), 402 (loss of H O), 218 (base peak). 2,5,8-Tris-(4-aminobutyl)-22-(1,4-
2
dioxo-1,2,3,4-tetrahydrophthalazin-6-yl)-4,7,10,14-tetraoxo-12-oxa-3,6,9,15,22-
pentaazatetracos- an-1-oic acid (14): to the isoluminol probe (12) (0.042 g,
0.01 mmol) in 5 ml of anhydrous methanol was added the coupling agent
DMTMM (0.041 g, 0.015 mmol) and the resulting mixture stirred under
ambient conditions for 30 min followed by addition of LysLysLys (13)
(0.040 g, 0.01 mmol). The reaction mixture was stirred for 5 h under ambient
conditions and then solvent was removed under vacuum. Flash
overnight. The solid obtained was recrystallized from toluene to afford 5-nitro-
1
isobenzofuran-1,3-dione (6); yield: 9.9 g (51%), mp 119 °C. H NMR (CDC1
3
,
,
1
3
3
7
00 MHz): d 8.90 (1H, s), 8.80 (1H, m), 8.40 (1H, m) ppm. C NMR (CDC1
+
3
5 MHz): d 162.3, 153.6, 136.8, 133.8, 127.2, 121.1 ppm. MS: m/z 193 (M ). 2-
Methyl-5-nitro-isoindole-1,3-dione (7): to 5-nitro-isobenzofuran-1,3-dione (6)
9.5 g, 49.2 mmol) in round-bottomed flask, was added 20 ml of 33%
methylamine solution in absolute ethanol and the mixture was stirred for
(
a
chromatography of the residue utilizing
a Sephadex G-25 column under