SUPPORTING INFORMATION
Full Title
Matrix-assisted laser desorption ionization tandem mass
spectrometry of N-glycans derivatized with isonicotinic hydrazide
and its biotinylated form
Stephanie Bank1, Eberhard Heller1, Elisabeth Memmel2, Jürgen Seibel2, Ulrike Holzgrabe1,
Petra Kapková1*
*
Correspondence to Dr. Petra Kapková Tel: +49 931 3189912
E-mail: p.kapkova@uni-wuerzburg.de
1
Institute of Pharmacy and Food Chemistry, Julius-Maximilians-University Würzburg, Am Hubland, 97074
Würzburg, Germany
2
Institute of Organic Chemistry, Julius-Maximilians-University Würzburg, Am Hubland, 97074
Würzburg, Germany
Synthesis of BINH
Starting from methyl 4-pyridine carboxylate 1 the N-oxide 2 was obtained in 99% yield by
treatment of 1 with m-chloroperbenzoic acid in CHCl3. According to Allen et al.,[36] the oxide
2 was heated up in acetic anhydrid by microwave irradiation to 185 °C for 2 h. The crude
product was further microwave irradiated for 30 min in MeOH to achieve 3 in 26% yield.
In the next step, 6-bromohexanol 4 was acetylated in the microwave to give the bromoester 5.
The pyridinone 3 and the bromoester 5 reacted in the presence of KOtBu in the microwave at
130 °C. The intermediate was treated with MeOH and strong acidic ion exchanger in a
microwave cavity to give compound 6 in 30% yield after a column chromatography.
The coupling of 6 to the biotin was reached with diisopropylcarbodimide and
hydroxybenzotriazole at 120 °C under microwave irradiation and leaded to compound 8.The
desired final compound BINH 9 was received after reaction of 8 (0.125 mmol, 60 mg) with
hydrazine hydrate (1.25 mmol, 63 mg). The mixture was heated up in microwave cavity
within 2 min to 80 °C and the temperature was hold for 10 min. After cooling to 25 °C, the
solvent was evaporated and the residue was washed with petrol ether to get 9 in nearly
quantitative yield (59 mg, 98%).
Experimental
All microwave reactions were carried out in Ethos 1600 microwave reactor (MLS GmbH,
Leutkirch, Germany). At or below the boiling point of the solvent the reactions were carried
out in an open flask, beyond the boiling point they were done in a sealed tube. The
temperature was measured with a fibred optic sensor. TLC was performed on silica gel 60
F254 aluminium sheets (Merck, Darmstadt, Germany). FTIR spectra were obtained using a
Bruker Optics Alpha FTIR spectrometer (Bruker Optik, Ettlingen, Germany). 1H- and 13CNMR spectra were measured on a Bruker Avance 400 instrument (Bruker Biospin, Ettlingen,
Germany). The mass spectrum was measured on Agilent 1100 LC/MSD Trap (Agilent
Technologies, Waldbronn, Germany) equipped with an electrospray ionization (ESI) source.
The conditions of the spray chamber were as follows: drying gas temperature, 350 °C;
nebulizer pressure, 50 psi; drying gas flow, 10 L/min.
4-(Methoxycarbonyl)pyridine 1-oxide 2:
100 mmol (11.8 mL) of the pyridine compound 1 was dissolved in 160 mL CHCl3. 105 mmol
(25.88 g) mCPBA (75%) was added and the solution was stirred at 25 °C. The progress of the
reaction was monitored by TLC (EtOAc, silica gel). After 30 min 1 g of mCPBA were added
and the mixture was stirred for 1 h. 25 g K2CO3 in 200 mL water were poured into the
solution and stirred for 30 min more. The layers were separated; the aqueous layer was
extracted with CHCl3 (2 x 140 mL) and the combined organic layers were dried with Na2SO4.
The solid was filtered off and the solvent was evaporated to get the N-oxide in 99% yield
(15.20 g). Rf = 0.24 (EtOAc, silica gel). FTIR (ATR): 3119, 3050 (=CH), 2952 (CH3), 1713
(C=O), 1608, 1544, 1482 (C=C aromatic), 1295 (N-oxide), 809 (out of plane) cm-1. 1H-NMR
is in accordance with ref. [37]. 13C-NMR (CDCl3): 163.7 (C=O), 139.5 (C-2, C-6), 126.9 (C4), 126.5 (C-3, C-5), 52.9 (OCH3) ppm.
Methyl 2-oxo-1,2-dihydropyridine-4-carboxylate 3:
The N-oxide (50 mmol, 7.66 g) was dissolved in 60 mL Ac2O and irradiated in the microwave
for 2 h at 185 °C (5 min from 25 °C to 185 °C) in a sealed tube. After cooling down to 30 °C,
the volatiles were evaporated and the residue was diluted with 200 mL of water and 100 mL
sat. NaHCO3 solution. The solution was extracted with CHCl3 (3 x 150 mL). The combined
organic layers were dried (K2CO3), filtered and the solvent was distilled i. vac. The residue
was “filtered” through silica gel (EtOAc) and the solution was evaporated. The residue was
dissolved in 60 ml MeOH and 5 g aluminium oxide acid was suspended in a sealed tube. The
mixture was heated up to 100 °C in microwave cavity for 1 h (3 min to 100 °C). After cooling
to 30 °C the solution was filtered, evaporated and re-crystallized from toluene and petrol ether
to get 2.61 g in 34% yield as a pale yellow solid. Analytical data refer with ref. [38].
6-Bromohexyl acetate 5:
6-Bromohexanol 4 (188 mmol, 34 g), Ac2O (200 mmol, 19.5 mL) and pyridine (190 mmol)
were mixed and stirred for 30 min at 25 °C. The solution was then irradiated for 30 min at 70
°C (2 min 25 °C to 70 °C). The mixture was poured in 200 mL ice water and treated with 150
mL 10% HCl. The solution was extracted with EtOAc (3 x 250 mL). The combined organic
layers were washed with 250 mL brine and 250 mL sat. NaHCO3, dried over Na2SO4, filtered
and the solvent was evaporated. The residue was distilled i. vac. to get 30 g of a colourless
liquid (72%). Bp: 115 °C/10 mbar. 1H-NMR and 13C-NMR is in accordance with ref. [39].
Methyl 1-(6-hydroxyhexyl)-2-oxo-1,2-dihydropyridine-4-carboxylate 6:
The pyridinone 3 (15.30 mmol, 2.34 g) was dissolved in 30 mL abs. acetonitrile, treated with
KOtBu (16.90 mmol, 1.90 g) and stirred at 25 °C for 30 min. The bromide 5 (16.00 mmol,
3.56 g) was dissolved in 10 mL abs. acetonitrile, poured into the pyridinone solution and
irradiated at 130 °C for 2 h (heating rate 4 min from 25 °C to 130 °C). The solution was
filtered and the solvent was evaporated. The residue was dissolved in 40 mL MeOH, treated
with 3.3 g strong acidic ion exchanger and heated up to 140 °C in a microwave cavity within
3 min. The temperature was hold for 1 h and the solution was allowed to cool down to 25 °C.
After evaporation of the solvent the residue was purified by column chromatography on silica
gel (eluent EtOAc) to get 6 as a pale yellow solid in 30% yield (30%). Rf: 0.16 (silica
gel/EtOAc). FTIR (ATR): 3303 (OH), 3035 (=CH), 2926, 2898, 2877, 2848 (-CH), 1720 (OC=O), 1664 (N-C=O), 1589 (C=C), 1254 (C-O), 814 (out of plane) cm-1. 1H-NMR (CDCl3):
7.31 (1 H, d, 6-H, J=7.0 Hz), 7.19 (1 H, d, 3-H, J=1.0 Hz), 6.55 (1 H, dd, 5-H, J1=7.0, J2=1.0
Hz), 3.97 – 3.93 (3 H, m, 6’-H, OH), 3.90 (3 H, s, OCH3), 3.63 (2 H, t, 1’-H, J=6.5 Hz), 1.76
(2 H, quintet, 5’-H, J=7.5 Hz), 1.56 (2 H, quintet, 2’-H, J=7.5 Hz), 1.49 – 1.39 (4 H, m, 3’-H,
4’-H) ppm. 13C-NMR (CDCl3): 165.1 (OC=O), 162.3 (C-2), 140.4 (C-4), 137.8, (C-6), 122.8
(C-3), 104.4 (C-5), 62.5 (C-1’), 52.8 (OCH3), 49.8 (C-6’), 32.4 (C-2’), 29.1 (C-5’), 26.2 (C3’), 25.4 (C-4’) ppm.
Methyl 2-oxo-1-(6-((5-((4R)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4yl)pentanoyl)oxy)hexyl)-1,2-dihydropyridine-4-carboxylate 8:
The pyridinone 6 (1.0 mmol, 250 mg), the biotin (1.0 mmol, 240 mg) and the
hydroxybenzotriazole (0.12 mmol, 16 mg) were dissolved in dry DMF under Ar. After all the
compounds were in solution diisopropylcarbodiimide (1.2 mmol, 0.19 mL) was added and the
solution was heated up to 120 °C for 1 h in the MW (2 min from 25 °C to 120 °C). After
cooling to 25 °C the solvent was distilled i. vac. and the residue was “filtered” through silica
gel with CHCl3/MeOH (9:1). The solvent was evaporated and the residue was purified by
column chromatography (silica gel, CHCl3/MeOH 25:1) to achieve 60 mg of 7 (yield 13%).
Rf: 0.16 (silica gel, CHCl3/MeOH 25:1). 1H-NMR (CDCl3): 7.31 (1 H, d, 6-H, J=7.0 Hz), 7.17
(1 H, d, 3-H, J=1.0 Hz), 6.50 (1 H, dd, 5-H, J1=7.0, J2=1.0 Hz), 5.82 (2 H, br, 2 x NH), 4.52 –
4.49 (1 H, m, H-7’’), 4.33 – 4.30 (1 H, m, H-3’’), 4.04 (2 H, t, H-6’, J=6.5 Hz), 3.93 (2 H, t,
H-1’, J=7.5 Hz), 3.89 (3 H, s, OCH3), 3.16 – 3.15 (1 H, m, H-2’), 2.90 (1 H, dd, Ha-8’’,
J1=12.8 Hz, J2=4.9 Hz), 2.74 (1 H, d, Hb-8’’, J=12.8 Hz), 1.76 – 1.60 (8 H, m, H-2’, H-5’, H9’, H-11’), 1.48 – 1.38 (6 H, m, H-3’, H-4’, H-10’) ppm. 13C-NMR (CDCl3): 173.7
(H2COC=O), 165.1 (H3COC=O), 163.5 (C-5’’), 162.2 (C-2), 140.4 (C-4), 137.9, (C-6), 122.8
(C-3), 104.4 (C-5), 64.2 (C-1’), 62.4 (C-3’’), 60.2 (C-7’’), 55.4 (C-8’’), 52.8 (OCH3), 49.8 (C6’), 33.9 (C-2’), 29.7, 29.0, 28.4, 28.2 (C-5’, C-8’, C-9’, C-11’), 26.4, 25.6, 24.8 (C-3’, C-4’,
C-10’) ppm.
6-(4-(hydrazinecarbonyl)-2-oxopyridin-1(2H)-yl)hexyl 5-((4R)-2-oxohexahydro-1Hthieno[3,4-d]imidazol-4-yl)pentanoate 9:
The biotin ester (0.125 mmol, 60 mg) was dissolved in 8 mL MeOH and treated with
hydrazine hydrate (1.25 mmol, 63 mg). The mixture was heated up in microwave cavity
within 2 min to 80 °C and the temperature was hold for 10 min. After cooling to 25 °C, the
solvent was evaporated and the residue was washed with petrol ether to get 9 in nearly
quantitative yield (59 mg, 98%).
FTIR (ATR): 3225, 3197 (NH2), 3100 (=CH), 2929, 2882, 2854, (C-H), 1685 (N-C=O), 1625,
1470, 1460, 1258 (C-O) cm-1. 1H-NMR (D2O): 7.82 (1 H, d, J=7.0 Hz, 6-H), 6.91 (1 H, d,
J=1.5 Hz, 3-H), 6.75 (1 H, dd, J1=7.0 Hz, J2=1.5 Hz, 5-H), 4.66 – 4.63 (1 H, m, 3’’-H), 4.48 –
4.44 (1 H, m, 7’’-H), 4.07 (2 H, t, J=7.2 Hz, 6’-H), 3.61 (2 H, t, J=6.6 Hz), 3.40 – 3.35 (1 H,
m, 2’’-H), 3.03 (1 H, dd, J1=13.0 Hz, J2=5.0 Hz, 8’’-Ha), 2.81 (1 H, d, J=13.0 Hz, 8’’-Hb),
2.26 (2 H, t, J=7.3 Hz, 8’-H), 1.81 – 1.36 (14 H, m, 2’-H, 3’-H, 4’-H, 5’-H, 9’-H, 10’-H, 11’H) ppm. 13C-NMR (D2O): 175.6 (C-7’), 165.4 (C-5’’), 164.2 (C-2), 153.7 (H2NHNC=O),
144.9 (C-4), 140.2 (C-6), 117.6 (C-3), 106.1 (C-5), 62.1 (C-7’’), 61.6 (C-1’), 60.2 (C-3’’),
55.3 (C-2’), 50.6 (C-6’), 39.8 (C-8’’), 33.4 (C-8’), 31.1 (C-5’), 28.3 (C-11’), 27.8 (C-9’), 27.6
(C-3’), 25.4 (C-2’), 24.8 (C-4’), 24.6 (C-10’) ppm. ESI-MS: [M+H]+ m/z 480.4.
References
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Counting of 9: