Substituent Effects on the pH Sensitivity of Acetals and Ketals and Their Correlation with Encapsulation Stability in Polymeric Nanogels

Article abstract of DOI:10.1021/jacs.6b11181

The effect of structural variations in acetal- and ketal-based linkers upon their degradation kinetics is studied through the design, synthesis, and study of six series of molecules, comprising a total of 18 different molecules. Through this systematic study, we show that the structural fine-tuning of the linkers allows access to variations in kinetics of degradation of more than 6 orders of magnitude. Hammett correlations show that the ρ value for the hydrolysis of benzylidene acetals is about ?4.06, which is comparable to an S_N1-like process. This shows that there is a strong, developing positive charge at the benzylic position in the transition state during the degradation of acetals. This positively charged transition state is consistent with the relative degradation rates of acetals vs ketals (correlated to stabilities of 1°, 2°, and 3° carboxonium ion type intermediates) and the observed effect of proximal electron-withdrawing groups upon the degradation rates. Following this, we studied whether the degradation kinetics study correlates with pH-sensitive variations in the host-guest characteristics of polymeric nanogels that contains these acetal or ketal moieties as cross-linking functionalities. Indeed, the trends observed in the small molecule degradation have clear correlations with the encapsulation stability of guest molecules within these polymeric nanogels. The implications of this fundamental study extend to a broad range of applications, well beyond the polymeric nanogel examples studied here.

Full text of DOI:10.1021/jacs.6b11181

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Article
Substituent Effects on the pH Sensitivity of Acetals and Ketals and
Their Correlation with Encapsulation Stability in Polymeric Nanogels
Bin Liu, and Sankaran Thayumanavan
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b11181 • Publication Date (Web): 20 Jan 2017
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Substituent Effects on the pH Sensitivity of Acetals and Ketals and Their
Correlation with Encapsulation Stability in Polymeric Nanogels
Bin Liu1 and S. Thayumanavan*1,2
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1Department of Chemistry, 2Center for Bioactive Delivery, Institute for Applied Life Sciences, University
of Massachusetts, Amherst, Massachusetts 01003, USA
Abstract
Effect of structural variations in acetal- and ketal- based linkers upon their degradation kinetics is studied
through the design, synthesis and study of six series of molecules, comprising a total of eighteen different
molecules. Through this systematic study, we show that the structural fine-tuning of the linkers allow
access to variations in kinetics of degradation of more than six orders of magnitude. Hammett
correlations show that the ρ value for the hydrolysis of benzylidene acetals is about -4.06, which is
comparable to an SN1-like process. This shows that there is a strong, developing positive charge at the
benzylic position in the transition state during the degradation of acetals. This positively charged
transition state is consistent with the relative degradation rates of acetals vs. ketals (correlated to
stabilities of 1°, 2°, and 3° carboxonium ion type intermediates) and the observed effect of proximal
electron-withdrawing groups upon the degradation rates. Following this, we studied whether the
degradation kinetics study correlates with pH-sensitive variations in the host-guest characteristics of
polymeric nanogels that contains these acetal or ketal moieties as crosslinking functionalities. Indeed, the
trends observed in the small molecule degradation have clear correlations with the encapsulation stability
of guest molecules within these polymeric nanogels. The implications of this fundamental study extend
to a broad range of applications, well beyond the polymeric nanogel examples studied here.
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Introduction
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Materials that predictably respond to a specific environment change through changes in their
physicochemical properties have attracted attention due to potential applications in areas ranging from
material science to biomedicine.1-3 The environmental change, i.e. the input signal, in these cases can be
physical (e.g. temperature, light, ultrasonic, magnetic, and electrical), chemical (e.g. pH, redox, and
ionic), or biological (e.g. protein, carbohydrates, and nucleic acids) in nature. Among this diverse range
of inputs that are pursued, pH has attracted significant attention mainly because of its simplicity and its
biological relevance. pH is simple in that the diffusion of protons inside nanomaterials is a relatively
facile process, because of its small size. pH is also very relevant in human biology, as specific pH
variations have been identified from sub-cellular levels to tissue levels.4 For example, the pH of the
typical extracellular environment is about 7.4, which decreases to ~5.5-6.5 inside the endosome and
further to 4.5~5.5 inside the lysosome.4-7 Also, while the pH is ~7.4 in the extracellular environment of
normal tissues, it has been shown that the pH around certain tumor tissues is ~6.5 due to the
hypoxia-induced production of excess lactic acid in the tumor microenvironments.8-11 Thus, pH
responsive materials have been explored for many biological applications, including sensing, diagnostics,
tissue engineering and targeted drug delivery.12-14
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pH responsive materials can be broadly classified into two main categories: (i) the responsive units
are based on non-cleavable, ionizable functional groups where pH-induced charge variations play a key
role (e.g. amines and carboxylic acids);12,15 (ii) the key functionalities are based on chemical linkages that
degrade due to pH variations (e.g. acetal, ketal, imine, hydrazine, cis-aconityl and beta-thiol ester).15-18
Cleavable linkers have been widely used as building blocks for polymers, nanoparticles, and
antibody-drug conjugates, as well as in protein purification and solid phase synthesis.19-21
Among pH-sensitive linkers, acetals and ketals have attracted significant attention, because they
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yield charge-neutral and potentially non-toxic by products upon cleavage. The mechanism of the
hydrolysis of the acetals and ketals has been a subject of interest since the 1960s.22-24 More recently, these
substituted methyleneglycol diether materials have been pursued in a variety of areas including controlled
release applications,25-27 nucleic acid delivery,28 degradable polymers,29-33 temporary masking agents for
polymers and nanoassemblies,26,34-36 and pro-drug formulations.37-39 Despite such widespread use, there is
no report that provide a systematic understanding on the structural factors that affect the pH sensitivity of
these functionalities. An understanding of the dynamic range, which each functional group variation
can provide, will fundamentally impact the utility of these functionalities in designing biomaterials, as
these will provide a robust and convenient framework for choosing the appropriate linker for a specific
application. This is important because the variations in pH in different locations are rather subtle and
the drug release profile requirements are quite different in various applications. In drug delivery
applications for example, extended release of drugs is required for the treatment of chronic diseases,40,41
while a targeted and burst release is needed for diseases such as cancer and certain acute inflammatory
diseases.42,43 On the other hand, in applications such as protein purification, it is critical that the cleavage
is achieved under mild reaction conditions in order to prevent possible denaturation. In this paper, several
series of acetals and ketals linker have been designed and studied for their relative pH sensitivity. As a
first step to test the fidelity of such a study in drug delivery applications, we incorporate these moieties
within polymeric nanoparticles using our previous emulsion-free method44,45 and evaluate their
pH-induced variations in encapsulation stability of guest molecules.
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Results and Discussion
Acetals and ketals are known to be quite stable under basic conditions, but readily hydrolyze to the
corresponding carbonyl compound (aldehyde and ketone) and alcohol under acidic conditions. To
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establish the design variations needed for this study, we first considered the mechanism of acid-catalyzed
cleavage of these diethers (Figure 1). In the hydrolysis process, formation of the intermediate of
resonance-stabilized carboxonium ion has been suggested to be the rate determining step.22 Therefore, the
stability of this carboxonium ion would dictate the hydrolysis rate of the corresponding acetal or ketal; i.e.
if the intermediate carboxonium ion is more stabilized, then the hydrolysis rate would be faster. Due to
the cationic character of the intermediate, electron-donating group substituents are generally expected to
stabilize the intermediate and thus accelerate hydrolysis. Also, in general, the R1 group is likely to have
greater contributions based on inductive effects, whereas R2 and R3 could have contributions from
inductive and resonance effects. Thus, a series of eighteen different ketal or acetal based molecules, with
different R1, R2 and R3 groups, were designed and synthesized. Structures of these molecules are shown
in Figure 2.
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Figure 1. The well-established mechanism of the hydrolysis of acetals and ketals. Formation of the
resonance-stabilized caboxonium ion intermediate, shown within the red color square bracket, is considered to be the
rate-determining step of this cleavage reaction. Therefore, structures of R1, R2, and R3 will all influence the kinetics
of this cleavage reaction.
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Figure 2. Structures of the acetal and ketal small molecules studies in this work.
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Note that a key objective of this work is to compare the small molecule sensitivity with the
corresponding functional groups being used as crosslinkers in polymeric nanoparticles. Since the
crosslinks in these nanoparticles will be based on amide moieties, the small molecules studied here are
also based on the corresponding amides. These molecules are divided into six different series, where each
of the series probes a specific structural correlation within these acetal or ketal based crosslinkers, as
shown in Figure 2. Syntheses of these molecules are detailed in the Supporting Information.
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Although the hydrolytic stabilities of these molecules are ideally studied in aqueous solutions, many of
these targeted molecules proved to be insoluble. In order to exclude variations in extraneous factors such
as hydrophobicity induced aggregation or solution heterogeneity, a homogeneous solution system was
chosen to monitor the intrinsic hydrolysis kinetics. In general, a mixture of 0.3 mL CD3CN and 0.1 mL
PB buffer in D2O was used as the solvent, where the concentration of the molecules under investigation
was kept at 25 mM at pH 5. The hydrolysis kinetics was assessed by monitoring the NMR of the solution
at different time intervals at 25 °C. The typical process and the data are exemplified using the ketal
1
molecule 3, as shown in Figure 3. Key changes in the evolution of the H NMR spectrum involve the
Figure 3. The hydrolysis process of molecule 3. A) the hydrolytic equation, B) the NMR spectra of the hydrolytic
process, C) the hydrolytic kinetics together with the first-order reaction fitting curve (solid line).
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gradual decrease in the peak at 1.29 ppm, with the concurrent increase in the intensity of the peak at 2.09
ppm. The peak at 1.29 ppm is attributed to the methyl group connected to ketal moiety, while the peak at
2.09 ppm represents the formation of the acetone product. Percentage degradation of the ketal moiety was
thus calculated either by the NMR signals from the rate of disappearance of the ketal (or acetal) substrate
moieties or from the rate of appearance of the ketone (or aldehyde) product functional groups. The
reactions obeyed first order reaction kinetics and hydrolysis rates were obtained through an exponential
fit with excellent correlation functions (typical R2>0.99). To quantify the relative rates of hydrolysis,
half-life of these pH-sensitive functional groups were extracted from these plots. In such a case, all the
experiments were performed three times and the error values of t1/2 represent the standard deviation from
these three trials. The half-life of 3 under these conditions was found to be 32.33±0.90 h. Further, the
effect of pH on the hydrolysis kinetics was performed and the results were shown in Figure 4. The
hydrolysis kinetics dramatically decreased with increasing pH. For instance, the hydrolysis rate decreased
by ~3 times, upon changing the pH from 5.0 to 5.5, and by another 3 times at pH=6.0. When the pH
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Figure 4. Relative hydrolytic kinetics of ketal 3 at different pH. The solid lines are the fitting curves.
was increased to 6.5, the hydrolysis rate was decreased another 6 times. This molecule was quite stable
and exhibited no measurable hydrolysis under pH=7.4 for 7 days.
In the first series of molecules, we vary the distance between the amide functional group and the
ketal moiety (Series A in Figure 2). The amide moiety is an inductively electron-withdrawing moiety
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and thus this series interrogates the effect of placing an electron-withdrawing group close to the
carboxonium ion intermediate. The half-life of ketal 1, formed between acetone and the amide derivative
6-amino-hexan-1-ol, was found to be about 24 minutes. When the six-carbon linker was changed to only
three carbons in 2, the half-life of the ketal increased by about an order of magnitude to more than 3
hours, indicating substantial increase in stability. This increased stability is attributed to the closer
proximity between the amide and the ketal moieties. Interestingly, when the linker length was decreased
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Figure 5. Relative hydrolysis rates of ketals based on the variation in the distance between the electron-withdrawing
amide moiety and ketal moiety. The solid lines are the fitting curves.
by one more carbon in 3, the half-life increased by yet another order of magnitude to about 32 hours. The
greater sensitivity at shorter distances is the classic hallmark of inductive effect based destabilization of
the carboxonium ion intermediate, which understandably raises the transition state energy.
Next, the effect of the intermediate being a primary, secondary or tertiary carboxonium ion upon the
rate of hydrolysis was investigated (Series B in Figure 2). In this case, molecules 4, 5, or 6 were
synthesized from the reaction of the phthalimide derivative of 2-amino-ethanol and acetone, acetaldehyde
or formaldehyde, respectively. Phthalimides were used as the protecting group in this series, because of
experimental difficulties in synthesizing the corresponding trifluoroacetamide derivatives. Also, the
electron-withdrawing ability of these two amino protecting groups were also found to be similar. The
hydrolytic half-life of the phthalimide-substituted ketal 4 was found to be about 33 h (Figure 6A), which
is very similar to that of the corresponding trifluoroacetamide-substituted ketal 3 (t1/2 = 32.33±0.90 h).
Within the Series B, the two acetals, 5 and 6, were too inert to be studied at the pH 5 conditions used for
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4. Therefore, the reaction was carried out in the presence of trifluoroacetic acid (TFA) and the results are
shown in Figure 6B and 6C. Under these conditions, a mixture of 0.3 mL CD3CN and 0.1 mL of 50 mM
TFA in D2O was used as the solvent, where the concentration of the molecules under investigation was
also kept at 25 mM. Even under these conditions, the formaldehyde acetal moiety in 6 did not show any
degradation, suggesting that the barrier for the formation of the formal primary carboxonium ion is quite
high. On the other hand, the half-life of the acetal from acetaldehyde in 5 was found to be about 4 hours.
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Figure 6. (A-C) Effect of the primary, secondary or tertiary carboxonium ion as the intermediate species upon
the hydrolysis rates of acetals and ketals. A and i were under pH=5 buffer condition; B and ii were under TFA
condition. (D and E) Effect of alkyl chain length upon the stability of secondary carboxonium ions. The solid lines
are the fitting curves.
Since the alkylidene acetals showed any appreciable cleavage only in the presence of TFA, these
conditions were also used to investigate the effect of alkyl chains upon the hydrolysis kinetics (Series C).
Not surprisingly, the alkyl chain length did not have any appreciable effect on the hydrolysis rate (Figure
6D and 6E).
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Similarly, the effect of alkyl group variations in ketals was also investigated by comparing ketals
formed from acetone, cyclopentanone and cyclohexanone at pH 5. Although all three molecules are
ketals and therefore result in a tertiary carboxonium intermediate, the half-lives were found to be
substantially different. Hydrolysis rate of the cyclopentyl ketal was found to be about two times slower,
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Figure 7. Relative hydrolysis rates of ketals from acyclic vs. cyclic ketones at pH 5. The solid lines are the fitting
curves.
while that of cyclohexanone was about seven times slower, compared to the acetone analog. The
difference between the five- and the six- membered ring can be attributed to the difference in torsional
energy strain between the ground state and the transition state in the reaction.24
Although secondary carboxonium intermediates exhibit slow hydrolysis rates, we were interested in
understanding the effect of replacing the alkyl substituent with an aryl moiety. The placement of the aryl
moiety also provides the opportunity to more reliably probe the effect of electron-withdrawing and
Figure 8. Substituent effects upon the hydrolysis of benzylidene acetals. A) hydrolysis kinetics and the first-order
reaction fitting curve, B) Hammett plot. The hydrolysis was performed under TFA condition. The solid lines are the
fitting curves
electron-donating substituents upon the stability of the carboxonium ion and thus on the hydrolysis rates
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of the acetals. The results of these findings are shown in Figure 8. Briefly, benzylidene acetals that are
unsubstituted or substituted with electron-withdrawing functionalities, exhibit slow hydrolysis under the
pH 5 conditions. Therefore, these experiments were carried out in the presence of TFA. Under these
conditions, the half-life of the phenyl-substituted acetal 13 was found to be about 4 minutes.
Incorporating a trifluoromethyl moiety, an inductively electron-withdrawing group, at the para- position
in 12 reduced the half-life by two orders of magnitude to about 12.3 hours. The presence of a
resonance-based electron-withdrawing group in 11 reduced the half-life by another 4 times.
Interestingly, furan-based acetal 14 seems to behave very similar to the unsubstituted benzylidene acetal
13. Incorporating an electron-donating p-methoxy moiety in 15 greatly enhanced the rate of hydrolysis in
that the reaction was too fast to be measured under these conditions. By comparison, the half-life of
acetal 15 under pH 5 conditions was found to be about 70.4 hours. In order to bridge the gap for the two
different hydrolytic conditions, the reduced TFA amount (1/5 of the initial amount) was used for the
hydrolytic study. For instance, a mixture of 0.3 mL CD3CN and 0.1 mL 10 mM TFA in D2O was used as
the solvent and the concentration of the molecules under investigation was also kept at 25 mM. In this
case, molecule 13 showed half-life of 4.08 min under typical TFA conditions (Figure 7), however, it
revealed the half-life of 25.33 min under these reduced TFA conditions (Figure S3). It meant the
reaction rate decreased by about 6 times under the milder acidic conditions. Further, acetal 18 has a
half-life of 4.82 min under reduced TFA condition (Figure S3), but it is increased to 425 h (Figure 8)
under the pH=5 buffer condition, suggesting that the difference in reaction rate between these two
conditions is about 5.29*103. Based on these, the reaction rate under our typical TFA conditions is
about 3.28*104 times faster than that under pH 5 buffer conditions. Thus, the hydrolytic rate for acetal
15 is estimated to be ~32 times faster than that of acetal 13. As all reactions showed very good
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first-order reaction kinetics, the reaction rate constant k equals 0.693/t1/2. A Hammett plot with 11, 12,
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13 and 15 from log(k/k0) vs. σ value46 of the substituents on the phenyl ring yielded ρ value of -4.06
(R2>0.99), as shown in Figure 8B. The reasonably high negative ρ value shows the importance of
stabilizing the carboxonium ion intermediate in these molecules to accelerate the degradation.
Since acetal 15 did exhibit reasonable hydrolysis rates at pH 5, we further probed the effect of
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Figure 9. Effect of varying the number and position of the electron-donating methoxy substituents upon the
hydrolysis of benzylidene acetals. The solid lines are the fitting curves.
varying the number and position of methoxy moieties in the aryl moiety. Accordingly, when the number
of methoxy moieties were increased to two, where these functionalities were incorporated at the para and
the ortho positions in 16, the hydrolysis rate increased by an order of magnitude. The half-life
decreased for 16 to ~8 hours, compared to ~70 hours for 15. Adding one more methoxy moiety to the
other ortho position in 17 decreased the half-life even further to about 1.8 hours. Interestingly, when the
substitution pattern is changed from 2,4,6-trimethoxy to 3,4,5-trimethoxy units, the hydrolysis rate
decreased by more than two orders of magnitude to ~425 hours. This is attributed to the fact that the
methoxy units at the meta- positions behave more as an inductively electron-withdrawing moiety with
respect to the carboxonium ion. The results of these findings are shown in Figure 9.
Based on all the kinetics of the different acetal/ketal molecules, the relative hydrolytic kinetics is
summarized and shown in Table 1. The relative hydrolysis rates are tabulated with the unsubstituted
benzylidene acetal 13 as the standard. Based on the cross-correlations mentioned above, where the
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same molecule has been subjected to different degradation conditions, all these relative rates have
been estimated as rates under the pH 5 conditions. The data in Table 1 show that the relative
hydrolysis rates of the acetal/ketal molecules can be varied by nearly seven orders of magnitude by
judiciously choosing the substituents. In addition to the range provided by the substituents studied
here, one can also imagine using the structure-property correlations here to logically expand this
range even further.
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Table 1. Relative hydrolysis rates of the acetal/ketals used in this study.
Since the inspiration behind the construction of most pH-sensitive materials is driven by the possible
utility in biological applications such as drug delivery and sensing, we were interested in testing whether
there is a correlation between the kinetics of hydrolysis of small molecule acetals and the behavior of the
same in nanomaterials. By designing acetal- and ketal- containing nanomaterials that are capable of
acting as hosts for non-covalently bound guest molecules, we were especially interested in understanding
the evolution of the fidelity of these nanomaterials as hosts at different pHs. We design nanogels,
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prepared by a recently-reported emulsion-free method,44,45 that are capable of sequestering hydrophobic
guest molecules. In addition to simply correlating the small molecule degradation with encapsulation in
polymeric nanoparticles, we also envisage that this study is important, because the microenvironments of
these functionalities in the nanogels are different from those in the bulk solution studied above with small
molecules.
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An amphiphilic random co-polymer precursor was first prepared, where the hydrophilic monomer
contained a PEG moiety as the hydrophilic side chain and the hydrophobic monomer was based on the
reactive pentafluorophenyl ester (PFPE) moiety. The amphiphilic polymer can self-assemble in aqueous
phase to form a nanoscopic amphiphilic aggregate, which was used as the template for the nanogels. The
active PFPE groups on the amphiphilic polymer can readily react with amine groups of the pH-sensitive
linker, generated by deprotecting the trifluoroacetyl protecting groups in Figure 2, to crosslink the
micellar aggregates in water through the amidation (Figure 10A). The structures of the deprotected
linkers were shown in Figure S2, the numbers of which correspond to the protected precursor molecules
with a prime on the number. The crosslinking process was conveniently monitored by FT-IR as shown
in Figure 10D. The activated carbonyl group in PFPE of the polymer precursor exhibits a strong,
characteristic peak at 1850 cm-1. Upon amidation based crosslinking, this peak disappears with the
concurrent appearance of new peaks at ~1650 cm-1 and at 3300 cm-1, attributed to the carbonyl and the
N-H functionalities of the amide moiety respectively. We found this reaction to be nearly quantitative in
all cases. When 0.5 equivalent of a linker was used for reaction, the peak attributed to the activated
ester completely disappears, accompanied by the appearance of a strong peak for the amide moiety. We
assume that this represents >95% crosslinking in these nanogels. Similarly, if only 0.25 equivalent of the
linker was consumed, the peak intensity for the activated ester decreased to about half of the original
precursor polymer. The quantitative nature of this crosslinking reaction, with all crosslinkers studied,
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facilitates comparisons between different crosslinkers. Also, since the precursor polymer assembly size
determines the size of the crosslinked polymer nanogel in these cases, sizes of the nanogel in all these
variations were also quite consistent. Size of the nanogel was found to be ~10 nm both by dynamic light
scattering (DLS) and transmission electron microscopy (TEM) as exemplified in Figure 10 and Figure
S5.
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Figure 10. Preparation and characterization of the nanogels: A) the process for synthesis of the nanogels, B) TEM
image of the nanogels from crosslinker 3’ (inset figure is magnified TEM image, scale bar=20 nm), C) the DLS of
the nanogels from crosslinker 3’, D) IR spectrum of the nanogels from different crosslinkers.
Encapsulation stability can be most rigorously defined as a measure of the dynamic exchange of the
encapsulated guest molecules with the bulk solvent and thus with another nanogel present in solution.47
We use the previously established fluorescence resonance energy transfer (FRET) method to interrogate
this dynamics.47 Briefly, a pair of hydrophobic FRET dyes (3,3′-dioctadecyloxacarbocyanine perchlorate
(DiO, donor) and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI, acceptor)) are
incorporated as guests into two separate nanogels. When aqueous solutions of these two nanogels are
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mixed, the dye molecules will stay within their respective nanogels without any temporal evolution of
FRET, if the encapsulation stability were very high. On the other hand, if the encapsulation stability
were low, there would be a temporal evolution of the donor and acceptor fluorescence. The kinetics of
this evolution is directly related to the magnitude of encapsulation stability, which is defined as the
leakage coefficient (Λ) for supramolecular assemblies.
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As shown in Figure 11A, the guest exchange was faster when the precursor polymer was used as the
Figure 11. The encapsulation stability of the nanogels under different crosslink degree at pH=7.4. A) 0%, B) 60%,
C) 100%; D) the FRET ratio change with time.
host without any crosslinking, where the emission of the donor peak at 508 nm decreases and the
emission peak of the acceptor at 568 nm increases with time. This suggests that this assembly is quite
leaky. Upon crosslinking the assembly with 0.3 equivalent crosslinker 3’, the 60% crosslinked nanogel
was less leaky, while the FRET ratio for the 100% crosslinked nanogel did not change with time. This
supports the idea that the un-crosslinked micelle-like assemblies are quite leaky, but the encapsulation
stability can be substantially increased with crosslinking. In all the subsequent studies that vary the
structure of the acetal or ketal based crosslinkers, 100% crosslinking was used. This degree of
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crosslinking was targeted to: (a) rule out the possibility of change in encapsulation stability due to the
slow hydrolysis of PFP moieties; (b) to insure high intrinsic stability of the nanogels in the absence of
pH-induced cleavage of the acetal- or ketal- based crosslinkers.
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Figure 12. pH-dependent variations in exchange dynamics of the nanogel N3, derived from the crosslinker 3’ or 4’
based on the ketal 3 and 4. The red lines in D are the linear fitting curves.
Since the acetone-based ketal provides reasonable susceptibility to pH, linker 3’ was first used for the
preparation of the nanogels to monitor the difference in encapsulation stability upon varying the pH of
the solution. As shown in Figure 12A and 12B, although there is no FRET evolution at pH 7.4, indicative
of high encapsulation stability, the nanogel becomes leaky at pH 6.5. The leakage coefficient at pH 6.5
was found to be 0.026 h-1, compared to ~0 at pH 7.4 (Figure 12D). This leakage increased further at pH 5
to 0.087 h-1, supporting the assertion that the pH-dependent ketal degradation kinetics can directly affect
the encapsulation stability of the nanogel.
To compare the relative effects of pH-induced cleavage
upon the nanogel’s encapsulation stability, we evaluated FRET evolution of nanogels containing different
acetal- or ketal- based crosslinkers at pH 5. Accordingly, we tested the nanogels made of the
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crosslinkers derived from ketals shown in Series A in Figure 2, where the effect of the
electron-withdrawing amide moiety is tested. As shown in Figure 13, the distance between the
pH-sensitive ketal moiety and the electron-withdrawing amide moiety does indeed affect the
encapsulation stability. The leakage coefficient for the nanogel N1 with a C6 linker was found to be 0.52
h-1, while it was found to be 0.15 and 0.087 h-1 for N2 and N3 respectively. Two factors, which could
potentially complicate the interpretation that the electron-withdrawing nature of the substituents are the
key players in this series, should be considered: (i) the increased distance between ketal and the amide
also increases the length of the crosslinker, which could make the nanogel based on 1’ to exhibit less
encapsulation stability. Although possible, this is less likely to be a factor, because all three nanogels
displayed no leakage at pH 7.4, where ketal degradation is not expected to occur. (ii) the C6-crosslinker
in 1’ endows the nanogel with a more hydrophobic interior, compared to the C2-crosslinker in 3’. In this
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Figure 13. Effect of varying the distance between the electron-withdrawing amide moiety and ketal moiety upon
guest encapsulation stability in nanogels N1-N3. The solid lines in the figure are the linear fitting curves.
case, the encapsulation stability is expected to be higher for N1, compared to N3. However, the
opposite trend is observed, which is consistent with the hydrolysis kinetics of the small molecule ketal.
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To further test whether the hydrolysis kinetics of the small molecules correlate with the encapsulation
stability in the nanogels, we tested Series B, where the linker lengths are identical, but the stability of the
carboxonium ions vary significantly. Indeed the formaldehyde acetal, which did not exhibit any cleavage
as a small molecule in 6, did not seem to degrade in the nanogel N6 either as the leakage coefficient was
found to be close to zero (Figure 14). On the other hand, the nanogel N5 based on the acetaldehyde-acetal
exhibits slow change in encapsulation stability with a leakage coefficient of 0.019 h-1, while the FRET
evolution was much faster in N4 (same as N3 in the nanogel) with a leakage coefficient of 0.087 h-1.
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Figure 14. Effect of the primary, secondary or tertiary carboxonium ion as the intermediate species upon the
encapsulation stability in nanogels N4-N6. The solid lines in the figure are the linear fitting curves.
Once again, the small molecule hydrolytic stability seems to qualitatively correlate with the encapsulation
stability in the nanogels. Note however that the difference in the hydrolytic rate constants between 4
and 5 is more than three orders of magnitude, while the leakage coefficient is different by only four to
five. Similar correlations were also found for the nanogels synthesized from Series D (see SI for
Figure 15. Substituent effects in benzylidene acetal based nanogels, N13, N15 and N16, upon their guest
encapsulation stability. The solid lines in the figure are the linear fitting curves.
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details).
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Finally, we explored the effect of substituents in benzylidene acetals on the guest encapsulation
stability of the corresponding nanogels. In Series E and F, the aryl moieties that contain
electron-withdrawing moieties were too slow to be monitored through the FRET exchange studies.
Therefore, we studied the phenyl- (N13) and compared them with methoxy- substituted nanogels, N15
and N16. Once again, the trend in encapsulation stability, shown in Figure 15, was consistent with that
found with small molecules. Interestingly, there was a small difference in encapsulation stability between
the phenyl- substituted and furan-substituted nanogels, while such difference did not exist in the case of
small molecules (Figure S9). This small difference is attributed to the fact that the furan ring is
considered to be a bit more hydrophilic compared to the phenyl ring, which is likely playing a role in the
encapsulation stability of these nanogels.
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Conclusion
In summary, a series of acetal- and ketal- based molecules have been synthesized and evaluated for
their pH-sensitive degradation properties. Upon studying six different series of these molecules, we arrive
at the following conclusions. (i) Hammett correlations of substituted benzylidene acetals show that the
ρ value for the degradation reaction is about -4.06. This shows that there is a strong positive charge
development in the transition state of this acid-catalyzed degradation process. While it is anticipated
that an acid-catalyzed process would have a developing positive charge, the large magnitude of this
influence was certainly surprising. As a point of comparison, the ρ+ value for the solvolysis of cumyl
chloride is -4.45,48 which is considered to be an SN1-like process, supporting the strong developing
positive charge at the benzylic position in the transition state in acetal degradation. (ii) The relative
positioning of the ketal moiety and the electron-withdrawing amido group can greatly influence its
degradation, in that there is a two orders of magnitude difference in the cleavage rates of 2- and 6- carbon
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linkers. (iii) Acetal derivatives of formaldehyde and acetaldehyde, and the ketal derivative of acetone,
have substantially different reactivities, presumably due to the variations in stabilities of 1°, 2°, and 3°
carboxonium ion type intermediates. (iv) Understandably, the length of the alkyl chain substituents in
the acetal carbon did not have a substantial influence upon the stability of carboxonium ion stability and
thus the rates of acetal hydrolysis. (v) Comparison of ketals derived from acetone, cyclopentanone, and
cyclohexanone show that torsional effects also have substantial influence upon the degradation kinetics.
(vi) Incorporation of these functional groups as crosslinkers within polymeric nanogels causes them to
exhibit pH-sensitive host-guest properties. Studying the effect of variations in the structure of the
crosslinker upon the encapsulation stability of the nanogels at lower pH reveals that the trends observed
in the small molecule degradation faithfully translate to the host-guest properties in the nanogels.
Overall, the study outlined here provides a comprehensive overview of structural variations that afford
the opportunity to tune the pH-sensitivity of acetal- and ketal- based linkers. We show that the structural
fine-tuning of the linkers allows access to variations in kinetics of degradation of more than six orders of
magnitude. The effect of structural variations upon the pH-sensitive host-guest characteristics of
polymeric nanogels constitutes one such example. The overall impact of this fundamental study is likely
to be much broader in a variety of applications, including drug delivery and tissue engineering.49-52
Utilizing these findings in biosensing and drug delivery applications includes part of the future efforts in
our own laboratories.
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Experimental section
Most of the experimental details are presented in the Supporting Information. Three of the key
procedures are outlined here.
Hydrolytic study of the acetal/ketal
0.01 mmol of the acetal/ketal molecules were dissolved in 0.3 mL CD3CN and then 0.1 mL PB D2O
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buffer (0.2 M, pH=5) was added for hydrolysis. The corresponding hydrolytic process was monitored by
400 MHz NMR at room temperature. The fast hydrolytic study was performed with acid as the catalyst.
0.01 mmol of the acetal/ketal molecule was dissolved in 0.3 mL CD3CN and then 0.1 mL CF3COOH
solution (in D2O, 50 mM) was added for hydrolysis. The reduced TFA hydrolytic condition was similar
except using 0.1 mL 10 mM CF3COOH solution (in D2O).
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Preparation of the nanogels
10 mg of polymer was dissolved in 1 mL pH=7.4 buffer. Then, the crosslinker (11.33 µmol for 100%
crosslink degree) and 4 µL of DIPEA were added under stirring for another 24 h. The solution was
further purified through dialysis under PBS buffer. If the nanogels was used to load dyes inside, the stock
solution of the dye was added into the polymer solution before the addition of the crosslinker and DIPEA.
The final nanogels were stored in PBS solution with the concentration of 2 mg/mL.
FRET study of the nanogels
FRET study was performed by the mixture of the DiI loaded nanogels and DiO loaded nanogels under
different pH. 0.1 mL DiI loaded nanogels and 0.1 mL DiO loaded nanogels were mixed in the presence of
0.8 mL buffer (0.2 M) with different pH. The fluorescence of the solution was recorded with the
excitation wavelength of 450 nm.
Data analysis
All the hydrolysis experiments were performed three times. The error values for t1/2 were reported as the
standard deviations of the three trials. The data points and error bars in the figures represent the average
and standard deviation at each data point respectively. The solid lines are the exponential fitting curve of
the data points.
Associated Content
Supporting information
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Experimental details, Figures S1-S10, and Table S1-2. This material is available free of charge via the
Internet at http://pubs.acs.org.
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Author Information
Corresponding Authors
thai@chem.umass.edu
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Notes
The authors declare no competing financial interest
Acknowledgment
We thank support from the Army Research Office (W911NF-15-1-0568) and National Institutes of
Health (CA-169140) for support.
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