M.A. Siyad, G.S.V. Kumar / Polymer 67 (2015) 80e91
81
dendrimers on solid supports [22e24]. Solid-phase synthesis can
2.2. Synthesis of dendron units
rectify a number of problems usually associated with dendrimer
preparation in solutions such as simple purification and use of
excess reagents to ensure reaction completion. We have already
synthesized and reported PEGylated poly(N,N-bisethylamine)
dendrimers for solid phase organic synthesis and the dendrons
were generated from cross-linker [25] as well as from backbone of
polystyrene supports [26,27]. In previously published works
The polymerization was carried out in a conventional suspen-
sion polymerization reactor as reported earlier [14]. The dendron
1,3,5-tris(hydroxymethyl)benzene was synthesized as follows. (a)
Trimethyl-1,3,5-benzenetricarboxylate: Trimesic acid (10 g,
47.5 mmol) was suspended in MeOH (30 mL) and heated to reflux.
2 4
When a clear solution was obtained, 1.6 mL of conc. H SO was
[
25,26] we have used bulky Schiff base dendrons for dendrimer
added drop wise and the mixture was allowed to reflux for 6 h.
After 3 h, white solid got precipitated from the solution. The
mixture was further cooled to RT, slowly neutralized with sat.
grouping and reactions involved were four stage reactions such as
grouping of Schiff base, acidolytic cleavage, diazotization and
chlorination. The present work describes the synthesis of poly(O-
benzyl ether) dendrimers to the third generation (G ) by
3
following successive Williamsons etherification and chlorination
NaHCO
aqueous layer turned neutral. It was then dried to yield a white
solid. (b) 1,3,5-Tris(hydroxymethyl)benzene: LiAlH tablets (5 g,
132 mmol) were suspended and refluxed in dry THF (100 mL) un-
der inert atmosphere of N gas. To this suspension, (10 g, 40 mmol)
3
solution filtered and washed with excess water until the
4
reaction. It was followed by PEGylation in which PEG having
different molar masses (PEG400 Da and PEG1500 Da) is grafted to G
3
2
dendrimer and used for peptide synthesis. The reactions such as
dendrimer grouping and PEG grafting were performed on styrene-
poly(ethylene glycol)dimethacrylate-vinylbenzylchloride (PS-
PEGDMA-VBC) support. Instead of using bulky Schiff base as
dendritic units, more symmetric and less crowded tri(methylol)
benzene produced from trimesic acid was preferred to dendrimer
creation. Instead of series of four stage reactions used in Schiff base
assemblage, more facile and simple two step reactions such as
Williamson's etherification and thionyl chloride reactions were
used throughout reactions. Here we tried to improve the hydro-
philicity of polymeric support by different alternatives such as us-
ing flexible poly(ethylene glycol)dimethacrylate cross-linker,
dendritic units connecting through ether linkages and by grafting
hydrophilic moieties such as PEG through dendrimeric hands so
that more number of PEG molecules can be accommodated with
optimum steric hindrances.
dissolved in 100 mL dry THF was added slowly over 1 h. The so-
lution was then refluxed for 24 h, cooled to 0 C and acidified with
ꢀ
1 M HCl. THF was removed under reduced pressure and the
aqueous suspension (100 mL) thus obtained was extracted with
ethyl acetate for 48 h in a liquideliquid extractor to yield 1,3,5-
tri(methylol)benzene which appeared as colorless crystals at
room temperature. The mother liquor was concentrated to yield
second fraction of same product. Both the fractions were NMR pure
and the combined yield obtained was 5.1 g. The formation of ligand
1
13
1
was confirmed by H and C NMR analysis. H NMR (300 MHz,
ꢀ
DMSO-d6, 25 C):
d
¼ 7.096 (s, 3H), 5.21 (t, J(H,H) ¼ 5.6 Hz, 3H), 4.45
13
ꢀ
(d, J ¼ 5.6 Hz, 6H); C NMR (63 MHz, DMSO-d6, 25 C):
d
¼ 142.9,
123.8, 63.9.
2.3. Assemblage of 1,3,5-tri(methylol)benzene to PS-PEGDMA-VBC
resin
All the reactions have been conducted in an atmosphere of inert
2
. Experimental section
2
N gas. Calculated amount of lyophilized 2 mol% cross-linked PS-
PEGDMA-VBC resin (4 g, 0.404 mmol) was allowed to swell in dry
THF (100 mL) for 1 h. A solution of sodium salt of 1,3,5-
tris(hydroxymethyl)benzene was prepared by dissolving weighed
(0.680 g, 4.04 mmol) amount of 1,3,5-trimethylolbenzene in
2
.1. Materials and methods
FTIR spectra of the PS-PEGDMA-VBC beads, PEG grafted beads,
ꢀ
and functional alterations were recorded using Nicolet 5700 FTIR
spectrometer. SEM samples were mounted onto a SEM sample
holder (Hitachi SS 2000) using graphite paint, and a thin coating of
gold (10 nm) was applied under high vacuum. Optical density (OD)
value of dibenzofulvene:piperidine adduct formed was measured
with a Shimadzu ultraviolet-visible spectrophotometer at 290 nm.
anhydrous THF and cooled to 0 C. Sodium hydride (2.04 g,
85 mmol) dissolved in dry THF has been transferred to the solution
with occasional swirling. After the complete addition of NaH, the
reaction condition was brought back to room temperature and
2
allowed to stand for 1 h.When the bubbling of H gas formation
ceases, the reaction mixture has been quantitatively transferred to
swelled PS-PEGDMA-VBC resin with excess addition of anhydrous
THF (10 mL) and NaH (0.24 g, 10 mmol) and refluxed for an over-
13
Carbon-13 nuclear magnetic resonance C NMR; cross-polarization
magic-angle spin (CP-MAS) spectra of the samples are taken using a
dsx 300 (75.47 MHz). The samples rotated at different frequencies
so that the side bands will be eliminated. CHN analysis has been
carried out using Elementar Vario EL III. High performance liquid
chromatography (HPLC) analysis was conducted using a Pharmacia
Akta purifier system using C-18 reverse phase semi preparative
HPLC column with binary gradient system (water and acetonitrile)
containing 0.1% TFA. The flow rate was 1 mL/min, and detection was
at 214 nm. The HPLC conditions used for all synthetic peptides were
same: C-18 column; buffer (A) 0.1% TFA in water: acetonitrile (19:1,
v/v) and buffer (B) 0.08% TFA in acetonitrile:water (4:1, v/v).
Gradient used 0% B in 5 min.100% B in 30 min and 100% B in 35 min.
Mass spectra of peptides were obtained with a Kratos MALDI TOF
MS instrument. All side chain protected Fmoc amino acids (L) with
different cysteine derivatives and 2-(1H-benzotriazol-1-yl)1,1,3,3
tetramethyluraniumhexafluorophosphate (HBTU) were purchased
from Peptide international company (USA). 1-Hydroxybenzotriazol
ꢀ
night reaction at 80 C. The polymer beads were transferred to
filtering flask and un-reacted sodium hydride was deactivated with
excess quantity of ethanol. The polymer beads were successively
washed with dilute HCl (5 ꢁ 10 mL), MeOH (10 ꢁ 10 mL), DCM
(10 ꢁ 10 mL), acetone (10 ꢁ 10 mL) and ether (5 ꢁ 5 mL). Solvent
extracted with THF and EtOH and dried at room temperature in
vacuum. The yield of the polymer obtained was 4.316 g.
2.4. Hydroxymethyl groups to chloromethyl groups
2 mol% hydroxymethyl O-benzyl ether resin (3.5 g, 0.700 mmol)
was suspended in double distilled DCM (50 mL). After 1 h, excess
solvent was filtered off. To the swelled resin thionyl chloride
(510
m
L, 7 mmol) was added in drop wise manner within 30 min.
ꢀ
The reaction mixture was heated at 55 C with occasional swirling
for overnight reaction. The mixture was cooled, allowed to stand
open at room temperature to remove un-reacted thionyl chloride,
(
HOBt) and 1-(2-mesitylenesulfonyl)-3-nitro-1,2,4-triazol (MSNT)
were purchased from Novabiochem Ltd.
washed with ethanol (5 ꢁ 15 mL), THF/H O (1:1, 5 ꢁ 15 mL), THF
2