Synthesis and mechanistic investigations of pH-responsive cationic poly(aminoester)s

Article abstract of DOI:10.1039/c9sc05267d

The synthesis and degradation mechanisms of a class of pH-sensitive, rapidly degrading cationic poly(α-aminoester)s are described. These reactive, cationic polymers are stable at low pH in water, but undergo a fast and selective degradation at higher pH to liberate neutral diketopiperazines. Related materials incorporating oligo(α-amino ester)s have been shown to be effective gene delivery agents, as the charge-altering degradative behavior facilitates the delivery and release of mRNA and other nucleic acids in vitro and in vivo. Herein, we report detailed studies of the structural and environmental factors that lead to these rapid and selective degradation processes in aqueous buffers. At neutral pH, poly(α-aminoester)s derived from N-hydroxyethylglycine degrade selectively by a mechanism involving sequential 1,5- A nd 1,6-O→N acyl shifts to generate bis(N-hydroxyethyl) diketopiperazine. A family of structurally related cationic poly(aminoester)s was generated to study the structural influences on the degradation mechanism, product distribution, and pH dependence of the rate of degradation. The kinetics and mechanism of the pH-induced degradations were investigated by ¹H NMR, model reactions, and kinetic simulations. These results indicate that polyesters bearing α-ammonium groups and appropriately positioned N-hydroxyethyl substituents are readily cleaved (by intramolecular attack) or hydrolyzed, representing dynamic dual function materials that are initially polycationic and transform with changing environment to neutral products.

Full text of DOI:10.1039/c9sc05267d

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Synthesis and mechanistic investigations of pH-
responsive cationic poly(aminoester)s†
Cite this: DOI: 10.1039/c9sc05267d
Timothy R. Blake, ‡a Wilson C. Ho, ‡a Christopher R. Turlington,a Xiaoyu Zang,a
All publication charges for this article
have been paid for by the Royal Society
of Chemistry
a
Melanie A. Huttner,a Paul A. Wenderab and Robert M. Waymouth
*
The synthesis and degradation mechanisms of a class of pH-sensitive, rapidly degrading cationic poly(a-
aminoester)s are described. These reactive, cationic polymers are stable at low pH in water, but undergo
a fast and selective degradation at higher pH to liberate neutral diketopiperazines. Related materials
incorporating oligo(a-amino ester)s have been shown to be effective gene delivery agents, as the
charge-altering degradative behavior facilitates the delivery and release of mRNA and other nucleic acids
in vitro and in vivo. Herein, we report detailed studies of the structural and environmental factors that
lead to these rapid and selective degradation processes in aqueous buffers. At neutral pH, poly(a-
aminoester)s derived from N-hydroxyethylglycine degrade selectively by
a mechanism involving
sequential 1,5- and 1,6-O/N acyl shifts to generate bis(N-hydroxyethyl) diketopiperazine. A family of
structurally related cationic poly(aminoester)s was generated to study the structural influences on the
degradation mechanism, product distribution, and pH dependence of the rate of degradation. The
1
kinetics and mechanism of the pH-induced degradations were investigated by H NMR, model reactions,
Received 18th October 2019
Accepted 4th February 2020
and kinetic simulations. These results indicate that polyesters bearing a-ammonium groups and
appropriately positioned N-hydroxyethyl substituents are readily cleaved (by intramolecular attack) or
hydrolyzed, representing dynamic “dual function” materials that are initially polycationic and transform
with changing environment to neutral products.
DOI: 10.1039/c9sc05267d
rsc.li/chemical-science
polymer backbone is cleaved by appropriately positioned
nucleophiles. Self-immolative polymers that undergo quinone
Introduction
(or azaquinone) methide elimination upon removal of an end-
cap have been investigated in detail by Shabat,10 Gillies,7,11
Phillips,12–14 and others.1,3 Various types of stimuli have been
explored to trigger the removal of the terminal end-group,
including pH changes, enzymatic activities, or oxidative
cleavage with H2O2.
Self-degrading polymers are important dynamic synthetic
materials that undergo a rapid degradation in response to
environmental stimuli.1–3 These stimuli-responsive degrada-
tions are now being leveraged in biomedical materials as
a means to initiate complexation and then release therapeutic
cargos under specic biological conditions/environments.1–9
These features provide an attractive platform for the delivery of
compounds, such as drugs, proteins or polynucleotides, which
must be protected until they reach their therapeutic destina-
tion. Several classes of stimuli-responsive degrading materials
have been reported in the literature: end-capped systems that
upon cleavage of a terminal group undergo an end-to-end (or
self-immolative) degradation, and systems in which the
The potential toxicity of the (aza)quinone methide byprod-
ucts15 has stimulated research into alternative chemistries for
stimuli-sensitive degradation processes,16 including disulde-
based self-immolative systems that degrade upon reduc-
tion,17,18 acid-labile polymers,19–22 or amine-functionalized
polyesters and polycarbonates that degrade either through
intramolecular nucleophilic attack or by hydrolysis.11,23–31
Cationic poly(aminoester)s are an attractive class of
materials due to the tunable lability of the ester linkage32
and the functional cationic ammonium repeat units that
can bind appropriate polyanionic cargoes and then
become neutral, enabling cargo release. Several classes of
cationic poly(aminoester)s have been shown to undergo pH-
sensitive degradation resulting from deprotonation of
embedded ammonium groups, which leads to nucleophilic
attack on the ester backbone.23–25,30,33 Cationic poly(b-amino-
ester)s have demonstrated good biocompatibility and are used
aDepartment of Chemistry, Stanford University, Stanford, CA 94305, USA. E-mail:
waymouth@stanford.edu
bDepartment of Chemical and Systems Biology, Stanford University, Stanford, CA
94305, USA
† Electronic supplementary information (ESI) available: Supplemental gures and
information regarding materials used, instrumentation, synthetic procedures,
degradation kinetics and modeling and characterization data. CCDC 1960011.
For ESI and crystallographic data in CIF or other electronic format see DOI:
10.1039/c9sc05267d
‡ These authors contributed equally.
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Fig.
1 Proposed mRNA binding and release by Charge-Altering
Releasable Transporters (CARTs). Selective degradation of the poly-
cationic poly(a-aminoester) block generates neutral diketopiperazines
and hydrolyzed N-hydroxyethyl glycines.
in a wide range of biomedical applications,34,35 highlighted by
recent advances in gene delivery vehicles for a variety of thera-
peutic indications.36–43
We recently reported a novel class of materials based on
cationic poly(a-aminoester) backbones that undergo a pH-
sensitive rapid and selective degradation to neutral amide and
amino acid byproducts in water.44 We have leveraged this
behavior in the design of novel cationic amphiphilic oligomers,
charge-altering releasable transporters (CARTs),33,45–47 that bind,
complex, and deliver oligonucleotides of wide ranging size
(mRNA, pDNA) into cells, in vitro and in vivo.33,45–49 At low pH,
these oligomeric amphiphiles are cationic and associate in
water with polyanionic oligonucleotides to form polyelectrolyte
complexes. At elevated pH these polyelectrolyte complexes
release the oligonucleotides, which was attributed to the
charge-altering degradative behavior of the cationic oligo(a-
amino ester)s (Fig. 1). These charge-altering releasable trans-
porters (CARTs)33,45–47 were shown to be effective for mRNA and
pDNA delivery to a variety of cell types in cell culture and live
animals, enabling multiple therapeutic vaccination strategies
for eradication of established tumors in mice.48,49
As the pH-induced charge-canceling degradation of the
cationic poly(a-aminoester) backbone was implicated in the
intracellular release of mRNA,33 herein we describe a detailed
mechanistic study directed at elucidation of the structural and
environmental factors that lead to the selective degradation
processes for the cationic poly(ammonium ester) homopoly-
mers in aqueous buffers. While not directly comparable to the
polyelectrolyte environments in which the amphiphilic CARTs
degrade,33,45–47 these studies provide fundamental insights on
the structural factors that contribute to the degradation
behavior of poly(amino ester)s in aqueous buffers. We report
the synthesis of a family of structurally related cationic poly-
aminoester homopolymers and investigate how their structural
variations inuence the mechanism, product distribution, and
pH dependence of their degradation.
Fig. 2 Synthesis of azalactone monomers and polymers.
as previously reported.44 The a-methyl substituted morpholi-
none 2 was prepared by a 3-step procedure in one ask;50 this
approach provides a general strategy for introducing a variety of
substituents at the a- and g-positions. Azacaprolactone 3 was
synthesized via the ring-expansion Baeyer–Villager oxidation of
the Boc-protected piperidone (1–2 steps), while N-glycyl mor-
pholinone 4 was made via Cu-catalyzed aerobic oxidative lac-
tonization (2 steps).51 As both of these latter reactions are
compatible with amides and carbamates, these pathways
provide access to a wide variety of N-substituents including the
proteinogenic amino acids.
Monomers 1–4 undergo controlled organocatalytic ring-
opening polymerization (OROP) with the thiourea/DBU cata-
lyst system at room temperature (Table 1). With initial mono-
mer concentrations of [M]0 ¼ 1.0 M in toluene, 1 and 3 reached
equilibrium conversions of 87% and >95% (see ESI†), respec-
tively, as determined by 1H NMR. a-Me morpholinone 2
required substantially higher initial monomer concentration
for polymerization (at [M]0 ¼ 4.0 M conversion reached 52%),
which we attribute to the less favorable thermodynamics of
ring-opening.44,52 The N-glycyl amide functionalized lactone 4
exhibited poor solubility in toluene and was thus polymerized
in dichloromethane (DCM). Although prior studies had shown44
that the ring-opening thermodynamics were less favorable in
DCM than in toluene, the polymerization of 4 in this solvent
proceeded to 63% conversion when [M]0 ¼ 1.0 M. The polymers
generated from 4 represent a new subclass of functionalized
polyesters derived from amino acids (Fig. 2, P4+). Deprotection
of each polymer 1–4 with triuoroacetic acid (TFA) in DCM
afforded cationic poly(ammonium ester)s that are water-soluble
and stable in unbuffered D2O for >24 hours.
Results
To investigate the inuence of poly(amino ester) structure on
the rate and mechanism of their degradation, a series of
Degradation of cationic poly(aminoester) P1+
monomers 1–4 were synthesized in 1–3 steps from commer- Treatment of the cationic poly(a-aminoester) P1+ (Mn ¼ 14.5
cially available reagents (Fig. 2 and Table 1). The morpholin-2- kDa, Đ ¼ 1.12) with saturated aqueous NaHCO3 (pH ꢀ 9) for 30
one 1 was generated by a Pd-catalyzed oxidative lactonization, minutes resulted in the rapid degradation of the cationic
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Table 1 Synthesis and OROP of new monomersa
Monomer synthesis
Monomer
Polymerizations
Yieldb (%)
Solvent
[M] (M)
[I] (M)
[C] (M)
DP
Mn (kDa)
Đ
1
2
3
4
60
29
73
76
PhMe
PhMe
PhMe
DCM
1.0
4.7
2.3
1.0
0.015
0.05
0.024
0.05
0.05
0.26
0.8
65
49
75
74
14.5
6.9
18.9
2.0
1.12
1.36
1.10
1.22
0.025
a
M ¼ monomer, I ¼ initiator (1-pyrenebutanol), and C ¼ catalyst (1 : 1 TU/DBU). DP was determined by 1H NMR end-group analysis. Mn and Đ were
determined by PS-calibrated gel permeation chromatography. b Based on the rst starting material shown in each scheme of Fig. 2.
polyester; the major product at the end of this degradation was copolymer P(VL-b-1+) was dissolved in phosphate-buffered
the diketopiperazine 5a, isolated in 72% yield. N-Hydrox- saline (PBS, pH 7.4). Aer
1 hour, analysis of the
yethylglycine (6a) formed by hydrolysis was also identied as resulting sample by gel-permeation chromatography (GPC)
1
one of the minor products (Fig. 3).
and H NMR revealed the predominant products to be 5a and
To assess the relative rate of degradation of the cationic poly(valerolactone) with an Mn of 6.4 kDa (Đ ¼ 1.30). These
poly(a-aminoester) P1+ relative to an analogous unfunctional- results showed that the aqueous degradation of P1+ was much
ized aliphatic polyester, we investigated the degradation of faster than that of the unfunctionalized polyester pVL. While
a diblock copolymer of poly(valerolactone) (pVL) and P1+ in the cationic segment P1+ had degraded completely, the valer-
aqueous solution (Fig. 4). The diblock copolymer P(VL-b-1) was olactone segment had only been partially hydrolyzed, as evi-
prepared from a pVL macroinitiator (Mn ¼ 8.3 kDa, Đ ¼ 1.16) denced by the slight decrease in the molecular weight and slight
and monomer 1 (Fig. 4a). Aer deprotection, the water-soluble broadening of the molecular weight distribution (dashed green
Fig. 3 Degradation products and base-triggered degradation of P1+ with saturated NaHCO3 in H2O.
Fig. 4 Degradation of P(VL-b-1+) diblock copolymer. (a) Synthesis and reaction scheme. (b) GPC traces of polymers. Poly(valerolactone)
macroinitiator (black line); protected P(VL-b-1) diblock (dotted red line); products after degradation of deprotected P(VL-b-1+) (dashed green
line).
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vs. solid black trace, Fig. 4b). Previous studies showed that P1+
also degrades much more quickly than a polycarbonate block in
an analogous copolymer.33
Degradation kinetics of cationic poly(aminoester) P1+
Prior studies33 of the highly selective degradation of P1+ impli-
cated a mechanism that involved an alternating 1,5- and 1,6-
cyclization cascade that liberated neutral diketopiperazines (e.g.
5, Fig. 5). In this proposed mechanism, as the pH increases,
some fraction of the ammonium groups are deprotonated to
nucleophilic amines which then undergo an intramolecular
rearrangement (1,5-O/N acyl shi)53 to generate hydroxyethyl
amides (A, Fig. 5). This rearrangement contracts the backbone,
allowing a subsequent 6-membered cyclization (1,6-O/N acyl
shi) to liberate the neutral diketopiperazine. This 1,5-1,6
rearrangement sequence repeats until much of the polymer is
consumed, producing 5 as the predominant nal product.
The kinetics of degradation of the cationic poly(aminoester)
P1+ (Mn ¼ 14.5 kDa, Đ ¼ 1.12) were investigated in aqueous
buffers as a function of pH. To facilitate kinetic modeling, we
employed an experimental approach11,27,29 that monitored the
decay of the N-hydroxyethylglycine ester repeat units in the
chain, as well as the appearance of identiable products (see
Fig. 6 Kinetics for the degradation of P1+ in D2O at pH 6.5: conversion
of starting material (blue dots); yield of diketopiperazines 5 (red
triangles); yield of acids 6 (green crosses).
By this time, 87% of the N-hydroxyethylglycine esters have
reacted to afford 5 (49% yield), 6 (29% yield), and other products
that are not readily identied. At higher pH, the rate of degra-
dation and the selectivity for the diketopiperazines increase
(Table 2).
These data reveal the strong inuence of pH on the rate of
degradation of the poly(a-aminoester) P1+ in aqueous solution.
At pH 7.0, 50% of the hydroxyethyl esters of P1+ have rearranged
or degraded in less than 3 minutes, whereas at pH 5.1 this takes
29 min. At all pH values, diketopiperazines are the major
product. Control experiments indicate that the rates and
selectivities of the degradation are not strongly inuenced by
the nature of the counteranion (CF3COOꢁ vs. Clꢁ, Table 2 entry
1 vs. 2).
1
below) as a function of time by H NMR.
Under buffered aqueous conditions, two major product types
could be quantied by 1H NMR: diketopiperazines 5 and a-
amino acids 6 (R0 ¼ H, C(O)R00), the latter arising from hydro-
lysis (Fig. 5). These products were identied by comparison to
1
the H NMR spectra of 5a and 6a (R0 ¼ H) prepared indepen-
dently. Minor amounts of linear N-hydroxyethylamides were
visible in the spectra but not readily quantied. The reaction
kinetics at pH 6.5 were monitored for approx. 3 hours, at which
point the polymer had degraded to diketopiperazines 5 (from
54% of the starting hydroxyethyl glycine units) and 6 (from 28%
of the starting units). Under these conditions, the disappear-
ance of repeat units in the chain occurred with an empirical
half-life of t1/2 ¼ 3.8 min (t1/2 ¼ time at which 50% conversion is
reached). A representative time course for the degradation of
P1+ at pH 6.5 is given in Fig. 6 and data for all experiments are
summarized in Table 2.
The degradation of P1+ can also be observed in methanol-d3
in the presence of Et3N (Fig. 7); under these conditions, two
major products could be identied: those derived from dike-
topiperazines (R0 ¼ H (5a) or C(O)R00) and those derived from N-
hydroxyethyl glycinate esters (R
diketopiperazyl).
¼
CD3 (7-d3) or
The degradation of the poly(a-aminoester) P1+ (Mn ¼ 10.6
kDa, Đ ¼ 1.42) in CD3OD in the presence of 2.5 eq. Et3N (125
mM) was studied by 1H NMR by monitoring the disappearance
of N-hydroxyethylglycine ester units as a function of time. The
reaction kinetics were monitored for approximately 3 h, at
which point 86% of the hydroxyethyl glycine repeating units
had been converted, predominantly to diketopiperazines (5,
85% yield, Table 2, entry 5). Under these conditions, the
disappearance of the repeating units in the chain occurred with
an empirical half-life of t1/2 ¼ 6.4 min (empirical t1/2, the time at
which half of the monomer units had been converted, was used
to simplify comparing mixed order degradation kinetics).
As only 85% of the repeat units transformed to diketopi-
perazines and conversion would halt aer this point, we
hypothesized that the structural requirements of the mecha-
nism (namely, that two unreacted ester units must be adjacent
to each other) were responsible for this limit on conversion. We
modeled the degradation kinetics using stochastic simula-
tions54,55 averaged over 1000 trials (see ESI†) under the
At the more acidic pH 5.1, the cationic polyester P1+
degrades more slowly (t1/2 ¼ 29 min) over the course of 3 hours.
Fig. 5 Proposed mechanism of degradation of P1+. Blue arrow, k5
5-membered cyclization. Red arrow, k6 ¼ 6-membered cyclization.
¼
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Table 2 Polymer degradation kineticsa
Entry
Polymerb
pHc
t1/2 (min)
Timed (h)
Conv.e (%)
Pdt. 1f (%)
Pdt. 2f (%)
1
2
3
4
5
6
7
8
P1+
5.1
5.1
6.5
7.0
Et3Ng
5.1
6.5
7.0
7.5
ꢀ9h
5.1
6.5
7.0
29
33
3.8
<3
6.4
77
7.6
201
42
3.0
3.0
1.5
1.0
3.0
87
84
100
96
86
87
94
69
69
100
56
5
5
5
5
49
48
54
59
85
—
—
35
52
85
45
87
95
6
6
6
6
6
8
8
10
10
10
12
12
12
29
34
28
15
0
85
97
33
18
<5
9
P(1+Clꢁ)
P1+
P1+
P1+
5
P2+
3.0
0.4
—
—
9
9
9
11
11
11
P2+
P3+
10.0
3.0
16.8
10.4
3.0
9
P3+
10
11
12
13
P3+
13
443
15
P4+
P4+
100
100
12
5
P4+
<3
1.0
a
Reactions done in duplicate at 25 ꢂC, [polymer]0 ¼ 40–60 mM (calculated with respect to repeat units) in a 1 : 1 mixture of buffer and D2O and
quantied by 1H NMR with an internal standard. Dashed entries unknown or not accurately quantied. Default counteranion triuoroacetate;
b
+
c
typical polymer lengths were P165+, P248+, P375+, and P448
.
All pH degradation experiments were conducted in deuterated NMR buffers. The
pH indicated were measured upon dilution of these buffers with distilled H2O. The pH 5.1 buffer was made with acetic acid-d4/NaOH, and the
d
pH 6.5, 7.0, and 7.5 buffers with KH2PO4/K2HPO4 in D2O (see ESI for details). Time at which the next three data columns are measured.
e
f
g
Percentage of starting esters that have disappeared. Percentage of starting esters incorporated into this product. Degradation carried out
with 2.5 eq. Et3N in CD3OD. h Saturated NaHCO3 in D2O, used without dilution.
assumption that only two processes had contributed to the
degradation in CD3OD: a 1,5-O/N acyl shi characterized by
the rate constant k5 and a subsequent 1,6-O/N acyl shi
characterized by the rate constant k6 (Fig. 5).
Three kinetic models were evaluated with two of the models
(S1 and S3) assuming that the initial 1,5-cyclization occurred
with equal probability at any hydroxyethyl unit in the polymer
chain (random simulations S1 and S3, DP ¼ 65) while the other
model constrained the rearrangements to occur only at the
hydroxyl chain terminus (end-to-end simulation S2, DP ¼ 64).
For simulations S1 and S2, we made the simplifying assumption
that the rate constant k6 was much larger than k5 (k6/k5 ¼ 106);
for S3 the rate constants k5 and k6 were set to be equal (k6/k5 ¼
1). The t to experimental data was carried out by adjusting the
magnitude of k5 so that the simulated and experimental
conversions would intersect at 50%.
Fig. 8 Stochastic kinetic simulations for the yield of diketopiperazine
5. S1 (green line, k6 [ k5 ¼ 0.0412 minꢁ1); S2 (dashed red line, k6 [ k5
¼ 1.44 minꢁ1); S3 (dotted blue line, k5 ¼ k6 ¼ 0.483 minꢁ1); Expt 1
(purple circles); Expt 2 (black triangles).
As shown in Fig. 8, the random model S1 (k6/k5 ¼ 106)
provides an excellent t to both sets of experimental data,
matching not only the yield but also the production of diketo-
piperazines as a function of time. In contrast, the end-to-end
model S2 (k6/k5 ¼ 106) predicts that all of the N-hydroxyethyl
glycine units of the chain should be converted to diketopiper-
azines and the random model S3 with k5 ¼ k6 predicts that the
yield of diketopiperazines should not exceed 28%.
Fig. 7 Degradation of P1+ in CD3OD/Et3N to liberate diketopiperazine 5a and N-hydroxyethyl glycinate esters (R ¼ CD3 (7-d3) or diketopi-
perazyl). The blue numbers indicate the 1H NMR shifts of representative proton resonances.
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These simulations suggest that the degradation of P1+ is product observed for P2+ at this pH was N-hydroxyethylalanine
initiated by random 1,5-O/N acyl shis that occur at any point (8) formed by hydrolysis in 97% yield; no diketopiperazine was
along the polymer chains and that the rate of the 1,6-O/N acyl observed (eqn (1)). This observation reveals that the presence of
shi is faster than that of the preceding 1,5-O/N acyl shi. As an a-methyl group (P2+ vs. P1+) signicantly inuences not only
the generation of 5 consumes two adjacent hydroxyethyl glycine the rate of degradation but also the mechanism; for P2+
repeat units and creates a break in the chain, these cyclization hydrolysis of the esters in the backbone is the predominant
events will randomly leave some of the N-hydroxyethyl glycine mechanism of degradation.
units in the chain “stranded” and incapable of generating
a diketopiperazine (Fig. 7). This is analogous to the situation
calculated by Flory for intramolecular reactions of adjacent
reactive units in polymer chains.56,57 The faster rate of the 1,6-
O/N acyl shi (relative to the 1,5-O/N acyl shi) suggested by
the simulations is needed to reproduce the high selectivity for
cyclization to the diketopiperazine 5 in competition with
consecutive 1,5-O/N acyl shis to yield the linear amides,
which are observed experimentally only in trace amounts.
(1)
The rate of formation of hydroxyethyl alanine units (8) from
P2+ differs signicantly from the formation of hydroxyethyl
glycine units (6) from P1+ (Fig. 10a vs. Fig. 6 and 8). At short
times, the rate of formation of 8 increases and then occurs at
a constant rate until approx. 75% conversion (Fig. 10a).
Inuence of structure on degradation of poly(aminoester)s
To assess the structural features that contribute to the rapid and
selective base-induced degradation of poly(aminoester)s, we
carried out a series of comparative experiments on the degra-
dation behavior of the poly(a-aminoester) P1+ with the a-methyl
substituted poly(a-aminoester) P2+, the poly(b-aminoester) P3+,
and the poly(a-amidoester) P4+ bearing a pendant glycine
(Fig. 1). All four cationic polymers P1+–P4+ degrade readily in
aqueous buffer, but the rates and products differed signicantly
as a function of the polymer structure (Table 2 and Fig. 9).
The rate of formation of 8 could be simulated using
stochastic simulations where hydrolysis of the esters in the
chain is assumed to be the only degradation process. Three
simulations were carried out: the rst (S1) with a single rate
constant for hydrolysis at any ester in the chain (kh), the second
(S2) where hydrolysis occurs exclusively at the chain-end (kend),
and the third (S3) where hydrolysis processes of different rate
constants occur both at the chain-ends (kend) and at internal
esters (kint). The kinetic data and simulated ts are shown in
Fig. 10. As shown in Fig. 10, random simulation S1 with a single
rate constant for hydrolysis yields a poor t to the experimental
data as this mechanism would predict a rst-order appearance
of hydroxyethyl alanine 8. The end-to-end simulation S2 yields
a better t to the data but does not capture the slight
a-Me substituted poly(a-aminoester) P2+
The degradation of P2+ is considerably slower than that of P1+ at
pH 5.1 (t1/2 ¼ 77 min vs. 29 min), but the difference is smaller at
pH 6.5 (7.6 min vs. 3.8 min). In stark contrast to P1+, the nal
Fig. 9 Summarized kinetic data (a) histogram of empirical half-lifes of polymers P1+–P4+ at different pH (*dashed line represents detection limit
for 1H NMR experiment). (b) Comparison of P1+, P2+, and P4+ at a single pH 6.5. (c) Data comparing degradation of P4+ at pH 5.1, 6.5 and 7.0.
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Fig. 10 Degradation simulation of P2+. (a) Yield of 8 vs. time. Expt. data (black triangles), stochastic simulations (S1 ¼ dashed blue, S2 ¼ dotted
red, S3 ¼ solid green line). (b) Model for hydrolysis of P2+, at chain-end vs. internal esters.
acceleration at early times or the decay in rate aer 80% be attributed to the diminished activating effect of the b-
conversion. The best t to the experimental data was obtained ammonium on the ester in P3+ relative to the a-ammonium in
with simulation S3 when the hydrolysis rate of the esters at the P1+.58
1
chain-end were faster (kend ¼ 0.602 minꢁ1) than those of
When the degradation of P3+ was monitored by H NMR in
internal esters in the polymer chain (kint ¼ 0.0144 minꢁ1).
D2O at pH 7.0, resonances corresponding to the hydroxyethyl b-
The results of these simulations imply that esters at the chain- ammonium esters at d ¼ 4.45 ppm decreased with the appear-
end are more reactive to hydrolysis than internal esters of the ance of a new resonance at d ¼ 4.40 ppm, which subsequently
polymer chain, as the simulations based on either exclusive (S2) decreased but more slowly, as shown in Fig. 11a. Upon
or accelerated hydrolysis at the chain-end (S3) yield much better a detailed comparison of the independently prepared model
ts than the simulation (S1) based on uniform hydrolysis at any complexes, the resonance at d ¼ 4.40 ppm is assigned to an N-
ester in the chain. While S3 yields a slightly better t to the data hydroxylethyl ammonium ester anked by an N-hydroxyethyl
than S2, the difference is not large enough to justify a specic amide (Fig. 11b). The faster decay of the resonances at d ¼
conclusion about how much larger kend is relative to kint
.
4.45 ppm relative to those at d ¼ 4.40 ppm suggests that esters
containing b-ammonium substituents react more rapidly than
those containing b-amide substituents.
Poly(b-aminoester) P3+
At pH 7.0 and 7.5, the total conversion of both types of
hydroxyethyl repeat units of P3+ plateaus at approximately 70%
over the course of several hours (Fig. 12a, black circles, Table 2);
the appearance of b-amino acids could also be observed
(Fig. 12a, black triangles). The complete conversion of the
hydroxyethyl units of P3+ required several days (data not
shown).
To simulate these kinetics, two models were evaluated. For
the rst model (S1), two competitive processes were assumed,
one involving a 1,5-O/N acyl shi to generate the linear amide
(with rate constant k5) and a second process involving the
hydrolysis of esters (with rate constant kh, Fig. 12b, dashed blue
lines in Fig. 12a). The second model (S2, Fig. 12a) assumed
a mechanism incorporating the observation that b-ammonium
esters are more reactive both for the 1,5-O/N acyl shi and
The aqueous degradation of P3+ is signicantly slower than that
of the poly(a-aminoester) P1+ (t1/2 (pH 7.0) ¼ 201 min vs. <3 min)
yielding a mixture of linear oligo(N-hydroxyethylamide)s (9) and
products from hydrolysis (10, eqn (2), Table 2). At higher pH, the
selectivity for the formation of linear amides increases; in
saturated sodium bicarbonate (NaHCO3), the linear amides
account for 85% of the products. Unlike the other 3 polymers,
the degradation products of P3+ are primarily oligoamides
instead of small molecules.
The formation of linear amides is consistent with a proposed
mechanism where 1,5-O/N acyl shis occur along the back-
bone. For P3+, the liberation of a cyclic dipeptide is not
observed, as a subsequent O/N acyl shi in the opposite
direction would require the formation of an 8-membered ring.
The much slower rate of P3+ degradation compared to P1+ can
(2)
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1
Fig. 11 (a) H NMR resonances corresponding to N-hydroxyethyl repeat units in polymer P3+ (D2O, pH 7.0) from three time points (activated
1
ester ¼ SMact, unactivated ester ¼ SMunact). (b) Structural assignments for H NMR resonances at d ¼ 4.45 and 4.40 ppm.
Fig. 12 (a) Plot of the percent conversion of hydroxyethyl ester repeat units of P3+ (black circles) and percent yield of 10 (black triangles). Simulations
with optimized rate constants are also plotted: S1 (blue dashed lines, k5 ¼ kh ¼ 1.44 ꢃ 10ꢁ3 minꢁ1); S2 (red solid lines, k5a ¼ 2.02 ꢃ 10ꢁ3 minꢁ1, k5u
¼
6.06 ꢃ 10ꢁ5 minꢁ1, kha ¼ 1.62 ꢃ 10ꢁ3 minꢁ1, khu ¼ 2.46 ꢃ 10ꢁ5 minꢁ1). (b) Scheme of degradation processes and rate constants used in simulations.
hydrolysis. For simulation S2, four rate constants were opti- the b-substituent (ammonium or amide respectively), and kha
mized to t the data (Fig. 12a): k5a and k5u for the rates of the and khu for the similarly dened rates of hydrolysis. Optimiza-
1,5-O/N acyl shi when the ester is activated or unactivated by tion over these parameters was conducted to simultaneously t
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the experimental curves of conversion and hydrolysis vs. time slower rate than P1+ (t1/2 ¼ 443 min), but the degradation rate
for pH 7.0 (Fig. 12a, solid red lines).
increases dramatically with increasing pH. At pH 7.0 P4+
As shown in Fig. 12a, simulation S2, which accounts for degrades with a rate comparable to that of P1+ (Table 2; both
different reactivities for the two types of esters, yielded better have t1/2 < 3 min). Compared to P1+, P4+ degrades with a higher
ts to the data. Simulation S1, with a single rate constant each selectivity for the diketopiperazine; at pH 7.0, 11 accounts for
for the O/N acyl shi and hydrolysis, would lead to >90% 95% of the products, at pH 6.5, for 87% of the products (Table 2
conversion of the polymer repeat units aer 1000 minutes, and Fig. S5a, ESI†). For P4+, only one 1,6-O/N acyl shi is
whereas S2 captures both the shape of the kinetics curves required to cleave the chain and liberate diketopiperazine 11,
throughout and the plateau in conversion at the end. Overall, S2 which likely contributes to the high selectivity for 11.
is consistent with the suggestion by 1H NMR (Fig. 11a) that the
esters containing b-ammonium groups are more reactive than
Controlling the rate of degradation: copolymers
those containing b-amide groups.
The degradation of P3+ in CD3OD in the presence of 2.5 eq.
As the rate of degradation of the poly(a-aminoester) P1+ is
considerably faster than that of P2+ at pH 5.1 (Table 2), we
investigated whether the rate of degradation could be tuned by
generating random copolymers from aminoester repeat units
that exhibit different rates of degradation. To that end, mono-
mers 1 and 2 were copolymerized to generate a series of random
copolymers (Mn ¼ 5.5–10.4 kDa, Đ ¼ 1.22–1.31, see ESI†), which
upon deprotection, afforded the cationic a-aminoester copoly-
mers with 35%, 54%, and 80% P2+ content by mole fraction.
Because of the fast degradation rates of P1+ and P2+ at pH 6.5
(t1/2 ¼ 3.8 min and 7.6 min, respectively), the kinetic studies
were carried out at pH 5.1. The conversion values were calcu-
lated relative to the sum of both components, and the empirical
half-life as a function of % P2+ is plotted in Fig. S6 (ESI† p. S17).
As shown in Table 2, the empirical half-life of N-hydrox-
yethylglycine P1+ homopolymer degradation is approximately
30 minutes at pH 5.1, whereas the empirical half-life for N-
Et3N selectively yields the linear polyamides
9 without
competing hydrolysis (Fig. 13a). The conversion of hydroxyethyl
repeat units in P3+ is shown in Fig. 13b. Attempts to model the
disappearance of the hydroxyethyl repeat units with a single rate
constant k5 for the O/N acyl shi (model S3, blue dashed line,
Fig. 13b) did not reproduce the data well, but the simulated ts
incorporating two rate constants k5a and k5u (dened as above,
Fig. 12b) provide excellent agreement with the experimental
data for two separate sets of experiments (model S4 red line;
exptl. data, black squares and triangles, Fig. 13b). These data
provide additional evidence that esters containing b-ammo-
nium groups are more reactive than those containing b-amide
groups.
N-Glycyl substituted poly(a-amidoester) P4+
The aqueous degradation of P4+ affords a nal mixture of the hydroxyethylalanine homopolymer P2+ is approximately 80
diketopiperazine 11 and the N-hydroxyethyl diglycine 12 from minutes. By incorporating increasing amounts of P2+ into the
hydrolysis (eqn (3), Table 2). At pH 5.1, P4+ degrades at a much copolymers, the rate of degradation of the copolymers could be
Fig. 13 (a) Scheme of degradation processes of P3+ in Et3N/CD3OD. (b) Experimental (triangle and squares are experimental replicates) and
simulated data (S1 ¼ dashed blue, S2 ¼ solid red) for the percent conversion of repeat units of P3+ in CD3OD with 2.5 equiv. of Et3N.
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(3)
tuned as a function of composition, as indicated by the increase rate of hydrolysis of the b-ammonium esters 17 and 18 (t1/2 ¼ 7–
of the empirical half-lives (t1/2) as the mol% of hydroxyethyl 9 days) relative to the b-amido ester 19 (t1/2 ¼ 42 days) reveals
alanine repeat units (P2+) in the PaAE copolymers increased that b-ammonium substituents exhibit a stronger activating
(Fig. S6†).
effect on ester hydrolysis than b-amides. Several mechanisms
have been proposed to rationalize these effects, including
inductive H-bonding and electrostatic activation of the ester by
the a-ammonium group.61,64 Our results provide a clear ratio-
nale for the much faster rate of degradation of the poly(a-
ammonium ester)s P1+ and P2+ relative to the poly(b-ammo-
nium ester) P3+ (Table 2).
The much faster rate of hydrolysis of N-hydroxyethyl gly-
cinate 7 (t1/2 ¼ 13.5 min) relative to glycinate 13, sarcosinate
14, and serinate 15 (t1/2 > 1000 min) illustrates the N-hydrox-
yethyl substituent's powerful activating effect on the ester
toward hydrolysis. This effect is less pronounced if the N-
hydroxyethyl group is in the b-position (17 vs. 18, t1/2 ¼ 8.8 vs.
7.1 days). The rate of hydrolysis of N-acetyl, N-hydroxyethyl
methyl glycinate 16 (t1/2 ¼ 147 min) is considerably slower
than that for 7, providing further support for the activating
effect of alpha ammonium substituents in activating the ester.
Moreover, 16 hydrolyzes much more rapidly than 13 or 14 (t1/2
¼ 147 min vs. t1/2 > 1000 min), again indicating that an
appropriately positioned N-hydroxyethyl group could activate
the adjacent ester for hydrolysis. These phenomena may be
a consequence of the neighboring group effect of the pendant
hydroxyl group,68 as the rate of hydrolysis of 7 is considerably
faster than that of 18.
Model systems to assess inuence of structure on rate and
mechanism of degradation
In an effort to provide further insight on the role of polymer
structure on the rate of degradation, model studies were carried
out with a series of methyl esters that were chosen to mimic the
structures of polymer repeat units or intermediates that were
implicated in the degradation mechanism. The role of appro-
priately positioned ammonium and hydroxyethyl groups on the
rates of hydrolysis for a series of methyl esters were carried out
in buffered D2O at pH 7.0. Under these conditions, hydrolysis of
the methyl esters afforded the corresponding acids with high
selectivity following rst order kinetics; thus, the rst order
half-lives provide a convenient measure for assessing the role of
structure on ester reactivity toward hydrolysis (Fig. 14).
As shown in Fig. 14, N-hydroxyethylglycinate 7 hydrolyzes
most rapidly with a half-life t1/2 ¼ 13.5 min at pH 7.0 in buffered
D2O. Methyl glycinate 13, methyl sarcosinate 14, and methyl
serinate 15 hydrolyze more slowly (t1/2 ¼ 1228, 1044, and
1825 min, respectively), but considerably faster than the N-
methyl b-aminopropionate 17 (t1/2 ¼ 8.8 days).
The faster hydrolysis rate for a- vs. b-ammonium esters (7 vs.
18, 13–15 vs. 18, Fig. 14) is consistent with prior studies on the
hydrolysis rates of a-amino esters59–64 and esters with quater-
nary ammonium substituents.65,66 Esters containing alkyl
Cyclodimerization model system studies
suldes in the alpha position also hydrolyze more rapidly than The above studies clearly demonstrate that N-hydroxyethyl
those substituted in the beta position.67 Nevertheless, the faster substituted methyl glycinate 7 is considerably more reactive to
Fig. 14 Model methyl esters and their first-order half-lives of hydrolysis at pH 7.0 in D2O.
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new insights on the factors that inuence the degradative
charge-neutralizing cationic amine-to-amide/lactam rear-
rangement which contributes to the charge-altering behavior
of the more complex polyelectrolyte assemblies that form
when the amphiphilic CART oligomers are complexed with
oligonucleotides.
Kinetics and mechanistic studies reveal that the poly(a-
ammonium ester) P1+ degrades rapidly and selectively to the
diketopiperazine 5 with rates that depend on pH. The base-
induced degradation of the cationic P1+ selectively generates
the neutral diketopiperazine 5 as the major product. The
cationic P1+ degrades much more rapidly than polyesters
bearing b-ammonium groups (P3+) or those lacking an ammo-
nium group (poly(valerolactone)). We had previously proposed
that the rapid degradation of the a-ammonium polyester P1+
was due in part to the activation of the ester carbonyl by
inductive and hydrogen-bonding interactions from the vicinal
ammonium (Fig. 16).33 The proposed 1,5-O/N acyl shi has
been reported for related processes in poly(aminoesters) (PAEs)
containing side-chain amines.25,69,70 The proposed 1,6-O/N
acyl shi is analogous to that reported by Almutairi and
coworkers for 6- and 7-membered acyl shis in PAEs featuring
side chain nucleophiles.23,26,27,30 However, the rapid and highly
selective sequence of alternating 1,5- and 1,6-cyclizations to
generate diketopiperazine 5 is a novel and characteristic feature
for the degradation of the poly(a-amino esters) such as P1+. As
diketopiperazines are common elimination products in the
degradation of peptides,71 the structure–activity relationships
described herein for O/N acyl shis may provide insights on
related chemistries of biopolymers (i.e. O/N or O/S acyl
shis).
The selective degradation of the cationic poly(N-hydroxyethyl
glycine) polymer P1+ to liberate the neutral diketopiperazine
can be attributed to the propitious positioning of the amine
following contraction of the chain by the rst 1,5-O/N acyl
shi (Fig. 16). Modeling of the kinetics suggests that this
selective cyclization cascade occurs randomly along the chain,
leaving some of the N-hydroxyethyl glycines “stranded”; this is
consistent with observations that the degradation of P1+ yields
the diketopiperazine as the major product (#85%) along with
the stranded hydroxyethyl glycinates ($15%), imposing
a stochastic limit (ꢀ85%) on the yield of DKP even under non-
aqueous conditions. Model studies (Fig. 14) and literature
precedent59–64 also indicate that the rapid rate at P1+
Fig. 15 Scheme of cyclodimerization experiments with 7 and 13 in
CD3OD.
hydrolysis than the unsubstituted methyl glycinate 13. To assess
if this relative reactivity was also observed with amine nucleo-
philes, the methyl glycinates 7 and 13 were each treated with
base (Et3N) in methanol-d3 at room temperature. When moni-
tored by 1H NMR under these conditions, the only new species
observed was the diketopiperazine dimerization product
(Fig. 15, see ESI† for other conditions). Both the conversion of
the methyl ester 7 and the yield of diketopiperazine 5 were
much greater than the analogous quantities for unsubstituted
glycinate 13. These experiments provide additional evidence for
the activating effect of the N-hydroxyethyl substituent on the
reactivity of a-ammonium esters.
Discussion
We recently discovered a class of environmentally-sensitive
novel cationic amphiphilic oligomers (charge altering releas-
able transporters, CARTs, Fig. 1) that are effective for the
delivery of mRNA and pDNA to a variety of cell types in cell
culture and live animals.33,45–49 These cationic amphiphilic
oligomers, when complexed with negatively-charged oligonu-
cleotides, form polyelectrolyte complexes that exhibit
dynamic, charge-altering behavior that facilitates the intra-
cellular release of the oligonucleotides.33 This dynamic
charge-altering behavior was attributed to the unique struc-
tural features of the cationic a-aminoester sequences44 of
these amphiphilic oligomers which exhibit a pH-dependent
selective degradation to neutral diketopiperazines.33 The
studies reported herein reveal that the rate and mechanism of
degradation of a series of cationic poly(aminoester) homo-
polymers in buffered water differ signicantly as a function of
the structure of the aminoester repeating unit and the solu-
tion pH (Fig. 9). These fundamental investigations provide
Fig. 16 Proposed mechanism of DKP formation; selectivity of 1,6- vs. 1,5-O/N acyl shift from intermediate A.
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degradation can be in part attributed to the activating effect of
The signicant activating effect of the a-ammonium group
the a-ammonium substituent, which predisposes the adjacent is in part responsible for the rapid rate of degradation of the
ester for nucleophilic attack.33 As the pH increases, more of the poly(a-ammonium ester)s P1+ and P2+; this effect likely
ammonium groups are deprotonated, facilitating cyclization by contributes to the rapid rate of degradation of the related
a 1,5-O/N acyl shi25,53,62,64,72,73 to generate an N-hydrox- poly(serine esters)25,47,69,80–83 and poly(hydroxyproline esters).24
yethylamide intermediate A (Fig. 16). The rapid rate of degra- In contrast to P1+, the aqueous degradation of P2+ occurs
dation of P1+ and the faster hydrolysis rate for a- vs. b-amino exclusively by hydrolysis to give the N-hydroxyethylalanine as
ester model compounds (Fig. 14) are consistent with prior a zwitterion. Both the faster degradation rate of P1+ and the
studies on the hydrolysis rates of a-amino esters59–64 and esters different degradation products than that of the N-hydrox-
with quaternary ammonium substituents.65,66 The activating yethylalanine polymer P2+ can be explained by the steric effect
effect of the a-ammonium group is a critical factor for the much of the a-methyl substituent that disfavors the 1,5-O/N acyl
faster rates of degradation for the poly(a-aminoester)s P1+ and shi. The kinetics of degradation are also different; kinetic
P2+ relative to the poly(b-amino ester) P3+ (Table 2).
modeling of the evolution of the N-hydroxyethylalanine as
As suggested by the kinetic modeling (Fig. 8), the selective a function of time (Fig. 10a) indicates that hydrolysis occurs
degradation of P1+ to diketopiperazine requires that aer the preferentially at the N-hydroxylethyl chain-end (Fig. 10b),
initial 1,5-O/N acyl shi to form A (Fig. 16), the subsequent which correlates well with the activating effect implicated for
1,6-O/N acyl shi to yield the diketopiperazine must the N-hydroxyethyl substituents on the reactivity of esters
happen at a rate much faster than the 1,5-O/N acyl shis bearing that functional group.
that yields linear amides. Literature precedent suggests that
The degradation of the b-ammonium polyester P3+ is both
for unsubstituted 1,4- and 1,5-aminoesters, the 6-membered slower and yields different products than either of the a-
cyclization should be slightly favored by 4–7 fold over the 5- ammonium polyesters P1+ and P2+. The aqueous degradation
membered cyclization.73 For the proposed N-hydrox- of the b-ammonium polyester P3+ yields the linear amide
yethylamide intermediate A (Fig. 16), the planarity of the poly-b-peptoids as the major products along with minor
amide linkage is likely to provide a further bias for 6- amounts of the N-hydroxyethyl esters (eqn (2)). The kinetics,
membered cyclization, leading to a high selectivity for the simulations, and model studies (Fig. 14) suggest that esters
diketopiperazine. The structural factors that favor the rapid bearing a-ammonium substituents are more reactive than
6-membered cyclization of intermediate A (Fig. 16) can also those bearing b-ammonium substituents, and that esters
explain the rapid and selective degradation of P4+, which containing b-ammonium groups are more reactive than those
bears a structural motif similar to that of intermediate A, as containing b-amide groups. The latter observation could be
its repeating monomer unit (eqn (3)).
incorporated into a kinetic model to rationalize the marked
In addition to the activating effect of the a-ammonium deceleration in the rate of degradation of P3+ over time
substituent, the observation from model studies that N- (Fig. 12a); as the b-ammonium esters are converted to b-
hydroxyethylglycine esters hydrolyze and dimerize much amido esters by 1,5-O/N acyl shis, the resulting b-amido
more rapidly than the corresponding glycinates or sarcosi- esters react more slowly.
nates (Fig. 14 and 15) indicates that an appropriately-
positioned N-hydroxylethyl group is key structural diketopiperazine 11 with higher selectivity (95%, pH 7.0) than
The aqueous degradation of P4+ liberates the unsubstituted
a
element that inuences both the rate and selectivity of the P1+, due to the positioning of the pendant amine that requires
degradation of these polymers. The N-hydroxyethyl substit- only one cyclization event to cleave the chain. In contrast, the a-
uent may exert its effect through neighboring group partici- methyl substituted polymer P2+ degrades selectively by hydro-
pation (Fig. 17), as has been suggested for analogous lysis, likely as a consequence of steric constraints that disfavor
reactions of peptides.74–79
a 1,5-O/N acyl shi.
Fig. 17 Proposed role of N-hydroxyethyl substituents in facilitating the 1,6-O/N acyl shift: neighboring group participation by intramolecular
lactonization.
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degradation mechanisms), NSF CHE848280 and NIH-CA031845
grants (P. A. W.). Stanford Cancer Translational Nanotech-
nology Training T32 Training Grant CA196585 funded by the
National Cancer Institute (T. R. B.). C.R.T. acknowledges
support from the National Institute of Biomedical Imaging and
Bioengineering of the NIH under Award F32EB021161. We
would like to thank Dr Andrey Rudenko for obtaining and
analyzing x-ray crystallography data.
Conclusions
This
work
demonstrates
that
water-soluble
cationic poly(aminoester)s bearing appropriately positioned a-
ammonium groups and N-hydroxyethyl substituents degrade
rapidly and selectively in aqueous solution as functions of
structure and pH. The cationic poly(a-amino ester) P1+ is stable
in acidic aqueous solutions, but at a pH where some of the
ammonium groups are deprotonated, this polymer degrades
rapidly and selectively to the diketopiperazine 5 as a conse-
quence of a facile cyclization cascade mediated by successive
1,5- and 1,6-O/N acyl shis. Mechanistic and comparative
studies with related poly(aminoester)s reveal the key role of
both the a-ammonium and N-hydroxyethyl substituents on both
the rate and mechanism of these selective degradation reac-
tions. These insights have provided a series of cationic water-
soluble polyesters whose rates of degradation can be tuned
from minutes to hours, depending on the pH.
In related studies, we have demonstrated that this pH-
induced degradative transformation of cationic polymers to
neutral small molecules provides a strategy to electrostatically
bind and release polyanions, such as mRNA and DNA. Oligo-
mers containing short repeat segments of degradable a-amino
esters and lipophilic domains constitute a class of charge-
altering releasable transporters (CARTs) for the delivery and
release of polyanionic genes in cells and live animals.33,45–49 The
studies reported herein provide key structural and mechanistic
understandings on how current CARTs function and inform the
criteria to design new pH sensitive polymers for diagnostic and
therapeutic delivery. More generally, these studies provide
a structural and mechanistic basis for the design of self-
immolative polymers or linkers for applications where appro-
priate environmental triggers can lead to selective degradation,
degradation product amplication, or payload release.1–3,5,8,16
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Acknowledgements
This research was supported by the U.S. Department of Energy,
Office of Science, Office of Basic Energy Sciences, under Award
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