4196 J. Agric. Food Chem., Vol. 50, No. 15, 2002
Korpima¨ki et al.
product was purified with RP-HPLC as above. The peak at 14.35 min
found 788.6 [M + H]+, C37H57N9O6S2 + H requires 788.3873. The
final product (Figure 3, referred to as biotinylated sulfamerazine from
here on) was dissolved in 1 mL of DMF.
DELFIA-Eu-N1-ITC (10.1 mg) was dissolved in aqueous 50 mM
CO3- (0.20 mL) and added to the solution of N1-{[4-(5-amino)pentyl]-
2-pyrimidyl}sulfanilamide (4.9 mg) in aqueous 50 mM CO3- (0.4 mL).
The reaction mixture was stirred overnight at room temperature. The
product was purified with RP-HPLC using a 250 × 4 mm, 5 µm
Hypersil ODS C 18 column (Thermo Hypersil), gradient from 95% A
and 5% B to 100% B in 30 min, flow rate of 1.0 mL/min, and detection
at 280 nm (A ) aqueous 20 mM TEEA and B ) 20 mM TEEA +
50% acetonitrile). The peaks were analyzed using the mass spectrometer
function of the instrument. The peak at 17.91 min found 968.2 [M +
H]+, C35H43EuN9O10S2 + H requires 967.1787. The final product
(Figure 3, referred to as Eu-sulfamerazine from here on) was dissolved
in 1 mL of DMF.
Cloning of Mab27G3 to scFv Format. Messenger RNA was
isolated from a frozen pellet of 107 hybridoma cells of the 27G3A9B10
line (2) using a Quickprep Micro mRNA purification kit (Amersham
Pharmacia Biotech, Espoo, Finland) as instructed by the manufacturer.
Complementary DNA was synthesized with a First-Strand cDNA
synthesis kit (Amersham Pharmacia Biotech) following the random
hexamer primer protocol of the kit. The variable domains of antibody
27G3 were amplified in PCR reactions using Pfu polymerase (Promega)
and the primer mixes provided in an RPAS mouse scFv module kit
(Amersham Pharmacia Biotech) as instructed by the manufacturer. PCR
products were cloned using the pGEM-T vector system (Promega) by
following the standard protocols provided with the kit. Several
individual clones of each variable chain were sequenced, and the correct
terminus sequences were deduced by comparison to the corresponding
germline gene sequences, which were retrieved from the GenBank
sequence database of the U.S. National Center of Biotechnology
Information. Primers matching the most closely related germline genes
were designed, and the variable chains were amplified again from cDNA
in PCR reactions containing 20 mM Tris-HCl (pH 8.8), 10 mM KCl,
10 mM (NH4)2SO4, 20 mM MgSO4, 0.1% Triton X-100, 0.1 mg/mL
bovine serum albumin (BSA), 200 µM dNTP, 5 µL of the cDNA
preparate, and 0.04 unit/µL of Pfu polymerase in a total volume of 50
µL. For the amplifications of the heavy chain, 5 pmol of HeavyFor
and HeavyRev primers were used, and for the case of the light chain,
5 pmol of LightFor and LightRev primers were used (Table 1). The
PCR program was 95 °C for 5 min, 7 cycles of 94 °C for 1 min, 63 °C
for 30 s, 58 °C for 50 s, and 72 °C for 1 min, and finally 23 cycles of
94 °C for 1 min, 63 °C for 30 s, and 72 °C for 1 min.
The obtained PCR products were connected to form a full-length
scFv gene [synthetic (Gly4Ser)4-linker and VL-VH orientation] in a strand
overlap extension PCR reaction (18). The reaction tube contained 20
mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 20 mM
MgSO4, 0.1% Triton X-100, 0.1 mg/mL BSA, 200 µM dNTP, 20 ng
of both amplified variable chains, and 0.04 unit/µL of Pfu polymerase
in a total volume of 50 µL. A PCR program of 95 °C for 3 min and 2
cycles of 94 °C for 1 min, 63 °C for 30 s, 58 °C for 50 s, and 72 °C
for 1 min was completed. Five picomoles each of SfiI site containing
primers ScFor and ScRev (Table 1) was added to the reaction vessel,
and the PCR was continued with the following program: 5 cycles of
94 °C for 1 min, 63 °C for 30 s, 58 °C for 50 s, and 72 °C for 1 min
and 23 cycles of 94 °C for 1 min, 63 °C for 30 s, and 72 °C for 1 min.
The resulting scFv27G3 gene was cloned to the pAK series of vectors
(Figure 2) using the introduced SfiI sites.
Primary Libraries. The primary libraries were constructed using
random mutagenesis by error-prone PCR (19, 20). The scFv27G3 gene
fragment was amplified in PCR reactions containing 10 mM Tris-HCl
(pH 9.0), 50 mM KCl, 0.1% Triton X-100, 0.01% gelatin, 2.5 mM
MgCl2, 20 µM each ATP and GTP, 100 µM each TTP and CTP, 0.5
mM MnCl2, 0.4 nM ScFor- primer, 0.4 nM ScRev primer (Table 1),
and 0.05 unit/µL Taq polymerase in a total reaction volume of 50 µL.
A high amount (600 ng) of template was used to obtain the low mutation
frequency library (library 1), and a low amount of template (6 ng) was
used to obtain the high mutation frequency library (library 4). This
was done by assuming that the mutation frequency is dependent on
Figure 3. Sulfonamide derivatives synthesized for use in phage display
and immunoassays: (A) biotinylated sulfamerazine derivative; (B) bio-
tinylated sulfathiazole derivative; (C) N1-europium chelate labeled sul-
famerazine derivative.
100 mL), dried (Na2SO4), and evaporated. The product was purified
with FC (silica gel, 30% ethyl acetate/petroleum ether).
{2-Amino-[4-(5-N-tert-butyloxycarbonylamino)pentyl]}pyrimidine (D).
A mixture of C (0.53 g) and guanidium nitrate (0.325 g) in ethanol
was heated in an oil bath until the guanidium nitrate was dissolved. A
solution of NaOH in water (0.213 g/0.70 mL) was added. The reaction
mixture was refluxed for 3 h and allowed to cool before evaporation.
The product was purified with FC (silica gel, 3% MeOH/dichloro-
methane).
N4-Acetyl-{N1 -[4-(5-N-tert-butoxycarbonylamino)pentyl]-2-pyrimidyl}-
sulfanilamide (E). N-Acetylsulfanilyl chloride (0.220 g) was dissolved
in dry pyridine and cooled in an ice bath. A cooled solution of D (0.240
g) in dry pyridine was added. The reaction mixture was stirred overnight
at room temperature, evaporated, and dissolved in dichloromethane (20
mL). The solution was washed once with brine (10 mL), dried (Na2-
SO4), and evaporated. The product was purified with FC (silica gel,
first 3/5/1 ) ethyl acetate/petroleum ether/triethylamine and finally the
product 10% MeOH/dichloromethane).
N1-{[4-(5-N-tert-Butoxycarbonylamino)pentyl]-2-pyrimidyl}sulfanil-
amide (F). E (0.039 g) was dissolved in 2 M NaOH (25 mL) and stirred
overnight at room temperature. NaCl was added to the reaction mixture
followed by dichloromethane (35 mL). The phases were separated, and
the water phase was extracted with dichloromethane (2 × 40 mL). The
combined organic phases were dried (Na2SO4) and evaporated. The
product was purified with FC (silica gel, 10% MeOH/dichloromethane).
N1-{[4-(5-Amino)pentyl]-2-pyrimidyl}sulfanilamide Monohydro-
chloride. F (0.024 g) was dissolved in 1 M HCl and stirred overnight
at room temperature. The reaction mixture was evaporated, and the
product was used without further purification: 1H NMR (200 MHz,
D2O) δ 1.12 (t, 2H, CH2), 1.42 (t, 4H, 2 × CH2), 2.53 (t, 2H, CH2),
2.78 (t, 2H, CH2), 6.75 (d, 2H, ArH), 6.87 (d, 1H, ArH), 7.67 (d, 2H,
ArH), 8.07 (d, 1H, ArH).
Biotinylation and Labeling of the Sulfonamide Derivatives. N1-
[4-(Carboxymethyl)-2-thiazolyl]sulfanilamide (3.24 mg) was first ac-
tivated with carbonyldimidazole (1.80 mg) in dry DMF (0.20 mL). The
mixture was stirred for 2 h and then added to a solution of Biotin-
XX-Hydratzide (4.81 mg) in dry DMF (0.30 mL). The reaction mixture
was stirred overnight at room temperature. The product was purified
with RP-HPLC using the LC/MS/MS instrument with a 150 × 4.6 mm,
5 µm HyPurity Elite C18 column (Thermo Hypersil, Runcorn, U.K.),
gradient from aqueous 0.1% TFA to acetonitrile in 30 min, flow rate
of 1.0 mL/min, and detection at 265 nm. The peaks were analyzed
using the mass spectrometer function of the instrument. The peak at
12.54 min found 780.6 [M + H]+, C33H43N9O7S2 + H requires
780.2917. The final product (Figure 3, referred to as biotinylated
sulfathiazole from here on) was dissolved in 1 mL of DMF.
Biotin-XX-NHS (5.88 mg) in dry DMF (0.4 mL) was added to the
solution of N1-{[4-(5-amino)pentyl]-2-pyrimidyl}sulfanilamide (3.85
mg) in dry DMF (0.2 mL). Diisopropylethylamine (2 µL) was added
to the reaction mixture and stirred overnight at room temperature. The