Beilstein J. Org. Chem. 2014, 10, 1570–1577.
and the organic layer was washed with brine (2 × 10 mL) and added slowly until the pH was ~7. This neutralized solution was
water (2 × 10 mL), dried over MgSO4, filtered and the solvent placed in a dialysis membrane (MW cutoff 3500 Da) and
was removed in vacuo. The residual product was taken up in dialyzed in 1 L of deionized water for 8 h. The water was
CH2Cl2 (50 mL) with 2-(2-isothiocyantoethoxy)ethanol (0.6 g, changed and let stand for a further 8 h twice more. The
4
mmol) and 4 Å molecular sieves. BF3OEt2 (0.6 g, 4 mmol) remaining liquid in the membrane was frozen and lyophilized to
was added to the mixture over 30 min at 0 °C, and the reaction give a white fluffy solid. This procedure for deacetylation was
tion (2 × 20 mL), brine (2 × 20 mL), and water (1 × 20 mL),
dried over MgSO4, filtered and the solvent was removed in NMR spectroscopy
vacuo. The oily residue was purified by silica gel column chro- 1H NMR spectra were recorded on Bruker DPX 300 (300 MHz)
matography with a 60:40 ethyl acetate/hexanes eluent, fol- and Bruker DPX-500 (500 MHz) spectrometers. Chemical
lowed by a 20:1 ethyl acetate/MeOH eluent to yield 2.6 g of shifts are reported in ppm from tetramethylsilane with the
product. 1H NMR (300 MHz, CDCl3) δ 5.33 (d, J = 3.1 Hz, 1H, residual protic solvent resonance as the internal standard (chlo-
H4’), 5.20 (app t, J = 9.3 Hz, 1H, H3), 5.09 (dd, J = 8.1 and roform: δ 7.25 ppm; dimethyl sulfoxide: δ 2.50 ppm). Data are
1
0.1 Hz, 1H, H2’), 4.93 (m, 2H, H2 and H3’), 4.50 (m, 3H, H1, reported as follows: chemical shift, multiplicity (s = singlet, bs
H1’ and H6), 4.06 (m, 4H), 3.89 (m, 6H), 3.78 (m, 3H), 3.63 = broad singlet, d = doublet, t = triplet, q = quartet, p = pentet,
m, 6H), 2.13 (s, 3H), 2.10 (s, 3H), 2.04 (s, 3H), 2.02 (m, 9H), m = multiplet, app = apparent), integration, coupling constants
1
.95 (s, 3H). As reported [37].
General procedure for the synthesis of acetyl-
protected lactose-functionalized dendrimers
MALDI–TOF mass spectrometry
An aqueous solution of amine terminated G(4)-PAMAM MALDI mass spectra were acquired using a Bruker Biflex-III
dendrimer (2.48 g of a 17% w/w solution in water, 421 mg, time-of-flight mass spectrometer. Spectra of all-functionalized
3
1.2 μmol) was lyophilized to leave a foamy residue. 7.02 mL dendrimers were obtained using a trans-3-indolacrylic acid
of DMSO was added to this residue to give a 60 mg/mL solu- matrix with a matrix:analyte ratio of 3000:1 or 1000:1. Bovine
tion of the dendrimer. 0.47 mL of a 300 mM solution of 1 serum albumin (Mw 66,431 g/mol), cytochrome C (Mw 12,361
(
184 mg, 141 μmol) was added to 0.5 mL of the 60 mg/mL g/mol), and trypsinogen (Mw 23,982 g/mol) were used as
G(4) PAMAM dendrimer (30 mg, 2.20 μmol) solution. The external standards. An aliquot corresponding to 12–15 pmol of
mixture was stirred for 8 h at which point a 75 μL aliquot was the analyte was deposited on the laser target. Positive ion mass
collected and lyophilized for MALDI–TOF and NMR analysis. spectra were acquired in linear mode and the ions were gener-
The remainder of the reaction mixture was lyophilized and ated by using a nitrogen laser (337 nm) pulsed at 3 Hz with a
subjected to the deacetylation procedure. This procedure for pulse width of 3 nanoseconds. Ions were accelerated at
a manner similar to our previously described procedure [29]. multiplier. Spectra (100 to 200) were summed into a LeCroy
lated by using the Polymer Module included in the software
General procedure for deacetylation to afford
lactose-functionalized dendrimers
To the lyophilized solid per-O-acetylated dendrimers, 1 mL of
1
:1 water/methanol was added, at which point the dendrimer Dynamic light scattering (DLS)
became a white precipitate. To this mixture was added 0.2 equiv DLS measurements were acquired with the Brookhaven 90Plus
of NaOMe (0.8 M in MeOH) for each peripheral carbohydrate, Particle Size Analyzer equipped with a 15 mW solid state,
and let stir for 3 h. If, at this time, the mixture had not become a 633 nm laser and upgraded APD detector. Scattered light was
clear solution a further 0.2 equiv of NaOMe (0.8 M in MeOH) detected at 90° incidence and optimized to a count rate of
was added and this step was repeated until the mixture became 200–400 kilocounts per second (kcps) through adjustment of a
a clear and colourless solution. Aqueous HCl (0.1 M) was then neutral density filter prior to the sample chamber. The intensity
1575