room temperature. The titled compound was isolated by reversed-
phase chromatography with 50 mM ammonium bicarbonate/
4.29 (m, 2H, CONHNH2); positive ion mode MALDI-TOF m/z
509.2 (M + H)+.
NR-(Benzyloxycarbonyl)tyrosinylarginine Hydrazide (ZYRh). To
a solution of NR-(benzyloxycarbonyl)tyrosine (788 mg, 2.5 mmol)
in dry THF (6 mL), we added N-methylmorpholine (330 µL, 3.0
mmol) and isobutyl chloroformate (393 µL, 3.0 mmol) at -20 °C.
The mixture was heated to room temperature and stirred for 15
min and was followed by the addition of a solution of the arginine
methyl ester dihydrochloride (784 mg, 3.0 mmol) and NaHCO3
(252 mg, 3.0 mmol) in H2O (3 mL) at 0 °C. The reaction mixture
was stirred for 1 h. Solvents were removed under reduced
pressure. The residue was chromatographed on a column of silica
gel using 5:1 CHCl3/MeOH to give (Z)-Tyr-Arg methyl ester (0.8
1
acetonitrile (80:20, vol) at a yield of ∼87%; H NMR (500 MHz,
DMSO-d6) δ 8.31-7.37 (m, 5H, Ph), δ 4.42 (m, 1H, R-CH), 4.24
(bs, 2H, CONHNH2); positive ion mode MALDI-TOF m/z 293.2
(M + H)+.
NR-(Benzyloxycarbonyl)arginine Hydrazide (ZRh). NaHCO3 (168
mg, 2 mmol) and Z chloride (313 µL, 1 mmol) was added to a
solution of R-OMe (261 mg, 1.0 mmol) in H2O (5 mL). The
mixture was stirred for 12 h at room temperature. The reaction
mixture was extracted with CHCl3, and the organic layer was
washed with saturated aqueous NaHCO3 and brine. The extraction
was concentrated, and the residue was purified by silica gel
chromatography, eluting it with CHCl3/MeOH (5:1) to give 0.3 g
1
g, 67%); H NMR (500 MHz, CD3OD) δ 8.53-7.09 (m, 9H, Ph),
1
4.95 (d, 1H, J ) 12.9 Hz, 1/2 × CH2Ph), 4.92 (d, 1H, J ) 12.9 Hz,
1/2 × CH2Ph), 4.23 (m, 2H, Tyr R- and Arg R-CH), 3.62 (s, 3H,
COOCH3). (Z)-Tyr-Arg methyl ester hydrochloride (5.2 mg,
10.7µmol) was dissolved in methanol (0.54 mL), and hydrazine
monohydrate (0.2 mL, 4.0 mmol) was added. The reaction mixture
was stirred for 6 h at room temperature. The titled compound
was isolated by reversed-phase chromatography with 50 mM
ammonium bicarbonate/acetonitrile (66:34, vol) at a yield of ∼66%;
1H NMR (500 MHz, DMSO-d6) δ 7.35-6.63 (m, 9H, Ph), 4.97 (d,
1H, J ) 13.4 Hz, 1/2 × CH2Ph), 4.92 (d, 1H, J ) 12.9 Hz, 1/2 ×
CH2Ph), 4.37 (m, 2H, CONHNH2), 4.20 (m, 2H, Tyr R- and Arg
R-CH); positive ion mode MALDI-TOF m/z 486.2 (M + H)+.
Digestion of Glycoproteins with Trypsin. Digestion of
glycoproteins with trypsin was performed according to the
procedure previously described.23 Briefly, glycoproteins were
solubilized with 0.1% (w/v) RapiGest SF (Waters, Milford, MA)
solution buffered with 50 mM ammonium bicarbonate (pH of 7.8)
at a concentration of 15 µM each. They were digested with 5 µg
of modified trypsin at 37 °C for 1 h. Heating the reaction mixture
at 100 °C for 3 min terminated the reaction.
Deglycosylation of Tryptic Digests by PNGase F. The
tryptic digest of each glycoprotein was incubated by directly
adding PNGase F (0.5 U) to the tryptic glycoprotein digest at 37
°C overnight. The reaction mixture was kept at -20 °C until
further analysis.
Derivatization of Standard Oligosaccharides with Labeling
Reagents. The derivatization reaction was modified from the
procedure for hydrazone formation, as previously described.24,25
For standard derivatization, 10 µL of each labeling reagent (200
µM) in 80% methanol and 1 µL of 10 µM oligosaccharide (NA2)
were added to 10 µL of 50 mM ammonium bicarbonate. The
mixture was then incubated at 90 °C for 1 h. After cooling the
reaction tube on ice, the reaction mixture was accordingly mixed
with DHB (10 g/L in 30% acetonitrile), and 0.5 µL of the resulting
mixture was subjected to MALDI-TOF mass analysis without any
purification.
(31% yield) of (Z)-arginine methyl ester ((Z)R-OMe); H NMR
(500 MHz, CDCl3) δ 7.27 (m, 5H, Ph), 5.08 (d, 1H, J ) 12.1 Hz,
1/2 × CH2Ph), 5.03 (d, 1H, J ) 12.1 Hz, 1/2 × CH2Ph), 4.23 (bs,
1H, R-CH), 3.67 (s, 3H, COOCH3). (Z)R-OMe (2.9 mg, 9.0µmol)
was dissolved in methanol (0.45 mL), and hydrazine monohydrate
(0.17 mL, 3.4 mmol) was added. The reaction mixture was stirred
for 6 h at room temperature. The titled compound was isolated
by reversed-phase chromatography with 50 mM ammonium
1
bicarbonate/acetonitrile (66:34, vol) at a yield of ∼68%; H NMR
(500 MHz, DMSO-d6) δ 7.37 (m, 5H, Ph), 5.01 (s, 2H, CH2Ph),
4.22 (bs, 2H, CONHNH2), 4.09 (bs, 1H, R-CH); positive ion mode
MALDI-TOF m/z 323.2 (M + H)+.
NR-(Benzyloxycarbonylphenylalanylarginine Hydrazide (ZFRh).
(Z)-Phe-Arg methyl ester, hydrochloride (2.7 mg, 5.3µmol) was
dissolved in methanol (0.27 mL), and hydrazine monohydrate (0.1
mL, 2.1 mmol) was added. The reaction mixture was stirred for
6 h at room temperature. The titled compound was isolated using
reversed-phase chromatography with 50 mM ammonium bicar-
1
bonate/acetonitrile (66:34, vol) at a yield of ∼77%; H NMR (500
MHz, DMSO-d6) δ 7.37 (m, 10H, Ph), 4.95 (d, 1H, J ) 13.0 Hz,
1/2 × CH2Ph), 4.91 (d, 1H, J ) 12.6 Hz, 1/2 × CH2Ph), 4.28 (m,
2H, Phe R- and Arg R-CH), 4.25 (bs, 2H, CONHNH2); positive
ion mode MALDI-TOF m/z 470.1 (M + H)+.
NR-(Benzyloxycarbonyl)tryptophanylarginine Hydrazide (ZWRh).
To a solution of NR-(benzyloxycarbonyl)tryptophan (846 mg, 2.5
mmol) in dry THF (6 mL), we added N-methylmorpholine (330
µL, 3.0 mmol) and isobutyl chloroformate (393µL, 3.0 mmol) at
-20 °C. The mixture was heated to room temperature and stirred
for 15 min, followed by the addition of a solution of the arginine
methyl ester dihydrochloride (784 mg, 3.0 mmol) and NaHCO3
(252 mg, 3.0 mmol) in H2O (3 mL) at 0 °C. The reaction mixture
was stirred for 1 h. Solvents were removed under reduced
pressure. The residue was chromatographed on a column of silica
gel using 5:1 CHCl3/MeOH to give (Z)-Trp-Arg methyl ester (1.1
1
g, 87%); H NMR (500 MHz, CD3OD) δ 8.55-7.18 (m, 10H, Ph
and indole), 4.93 (s, 2H, CH2Ph), 4.33 (m, 2H, Trp R- and Arg
R-CH), 3.63 (s, 3H, COOCH3). (Z)-Trp-Arg methyl ester (5.4 mg,
10.6µmol) was dissolved in methanol (0.53 mL), and hydrazine
monohydrate (0.2 mL, 4.0 mmol) was added. The reaction mixture
was stirred for 6 h at room temperature. The titled compound
was isolated using reversed-phase chromatography with 50 mM
ammonium bicarbonate/acetonitrile (66:34, vol) at a yield of ∼76%;
1H NMR (500 MHz, DMSO-d6) δ 8.13-6.95 (m, 10H, Ph and
indole), 4.94 (s, 2H, CH2Ph), 4.29 (m, 2H, Trp R- and Arg R-CH),
Derivatization of Deglycosylated Tryptic Digests with
Labeling Reagents. Typically, 1 µL of deglycosylated tryptic
digest of ovalbumin, asialofetuin, ribonuclease B, or R1-acid
(23) Yu, Y. Q.; Gilar, M.; Lee, P. J.; Bouvier, E. S.; Gebler, J. C. Anal. Chem.
2003, 75, 6023-6028.
(24) Shinohara, Y.; Sota, H.; Kim, F.; Shimizu, M.; Gotoh, M.; Tosu, M.;
Hasegawa, Y. J. Biochem. (Tokyo) 1995, 117, 1076-1082.
(25) Shinohara, Y.; Sota, H.; Gotoh, M.; Hasebe, M.; Tosu, M.; Nakao, J.;
Hasegawa, Y.; Shiga, M. Anal. Chem. 1996, 68, 2573-2579.
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