Quantification of Protein Glucosylation by Stable Isotope Dilution Assay
J. Agric. Food Chem., Vol. 47, No. 12, 1999 5087
(2) Nꢀ-tert-Butoxycarbonyl-L-lysyl-L-leucyl-L-glycine tert-Bu-
tyl Ester [Nꢀ-BOC-Lys-Leu-Gly-OtBu]. Following the classical
procedure for removal of the FMOC protection group (Carpino
and Han, 1972), morpholine (3.24 mmol) was added to NR-
FMOC-Lys(Nꢀ-BOC)-Leu-Gly-OtBu (0.86 mmol) dissolved in
a mixture of dimethylformamide (2.1 g) and methanol (0.2 g).
Cleavage was monitored by RP-TLC using methanol/water (8:
2, v/v) as the mobile phase. After a reaction time of 2 h at
room temperature, the target compound (Rf ) 0.7) was
detected upon spraying of the plate with ninhydrin solution
(5% ninhydrin in ethanol). The volatiles were removed in
vacuo, and the residual white solid was taken up in aqueous
hydrochloric acid (0.1 mol/L; 60 mL) and washed with diethyl
ether (3 × 30 mL). After the pH had been adjusted to 10.0 by
addition of sodium hydroxide (30% in water), the precipitate
formed was dissolved in diethyl ether (90 mL). Drying of the
organic layer over Na2SO4, filtration, and concentration in
vacuo afforded Nꢀ-BOC-Lys-Leu-Gly-OtBu as a white solid
(0.72 mmol, yield ) 83%).
solid of NR-FMOC-Ala-Lys-Leu-Gly‚CF3-COOH (0.25 mmol,
yield ) 95%).
LC/MS/ESI m/z (%): 610 (100, [M + H]+), 1219 (39, [M2 +
H]+), 632 (12, [M + Na]+), 535 (4, [M + H - Gly]+), 517 (2,
[M + H - Gly - H2O]+).
1H NMR (360 MHz, CD3OD): δ 7.8 (d, 2H, J ) 7.52 Hz,
FMOC-aryl), 7.7 (dd, 2H, J ) 2.27 Hz, FMOC-aryl), 7.4 (t, 2H,
J ) 7 Hz, FMOC-aryl), 7.3 (t, 2H, J ) 7.51 Hz, FMOC-aryl),
4.45 (t, 1H, J ) 7.52 Hz, Leu-CH), 4.35 (m, 1H, FMOC-CH2),
4.31 (m, 1H, Ala-CH), 4.2 (t, 1H, J ) 6.63 Hz, FMOC-CH),
4.07 (m, 1H, Lys-CH), 3.86 (d, 1Ha, J ) 18 Hz, Gly-CH2), 3.72
(d, 1Hb, J ) 18 Hz, Gly-CH2), 3.34 (m, 2H, Lys-CH2), 1.87 (m,
2H, Lys-CH2), 1.72 (m, 2H, Lys-CH2), 1.63 (m, 2H, Leu-CH2),
1.61 (m, 1H, Leu-CH), 1.41 (d, 3H, Ala-CH3), 1.36 (m, 2H, Lys-
CH2), 0.9 (m, 6H, 2 × Leu-CH3).
(5) NR-9-Fluorenylmethoxycarbonyl-L-alanyl-Nꢀ-(1-deoxy-D-
fructos-1yl)-L-lysyl-L-leucyl-L-glycine (NR-FMOC-Ala-DFLys-
Leu-Gly) and NR-9-Fluorenylmethoxycarbonyl-L-alanyl-Nꢀ-
([13C6]-1-deoxy-D-fructos-1-yl)-L-lysyl-L-leucyl-L-glycine (NR-
FMOC-Ala-[13C6]-DFLys-Leu-Gly). NR-FMOC-Ala-Lys-Leu-Gly
(as trifluoroacetic acid salt; 0.1 mmol) and triethylamine (0.2
mmol) were dissolved in anhydrous methanol (2 mL) and
stirred for 20 min. Anhydrous D-glucose or [13C6]-D-glucose
(0.40 mmol), respectively, was then added, and the mixture
was stirred at 64 °C under an atmosphere of nitrogen. The
formation of the glycosylated, bound DFLys (Rf ) 0.3) was
monitored by RP-TLC using a mixture of water/methanol (20:
80, v/v) as the mobile phase and spraying the plate with
ninhydrin (5% in ethanol). After 3 h, the solvent was removed
in vacuo, and the yellow residue was taken up in water and
then fractionated by column chromatography (20 × 200 mm)
on RP-18 silica (Lichroprep 25-40 µm; 10.0 g; Merck) sus-
pended in water. After flushing with water (50 mL), the target
compound was eluted with water/methanol (80:20, v/v; 100
mL). After removal of the methanol in vacuo, the aqueous
solution was freeze-dried to give NR-FMOC-Ala-DFLys-Leu-
Gly or NR-FMOC-Ala-[13C6]-DFLys-Leu-Gly, respectively, as
a pale yellow powder (0.04 mmol, yield ) 35%).
LC/MS/ESI m/z (%): 495 (100, [M + Na]+), 439 (7, [M +
Na - C2H4]+), 473 (5, [M + 1]+), 968 (2, [M2 + Na]+).
1H NMR (360 MHz, DMSO-d6): δ 4.46 (t, 1H, J ) 7.52 Hz,
Leu-CH), 4.08 (m, 1H, Lys-CH), 3.88 (d, 1Ha, J ) 18 Hz, Gly-
CH2), 3.74 (d, 1Hb, J ) 18 Hz, Gly-CH2), 3.03 (m, 2H, Lys-
CH2), 1.87 (m, 2H, Lys-CH2), 1.72 (m, 2H, Lys-CH2), 1.65 (m,
2H, Leu-CH2), 1.62 (m, 1H, Leu-CH), 1.4 (s, 9H, 3 × BOC-
CH3), 1.38 (s, 9H, 3 × t-Bu-CH3), 1.36 (m, 2H, Lys-CH2),0.9
(m, 6H, 2 × Leu-CH3).
(3) NR-9-Fluorenylmethoxycarbonyl-L-alanyl-Nꢀ-tert-butoxy-
carbonyl-L-lysyl-L-leucyl-L-glycine tert-Butyl Ester [NR-FMOC-
Ala-Lys(Nꢀ-BOC)-Leu-Gly-OtBu]. 1-Hydroxybenzotriazole (1
mmol) and dicyclohexylcarbodiimide (1 mmol) were added to
a solution of FMOC-Ala (0.7 mmol) in a mixture of dichlo-
romethane (2.2 mL) and dimethylformamide (0.3 mL). After
30 min of stirring at 25 °C, Nꢀ-BOC-Lys-Leu-Gly-OtBu (0.44
mmol) and triethylamine (0.40 mmol) were added and the
mixture was stirred at 25 °C. Reaction was monitored by silica
TLC using pentane/ethyl acetate (20:80, v/v) as the mobile
phase. After 4 h, the educt was completely converted into the
target compound (Rf ) 0.6). The reaction was then filtered,
and the filtrate was diluted with dichloromethane (60 mL) and
was then washed with with aqueous hydrochloric acid (0.1 mol/
L, 2 × 20 mL), followed by an NaHCO3 solution (5% in water;
20 mL) and brine (2 × 20 mL). After drying over Na2SO4,
concentration in vacuo yielded a white solid, which was washed
with diethyl ether (3 × 50 mL). Crystallization of the residue
from methanol afforded NR-FMOC-Ala-Lys(Nꢀ-BOC)-Leu-Gly-
OtBu as a white solid (0.35 mmol, yield ) 80%).
LC/MS/ESI of NR-FMOC-Ala-DFLys-Leu-Gly m/z (%): 772
(100, [M + H]+), 794 (51, [M + Na]+), 754 (4, [M + H - H2O]+),
1543 (3, [M2 + H]+), 610 (3, [M + H - Glu]+), 736 (2, [M +
H - 2H2O]+), 718 (1, [M + H - 3H2O]+).
LC/MS-ESI of NR-FMOC-Ala-[13C6]-DFLys-Leu-Gly m/z
(%): 778 (100, [M + H]+), 800 (46, [M + Na]+), 1556 (8, [M2 +
H]+), 616 (5, [M + H - Glu]+), 760 (4, [M + H - H2O]+), 742
(3, [M + H - 2H2O]+), 724 (2, [M + H - 3H2O]+).
1H NMR (360 MHz, CD3OD) of labeled and unlabeled NR-
FMOC-Ala-DFLys-Leu-Gly, respectively: δ 7.8 (d, 2H, J ) 7.52
Hz, FMOC-aryl), 7.7 (dd, 1H, J ) 2.27 Hz, FMOC-aryl), 7.4
(t, 2H, J ) 7 Hz, FMOC-aryl), 7.3 (t, 2H, J ) 7.51 Hz, FMOC-
aryl), 4.46 (t, 1H, J ) 7.52 Hz, Leu-CH), 4.35 (m, 1H, FMOC-
CH2), 4.33 (m, 1H, Glu-CH), 4.3 (m, 1H, Ala-CH), 4.2 (t, 1H,
J ) 6.63 Hz, FMOC-CH), 4.07 (m, 1H, Lys-CH), 3.87 (m, 1Ha,
Glu-CH2), 3.84 (m, 1H, Glu-CH), 3.82 (m, 1Hb, Glu-CH2), 3.80
(d, 1Ha, J ) 18 Hz, Gly-CH2), 3.77 (m, 1H, Glu-CH), 3.74 (d,
1Hb, J ) 18 Hz, Gly-CH2), 3.3 (m, 2H, Lys-CH2), 3.1 (s, 2H,
Lys-CH2-Glu), 1.87 (m, 2H, Lys-CH2), 1.72 (m, 2H, Lys-CH2),
1.64 (m, 2H, Leu-CH2), 1.61 (m, 1H, Leu-CH), 1.4 (d, 3H, Ala-
CH3), 1.36 (m, 2H, Lys-CH2), 0.9 (m, 6H, 2 × Leu-CH3).
(6) L-Alanyl-Nꢀ-(1-deoxy-D-fructos-1-yl)-L-lysyl-L-leucyl-L-gly-
cine (Ala-DFLys-Leu-Gly) and L-Alanyl-Nꢀ-([13C6]-1-deoxy-D-
fructos-1-yl)-L-lysyl-L-leucyl-L-glycine (Ala-[13C6]-DFLys-Leu-
Gly). A mixture of morpholine (2 mmol) and NR-FMOC-Ala-
DFLys-Leu-Gly or NR-FMOC-Ala-[13C6]-DFLys-Leu-Gly (0.04
mmol), respectively, was stirred in dimethylformamide (1.2
mL)/methanol (0.8 mL). The cleavage was followed by RP-TLC
using water/methanol (80:20, v/v) as the mobile phase and
ninhydrin (0.5% in ethanol) as the detection reagent. After 2
h of stirring at room temperature, the starting material was
converted into the target compound (Rf ) 0.6). Solvent and
morpholine were then removed under high vacuum, and the
residue was suspended in diethyl ether (20 mL). Filtration
afforded Ala-DFLys-Leu-Gly or Ala-[13C6]-DFLys-Leu-Gly, re-
spectively, as a white solid (0.036 mmol, 91%) in a purity of
∼96%.
LC/MS/ESI m/z (%): 788 (100, [M + Na]+), 732 (15, [M +
Na - C2H4]+), 1554 (8, [M2 + Na]+), 676 (5, [M + Na -
2C2H4]+), 767 (1, [M + 1]+).
1H NMR (360 MHz, CD3OD): δ 7.8 (d, 2H, J ) 7.52 Hz,
FMOC-aryl), 7.7 (dd, 2H, J ) 2.27 Hz, FMOC-aryl), 7.4 (t, 2H,
J ) 7 Hz, FMOC-aryl), 7.3 (t, 2H, J ) 7.51 Hz, FMOC-aryl),
4.45 (t, 1H, J ) 7.52 Hz, Leu-CH), 4.35 (m, 1H, FMOC-CH2),
4.3 (m, 1H, Ala-CH), 4.2 (t, 1H, J ) 6.63 Hz, FMOC-CH), 4.08
(m, 1H, Lys-CH), 3.87 (d, 1Ha, J ) 18 Hz, Gly-CH2), 3.75 (d,
1Hb, J ) 18 Hz, Gly-CH2), 3.3 (m, 2H, Lys-CH2), 1.88 (m, 2H,
Lys-CH2), 1.72 (m, 2H, Lys-CH2), 1.64 (m, 2H, Leu-CH2), 1.61
(m, 1H, Leu-CH), 1.44 (s, 9H, 3 × BOC-CH3), 1.43 (s, 9H, 3 ×
t-Bu-CH3), 1.41 (d, 3H, Ala-CH3), 1.36 (m, 2H, Lys-CH2), 0.9
(m, 6H, 2 × Leu-CH3).
(4) NR-9-Fluorenylmethoxycarbonyl-L-alanyl-L-lysyl-L-leucyl-
L-glycine (NR-FMOC-Ala-Lys-Leu-Gly). Following a procedure
of Kemp and Carey (1989), NR-FMOC-Ala-Lys(Nꢀ-BOC)-Leu-
Gly-OtB (0.27 mmol) was stirred in a solution of trifluoroacetic
acid (16 mL) in dichloromethane (6 mL) under an atmosphere
of nitrogen at room temperature. The cleavage was followed
by RP-TLC using a mixture of methanol/water (80:20) as the
mobile phase. After 2 h, the educt was completely converted
into the target compound, which was detected at Rf ) 0.4 upon
spraying with ninhydrin (5% in ethanol). The volatiles were
then removed under high vacuum, and the residual yellow oil
was dissolved in water. Freeze-drying afforded a pure white