Antibody Sensors
1006±1012
meter from SPEX Industries, and a Cytofluor II Plate-Reader from
Perseptive Biosystems.
(m, 4H), 2.87 (s, 3H), 2.23 (t, 7.4 Hz, 2H), 1.82 (m, 4H), 1.63 (m, 1H), 1.51
13
(m, 1H), 1.45 (m, 2H), 0.70 (t, J 7.4 Hz, 3H); C NMR (D
2 3
O/CD OD):
d 177.51, 170.74, 136.68, 134.36, 131.51, 128.63, 68.10, 63.91, 61.99, 47.50,
N-{[4'-(Hydroxyethyl)carbamido]phenyl}methyl-N-[3-(N'-acridonyl)-1''-
propyl]-N,N-dimethylpiperidinium trifluoroacetate (3): N-(3-dimethylami-
3
9.89, 36.68, 21.62, 20.63, 18.96, 13.75; HRMS (LSIM): C20
H
31
N
2
O
3
calcd
[30]
347.2327, found 347.23343.
no-1-propyl)-acridone (20 mg, 0.07 mmol) and N-hydroxyethyl-4-chloro
methyl-benzamide[ (30 mg, 0.14 mmol) were stirred in DMF (0.2 mL) for
two days at 208C. Dilution with water and purification by preparative RP-
HPLC gave 3 as the trifluoroacetate salt (40 mg, 0.07 mmol, 100%). Pale
26]
Table 3. HPLC Conditions for substrates and products.[a]
%
A
% B
t
R
[min]
HPLC purity
1
yellow crystalline solid, m.p. 206 ± 2088C (decomp); H NMR (300 MHz,
3
3
[b]
[b]
[c]
[b]
D
2
O): d 8.13 (dd, J 8.5, 2 Hz, 2H), 7.72 (ddd, J 9, 7, 2 Hz, 2H), 7.47
1
2
3
6
7
90
90
50
90
70
80
10
10
50
10
30
20
4.99
> 99%
> 98%
> 99%
> 99%
> 99%
ꢀ 80%
3
3
3
(
d, J 9 Hz, 2H), 7.33 (d, J 8.5 Hz, 2H), 7.26 (t, J 7 Hz, 2H), 7.01 (d,
7.53
3.80
4.73
8.03
3
3
3
J 8.5 Hz, 2H), 4.40 (t, J 7 Hz, 2H), 4.31 (s, 2H), 3.80, 3.53 (2t, J
1
3
6
CD
1
1
Hz, 2 Â 2H), 2.96 (s, 8H), 2.28 (m, 2H); C NMR (100 MHz, D
2
O 30%
[
[
b]
b]
3
OD): d 179.3, 170.1, 141.7, 136.5, 136.1, 133.4, 131.0, 128.5, 127.5,
23.2, 121.6, 116.0, 68.5, 61.0, 51.3, 43.1, 42.1, 21.9; IR (KBr): nÄ 3412, 1682,
10
11.40
�
1
652, 1598, 1558, 1500, 1462, 1292, 1182, 1122, 754, 674 cm ; HRMS
[a] Isocratic elution at 1.5 mLmin 1, detection by UV at 230 nm, A 0.1%
�
(
FAB ): C28
H
32
N
3
O
3
calcd 458.2444, found 458.2461.
TFA in H
2
O, B 50:50 CH
3 2
CN/H O, analytical column: [b] Vydac 218TP-
1
-(2-Aminoethyl)-b-d-galactopyranoside (9):[31] The product was obtained
by aminolysis of 2-chloroethyl-tetra-O-acetyl-b-d-galactopyranoside
5
3
4 (C18, poresize 300 ) or [c] Microsorb-MV 86 ± 203-C5 (C18, poresize
00 ), 0.45 Â 22 cm. Preparative HPLC was performed at 100 mLmin
[
32]
� 1
(
1.22 g, 2.97 mmol) in aqueous ammonium hydroxyde (25%, 20 mL) at
� 1
on a Waters prepak cartridge 500 g, gradient 1% Bmin starting with
658C over 3 days in a pressure vial. After evaporation of solvent, the crude
1
0 ± 15% less B than the isocratic conditions.
product was dissolved in water (20 mL) and purified on Dowex 50X8
SO H-form, washing with water and water/MeOH 1:1, elution with 5%
and 25% NH in water). After lyophilization 9 (473 mg, 2.12 mmol, 71%)
(
3
Antibodies: Monoclonal antibodies against the KLH conjugate of 1 were
3
produced by standard procedures, and were obtained from ascites fluid
was obtained. Spectral data corresponded to published data.
[33]
grown from the individual hybridoma cell lines.
Each antibody was
N-{[4'-(b-d-Galactosyloxyethyl)carbamido]phenyl}methyl-N-methylpiper-
idinium trifluoroacetate (6): 1-(4-carboxy-benzyl)-1-methylpiperidinium
chloride 2 (584 mg, 2 mmol) in DMF (6 mL) and water (0.6 mL) was
treated overnight with N-ethyl-N'-diethylaminopropylcarbodiimide hydro-
chloride (EDC; 766 mg, 4 mmol) and N-hydroxysuccinimide (460 mg,
purified to homogeneity by ammonium sulfate precipitation, followed by
anion exchange and protein G chromatography, dialyzed into phosphate
(10mm) and NaCl (160mm) with pH 7.4, and its concentration estimated by
UV at 280 nm as c (mgmL ) Abs/1.4.
�
1
Fluorescence measurements: Fluorescence measurements were carried out
at 208C in a 1 Â 1 cm cuvette with lexc 356 nm, lem 445 nm, set to give
maximum fluorescence reading (2.0) at 445 nm for 2mm free 3. All
measurements were taken at 208C in PBS (aqueous 160mm NaCl, 10mm
phosphate, pH 7.4). Kinetic assays were also carried out in 96-well
polypropylene round-bottom plates by use of a Cytofluor II Plate-Reader
from Perseptive Biosystems, with emission filter lex 360 Æ 20 nm, and
emission filter lem 440 Æ 20 nm. Results were identical to measurements
done in 1 Â 1 cm cells.
Equilibrium measurements (data in Table 1 and Figure 1): Aliquots of
properly prediluted solutions of analyte 1, 2, 5, 6 and 7 were added to the
sensor (2 mL; 1mm 3 0.5mm antibody 34F7 and/or 0.5mm antibody 83B8).
The solution was then mixed with a 1 mL pipetter and allowed to stand for
2 minutes before recording fluorescence.
4
mmol) at 208C. Purification by preparative HPLC and lyophilization of
the product-containing fractions gave 10 (549 mg, 1.23 mmol, 62%) as a
colorless solid. The product contained 15 ± 20% (according to HPLC
analysis) of 2 and was used as such.
Spectral data for 10: 1H NMR (300 MHz, D
H), 7.45 (d, J 8.5 Hz, 2H), 4.34 (s, 2H), 3.15 (m, 4H), 2.75 (s, 3H), 2.74
O): d 7.86 (d, J 8.5 Hz,
2
2
(
D
2
1
3
s, 4H), 1.68 (m, 4H), 1.47 (m, 1H), 1.34 (m, 1H); C NMR (100 MHz,
O/CD
OD): d 176.7, 173.3, 169.7, 135.1, 134.8, 131.7, 127.3, 61.1, 47.5,
6.5, 26.3, 21.5, 20.6.
Amine 9 (112 mg, 0.43 mmol), activated ester 10 (266 mg, 0.6 mmol), and
NaHCO (80 mg, 1 mmol) were stirred overnight. HPLC analysis showed
2
3
3
complete consumption of active ester. Preparative HPLC and lyophiliza-
tion of the product containing fractions gave 6 as colorless sirup (88 mg,
1
0
2
3
.16 mmol, 37%). H NMR (300 MHz, D
2
O): d 7.75, 7.53 (2d, J 8.5 Hz,
Kinetics of exchange (data in Figure 2): The reaction was initiated by adding
a prediluted solution of hapten 1 (1 mL) to the sensor solution (1 mL; 1mm
 2H), 4.44 (s, 2H), 4.33 (d, J 7.7 Hz, 1H), 3.98 (m, 1H), 3.80 (m, 2H),
.64 ± 3.49 (m, 6H), 3.43 (dd, J 7.7, 9.9 Hz, 1H), 3.30 (m, 4H), 2.87 (s, 3H),
{
3 ´ 34F7} complex) in the fluorometer cell and mixing for 5 seconds.
3
1
.82 (m, 4H), 1.62 (m, 1H), 1.54 (m, 1H); 1 C NMR (100 MHz, D
2
O/
Enzyme kinetics (data in Figures 4 and 5, Table 2): Assay mixtures were
initiated by adding an enzyme stock solution to PBS (2 mL) containing
sensor {3 ´ 34F7} (1mm) and substrate 6 or 7 at the given concentration in
CD
3
OD): d 171.03, 136.59, 134.31, 131.45, 128.71, 104.11, 76.14, 73.71,
7
1.78, 69.58, 69.42, 68.15, 61.93, 61.90, 47.51, 41.04, 21.57, 20.61; HRMS
(
EI ): C22
H N O [M ] calcd 439.2435, found 439.24597.
35 2 7
�
1
PBS. b-galacotsidase (Fluka 48274) contained 540 Umg {1 U (U unit)
N-(Butanoyloxyethyl)-4-chloromethylbenzamide (11): Triethylamine
� 1
hydrolyses 1 mmolmin 4-nitrophenyl-b-d-galactoside at pH 7.8, 378C).
(
(
160 mg, 1.6 mmol), catalytic amounts of DMAP and butyroyl chloride
� 1
Hog-liver esterase (Fluka 46058) contained 240 Umg (1 U hydrolyses
173 mg, 1.6 mmol) were added to a suspension of N-(hydroxyethyl)-4-
� 1
1
mmolmin ethyl valerate at pH 8.0, 258C).
[26]
chloromethylbenzamide (213 mg, 1 mmol) in dry toluene (20 mL). The
mixture was stirred for 90 min at 208C and then diluted with toluene
(
20 mL), washed (3 Â aq. sat. NaHCO
3 2 4
), and dried over Na SO . Solvent
evaporation and chromatography (hexane/EtOAc 1:3) gave 11 (256 mg,
0
(
3
Acknowledgment
1
.9 mmol, 90%) as colorless oil. H NMR (300 MHz, CDCl
3
): d 7.73, 7.42
This work was supported by the Deutsche Forschungsgemeinschaft, the
Ciba Jubiläums Stiftung (P.G.), the Humboldt Stiftung (N.B.), the Swiss
National Science Foundation, the Koordinationsgruppe für Forschungs-
fragen der Basler chemischen Industrie (KGF), and the Wander Stiftung.
2d, J 8.5 Hz, 2 Â 2H), 6.65 (s, 1H), 4.58 (s, 2H), 4.28 (t, J 5.15 Hz, 2H),
.69 (m, J 5.15, 5.51 Hz, 2H), 2.30 (t, J 7.7 Hz, 2H), 1.63 (m, J 7.7,
7
.4 Hz, 2H), 0.91 (t, J 7.4 Hz, 3H); HRMS (LSIM): C14
H
18ClNO
3
[M ]
calcd. 284.10491 found 284.10516; IR (CHCl
3
): nÄ 3018, 2968, 1730, 1654,
�
1
1
534, 1504, 1458, 1266, 1182, 1094, 668 cm .
N-{4'-[(Butanoyloxyethyl)carbamido]phenyl}methyl-N-methylpiperidini-
um trifluoroacetate (7): Butyrate 11 (256 mg) in N-methylpiperidine
[1] a) P. G. Schultz, R. A. Lerner, Science 1995, 269, 1835; b) S. Borman,
Chem. Eng. News 1996, 74(45), 37.
(
1 mL) and DMF (1 mL) was stirred for 60 min at 408C. After solvent
evaporation the residue was dissolved in water (30 mL) and purification by
[2] a) D. S. Tawfik, B. S. Green, R. Chap, M. Sela, Z. Eshhar, Proc. Natl.
Acad. Sci. USA 1993, 90, 373; b) G. MacBeath, D. Hilvert, J. Am.
Chem. Soc. 1994, 116, 6101; c) F. Benedetti, F. Berti, F. Massimiliano,
M. Resmini, E. Bastiani, Anal. Biochem. 1998, 256, 67.
preparative RP-HPLC gave substrate 7 (255 mg, 0.554 mmol, 62%) as
colorless solid. 1H NMR (300 MHz, D
O): d 7.72, 7.54 (2d, J 8.1 Hz,
2
2x2H), 4.45 (s, 2H), 4.21 (t, J 5.2 Hz, 2H), 3.57 (t, J 5.2 Hz, 2H), 3.27
Chem. Eur. J. 1999, 5, No. 3
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1999
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1011