FLUORINE-18 LABELING OF HUMAN C-PEPTIDE
513
LC-10AD pumps, a Gilson 115 UV absorbance detector at 220 nm and
a GM-tube (Alnor mini-monitor, series 900).
1
13
H- and C-NMR (90 MHz) were recorded using a JEOL FX90Q
spectrometer and chemical shifts are reported in ppm downfield from
internal tetramethylsilane (0.00 ppm) or from the known shift of the
solvent used.
4-Trimethylammnoium-benzonitrile trifluoromethanesulfonate was
synthesized from 4-(dimethylamino)benzonitrile using literature meth-
1
2,13
12
1
ods
d6) d 3.65 (s, 9 H), 8.2 (s, 4 H).
-Fluorobenzoic acid was produced by alkaline hydrolysis of the
; as intra m.p. 157–1598C (lit. 156–1588C ). H-NMR (DMSO-
4
14
corresponding nitrile. 4-Fluorobenzonitrile (6.06 g, 50.0 mmol) was
heated to 1108C with NaOH (2.5 M, 90 ml) and refluxed for 1 h, until
the liquid contained no oily drops and TLC (eluent CHCl ; Rf
3
acid ꢁ 0.0–0.1, R nitrile ꢁ 0.75) indicated that essentially all nitrile
f
had been consumed. Trace amounts of starting material were removed
by extraction with CHCl . HCl (12 M) was added to the aqueous phase
3
until acidic pH. The white precipitate was filtered and dissolved in
ethanol prior to azeotropic evaporation of the remaining water to give
the product (6.5 g, 46.4 mmol, 93% yield); m.p. 181–1838C (182.68C,
1
according to the Merck Index). H-NMR (MeOH-d4) d 7.05–7.35
1
3
(
m, 2 H), 7.95–8.2 (m, 2 H). C-NMR (MeOH-d4) d 115, 116, 133, 134,
1
62, 169, 173.
N-Succinimidyl 4-fluorobenzoate (SFB) was prepared from 4-
fluorobenzoic acid, N-hydroxysuccinimide and DCC according to
8
8
1
literature procedures ; m.p. 104–1068C (lit. 111–1138C ). H-NMR
(
CDCl ) d 2.90 (s, 4 H), 7.05–7.30 (m, 2 H), 8.05–8.30 (m, 2 H).
3
N-4-Fluorobenzoyl-C-peptide (FB-C-peptide) was synthesized by
reaction of C-peptide (2.25 mg, 0.745 mmol) with a 250-fold excess of
SFB (43.5 mg, 0.1835 mmol) in a mixture of NaHCO buffer(100 mM,
3
pH 8.3) and MeCN (ratio 1:1, 4.5 ml). After reaction for 10 min, a
hydroxylamine solution (pH 8.3) was added to a final concentration of
1
00 mM and reaction was continued for 10 min. The reaction mixture
was diluted with waterto a final MeCN concent ar tion of 20% and
clarified by centrifugation of 20 000 g for 10 min before FB-C-peptide
was isolated by repeated HPLC preparations, using a Kromasil C8
column (4.6 ꢀ 250 mm, 7 mm). Elution was performed at 1 ml/min with a
gradient of MeCN (containing 0.1% TFA) in dH O with MeCN
2
increasing linearly from 20 to 42.5% during 15 min. The absorbance was
monitored at 220 nm. Fractions containing FB-C-peptide were pooled,
Copyright # 2001 John Wiley & Sons, Ltd.
J Labelled Cpd Radiopharm 2001; 44: 509–519