Hetero-difunctional dimers as building blocks for the synthesis of poly(amidoamine)s with hetero-difunctional chain terminals and their derivatives

Article abstract of DOI:10.1002/pola.26325

This article reports on a simple and straightforward preparation method of poly(amidoamine)s (PAAs) with hetero-difunctional chain ends as well as of several up to now hardly obtainable PAA derivatives of biotechnological interest, such as for instance PAAs of controlled molecular weight and narrow polydispersity mono-functionalized at one end with an acrylamide group, PAAs with star-like molecular architecture, graft-PAA-protein conjugates, "tadpole-like" PAA conjugates with hydrophobic moieties able to self assemble into nanoparticles in aqueous media. The key step was to design suitable building blocks consisting of hetero-difunctional dimers (HDDs). In particular, the HDDs considered were the mono-addition products of bis-sec-amines and bisacrylamides expected to give PAAs of proven biomedical potential and were obtained as hydrochlorides or trifluoroacetates. In this form, they could be indefinitely kept dormant at 0-5 °C in the dry state, whereas at room temperature and in aqueous media, they polymerized at pH > 7.5. The preparation of the above-cited PAA derivatives did not necessarily involve the preliminary synthesis of hetero-difunctional PAAs but was directly achieved by one-pot polymerization of HDDs in the presence of the substrates of interest.

Full text of DOI:10.1002/pola.26325

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Hetero-Difunctional Dimers as Building Blocks for the Synthesis of
Poly(amidoamine)s With Hetero-Difunctional Chain Terminals and
Their Derivatives
ꢀ
Paolo Ferruti, Nicolo Mauro, Amedea Manfredi, Elisabetta Ranucci
ꢀ
Dipartimento di Chimica, Universita degli Studi di Milano, via Golgi 19, 20133 Milano, Italy
Correspondence to: P. Ferruti (E-mail: paolo.ferruti@unimi.it)
Received 28 June 2012; accepted 28 July 2012; published online
DOI: 10.1002/pola.26325
ABSTRACT: This article reports on a simple and straightforward
preparation method of poly(amidoamine)s (PAAs) with hetero-
difunctional chain ends as well as of several up to now hardly
obtainable PAA derivatives of biotechnological interest, such
as for instance PAAs of controlled molecular weight and nar-
row polydispersity mono-functionalized at one end with an ac-
rylamide group, PAAs with star-like molecular architecture,
graft-PAA-protein conjugates, ‘‘tadpole-like’’ PAA conjugates
with hydrophobic moieties able to self assemble into nanopar-
ticles in aqueous media. The key step was to design suitable
building blocks consisting of hetero-difunctional dimers
(HDDs). In particular, the HDDs considered were the mono-
addition products of bis-sec-amines and bisacrylamides
expected to give PAAs of proven biomedical potential and
were obtained as hydrochlorides or trifluoroacetates. In this
form, they could be indefinitely kept dormant at 0–5 ꢀC in the
dry state, whereas at room temperature and in aqueous media,
they polymerized at pH > 7.5. The preparation of the above-
cited PAA derivatives did not necessarily involve the prelimi-
nary synthesis of hetero-difunctional PAAs but was directly
achieved by one-pot polymerization of HDDs in the presence of
C
V
the substrates of interest.
2012 Wiley Periodicals, Inc.
J Polym Sci Part A: Polym Chem 000: 000–000, 2012
KEYWORDS: amphiphiles; nanoparticles; polyamines; poly(ami-
doamine)s; protein grafting; star polymers
INTRODUCTION Poly(amidoamine)s (PAAs) are synthetic
polymers obtained in linear form by Michael-type polyaddi-
tion of prim-mono-amines or bis-sec-amines to bisacryla-
mides (Scheme 1).1
cellular delivery of genes and toxins.4,7 Amphoteric PAAs car-
rying carboxyl groups as side substituents may be nearly as
biocompatible as dextran, and one of them, named ISA23,
when injected in animals exhibited ‘‘stealth-like’’ properties
and passively concentrated in solid tumors by the so-called
enhanced permeation and retention effect.5 Recently, PAAs
containing disulphide bonds in the main chain have been
prepared from N,N0-bis acryloylcystamine and various amines
and found to be amenable of bio-reductive degradation,
hence useful both in linear and crosslinked form as carriers
for selectively delivering bioactive substances in particular
body districts.8–12
PAAs are characterized by the presence of amide (a) and
tert-amine groups (b) regularly arranged along the polymer
chain according to sequences
a ꢁꢁa ꢁꢁb
or
a ꢁꢁa ꢁꢁb ꢁꢁb
The polymerization reaction takes place in aqueous or alco-
holic solutions, at room temperature and without added cat-
alysts. Since under these conditions many functional groups,
if present, do not interfere in the Michael reaction, the struc-
ture and the physico-chemical properties of PAAs, including
acid-base properties, can be tuned within ample limits.2
Most PAAs are degradable in aqueous media at pH > 7 and
biocompatible even as polycations.3–6 Some purposely
planned PAAs are amenable to intracellular localization and
act as endosomolytic polymers with potential for the intra-
The traditional synthesis of PAAs has a serious limitation. It
is apparent from Scheme 1 that to obtain high-molecular
weight products the functions involved, that is, activated
double bonds and amine hydrogens, must be stoichiometri-
cally balanced. A perfectly balanced reacting mixture will
ꢂꢂꢂꢂꢂꢂ
contain three types of macromolecules, a ꢁꢁ ꢁꢁ a,
ꢂꢂꢂꢂꢂꢂ
ꢂꢂꢂꢂꢂꢂ
b ꢁꢁ ꢁꢁ b, and a ꢁꢁ ꢁꢁ b in 1:1:2 ratio. Unbalanced
mixtures will contain the same molecular species, albeit in
different ratios, until the minority function is completely con-
sumed. Only at this point, the product will be entirely
Additional Supporting Information may be found in the online version of this article.
C
V
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hydrophobic moieties forming in aqueous media liposomes
or nanoparticles can be prepared as functional drug carriers.
Based on this premise, the aim of this article is to report on
the preparation of some model HDDs corresponding to PAAs
extensively studied for biomedical applications and to pro-
vide selected factual examples of their synthetic potential.
SCHEME 1 Traditional synthesis of linear PAAs by Michael-
EXPERIMENTAL
type polyaddition.
Materials
2,2-Bis(acrylamido)acetic acid (BAC) and 1,4-bis(acryloyl)pi-
perazine (BP) were synthesized as previously described,14,15
lithium hydroxide monohydrate (98%), piperazine hexahy-
drate (P) (99%), 2-(R, S)-methylpiperazine (MP) (95%),
1,4,8,11-tetrazacyclotetradecane (Cyclam) (98%), di-(tert-
butyl)dicarbonate (97%), thiocholesterol (TC), bovine serum
albumin (BSA) (96%), 4-acryloylmorpholine (97%), morpho-
line (99.5%), guanidine hydrochloride (98%), glacial acetic
acid, hydrochloric acid (37%), sodium hydroxide (98.5%),
sodium chloride (99%), dichloromethane (reagent grade),
constituted of molecules doubly terminated by the excess
function. There is no way, by the traditional method, to
straightforwardly obtain PAAs with controlled hetero-difunc-
tional chain terminals, that is, PAAs solely containing mole-
ꢂꢂꢂꢂꢂꢂ
cules of ‘‘a ꢁꢁ ꢁꢁ b’’ type. This precluded to PAAs the
access to the remarkable number of biotechnological applica-
tions, for instance liposome preparation, drug conjugation,
and protein modification, which have been so far nearly
uniquely mastered by hetero-difunctional PEGs that, how-
ever, are far to be endowed with the functional versatility of
PAAs. In the past, a PAA-albumin conjugate was prepared by
employing a large excess of doubly vinyl-terminated, low-
molecular weight PAA apt to react with the exposed protein
thiol and amine groups. However, despite tedious purifica-
tion procedures, the presence of ‘‘bridged’’ protein molecules
could not be excluded.13
R
trifluoroacetic acid, DOWEXV MAC-3 ion exchange resin were
purchased from Sigma-Aldrich and used as received.
Methods
Nuclear Magnetic Resonance (NMR) Spectra
1H NMR, 13C NMR, distortionless enhanced by polarization
transfer (DEPT), 1H-1H correlation (COSY), heteronuclear
multiple bond correlation (HMBC), and heteronuclear corre-
lation (HETCOR) spectra were run at room temperature on a
Bru¨ker Advanced 400 spectrometer operating at 400.132
and 100.623 MHz, respectively.
Two strategies exist, in principle, for unequivocally preparing
hetero-diterminated PAAs, either the ring-opening polymer-
ization of cyclic precursors or the self-polyaddition of het-
ero-difunctional (‘‘a ꢁꢁa ꢁꢁb ꢁꢁb’’ or ‘‘a ꢁꢁa ꢁꢁb’’) dimers
(HDDs). The first strategy was ruled out because it was
thought difficult to design a cyclic PAA precursor amenable
to thermal or catalytic ring-opening polymerization, espe-
cially in the case of purpose-tailored PAAs carrying reactive
side-substituents, as for instance carboxyl groups, liable to
interfere in these processes. Therefore, the preparation of
HDDs, in particular those of the ‘‘a ꢁꢁa ꢁꢁb ꢁꢁb’’ type,
expected to polymerize according to Scheme 2, was pre-
ferred. We limited our investigation to ‘‘a ꢁꢁa ꢁꢁb ꢁꢁb’’ type
HDDs because the procedure adopted in this investigation
was not easily applicable to the preparation of the
‘‘a ꢁꢁa ꢁꢁb’’ ones. A different approach would be actually
needed, hopefully to be reported in a future article.
Size Exclusion Chromatography
Size exclusion chromatography (SEC) traces were obtained
using a Knauer Pump 1000 equipped with a Knauer Auto-
sampler 3800, TKSgel G4000 PW, and G3000 PW TosoHaas
columns connected in series, light scattering/viscometer Vis-
cotek 270 Dual Detector and a refractive index detector
Waters model 2410. The mobile phase was a 0.1 M Tris
buffer pH 8.1 6 0.05 with 0.2 M sodium chloride. The sam-
ple concentration was 2% (w/v) and the flow rate 1 mL/
min. Triple detector SEC allowed determining, besides the
molecular weight, the long chain branching (LCB) frequency
of star-like PAAs by means of the dedicated Viskotek soft-
ware, which compared their Mark–Houwink plots with those
of samples of their linear counterparts of approximately the
same molecular weight and used common Zimm–Stockmayer
equations and methods.16
The synthetic scope of HDDs is manifold. They can be poly-
merized to high-molecular weight PAAs without bothering
with stoichiometric balance. PAAs of controlled average mo-
lecular weight and mono-functionalized with an acrylamide-
or a sec-amine group at one end can be prepared by adding,
respectively, a controlled amount of mono-functional acryla-
mides or sec-amines, whereas the addition of multifunctional
acrylamides or amines will lead to star-like PAAs. Block PAA-
PAA or PAA-PEG copolymers with controlled structure will
be easily obtained. ‘‘Velvety-like’’ grafting of PAA chains to
properly functionalized surfaces can be achieved. PAA chains
of controlled average length can be grafted to proteins with
no risk of undesirable side reactions such as protein–protein
coupling or crosslinking. ‘‘Tadpole-like’’ PAA conjugates with
Dynamic Light Scattering
Dynamic light scattering (DLS) analysis was performed to
evaluate the hydrodynamic radius of polymeric micelles
using a Malvern NanoZS instrument (Malvern Instruments,
Worcestershire, UK) with a laser fitted at 532 nm and fixed
SCHEME 2 Self-polymerization of the ‘‘a ꢁꢁa ꢁꢁb ꢁꢁb’’ type
HDDs.
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173ꢀ scattering angle. DLS measurements were performed
on two sets of experiments. In the first, samples were pre-
pared in phosphate buffur solution (PBS) saline in the con-
centration range of 0.1–1 mg/mL. In the second one, the
sample was prepared in PBS saline with concentration of 1
mg/mL, and the measures were performed varying the tem-
perature: 20, 25, 30, 35, 40, 45, 50, 55, and 60 ꢀC. Before
each analysis, the samples were filtered through a syringe fil-
ter (5 mm pore size; Watmann, UK) to remove the dust. All
data were evaluated as numeral distribution of the hydrody-
namic diameter.
0.129 mol), glacial acetic acid (7.6 g, 0.127 mol), di-(tert-
butyl)dicarbonate (23.2 g, 0.103 mol), and tap water (1.2 L).
Yield: 87%.
1H NMR (CDCl3, d, ppm): 1.47 (s, (CH3)3C), 1.82 (br,
NHCH2CH2NBoc), 2.81 (m, HNCH2CH2NBoc), 3.40 (m,
NHCH2CH2NBoc). 13C NMR (D2O, d, ppm): 28.97 ((CH3)3CO),
45.21 (HNCH2CH2N), 79.52 ((CH3)3CO), 154.84 (NCO). Anal.
Calcd for: C9H18N2O2: C, 58.04; H, 9.74; N, 15.04. Found: C,
56.12; H, 10.78; N, 14.46.
Synthesis of BAC-P
To a vigorously stirred solution of N-Boc-piperazine (1.3200
g, 7.042 mmol) in bidistilled water (15 mL) cooled at 0 C, a
Transmission Electron Microscopy
ꢀ
Transmission electron microscopy (TEM) analysis of self-
assembled polymeric BAC-MP-TC micelles was captured
using the negative staining technique. A solution of BAC-MP-
TC (1 mg/mL) in ultrapure water at pH 7.4 was prepared;
then a 5 mL aliquot was deposited onto a Formvar-coated Cu
grid (400 mesh). After adsorption, the grid was stained with
1% uranyl acetate solution (5 mL), wiped away by means of
a paper filter and dried at room temperature. Observation
was made by using a LEO 912 AB energy-filtering transmis-
sion electron microscopy (EFTEM; Carl Zeiss, Obercochen,
Germany) operating at 80 kV. Digital images were collected
by Proscan 1K slow-scan charge-coupled device camera (Pro-
scan Scheuring, Germany).
solution of 2,2-bis(acrylamido)acetic acid (BAC) (2.8000 g,
14.084 mmol) and LiOHꢂH2O (0.5963 g, 14.084 mmol) in
bidistilled water (30 ꢀmL) was added dropwise. The reaction
was maintained at 0 C and left under stirring for 2 days. Af-
ter this period, the crude reaction mixture was purified by
ionic exchange chromatography on Dowex Mac-3 (8 ꢃ 60
cm) using bidistilled water (500 mL), and then an acid con-
centration elution gradient passing from 10ꢁ4 M to 0.1 M hy-
drochloric acid (1000 mL total). The product obtained was
freeze-dried then dissolved in trifluoroacetic acid to obtain a
1 M solution maintained for 3 h under gentle stirring in
nitrogen atmosphere. The product was precipitated by addi-
tion of diethyl ether (10 mL), washed with fresh solvent
(3 ꢃ 10 mL), and dried under vacuum affording a white and
deliquescent solid. Yield: 55%.
Synthesis of 4-N-Boc-2-(R, S)-methylpiperazine (MP-Boc)
Typically, 2-(R, S)-methylpiperazine (MP) (21.1 g, 0.200 mol)
was dissolved in tap water (1.2 L) and glacial acetic acid
(12.0 g, 0.197 mol) was added. The mixture was gently
1H NMR and 13C-NMR (D2O) are reported in Supporting In-
formation (Fig. S1, Table S1). Anal. Calcd for: C12H20N4O4: C,
50.69; H, 7.09; N, 19.71. Found: C, 49.46; H, 7.31; N, 18.83.
ꢀ
stirred for 15 min then cooled to 0 C. After this time, a so-
lution of di-(tert-butyl)dicarbonate (34.9 g, 0.160 mol) in
methanol (150 mL) was added dropwise in 30 min to the re-
active mixture. During this time, the reaction was vigorously
stirred and the temperature gradually warmed up to room
temperature. The reaction was maintained under this condi-
tion for further 20 min, then the precipitated formed filtered
and the pH was adjusted to 5.5 with 1 M HCl. The aqueous
phase was extracted with CH2Cl2 (2 ꢃ 100 mL) to separate
the disubstituted derivative then the pH was adjusted to 9.5
with 1 M NaOH. The desired product was recovered by
repeated extraction with CH2Cl2 (5 ꢃ 200 mL), the organic
layers collected and dried over anhydrous sodium sulfate.
The solvent was evaporated to afford the crude solid. Yield:
90%.
Synthesis of BAC-MP
The reaction and purification of BAC-MP were performed as
previously described for BAC-P using BAC (2.8000 g, 14.084
mmol), LiOH.H2O (0.5963 g, 14.084 mmol) and 4-N-Boc-2-
methylpiperazine (1.4100 g, 7.042 mmol) as reagents. Yield:
60%.
1H NMR and 13C-NMR (D2O) are reported in Supporting In-
formation (Fig. S2, Table S1). Anal. Calcd for: C13H22N4O4: C,
52.34; H, 7.43; N, 18.78. Found: C, 50.47; H, 7.62; N, 17.89.
Synthesis of MBA-P
To a solution of MBA (4.7438 g, 30.77 mmol) in bidistilled
water (220 mL) a solution of N-Boc-piperazine (1.9297 g,
10.46 mmol) in bidistilled water (50 mL) was added drop-
wise under stirring at 0 ꢀC. The reaction was maintained at
1H NMR (CDCl3, d, ppm): 0.99 (d, HNCH2CHCH3), 1.41 (s,
(CH3)3C), 1.57 (br, NHCH2CH), 2.34 (br, axial NHCH2CH),
2.64 (m, NHCH2CH), 2.72 (m, axial NHCH2CH2NBoc), 2.90
(m, equatorial NHCH2CH), 2.95 (br, equatorial NHCH2CH2N).
13C NMR (D2O, d, ppm): 19.16 (CH3CH), 28.15 ((CH3)3CO),
45.71 (HNCH2CH2N, CH3CHCH2N), 51.65 (CH3CH), 79.51
((CH3)3CO), 154.70 (NCO). Anal. Calcd for: C10H20N2O2: C,
59.97; H, 10.07; N, 13.99. Found: C, 59.12; H, 10.13; N,
13.75.
0
ꢀC for 48 h and monitored by thin layer chromatography
(TLC) using 2-propanol/water/0.15 M acetate buffer pH 5.5
¼ 2:2:6 (v/v) as eluent. During this time, a precipitate was
formed that was filtered off and the solution acidified at pH
3.0 using 1 M hydrochloric acid. Unreacted MBA was suc-
cessfully eliminated by repeated extraction with diethyl ether
(5 ꢃ 100 mL) and ethyl acetate (2 ꢃ 150 mL). The aqueous
solution was treated with 5% hydrochloric acid (4.6 mL)
and stirred for 24 h. The mixture was then freeze dried and
the product obtained as a white solid. Yield: 70%.
Synthesis of N-Boc-piperazine (P-Boc)
P-Boc was synthesized following the same procedure above
described for MP-Boc using piperazine hexahydrate (25.0 g,
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1H NMR and 13C-NMR (D2O) are reported in Supporting In-
formation (Fig. S3, Table S1). Anal. Calcd for: C11H20N4O2: C,
54.98; H, 8.39; N, 23.32. Found: C, 49.98; H, 8.83; N, 21.14.
5 by the addition of 1 M HCl. The final product was obtained
after freeze drying. Yield: 97%.
Mn ¼ 3200, Mw ¼ 4100, Mw/Mn ¼ 1.28, Rh ¼ 6.8 nm, [g] ¼
0.234 dL/g, a ¼ 0.781, log K ¼ ꢁ3.74. 1H NMR (D2O) is
reported in Supporting Information (Fig. S7).
Synthesis of MBA-MP, BP-P, and BP-P
The same procedure above described for MBA-P was followed.
MBA-MP. Yield: 65%.
Synthesis of BAC-P-grafted-BSA
Guanidine hydrochloride (0.5056 g, 5.190 mmol) was dis-
solved in bidistilled water (5 mL), BSA (1.5065 g, 0.023
mmol) was added, and the mixture was vigorously stirred
for 10 min until a clear solution was obtained. BAC-P
(0.3190 g, 1.122 mmol) was then added to the mixture, and
the pH was adjusted at 8.0 by the addition of solid Na2CO3.
The reaction was kept under nitrogen atmosphere and gentle
stirring for 5 days. The mixture was purified by dialysis
against water (membrane cut-off 50,000) and the pure prod-
uct collected after freeze-drying. Yield: 72%.
1H NMR and 13C-NMR (D2O) are reported in Supporting In-
formation (Fig. S4, Table S1). Anal. Calcd for C12H22N4O2: C,
56.67; H, 8.72; N, 22.03. Found C, 51.92; H, 9.11; N, 19.74.
BP-P. Yield: 81%.
1H NMR and 13C-NMR (D2O) are reported in Supporting In-
formation (Fig. S5, Table S1). Anal. Calcd for C14H24N4O2: C,
59.98; H, 8.63; N, 19.98. Found: C, 59.43; H, 8.73; N, 19.59.
BP-MP. Yield: 75%.
1H NMR and 13C-NMR (D2O) are reported in Supporting In-
formation (Fig. S6, Table S1). Anal. Calcd for C15H26N4O2: C,
61.20; H, 8.90; N, 19.03. Found: C, 61.25; H, 9.02; N, 18.98.
Molecular weight and viscometric data are reported in
Table 2.
Synthesis of BAC-MP-grafted-BSA
The reaction was performed following the same procedure
described for BAC-P-grafted-BSA using guanidine hydrochlor-
ide (0.5056 mg, 5.190 mmol), BSA (1.5065 g, 0.023 mmol),
BAC-MP (0.3347 g, 1.122 mmol). The pure product was
obtained after the usual ultrafiltration procedure and freeze
drying. Yield: 84%.
Synthesis of BAC-MP Homopolymer (ISA23)
To a solution of BAC-MP (0.4 g, 1.3408 mmol) in bidistilled
water (450 lL), 1 M NaOH was added dropwise until pH 9,
and the reaction kept under stirring at room temperature for
5 days in nitrogen atmosphere. After this period, the mixture
was acidified to pH 4.4–5.5 with 37% HCl, filtered through a
paper filter then ultrafiltered through a membrane with
nominal cut-off of 3000. The product was obtained after
freeze-drying as a yellowish solid. Yield: 79%.
Molecular weight and viscometric data are reported in
Table 2.
Synthesis of BP-P-grafted BSA
Mn ¼ 38,000, Mw ¼ 50,000, Mw/Mn ¼ 1.32, Rh ¼ 7.4 nm,
1
The same procedure as in the previous case was followed
using guanidine hydrochloride (0.5056 mg, 5.190 mmol), BSA
(1.5065 g, 0.023 mmol), and BP-P (0.3146 mg, 1.122 mmol).
The usual work-up afforded the pure product. Yield: 79%.
[g] ¼ 0.567 dL/g, a ¼ 0.773, log K ¼ ꢁ3.855. H NMR (D2O,
d, ppm): 1.26 (d, CH3CH), 2.64 (m, CH2CONH), 2.85 (m,
CHCH3), 3.07 (br, NCH2 MP ring), 3.28 (br, equatorial
NCH2CH2 MP ring), 3.28 (br, axial NCH2CH2N MP ring,
NCH2CH2), 5.51, 5.45 (ds, CHCOOH and CHCOOꢁ).
Molecular weight and viscometric data are reported in
Table 2.
Synthesis of BP-P Homopolymer
The reaction was performed as above reported, starting from
BP-P (0.7200 g, 5.569 mmol) dissolved in bidistilled water (1.4
mL). After 5 days under gentle stirring, the mixture was acidified
with concentrated HCl, filtered through a paper filter then ultra-
filtered through a membrane with nominal cut-off of 3000. The
pure product was recovered after freeze-drying. Yield: 72.5%.
Synthesis of Star-like BAC-MP
To a solution of cyclam (2.0 mg, 0.001 mmol) in 0.5 M NaOH
(150 mL), BAC-MP (100.0 mg, 0.335 mmol) was added, and
the reaction left 5 days at pH 8 under gentle stirring. After
this time, the mixture was freeze-dried and the dry product
recovered. Yield: 98%.
Mn ¼ 30,900, Mw ¼ 38,000, Mw/Mn ¼ 1.13, Rh ¼ 3.808 nm,
[g] ¼ 0.294 dL/g, a ¼ 1.01, log K ¼ ꢁ4.703. 1H NMR (D2O,
d, ppm): 2.58 (br, CH2CONH), 3.06 (s, NCH2 piperazine ring),
3.12 (m, NCH2CH2), 3.31 (br, CH2NCO piperazine ring), 3.38
(br, CH2NCO piperazine ring).
Mn ¼ 10,800, Mw ¼ 11,660, Mw/Mn ¼ 1.08, [g] ¼ 0.125 dL/g,
a ¼ 0.607, log K ¼ ꢁ3.706, branching frequency ¼ 2.17. 1H
NMR (D2O) is reported in Supporting Information (Fig. S8).
Synthesis of Star-like BP-MP
The same procedure above described was followed using
cyclam (20.4 mg, 0.102 mmol), 0.5 M sodium hydroxide
(550 mL), and BP-MP (300.0 mg, 1.020 mmol). The reaction
was filtered through a paper filter and the crude product
freeze-dried. Yield: 97%.
Synthesis of BAC-MP-Acryloylmorpholine Conjugate
Acryloylmorpholine (4.4 mg, 0.035 mmol) was dissolved in
0.1 M sodium hydroxide (150 lL) and BAC-MP (100.0 mg,
0.335 mmol) was added under vigorous stirring until a clear
solution was obtained. Then, 1 M NaOH was dropped into
the reaction mixture until pH 8.5 was reached, and the reac-
tion was kept 3 days at room temperature under nitrogen
atmosphere. After this time, the mixture was acidified at pH
Mn ¼ 3300, Mw ¼ 3900, Mw/Mn ¼ 1.21, [g] ¼ 0.128 dL/g, a
¼ 0.521, log K ¼ ꢁ3.823, branching frequency ¼ 2.08. 1H
NMR (D2O) is reported in Supporting Information (Fig. S9).
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FIGURE 1 Structures of the HDDs synthesized.
Synthesis of Hetero-difunctional BAC-MP-TC
Anal. Calcd for (C15H26N4O2)10-(C27H46S): C, 63.53; H, 9.21;
N, 16.74. Found: C, 62.75; H, 9.11; N, 17.50.
To a solution of BAC-MP (103.3 mg, 0.251 mmol) in bidis-
tilled water (1 mL), TC (10.1 mg, 0.025 mmol) dissolved in
diethyl ether (0.1 mL) was added, and the formation of a
macroemulsion was observed. The pH was adjusted to 7.0
by the addition of 3 M NaOH, and the mixture vigorously
stirred for 2 days. Then, 1 M NaOH was added until pH 9
and the reaction maintained in this condition under stirring
for further 5 days. After this time, the mixture was acidified
to pH 5 by the addition of 1 M HCl and purified by dialysis
against water (membrane cut-off 3500) for 2 h. The crude
product collected after freeze drying was suspended in
diethyl ether (2 mL) to remove unreacted TC, and dried in
vacuum. Yield: 38%.
RESULTS AND DISCUSSION
The aim of this work was to prepare hetero-diterminated
PAAs as well as some novel PAA derivatives not previously
obtained by simple methods. HDDs were the simplest build-
ing blocks capable of exactly reproducing the same sequence
of amide-and tert-amine groups along the polymer chain of
PAAs, while unequivocally ensuring hetero-difunctional chain
terminals. As mentioned in Introduction, linear PAAs can be
prepared by the stepwise polyaddition of bisacrylamides and
either prim-monoamines or sec-bisamines, which yields PAAs
with different chain structures. The investigation reported in
this paper concerned only HDDs leading to PAAs with
‘‘a ꢁꢁa ꢁꢁb ꢁꢁb’’ sequence of amide- and amine groups
along the polymer chain, that is, HDDs obtained by the
mono-addition of bis-sec-amines to bisacrylamides (Scheme
2). Six HDDs were prepared starting from 2,2-bisacrylamido-
acetic acid (BAC), 1,4-bisacryloylpiperazine (BP), and N,N0-
methylenebisacrylamide (MBA) as bisacrylamides and piper-
azine (P) and 2-methylpiperazine (MP) as amines. BAC was
selected because, in combination with MP, it gives ISA23, the
highly biocompatible ‘‘stealth-like’’ amphoteric PAA men-
tioned in Introduction. BP and MBA were chosen because
with P and MP they give PAAs extensively studied as compo-
nents of bioactive hydrogels (data not shown). Moreover, P
and MP are endowed with high reactivity toward Michael
polyaddition, coupled with limited basicity leading, as a rule,
to good biocompatibility of the resultant PAAs.1,5,6,7,14 The
six HDDs obtained by combining the above building blocks
were named BAC-P, BAC-MP, BP-P, BP-MP, MBA-P, and MBA-
MP after their parent compounds. Their structures, together
with some relevant characterizations, are reported in Figure 1
and Table 1.
Anal. Calcd for (C13H22N4O4)10-(C27H46S): C, 55.69; H, 7.92,
N, 16.55. Found: C, 51.98; H, 8.46, N, 18.15.
Synthesis of Hetero-difunctional MBA-P-TC
The reaction was performed following the same procedure
as in the previous case using MBA-P (60.3 mg, 0.251 mmol)
and TC (10.1 mg, 0.025 mmol). Yield: 29%.
Anal. Calcd for (C11H20N4O2)10-(C27H46S): C, 58.64; H, 8.84;
N, 19.97. Found: C, 58.00; H, 8.76; N, 20.56.
Synthesis of Hetero-difunctional BP-P-TC
The reaction was performed following the same procedure
as in the case of ISA23-TC using: BP-P (70.4 mg, 0.251
mmol) and TC (10.1 mg, 0.025 mmol). Yield: 44%.
Anal. Calcd for (C14H24N4O2)10-(C27H46S): C, 62.56; H, 8.99;
N, 17.48. Found: C, 61.42; H, 8.83; N, 18.60.
Synthesis of Hetero-difunctional BPMP-TC
The reaction was performed following the same procedure
as in the case of ISA23-TC using: BP-MP (73.9 mg, 0.251
mmol) and TC (10.1 mg, 0.025 mmol). Yield: 36%.
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TABLE 1 C/N Ratio of HDDs
ophilized and pure BAC-P and BAC-MP finally obtained by
deprotection with trifluoroacetic acid.
C/N Ratio
The addition reactions of MBA or BP with Boc-P or Boc-MP
gave protected MBA-P, MBA-MP, BP-P, and BP-MP, respec-
tively. The reaction mixtures, containing dimers and residual
bisacrylamide, were acidified to pH 3 and the bisacrylamides
fractionally extracted with organic solvents. Under these con-
ditions, the protected dimers, being ionized, remained in the
aqueous phase and were retrieved by freeze-drying. As the
products were insoluble in TFA, the deprotection step was
performed with 5% hydrochloric acid.
HDD
Calculated
Found
BAC-P
2.57
2.79
2.35
2.57
3.00
3.22
2.63
2.82
2.36
2.63
3.04
3.23
BAC-MP
MBA-P
MBA-MP
BP-P
BP-MP
All protected HDDs were characterized by 1H NMR. All
deprotected HDDs were fully characterized by elemental
analysis, 1H NMR, 13C NMR, DEPT, 1H-1H COSY, HMBC, and
HETCOR (see Experimental and Supporting Information, Ta-
ble S1). The results of these characterizations were in agree-
ment with the proposed structures. C/N ratios by elemental
analysis are reported in Table 1. As all HDDs were isolated
as trifluoroacetates or hydrochlorides and vacuum dried or
lyophilized, some loss of acid was inevitable and, therefore,
the absolute values of elemental analyses were not fully reli-
able. However, the C/N ratios were not liable to be signifi-
cantly affected by the isolation procedures and provided a
reliable confirmation that the product structures were as
expected.
The general synthetic procedure adopted for preparing
HDDs is depicted in Scheme 3. It involved the synthesis of
mono-Boc-protected P and MP [Step (a)] followed by reac-
tion with moderate excess bisacrylamide, isolation and puri-
fication of the mono-addition product, and cleavage of the
protecting group [Step (b)].
Both steps deserve comments. In Step (a), Boc-P and Boc-MP
were prepared from quasi-stoichiometric amounts of di-(tert-
butyl)dicarbonate and mono-protonated P and MP, in turn
obtained by in situ adding acetic acid to the amine in 1:1
molar ratio. Only a slight excess of amine mono-acetate was
required to maximize the yield of the mono-substituted prod-
ucts, since the two pKa values of both amines differ by ꢄ four
orders of magnitude,17 and under the conditions adopted the
amount of doubly protonated molecules and, correspondingly,
of uncharged molecules amenable to react twice was minimal.
In Step (b), the addition reactions of Boc-P and Boc-MP to
BAC, BP, and MBA were performed for 2 days in water at
All HDDs, as either hydrochlorides or trifluoroacetates, could
be stored for a long time at 0–5 ꢀC, if protected from mois-
ture and did not polymerize at room temperature. In aque-
ous solution, polymerization was triggered by rising pH
above 7.5 with bases inert toward Michael addition, such as
inorganic hydroxides or tertiary amines. The molecular
weights of the resultant polymers were usually high and
0
ꢀC, using two- to threefold excess bisacrylamide. Under
these conditions, the double addition to the same bisacryla-
mide molecule was minimized. It may be noticed that the
excess bisacrylamide required to achieve this result was sig-
nificantly lower than expected from purely statistical calcula-
ꢀ
tions. It would appear that at 0 C the amine addition to one
of the two double bonds biased the reactivity of the other
notwithstanding the fairly long distance between them. How-
ever, at room temperature and with the same stoichiometric
ratios, significant amounts of the double-addition products
were formed. The product work-up depended on the bisacry-
lamide used. In the case of BAC, the reaction mixtures con-
tained excess BAC, protected BAC-P (or BAC-MP) and small
amounts of di-addition products, that is, di-protected trimers
of the ‘‘b ꢁꢁb ꢁꢁa ꢁꢁa ꢁꢁb ꢁꢁb’’ type. All these products
were highly polar and hardly extractable from water with or-
ganic solvents. Therefore, they were isolated by ion-exchange
chromatography using a weakly acid (carboxylated) resin and
following a two-step elution protocol. The first step was
performed with bidistilled water as eluent, the second step
with an acid concentration elution gradient passing from
10ꢁ4 M to 0.1 M hydrochloric acid (see Fig. 2). Unreacted
BAC was eluted in the first step. In the second step, the
mono-addition products were eluted first, followed by the di-
substituted products, which were discarded. The combined
fractions containing the mono-addition products were then ly-
SCHEME 3 General synthetic pathway for the preparation of
HDDs. Step (a): synthesis of mono-Boc-protected P and MP.
Step (b): Michael-type reaction with bisacrylamide, and cleav-
age of the protecting group.
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tion progress was monitored by 1H NMR until signals at 5.78,
6.13, and 6.67 ppm, relative to the acrylic hydrogens, fell below
the detection threshold of the instrument. The products had,
respectively, Mn 38,000 and 30,900, with Mw/Mn 1.32 and 1.13.
These molecular weight values were significantly higher than
those of the corresponding PAAs obtained by the traditional meth-
ods after the same reaction times, with lower polydispersities.
The polymerization of HDD mixtures with either monofunc-
tional acrylamides or amines with approximately the same
reactivity as the corresponding function present in HDDs,
leads at 100% conversion to an average polymerization
degree equal to:
NHDD;0
Xn ¼
(1)
Nmonofunctional;0
where NHDD,0 is the initial number of HDD molecules and
Nmonofunctional,0 is the initial number of mono-functional com-
pound. Obviously, Mn is given by the product of Xn times the
molecular weight of the HDD used, added by the molecular
weight of the mono-functional compound. The resultant
polymer will be terminated at one terminus with the unreac-
tive group and at the opposite terminus with the excess re-
active function. The preparation of an ISA23 oligomer mono-
functionalized at one terminus with an activated double
bond by polymerization of BAC-MP in the presence of
4-acryloylmorpholine is reported in Scheme 4.
FIGURE 2 Equipment adopted to obtain the hydrochloric acid
gradient used in the second elution step of BAC-P and BAC-MP
isolation. Column C was connected through Valve 1 to Flask B
containing 10ꢁ4 M hydrochloric acid, in turn connected through
Valve 2 to Flask A containing 10ꢁ1 M hydrochloric acid. Each
drop flowing out the column drew a drop from flask B, imme-
diately replaced by a drop from flask A, thus obtaining the
desired acid concentration gradient.
The reaction was performed adopting a 10/1 BAC-MP/
4-acryloylmorpholine molar ratio. The reaction progress was
monitored by 1H NMR by comparing the signals at 6.22 and
5.62 ppm, relative to the end-chain acrylic group and the
CH-COOꢁ hydrogen of BAC, respectively (see Supporting Infor-
mation Fig. S7). The reaction was stopped when in the NMR
spectrum, the ratio of the reference peaks’ integrals remained
constant with time and was consistent with the presence of a
double bond every 10 BAC-MP units, that is, the reaction yield
had approached 100%. The Mn of the product was 3200, with
Mw/Mn 1.28. This molecular weight value was very close to
the expected value of 3150, as calculated from eq 1.
obviously related to the reaction time, whereas all practical
difficulties to achieve a precise balance among the reactant
functions were got round.
The synthetic potential of HDDs was assessed by performing
model reactions, namely self-polyaddition, preparation of
monofunctional PAA oligomers with controlled molecular
weight, of star-like PAA architectures, of protein-PAA conju-
gates and of tadpole-like PAA-cholesterol amphiphiles. The
aim was to test the general aptitude of HDDs to be used for
these reactions and was limited to a few examples involving
only some of the prepared dimers at a time. It may be
noticed that most of the reaction products were hardly
obtainable by the traditional PAA preparation method.
The same derivative, as well as the cholesteryl-terminated
PAA (see below) could be also prepared by reacting 4-acryl-
oylmorpholine and TC with presynthesized HDDS polymers.
However, the direct polymerization of HDDs in presence of
the same monofunctional compounds presented two distinct
advantages: it could be performed in one-pot, one step reac-
tion; moreover, both 4-acryloylmorpholine and TC acted in
the mean time as chain regulators.
The self-polyadditions of BAC-MP and BP-P were performed as
model syntheses of PAAs via the corresponding HDDs. The reac-
SCHEME 4 Synthesis of the mono-functionalized ISA23 oligomer by polymerization of BAC-MP in the presence of 4-
acryloylmorpholine.
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where f is the number of reactive functions of the multifunc-
Na
þf
tional monomer (four for cyclam), r ¼
0 , Na ¼ Nb is the
molar amount of HDD and Nb’ is theNmb olNabr amount of the
multifunctional monomer. In the case of BAC-MP-C, the
adopted HDD/cyclam molar ratio was 34.2. The product had
Mn 10,800 with Mw/Mn 1.08, to be compared with the calcu-
lated values of 10,700 and 1.26. It may be observed that Mn
was in excellent agreement with the expected value, whereas
the Mw/Mn ratio was remarkably lower. In fact, the product
obtained was quasi-monodisperse. Interestingly, this effect
was not observed with BP-MP, suggesting that the zwitterionic
nature of BAC-MP was largely responsible for it. Possibly,
BAC-MP reversibly interacted with cyclam, resulting in a local
catalytic activity of the latter favoring the growth of the
chains attached to it over that of ‘‘free’’ molecular species.
Thus, the behavior of the BAC-MP/cyclam system as regards
the molecular weight distribution of the resultant polymer
approached that of a cyclic monomer growing on a multifunc-
tional center. The LCB frequency of BP-MP-C and BAC-MP-C
was determined by SEC connected in series with refractive
index (RI), light scattering, and viscometric detectors (see Ex-
perimental) and found in both cases of ꢄ two side chains per
macromolecule. This LCB frequency value was consistent with
the expected four-arm architecture originating from a cyclam
core.
SCHEME 5 Synthesis of four-arm star-like PAAs from BP-MP
and BAC-MP and cyclam.
Four-arm star-like PAAs (BP-MP-C and BAC-MP-C) were
synthesized from BP-MP and BAC-MP and 1,4,8,11-tetraa-
zacyclotetradecane (cyclam; Scheme 5). The polymeriza-
tion reactions were performed, as usual, in aqueous solu-
tion at pH 9 and monitored by 1H NMR. The products
were isolated when the signals attributed to the double
bond hydrogens fell below the instrument’s detection
threshold.
BAC-P, BAC-MP, BP-P, and BSA were chosen as model HDDs
and protein for the preparation of PAA-protein graft copoly-
mers. This was simply achieved by triggering the HDD poly-
merization in aqueous medium in the presence of BSA that
participated in the polymerization through its exposed NH2
and SH groups. To give an example, the synthesis of BAC-
MP-g-BSA is reported in Scheme 6.
In the case of BP-MP-C, the adopted HDD/cyclam molar ratio
was 10. The Mn of the product was 3300, with Mw/Mn 1.21.
These values were in close agreement with the values of
3400 and 1.29 calculated from the classic literature’s formu-
las18,19 extrapolated for p ¼ 1 (eqs 2 and 3):
The occurrence of PAA grafting onto BSA was evaluated by
SEC connected in series with RI, light scattering, and visco-
metric detectors. The data, reported in Table 2, provided clear
evidence of the increasing of the molecular weight of copoly-
mers with respect to native BSA, always accompanied by low
polydispersities and higher values of the Mark–Houwink con-
ðfrp þ 1 ꢁ rpÞ
ꢁ
Xn ¼
(2)
(3)
ð1 ꢁ rpÞ
ꢁ
Xw
ꢁ
Xn
frp
¼ 1 þ
2
stants, hence higher hydrodynamic radii.
A
further
ðfrp þ 1 ꢁ rpÞ
SCHEME 6 Synthesis of BAC-MP-g-BSA by polymerization of BAC-MP in the presence of BSA.
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TABLE 2 Molecular Weight and Viscometric Data of
PAA-Grafted BSA Samples Compared with BSA
[g]c
Rh
f
a
b
Sample
BSA
Mn
67,000
Mw/Mn (dL/g) ad
log Ke (nm)
1.04
0.054 0.034 ꢁ1.45 3.92
0.067 0.203 ꢁ2.74 6.1
BAC-MP-g-BSA 1,01,000 1.07
BAC-P-g-BSA
BP-P-g-BSA
a
84,900
88,100
1.17
1.14
0.053 0.265 ꢁ2.58 4.11
0.083 0.163 ꢁ2.04 5.90
Number average molecular weight.
Polydispersity index.
b
c
Intrinsic viscosity.
d,e
Mark–Houwink constants.
f
Hydrodynamic radius.
FIGURE 3 Potentiometric titration curves of BP-P-g-BSA copol-
ymer and of BSA/linear BP-P mixtures of known composition.
confirmation was provided by the far higher solubility in
aqueous media of the copolymers compared with virgin BSA.
These reaction systems closely resembled the preparation of
mono-functional PAAs by copolymerization of HDDs with
monofunctional amines or acrylamides, as the SH group
reacts with activated double bonds in the same way as the
amine groups.20,21 Being TC insoluble in water, the prepara-
tions of HDD-TC conjugates were performed in a diethy-
lether/water slurry. TC resided in the former solvent and the
HDDs in the latter. The two solutions were vigorously stirred
under nitrogen at room temperature in a closed vessel until
the signals due to the acrylic hydrogens of bisacrylamides
were no longer detectable in the 1H NMR spectra of the
aqueous phases. The feasibility of this process was ascer-
tained with BAC-MP, MBA-P, BP-P, and BP-MP. A 10:1 HDD/
TC molar ratio was used for obtaining polymers with 10
repeating units of HDD per TC molecule. This value was cho-
sen because literature data22,23 suggested it as leading to a
suitable hydrophilic/lipophilic balance to form and hydro-
phobically stabilize polyelectrolyte emulsions. In the case of
BAC-MP-TC and MBA-P-TC, their amphiphilic nature led to
phase segregation both in aqueous media and organic sol-
vents. Consequently, their 1H NMR spectra showed neither
traces of the PAA backbone in deuterated water, nor of the
cholesterol moiety in deuterated chloroform, as expected.
BP-P turned to be hardly soluble in both solvents, probably
due to the tendency of the BP-P chain to crystallize (data not
shown). Only in the case of BP-MP-TC both components were
soluble in deuterated chloroform. This allowed obtaining
The BP-P-g-BSA copolymer was further characterized by
comparing the tracing of its potentiometric titrations with
those of virgin BSA/linear BP-P mixtures of known composi-
tion (Fig. 3), as previously reported.13 The titration curves of
the copolymer and of the BSA/BP-P mixtures exhibited two
evident inflections at pH values close to the inflections of the
BP-P titration curve, whereas the BSA curve was character-
ized by a semicontinuous decrease of pH on acid addition
without clearly detectable inflections. Considering that the
amine character of the albumin ANH2 groups involved in the
grafting reaction is preserved, we may reasonably suppose
that PAA/BSA mixtures are good models for PAA-grafted al-
bumin, as far as titration is concerned. Therefore, from the
family of titration curves reported in Figure 3, the PAA con-
tent in the grafted product can be determined as ꢄ 20% by
weight. This value was consistent with the molecular weight
difference of the BP-P-g-BSA copolymer with respect that of
BSA (calculated 18,000, experimentally found 21,100).
Assuming that no BP-P was present, as the copolymer had
been extensively ultrafiltered through a membrane with mo-
lecular weight cut-off 50,000, this correspondence confirmed
that grafting reaction occurred as expected. No titration
experiments were performed with BAC-MP-g-BSA and BAC-
P-g-BSA as the acid–base properties of BAC-MP and BAC-P
are close to those of BSA.
1
clear homogeneous solutions and running H NMR spectra in
‘‘Tadpole-like’’ PAA-cholesterol amphiphiles were obtained by
performing the polymerization of HDDs in the presence of
TC, which participates in the Michael polyaddition through
its SH group (Scheme 7).
this solvent. These spectra did not significantly differ from
those of a previously synthesized BP-MP PAA carrying pendant
TC moieties24 and showed the presence of the diagnostic peaks
of both components. The calculated and experimentally
SCHEME 7 Synthesis of ‘‘tadpole-like’’ PAA-cholesterol amphiphiles by polymerization of HDDs in the presence of TC.
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TABLE 3 C/N Ratio of ‘‘Tadpole-like’’ PAA-Cholesterol
Amphiphiles
Sample
C/Na
C/Nb
HDD/TCc
BAC-MP-TC
MBA-P-TC
BP-P-TC
3.21
2.94
3.58
3.80
2.98
2.82
3.30
3.59
4.52
7.97
5.17
6.61
BP-MP-TC
a
Calculated.
b
Found.
c
Molar ratio between the HDD repeat unit and the TC moiety as calcu-
lated from C/N found values, to be compared with a 10:1 HDD/TC molar
ratio in the reaction feeding.
FIGURE 5 PAA-TC nanoparticle diameters in PBS solution pH
7.4 at 25 ꢀC as a function of concentration: BAC-MP (
);
MBA-P ( ); BP-P ( ); BP-MP ( ).
determined C/N ratios for all the PAA-TC polymers synthesized
are reported in Table 3. It may be observed that the experi-
mental values were invariably lower than the theoretical ones,
pointing to cholesterol content somewhat lower than expected,
doubtless due to the heterogeneous reaction conditions
adopted. All PAA-TC samples formed nanoparticles in aqueous
media, as confirmed by DLS and TEM analyses. As an example,
the DLS curves of BAC-MP-TC are reported in Figure 4.
their size increased sharply, reached a maximum at about
twice these values, then remained approximately constant
and finally decreased for concentrations higher than 1 mg/
mL. Consequently, the critical micelle concentration (CMC)
deduced from the graphs varied from 0.16 to 0.7 mg/mL, fol-
lowing the trend BP-P < BP-MP ꢅ MBA-P << BAC-MP. This
trend is consistent with the relative hydrophilicities of the
PAA portions. The apparent exception provided by BP-P-TC
can be explained by the previously mentioned structure-
forming tendency of its PAA portion. Nanoparticle average
sizes at 1 mg/mL concentration varied in the range 13–28
nm and, not surprisingly, followed the same trend as the
CMC, the most hydrophilic ones giving larger nanoparticles.
The temperature ꢀdependence of nanoparticle average size in
the range 25–60 C are reported in Figure 6. All curves, leav-
ing apart BAC-MP, show qualitatively the same trend. The av-
erage size initially decreased until about 30 ꢀC, remained
approximately constant in the range 30–45 ꢀC and ꢀ then
increased until the maximum tested temperature of 60 C. In
the case of BAC-MP, the trend was initially the same and the
It may be noticed that the correlogram [Fig. 4(a)] is consist-
ent with a neat homogeneous monomodal size distribution.
The other PAA-TC samples gave similar results. The average
PAA-TC nanoparticle sizes in PBS solution pH 7.4 as a func-
tion of concentration and temperature are reported in
Figures 5 and 6, respectively.
It may be observed that particle formation at 25 ꢀC in PBS
started at concentrations in the range 0.15–0.60 mg/mL,
ꢀ
average size decreased until about 30 C, but immediately af-
ter started to increase until 60 ꢀC. TEM analysis showed a
quasispherical shape with no evidence of dual distribution,
as for example in the case of BAC-MP reported in Figure 7.
FIGURE 4 DLS analysis of BAC-MP-TC nanoparticles in PBS (1
mg/mL) at 25 ꢀC. Panel (a): correlogram; panel (b): number
size-distribution.
FIGURE 6 PAA-TC nanoparticle diameters in PBS solution pH
7.4 (1 mg/mL concentration) as a function of temperature: BAC-
MP (
); MBA-P (
); BP-P (
); BP-MP (
).
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ꢂꢂꢂꢂꢂꢂ
that is, PAAs solely containing molecules of a ꢁꢁ ꢁꢁ b type.
This precluded to PAAs the access to a number of derivatives,
such as for instance PAAs of controlled average molecular
weight and mono-functionalized with an acrylamide- or a sec-
amine group at one end, PAAs with a star-like molecular
architecture, graft-PAA-protein conjugates, ‘‘tadpole-like’’ PAA
conjugates with hydrophobic moieties able to self-assemble
into nanoparticles in aqueous media. The devised strategy to
overcome this problem was to prepare HDDs of
‘‘a ꢁꢁa ꢁꢁb ꢁꢁb’’ type, that is, the mono-addition products of
a bis-sec-amine to a bisacrylamide, which as strong acid salts
12 Vader, P.; van der Aa, L. J.; Engbersen, J. F. J.; Storm, G.;
Schiffelers, R. M. Pharm. Res. 2012, 29, 352–361.
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but polymerized in aqueous media at pH > 7.5. In this article,
we report on the preparation of a selected number of HDDs
and provide evidence of their synthetic potential by perform-
ing model reactions following the above cited lines. It should
be noticed that the specificity of the Michael addition of
amines to bisacrylamides in aqueous media, shared only by
thiols under the PAA preparation conditions, allowed prepar-
ing most of the above derivatives by simply performing the
polymerization of HDDs in the presence of the substrates of
interest with no need of presynthesizing PAAs mono-function-
alized at one end with an activated double bond. This greatly
simplified the synthetic processes negligibly affecting the
products’ characteristics. On the whole, it can be concluded
that the aim of this research has been adequately fulfilled,
thus demonstrating the wide synthetic potential of HDDs.
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