Arborescent polypeptides from γ-benzyl l -glutamic acid

Article abstract of DOI:10.1002/pola.26958

The synthesis of arborescent polymers with poly(γ-benzyl L-glutamate) (PBG) side chains was achieved through successive grafting reactions. The linear PBG building blocks were produced by the ring-opening polymerization of γ-benzyl L-glutamic acid N-carboxyanhydride initiated with n-hexylamine. The polymerization conditions were optimized to minimize the loss of amino chain termini in the reaction. Acidolysis of a fraction of the benzyl groups on a linear PBG substrate and coupling with linear PBG using a carbodiimide/ hydroxybenzotriazole promoter system yielded a comb-branched or generation zero (G0) arborescent PBG. Further partial deprotection and grafting cycles led to arborescent PBG of generations G1 to G3. The solvent used in the coupling reaction had a dramatic influence on the yield of graft polymers of generations G1 and above, dimethylsulfoxide being preferable to N,N-dimethylformamide. This grafting onto scheme yielded well-defined (M_w/M_n ≤ 1.06), high molecular weight arborescent PBG in a few reaction cycles, with number-average molecular weights and branching functionalities reaching over 10⁶ and 290, respectively, for the G3 polymer. α-Helix to coiled conformation transitions were observed from N,N-dimethylformamide to dimethyl sulfoxide solutions, even for the highly branched polymers.

Full text of DOI:10.1002/pola.26958

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Arborescent Polypeptides from c-Benzyl L-Glutamic Acid
Greg Whitton, Mario Gauthier
Department of Chemistry, Institute for Polymer Research, University of Waterloo, 200 University Avenue West, Waterloo,
Ontario N2L 3G1, Canada
Correspondence to: M. Gauthier (E-mail: gauthier@uwaterloo.ca)
Received 15 May 2013; accepted 18 September 2013; published online 8 October 2013
DOI: 10.1002/pola.26958
ABSTRACT: The synthesis of arborescent polymers with poly(c-
benzyl L-glutamate) (PBG) side chains was achieved through
successive grafting reactions. The linear PBG building blocks
were produced by the ring-opening polymerization of c-benzyl
L-glutamic acid N-carboxyanhydride initiated with n-hexyl-
amine. The polymerization conditions were optimized to mini-
mize the loss of amino chain termini in the reaction. Acidolysis
of a fraction of the benzyl groups on a linear PBG substrate
and coupling with linear PBG using a carbodiimide/hydroxy-
benzotriazole promoter system yielded a comb-branched or
generation zero (G0) arborescent PBG. Further partial deprotec-
tion and grafting cycles led to arborescent PBG of generations
G1 to G3. The solvent used in the coupling reaction had a dra-
matic influence on the yield of graft polymers of generations
G1 and above, dimethylsulfoxide being preferable to N,N-
dimethylformamide. This grafting onto scheme yielded well-
defined (Mw/Mn ꢀ 1.06), high molecular weight arborescent
PBG in a few reaction cycles, with number-average molecular
weights and branching functionalities reaching over 106 and
290, respectively, for the G3 polymer. a-Helix to coiled confor-
mation transitions were observed from N,N-dimethylforma-
mide to dimethyl sulfoxide solutions, even for the highly
C
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branched polymers.
2013 Wiley Periodicals, Inc. J. Polym.
Sci., Part A: Polym. Chem. 2013, 51, 5270–5279
KEYWORDS: arborescent polymers; branched; chain conforma-
tion; conformational analysis; dendrigraft polymers; dynamic
light scattering; polypeptides; ring-opening polymerization
amide-like structures was considerably expanded by Tomalia
with the synthesis of polyamidoamine (PAMAM) dendrimers,
now commercially available and being investigated in a num-
ber of biomedical applications.8 Other examples of poten-
tially biocompatible dendrimers reported in the literature
include triazine (melamine) dendrimers,14 oligosaccharide–
polypeptide dendrimers,15 glycodendrimers,16 and PAMAM-
poly(L-glutamic acid) dendrimers.17 Hyperbranched dendritic
systems in that category have also been reported, including
polyglycerols18 and polyesters.19
INTRODUCTION Dendritic polymers have attracted much
attention due to their intriguing structure and unusual prop-
erties. Many methods have been suggested to synthesize dif-
ferent families of dendritic macromolecules including
dendrimers, hyperbranched polymers, and dendrigraft (arbo-
rescent) polymers from a wide range of monomers.1–5 This
allows tailoring of the properties of these materials to opti-
mize their performance in different applications, among
which the biomedical field certainly represents a major area
of interest.6–8 A primary concern for most biomedical uses is
biocompatibility, typically requiring a lack of toxicity, immu-
nological response, or other physiological reactions. For cer-
tain applications such as the intravenous delivery of drugs,
the carriers ideally should be monodispersed and in the 10
to 100 nm size range, to enhance their ability to cross epi-
thelial cell membranes and to avoid rapid clearance from the
body through the liver or the spleen.9 A narrow size distri-
bution should also lead to more reproducible pharmacoki-
netic behavior.10
Apart from dendritic architectures, star-branched polypep-
tides with a wide range of branching functionalities, high
molecular weights, and (in some cases) narrow MWDs have
been synthesized by different methods. Hadjichristidis20 thus
obtained 3-arm star-branched polypeptides, with molecular
weights reaching 1.8 3 105 and Mw/Mn 5 1.08, by coupling
polypeptides derived from c-benzyl L-glutamic acid N-car-
boxyanhydride (Glu-NCA) and e-benzyloxycarbonyl L-lysine
NCA with a triphenylmethane-4,40,400-triisocyanate linker. The
synthesis of peptide-based star polymers with branching
functionalities f 5 3–349 and Mw/Mn 5 1.20–1.83, obtained
mainly by an arms-first microgel-like coupling methodology,
The earliest examples of dendritic structures included den-
drimers analogous to globular polypeptides, derived from L-
lysine building blocks.11–13 The concept of dendrimers with
Additional Supporting Information may be found in the online version of this article.
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SCHEME 1 Synthesis of G0 arborescent PBG, with a comb-branched structure. G1–G3 dendritic structures are obtained by repeti-
tion of the partial acidolysis and grafting steps.
was reviewed recently.21,22 Another strategy relied upon the
initiation of Glu-NCA polymerization with poly(propylene
imine) dendrimers carrying up to 64 primary amine func-
tionalities, depending on the dendrimer generation number
used.23 Variations in the dendrimer to NCA ratio led to
molecular weights of up to 5 3 105 and polydispersities
quoted as “less than 1.2” for all the samples. A similar
approach used silica nanoparticles functionalized with pri-
mary amine groups as initiator for Glu-NCA to generate star
polypeptides with a silica core.24,25
trifluoroacetyl-protected L-lysine NCA.29 In this case the
polymerization was carried out in mildly acidic (pH 6.5)
water, and the polypeptide was deprotected with ammonia
to afford short linear poly(L-lysine trifluoroacetate) segments
with a number-average degree of polymerization Xn 5 8. The
fully deprotected linear substrate served as polyfunctional
initiator for the growth of protected poly(L-lysine) side
chains, in analogy to the Klok procedure. Subsequent cycles
of deprotection and side chain growth led to dendrigraft
polymer structures of generations up to G3, with Mn ꢀ 1.72
3 105 and Mw/Mn 5 1.36–1.46. Apart from the examples
provided above, the number of potentially biocompatible
dendrigraft polymers currently available, and particularly
those with peptide-like structures, remains limited.
The arborescent (or dendrigraft) systems are graft polymers
incorporating polymeric chain segment building blocks simi-
lar to star-branched molecules, but they have a multilevel
(dendritic) branched architecture arising from successive
grafting reactions, in analogy to dendrimer generations. This
graft-upon-graft strategy can yield relatively large (10–100
nm) macromolecules in a few synthetic cycles (generations),
while maintaining low polydispersity indices (Mw/Mn ꢁ 1.1
We now report the synthesis of well-defined (Mw/Mn < 1.1)
arborescent polymers from poly(c-benzyl L-glutamate) (PBG)
building blocks. This method is distinct from the ones
described above, in that in relies upon a generation-based
grafting onto methodology analogous to those reported for
the synthesis of arborescent polymers from different vinyl
monomers.1,26 This approach enables the full structural char-
acterization of the branched polymers (side chain and overall
molecular weight, and branching functionality), while main-
taining a narrow MWD over successive grafting cycles. The
approach used is summarized in Scheme 1. Linear PBG
chains are obtained by ring-opening polymerization of c-
benzyl L-glutamic acid N-carboxyanhydride (Glu-NCA) with a
primary amine initiator. Partial deprotection of linear PBG
provides a grafting substrate with carboxylic acid moieties,
that can be reacted with the primary amine terminus of PBG
by standard peptide coupling techniques to create a genera-
tion zero (G0) or comb-branched polypeptide. Subsequent
generations of arborescent polypeptides are obtained by suc-
cessive cycles of partial deprotection and grafting reactions.
The arborescent PBG molecules obtained should be
typically).26
A convenient grafting from technique was
reported by Klok et al. using L-lysine derivatives to synthe-
size dendrigraft polypeptides.27 This synthetic scheme
involved the polymerization of a protected L-lysine NCA to
produce a linear substrate, which was subsequently depro-
tected to generate primary amine moieties serving as initiat-
ing sites for the next generation of side chains. The method
of Klok et al. yielded large dendrigraft structures (up to gen-
eration G2) but suffered from significant molecular weight
distribution (MWD) broadening over successive cycles due
to side reactions. A variation of this technique used the
copolymerization of a protected L-lysine NCA with another
amino acid NCA.27,28 Selective deprotection of the L-lysine
units provided control over the branching density of the
molecules in this scheme, but the MWD broadening issue
remained unsolved. The grafting from technique developed
by Klok was subsequently modified by Collet et al., using
S1
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interesting as model compounds for globular proteins, and
can serve as intermediates in the preparation of unimolecu-
lar micelles potentially useful for controlled drug delivery
applications.
used in the grafting reactions. The concentration of the sam-
ples was 30 to 35 mg/mL and 64 scans were averaged.
Titrations were performed for selected linear and arbores-
cent partially deprotected substrates, to confirm the depro-
1
tection levels determined by H NMR analysis. The substrate
EXPERIMENTAL
(50 mg) was added to a mixture of DMF (10 mL) and water
(5 mL) with 3 drops of phenolphthalein indicator (0.5% w/v
in methanol). The solution was quickly titrated to a pink col-
oration (stable over 30 s) with a 0.1 N NaOH solution in
methanol, to minimize interference from atmospheric CO2.
Materials
N,N’-dimethylformamide (DMF; Aldrich, peptide synthesis
grade) was purified by distillation under reduced pressure
and was stored in the dark to prevent degradation due to pho-
tochemical reactions. Dimethyl sulfoxide and n-hexylamine
were purified by stirring overnight with CaH2 and distillation
under reduced pressure. Ethyl acetate (Fisher, 99.9%) was
distilled from LiAlH4 under nitrogen. The purified compounds
were stored under nitrogen in round-bottomed flasks (RBF)
over 3A molecular sieves (EMD). c-Benzyl L-glutamic acid
(Bz-Glu; Bachem, >99%), HBr solution (Aldrich, 33% in acetic
acid), N,N’-diisopropylcarbodiimide (DIC; Aldrich, 99%), 1-
hydroxybenzotriazole (HOBt; Fluka, water content ca. 15%
w/w), trifluoroacetic acid (TFA, Caledon), methanol (Omni-
solv), diethyl ether (Omnisolv), triethylamine (TEA, EMD),
acetic anhydride (Caledon), tetrahydrofuran (THF, Omnisolv),
triphosgene (Aldrich, 98%), and LiAlH4 (Aldrich, 95%) were
used as received from the suppliers.
Dynamic laser light scattering (DLS) measurements were
carried out to determine the hydrodynamic diameter of the
arborescent PBG molecules in DMF and in DMSO. The con-
centration of the samples ranged from 0.5–2% w/v (depend-
ing on the generation number) and LiCl (0.05% w/v) was
added to prevent aggregation. The measurements were done
on a Brookhaven BI-200 SM instrument at a temperature of
ꢂ
25 C and a scattering angle of 90ꢂ. The 256-channel correla-
tor was operated in the exponential sampling mode, the last
four data acquisition channels being used for the baseline
measurements. The z-average translational diffusion coeffi-
cients used for the hydrodynamic diameter calculations were
determined from first- and second-order analysis of the nor-
malized electric field correlation function.
Characterization
Synthesis of c-Benzyl L-Glutamic Acid N-Carboxyanhydride
Analytical SEC measurements were done on a system with a
Waters 510 HPLC pump, a 50 mL injection loop, and a
Waters 2410 differential refractometer (DRI) detector. A
Wyatt MiniDAWN laser light scattering detector operating at
a wavelength of 690 nm served to determine the absolute
molecular weight of the graft polymers. The column used
was a 500 mm 3 10 mm Jordi Gel DVB Mixed Bed model
with a linear polystyrene molecular weight range of 102 to
107. N,N-dimethylacetamide (DMA) with LiCl (1 g/L, added
to minimize adsorption of the polymer on the column) at a
flow rate of 1.0 mL/min served as the mobile phase at room
temperature.
(Glu-NCA)
30
ꢀ
The procedure used was adapted from Poche et al. Bz-Glu
(10.0 g; 42.0 mmol) was suspended in 300 mL of dry ethyl
acetate in a 1 L RBF fitted with a refluxing condenser and a
gas bubbler. The flask was purged with N2 and heated to
reflux. Triphosgene (4.8 g, 16 mmol) was then added and
refluxing was continued for 3 h. Caution: triphosgene is
highly toxic, so the whole procedure should be carried out in
a well-vented fume hood. The flask was then removed, stop-
ꢂ
pered, and cooled in a freezer (210 C) for 1 h. The solution
was transferred to a cold separatory funnel and quickly
washed successively with 100 mL of ice-cold water and 100
mL of chilled 0.5% aqueous NaHCO3 solution. The organic
phase was dried over anhydrous MgSO4, filtered, and concen-
trated to 100–120 mL on a rotary evaporator. An equal vol-
ume of cold hexane was then added to induce crystallization
of the product. The mixture was left in the freezer overnight
and the solid product was recovered by filtration in a
Schlenk funnel under N2. It was then dried overnight under
Preparative SEC was carried out on a system consisting of a
Waters M45 HPLC pump, a 2 mL sample injection loop, a
Waters R401 differential refractometer detector, and a Jordi
Gel DVB 1000 Å 250 mm 3 22 mm preparative SEC column.
N,N-Dimethylacetamide with 0.2 g/L LiCl served as the
mobile phase. The crude polymer was injected as a 30 mg/
mL solution and the SEC system was operated at room tem-
perature at a flow rate of 3.0 mL/min.
ꢂ
vacuum, and stored under N2 in a refrigerator (4 C).
1H NMR spectroscopy served to determine the degree of
polymerization of the linear polymers, to monitor the depro-
tection level of the substrates, and to analyze the conforma-
tion of the polypeptide chains. The instruments used were
Bruker 300 MHz and 500 MHz spectrometers; the latter was
employed only in conformation analysis. The concentration
of all the samples was 20 mg/mL and 16 scans were aver-
aged on both instruments. 19F NMR spectroscopy on a
Bruker 300 MHz instrument was used to determine the
chain end primary amine functionality, fNH2, of the polymers
Yield 5 10.2 g (92%); 1H NMR (300 MHz, CDCl3) d: 7.58–
7.24 (s, 5H), 6.75 (s, 1H), 5.11 (s, 2H), 4.38–4.33 (t, 1H),
2.59–2.53 (t, 2H), 2.35–2.21 (m, 1H), 2.21–2.02 (m, 1H).
Synthesis of Poly(c-benzyl L-glutamate)
A linear polymer serving as side chain material (sample
PBG-41) was synthesized by dissolving Glu-NCA (8.00 g, 30.4
mmol) in dry DMF (20 mL) in a 50 mL RBF at 0 ꢂC. After
the monomer was dissolved n-hexylamine (200 mL, 1.52
mmol, for a target Xn 5 20) was added with rapid stirring.
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The evolution of CO2 was noticeable at the beginning, and
the reaction was allowed to proceed for 2.5 to 3 days at 0
ꢂC. The linear polymer was recovered by precipitation in
methanol and suction filtration, and dried under vacuum
overnight.
ing as side chains [PBG-64, 1.10 g, 0.275 mmol chains] were
dissolved in 6 mL of dry DMF in a 25-mL RBF. The peptide
coupling reagents DIC (1.72 mL of 10% v/v solution in DMF,
1.10 mmol) and HOBt (0.149 g, 1.10 mmol) were then added
to the reaction with TEA (0.8 mL, 5.5 mmol). The reaction
was allowed to proceed for 24 h at room temperature before
adding n-hexylamine (0.50 mL, 4.94 mmol), to deactivate
residual activated carboxylic acid sites. After 1 h the product
was precipitated in methanol and recovered by suction filtra-
tion. Linear PBG contaminant was removed from the G0
crude polymer by preparative size exclusion chromatography
(SEC). The purified G0 polymer was recovered by evapora-
tion to dryness under high vacuum, dissolution in TFA, and
precipitation in methanol.
Yield 5 80%, Mw/Mn 5 1.10. 1H NMR (300 MHz, d6-DMSO):
Xn 5 26.0, d: 8.04 (b, 26H), 7.48–7.20 (s, 130H), 5.02–4.89 (s,
52H), 4.10–3.88 (b, 26H), 2.34–1.91 (b, 104H), 1.33–1.18 (b,
10H), 0.78–0.76 (s, 3H).
Quantification of Primary Amines by 19F NMR Analysis
The linear polymers were analyzed for their terminal primary
amine content by 19F NMR spectroscopy. For example, a linear
PBG sample (PBG-A; 0.119 g, Mn 5 5300, 2.25 3 1025 mol
chains) was dissolved in 3 mL of deuterated DMSO (d6-
DMSO). A solution of trifluorobenzaldehyde (TFBA, 0.1191 g,
6.84 3 1024 mol) and benzotrifluoride (BTF, 0.1014 g, 8.15 3
1024 mol; internal standard) in 2 g of d6-DMSO was prepared.
The reagent solution (0.2306 g, 7.07 3 1025 mol TFBA, 7.18
3 1025 mol BTF) was added to the polymer solution and
stirred for 2 h; a 0.5 mL sample was then transferred to an
NMR tube for analysis. The peak areas from the 19F NMR
spectra were used to determine the fNH2 values as described
in the Results and Discussion section.
Synthesis of Upper Generation (G1-G3) Arborescent PBG
The purified G0 polymer (G0–52, 0.400 g, 1.82 mmol Bz-Glu
units) was first partially deprotected by dissolution in TFA
(4 mL), and 0.13 mL of 33% (w/w) HBr solution in acetic
acid (0.044 g HBr, 0.30 equiv HBr per Bz-Glu residue) was
added with stirring. After 3 h the polymer was recovered
and further purified as described for the linear sample.
Yield 5 0.240 g, (68%), 32 mol % glutamic acid moieties.
The deprotected G0 polymer (G0–52-CO2H, 0.212 g, 0.356
mmol –CO2H) was coupled with linear side chains [PBG-64,
1.90 g, 0.445 mmol chains] by the same method described
for the G0 reaction, but using DMSO (8 mL) rather than DMF
as solvent. The crude G1 polypeptide was purified by prepa-
rative SEC as described for the G0 polymer. The G2 and G3
arborescent PBG samples were synthesized and purified as
described for the G1 sample.
Synthesis of PBG Precursor for Grafting Substrate
A linear PBG sample (PBG-34) serving as substrate for the
preparation of a G0 (comb-branched) polypeptide was synthe-
sized as described above with minor modifications: The target
Xn was 50, the reaction was performed at room temperature,
and it was quenched with acetic anhydride to deactivate the
terminal amine moiety. The sample was synthesized from Glu-
NCA (2.0 g, 7.6 mmol) in 5.0 mL of DMF and n-hexylamine
(20 mL, 0.15 mmol) at room temperature. After 4 h acetic
anhydride (290 mL, 3.1 mmol) was added and the reaction
was stirred for 1 h before sample recovery.
RESULTS AND DISCUSSION
Several methods reported in the literature for the synthesis
of PBG have led to different results in terms of yield and
their ability to minimize side reactions. The PBG building
blocks serving in the synthesis of arborescent polypeptides
should ideally be obtained in high yield, have a narrow
MWD and a predictable Xn, and preserve their primary
amine terminus. Beyond the influence of monomer purity
and the activated monomer polymerization mechanism,
Mitchell et al. proposed as early as 1957 that cyclization into
a lactam structure (Scheme S1, Supporting Information) dur-
ing the polymerization or sample storage was the dominant
side reaction affecting the primary amine terminus of linear
PBG.31 Preservation of the amino group is essential for the
grafting reaction: Deactivation of this moiety by side reac-
tions yields linear PBG chains incapable of coupling with the
substrate.
Yield 5 1.5 g (90%), 1H NMR (300 MHz, d6-DMSO): Xn 5 51,
d: 8.04 (b, 51H), 7.48–7.20 (s, 255H), 5.02–4.89 (s, 102H),
4.10–3.88 (b, 51H), 2.34–1.91 (b, 204H), 1.33–1.18 (b, 10H),
0.78–0.76 (s, 3H), SEC: Mw/Mn 5 1.19.
Partial Deprotection of Linear PBG Substrate
PBG-34 (Xn 5 51, 1.46 g, 6.66 mmol Bz-Glu units) was dis-
solved in TFA (15 mL) and 0.35 mL of 33% (w/w) HBr solu-
tion in acetic acid (0.14 g HBr, 0.25 equiv HBr per Bz-Glu
residue) with stirring. After 3 h the polymer was precipi-
tated in diethyl ether and recovered by suction filtration to
give an orange solid. After drying the polymer was dispersed
in 10 to 12 mL of THF and enough DMF (ca. 1–2 mL) was
added to obtain a clear solution. The polymer was precipi-
tated again in diethyl ether to obtain a white product.
Yield 5 0.92 g (72%), 31 mol % of free glutamic acid
moieties.
One approach suggested to minimize side reactions in the
polymerization of NCA monomers was to use the hydrochlor-
ide salt form of amine initiators, to decrease the reactivity of
the primary amine propagating center.32,34 For example,
block copolymers with poly(E-benzyl carbamate L-lysine)
contents of 66–70% by weight were synthesized at 40 to 80
ꢂC from an amine-terminated polystyrene macroinitiator in
the hydrochloride form (PS-NH2ꢃHCl, Xn 5 52), Mw/Mn < 1.03
Synthesis of G0 Arborescent PBG
The partially deprotected polymer serving as substrate [PBG-
34-CO2H, 0.141 g, 0.220 mmol –CO2H] and the polymer serv-
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