Facile synthesis of acid-labile polymers with pendent ortho esters

Article abstract of DOI:10.1021/bm201410c

This work presents a facile approach for preparation of acid-labile and biocompatible polymers with pendent cyclic ortho esters, which is based on the efficient and mild reactions between cyclic ketene acetal (CKA) and hydroxyl groups. Three CKAs, 2-ethylidene-1,3-dioxane (EDO), 2-ethylidene-1,3-dioxolane (EDL), and 2-ethylidene-4- methyl-1,3-dioxolane (EMD) were prepared from the corresponding cyclic vinyl acetals by catalytic isomerization of the double bond. The reaction of CKAs with different alcohols and diols was examined using trace of p-toluenesulfonic acid as a catalyst. For the monohydroxyl alcohols, cyclic ortho esters were formed by simple addition of the hydroxyl group toward CKAs with ethanol showing a much greater reactivity than iso-propanol. When 1,2- or 1,3-diols were used to react with the CKAs, we observed the isomerized cyclic ortho esters besides the simple addition products. Biocompatible polyols, that is, poly(2-hydroxyethyl acrylate) (PHEA) and poly(vinyl alcohol) (PVA) were then modified with CKAs, and the degree of substitution of the pendent ortho esters can be easily tuned by changing feed ratio. Both the small molecule ortho esters and the CKA-modified polymers demonstrate the pH-dependent hydrolysis profiles, which depend also on the chemical structure of the ortho esters as well as the polymer hydrophobicity.

Full text of DOI:10.1021/bm201410c

Article
pubs.acs.org/Biomac
Facile Synthesis of Acid-Labile Polymers with Pendent Ortho Esters
†
†
‡
,†
,†
Jing Cheng, Ran Ji, Shi-Juan Gao, Fu-Sheng Du,* and Zi-Chen Li*
†Beijing National Laboratory for Molecular Sciences, Key Laboratory of Polymer Chemistry and Physics of Ministry of Education,
College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, People's Republic of China
‡
CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences,
Beijing 100101, People's Republic of China
*
S Supporting Information
ABSTRACT: This work presents a facile approach for preparation of
acid-labile and biocompatible polymers with pendent cyclic ortho
esters, which is based on the efficient and mild reactions between cyclic
ketene acetal (CKA) and hydroxyl groups. Three CKAs, 2-ethylidene-
1
,3-dioxane (EDO), 2-ethylidene-1,3-dioxolane (EDL), and 2-ethyl-
idene-4- methyl-1,3-dioxolane (EMD) were prepared from the corre-
sponding cyclic vinyl acetals by catalytic isomerization of the double
bond. The reaction of CKAs with different alcohols and diols was
examined using trace of p-toluenesulfonic acid as a catalyst. For the
monohydroxyl alcohols, cyclic ortho esters were formed by simple addition of the hydroxyl group toward CKAs with ethanol
showing a much greater reactivity than iso-propanol. When 1,2- or 1,3-diols were used to react with the CKAs, we observed the
isomerized cyclic ortho esters besides the simple addition products. Biocompatible polyols, that is, poly(2-hydroxyethyl acrylate)
(
PHEA) and poly(vinyl alcohol) (PVA) were then modified with CKAs, and the degree of substitution of the pendent ortho
esters can be easily tuned by changing feed ratio. Both the small molecule ortho esters and the CKA-modified polymers
demonstrate the pH-dependent hydrolysis profiles, which depend also on the chemical structure of the ortho esters as well as the
polymer hydrophobicity.
INTRODUCTION
characteristics including the good biocompatibility, pH-
27,28
■
dependent surface degradation, and reproducible production.
pH-Sensitive polymers and the relevant materials have been
extensively studied regarding their applications in the fields
of smart drug/gene delivery and diagnosis because of the
Ortho esters have also been applied to prepare pH-sensitive
lipids or acid-degradable polymers that showed potential for
29−34
1
−3
drug/gene delivery.
While most of the aforementioned
numerous pH gradients in human body.
In general, pH-
poly(ortho ester)s have ortho ester units in the backbone, in
recent years, we and others reported a family of acid-labile
sensitive polymers can be categorized into two groups: those
with ionizable weak acid or base and those with acid-cleavable
35−39
4
polymers with pendent cyclic ortho esters.
Although these
linkage. The polymers of the later category are usually neutral
polymers exhibit promising properties as potential drug carriers,
they have to be prepared by (co)polymerization of the indi-
vidual monomers which are synthesized through multistep
reactions with relatively low yields.
and may have better biocompatibility. Various acid-labile linkers,
such as hydrazone, imine, cis-aconitic amide, acetal/ketal, ortho
ester, and so on, have been used to build up the versatile pH-
sensitive polymers, polymer−drug conjugates, hydrogels, and
5
−9
Ketene acetals (KAs) represent a well-known family of com-
micro/nanoparticles. Of these, the acid-degradable polymers
or nanoparticles containing ketal/acetal linkages in the
backbone or as the pendent groups have attracted great inte-
rest partly due to their tunable hydrolysis profile and the
neutral degradation products, which is very important for
pounds which have been used as reagents for organic synthesis
40−42
or as monomers for polymerization.
KAs have extra-
ordinary reactivity with compounds containing active hydrogen
including alcohols, however, the structural effect of the alcohols
43
1
0−23
on their reactivity toward KAs has not been studied in detail.
curing the acute inflammatory diseases.
Recently, acetal-
On this basis, we report a facile method to synthesize acid-labile
polymers with pendent cyclic ortho esters by modifying
multihydroxyl polymers (polyols) with cyclic ketene acetals
(CKAs) (Scheme 1). Various synthetic or natural biocompat-
ible polyols with primary or secondary hydroxyl group, such
poly(vinyl alcohol), poly(2-hydroxyethyl acrylate), dextran, and
derivatized dextran or cyclodextrin were synthesized by the
reaction of dextran or cyclodextrin with 2-methoxypropene,
providing a facile approach to prepare acid-labile polymers with
2
4,25
pendent ketal/acetals.
Ortho ester is one of the most acid-labile functionalities
available. Generally speaking, ortho ester is more susceptible to
a weakly acidic environment than ketal or acetal with a similar
2
6
structure. Poly(ortho ester)s, an important family of the acid-
degradable polymeric biomaterials, have been extensively in-
vestigated over the past 40 years because of their unique
Received: October 10, 2011
Revised: December 11, 2011
Published: December 16, 2011
©
2011 American Chemical Society
173
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For the reaction of CKAs with 1,2- or 1,3-diols, the diol (1.05 mmol)
and TsOH were dissolved in THF first, followed by the addition of
CKA (1.0 mmol) under magnetic stirring. The reaction mixture was
stirred at 30 °C for 15 min. After removal of the evaporated organic
Scheme 1. Modification of Polyols by CKA
solvents at reduced pressure, ∼10 μL of the mixed ortho esters was
1
dissolved in 0.5 mL of CDCl and analyzed by H NMR spectroscopy.
3
Then, 50 μL of deuterated hydrogen chloride (DCl) in D O (20%) was
2
charged into the NMR tube, which was shaken for 30 s and incubated
for 5 min at ambient temperature to completely hydrolyze the ortho
1
esters. The tube was directly used for H NMR analysis. Our preli-
minary experiments showed that more than 95% of the diol
monoesters remained in the CDCl phase while most part of the
3
diols were extracted into the D O phase.
2
so on, can be used as the backbones. Because CKA reacts
quickly with the hydroxyl group under gentle conditions, a large
number of ortho ester based bioerodable polymers with a wide
range of properties can thus be easily prepared in a high-
throughput way. In addition, the hydrolysis products of the
acid-labile polymers will be nontoxic and biometabolized if
appropriate CKAs are used for the modification.
Acid-Triggered Hydrolysis of the Ortho Esters. To character-
ize the structure of the hydrolysis products, ∼10 μL of cyclic ortho
ester was dissolved in 0.5 mL of D O in a NMR tube, to which 20 μL
2
of DCl in D O (20%) was added. After being shaken for 30 s and
2
incubated at ambient temperature for an additional 5 min, the tube
1
was analyzed by H NMR spectroscopy. In the case of hydrolysis
kinetics measurement, 10 mg of the ortho ester was dissolved in
0
.5 mL of deuterated phosphate buffer (PB, 1.0 mM, pD 8.4), and the
1
measured H NMR spectrum was used as that of the 0 time point.
After adding 10 μL of 0.5 M deuterated buffer (pD: 5.4, 6.5 and 7.5),
the solution was quickly mixed and the 1H NMR spectra were
recorded at specific time points. For pD 8.4, the hydrolysis experiment
was carried out in 1.0 mM deuterated phosphate buffer.
EXPERIMENTAL SECTION
Materials. p-Toluenesulfonic acid (TsOH), D O, DCl, D PO (85
■
2
3
4
6
6
wt % in D O), NaOD (40 wt.% in D O), DMSO-d , acetone-d (J&K
2
2
Chemical Ltd.), 2,2-dimethyl-1,3- propanediol, 1,3-propanediol, 2,4-
pentanediol, and tris(triphenylphosphine) ruthenium(II) dichloride
Modification of Polyols with CKAs. Two polyols, poly(2-
hydroxyethyl acrylate) (PHEA) and poly(vinyl alcohol) (PVA), were
modified with CKAs, and the reaction was carried out in anhydrous
DMSO at 30 °C for 1 h. Take the reaction of PHEA with 2-ethylidene-
(
(Ph P) RuCl , Alfa Aesar) were used as received. CDCl (J&K
3
3
2
3
Chemical Ltd.) was treated with anhydrous Na CO prior to use.
2
3
Acrolein, ethylene glycol, 1,2-propanediol, ethanol, iso-propanol,
triethyl amine (TEA), and other solvents were purchased from Beijing
Chemical Reagent Company. Regarding the synthesis and character-
ization of CKAs and ortho esters, all the solvents were distilled under
sodium or calcium hydroxide prior to use.
1
,3- dioxolane (EDL) as an example. PHEA (1.0 g) was azeotropically
dried with toluene prior to use and thoroughly dissolved in 10 mL of
anhydrous DMSO in a flame-dried flask. After sequential addition of
TsOH (1.0 mg in 0.1 mL THF) and 0.52 g EDL (5.2 mmol in 1.5 mL
THF), the mixture was stirred at 30 °C for ∼30 min followed by
adding one drop of TEA to quench the reaction. Then, the reaction
mixture was dialyzed against water containing 0.1% TEA for 5 h at
ambient temperature (MWCO: 3500) and lyophilized. The crude
viscous product was dissolved in 10 mL of CH Cl and precipitated
Stock solution of TsOH in anhydrous THF (10 mg/mL) was
prepared for convenient use. The deuterated buffers (pD 5.4, 6.5, 7.5,
and 8.5) were prepared using D O, D PO , and NaOD and calibrated
2
3
4
by pH meter.
Synthesis of Cyclic Ketene Acetals. Acrolein (0.5 mol), diol (0.5
mol), toluene (250 mL), and TsOH (40 mg) were placed in a 500 mL
flask equipped with a magnetic stirrer, Dean−Stark trap, and
condenser. A total of 10 mL of saturated NaCl solution with NaCl
crystal (∼2 g) was placed in the trap to avoid the acrolein dissolved in
the aqueous phase. The mixture was refluxed in an oil bath (120 °C)
for 6 h until about 9 mL of water was collected in the trap. After
cooling, the solution was removed by rotary evaporation and 200 mL
of CH Cl was added. The solution was washed with 30 mL of 1%
2
2
from a mixed solvent of diethyl ether and hexane containing 1% TEA.
The precipitate was dried in vacuum, yielding 1.0 g of the purified
polymer (EDL-PHEA-1). Other CKA-modified PHEAs or PVAs were
prepared by the similar procedure with yields of 60−80% and
1
characterized by H NMR spectroscopy.
pH-Dependent Hydrolysis of the CKA-Modified PHEAs.
Hydrolysis of the CKA- modified PHEAs was monitored by
NMR spectroscopy in a mixed solvent of deuterated acetone and D O
1
H
2
2
2
Na CO thrice and with 30 mL of saturated NaCl solution, respec-
2
3
(1:4, v/v) at 26 °C. Briefly, the polymer (80 mg) dissolved in
tively. Then, the separated organic phase was dried over anhydrous
deuterated acetone (0.5 mL) was added dropwise into 2.0 mL of
deuterated PB (1.0 mM, pH 8.5) under stirring. Then, 0.6 mL of the
formed emulsion was charged into a NMR tube, and the H NMR
spectrum was measured, which was used as that at the 0 time point.
After addition of 10 μL of 0.5 M deuterated buffer (pD: 5.4 and 7.5),
the solution was quickly mixed and the 1H NMR spectra were
recorded at specific time points. For the hydrolysis at pD 8.4, the
experiment was carried out in 1.0 mM deuterated PB.
K CO , and distilled at reduced pressure to afford vinyl acetal. The
2
3
yields of various vinyl acetals were in the range of 40−65%.
1
For the catalytic isomerization, vinyl acetal (0.2 mol) and
Ph P) RuCl (50 mg) were charged into a 50 mL flask fitted with
3 3 2
(
a magnetic stirrer, argon inlet, and refluxing condenser. The reaction
mixture was bubbled with argon gas for 10 min and heated to and kept
at 125 °C in an oil bath for 5 h until the isomerization finished, as
1
monitored by H NMR spectroscopy. Then, the mixture was distilled
to give the ketene acetal under reduced pressure (∼10 mmHg). The
isomerization yield of various CKAs were in the range of 70−90%. All
the CKAs and their precursors (vinyl acetals) were characterized by
RESULTS AND DISCUSSION
■
1
H NMR spectroscopy (Figure S1).
Although different methods have been used to prepare ketene
acetals, the isomerization of the double bond of the α,β-unsaturated
aldehyde-derived acetals in the presence of (Ph P) RuCl
developed by Crivello is one of the simple approaches. We
first synthesized three cyclic ketene acetals (CKAs) following
Crivello’s method. They are different in the number of cyclic
atoms (EDO vs EDL) or the substituted groups (EDL vs EMD)
(Scheme 2). The structures of these CKAs were confirmed by the
Reaction of CKAs with Alcohols or Diols. CKA (1.0 mmol) and
quantitative alcohol were thoroughly dissolved in 1.0 mL of anhydrous
THF (in a 5 mL flask) under magnetically stirring. After adding 10 μL
of p-toluenesulfonic acid (TsOH) in THF (10 mg/mL), the solution
was stirred in an oil bath (30 °C) for the designed time, and the
reaction was quenched by adding one drop of triethylamine (TEA).
After removal of the organic solvent at reduced pressure by rotary
3
3
2
44
1
evaporation, ∼10 μL of the residue was taken for H NMR analysis
1
(
in 0.5 mL of CDCl ).
H NMR spectra (Figure S1).
3
1
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1
Scheme 2. Synthetic Route of 2-Ethylidene-1,3-dioxane
isomerized ortho ester A1 (Scheme 4). The H NMR spectrum
revealed that both A1 and A′1 were formed, however, it was
(
EDO), 2-Ethylidene-1,3-dioxolane (EDL), and 2-
Ethylidene-4-methyl-1,3-dioxolane (EMD)
a
a
Scheme 4
a
Reagents and conditions: (i) acrolein, TsOH, toluene, 120 °C, 5 h;
(
ii) (Ph P) RuCl , 125 °C, 4 h.
3 3 2
Addition Reaction of CKAs with Different Mono-
alcohols. Ketene acetals (KA) have extraordinary reactivity
a
Reaction conditions: (i) TsOH, THF, r.t., 30 min; molar ratio of
with compounds containing active hydrogen, such as H O and
2
CKA to the diol: 1.0/1.05. (ii) CDCl /20% DCl in D O (10:1, v/v),
r.t., 5 min.
3
2
alcohols, due to the presence of the strong nucleophilic center
in the KA molecule. KAs react readily with primary or sec-
ondary alcohols to form ortho esters, while they are inert to the
4
0,43
difficult to directly determine their ratio because of the poorly
resolved spectrum (Figure 1A). According to literature and our
tertiary alcohols.
However, the effect of the structure of the
alcohols on their reactivity toward KAs has not been studied in
detail. Here, we investigated the reactions between EDO and
two alcohols (ethanol and iso-propanol), and the results are
summarized in Scheme 3. EDO reacted rapidly with ethanol
Scheme 3. Schematic Illustration of the Reactions between
a
CKA or Cyclic Ortho Ester with Monohydroxyl Alcohol
a
Concentration of TsOH: 0.1 mg/mL; solvent: THF.
(
EtOH) or iso-propanol (i-PrOH) at 30 °C in THF, and the
reactions were completed in 20 min. When a mixture of ethanol
and iso-propanol reacted with EDO in stoichiometry (1:1:1 in
molar ratio), the main product was 2-ethoxy-2-ethyl-1,3-
dioxane (Ia) formed from ethanol, with little 2-ethyl-2-iso-
propoxy-1,3- dioxane (Ib) detected (Figure S2). This indicates
that the less sterically hindered primary alcohol is much more
active than the secondary alcohol. Once the cyclic ortho ester
1
Figure 1. H NMR spectra of the cyclic ortho esters prepared by the
reaction of EDO with 2,4-pentandiol (A), and the in situ acid-
hydrolyzed products (B) in CDCl . Peak intensities of proton j′ (I ∼
3
j′
5
.1 ppm) and proton b′ (I ∼ 2.3 ppm) are used to calculate the molar
b′
ratio of the two monoesters: A1% = (2I /I ) × 100% = 65%. *Denotes
j′
b
(
Ib) was formed, however, the iso-propoxy group was not easily
the proton signals coming from 1,3-propanediol monopropionate, the
hydrolyzed product of EDO. EDO rapidly reacts with trace of water in
the solvent.
replaced by an ethoxy group.
We also examined the effects of temperature (15−40 °C in
THF) and solvent (CHCl and DMSO at 30 °C), and the
3
reaction between EDO and ethanol can be completed in
results, when 2-alkyloxy-1,3-dioxalane or 2-alkyloxy-1,3-dioxane
are hydrolyzed in a mildly acidic solution, cleavage of the
exocyclic alkyloxy takes place preferentially, resulting in the
formation of glycol monoester or 1,3-propanediol mono-
ester, but with little formation of the corresponding diols
3
0 min. Similar results were obtained for the other two CKAs
1
(
EDL and EMD). The H NMR spectra of the obtained cyclic
ortho esters are shown in Figures S3−S5.
Addition Reaction of CKAs with 1,2- or 1,3-Diols. Some
polyols, such as poly(vinyl alcohol) (PVA) and dextran, have
2
7,37,45,46
(
Figures S3−S5).
that the hydrolysis of cyclic ortho ester A1 produces only
,4-pentanediol monoester (H-A1), while 1,3-propanediol
Thus, we can reasonably assume
1,3- or 1,2-diol units. When CKAs are used to modify these
polyols, the isomerized structures may be formed besides the
simple addition product of hydroxyl group to CKA. To demon-
strate this issue, EDO was first reacted with 2,4-pentanediol, a
model compound of 1,3-diol. There are two possible cyclic ortho
esters resulting from the reaction of EDO and 2,4-pentanediol;
one is the simple addition product A′1, and the other is the
2
monoester (H-A′1) comes from the ortho ester A′1 (Scheme 4).
In other words, the ratio of the two cyclic ortho esters (A1 and
A′1) can be obtained by measuring the molar ratio of the two
monoesters.
1
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a
Table 1. Dominating Cyclic Ortho Esters by Reaction of the CKAs with Different 1,3- or 1,2-Diols
a
The value represents the percent molar content of the dominating ortho ester.
Scheme 5. Reaction of the CKAs with Different 1,2- or 1,3-Diols
As shown in Figure 1B, after in situ acid-triggered hydrolysis
of the cyclic ortho esters, the ratio of the two monoesters can
be determined by comparing the intensities of peaks at ∼5.1
and ∼2.3 ppm; and accordingly, the molar ratio of A1 to A′1
was 0.65:0.35, in favor of the isomerized ortho ester A1. We
further used a variety of 1,3- or 1,2-diols to react with the CKAs
following a similar condition and analyzed the formed cyclic
ortho esters by in situ hydrolysis method (Figures S6−S9). The
preferred cyclic ortho ester and its percent molar content in the
mixture of the two competing cyclic ortho esters for each of the
reactions are summarized in Table 1. When EDO was used as
the CKA, the isomerized cyclic ortho esters dominated, and the
main products were the ortho esters with substituted 1,3-
dioxane or 1,3-dioxalane structures. By contrast, in the case of
EMD, the predominant products were the cyclic ortho esters
with 4-methyl-1,3-dioxalane ring, which were produced by
simple addition of the diols to EMD. Interestingly, the domi-
nating ortho esters by reactions of EDO with 1,2-propanediol
and EMD with 1,3-propanediol were identical (A3 vs C2),
having almost the same percentage.
Table 2. Half-Life Time (t1/2, min) of the pH-Dependent
Hydrolysis of Various Cyclic Ortho Esters
a
pH
ortho ester
temperature (°C)
5.4
6.5
7.5
8.5
50
Ia
15
15
15
23
23
23
23
∼1
∼2
∼2
∼3
14
20
36
15
∼8
12
17
IIa
IIIa
IIIa
Ib
120
b
∼1900
∼720
28
b
IIb
IIIb
80
b
∼560
a
Nomenclature: Ia, 2-ethoxy-2-ethyl-1,3-dioxane; IIa, 2-ethoxy-2-
ethyl-1,3-dioxalane; IIIa, 2-ethoxy- 2-ethyl-4-methyl-1,3-dioxalane; Ib,
-ethyl-2-iso-propoxy-1,3-dioxane; IIb, 2-ethyl-2-iso-propoxy- 1,3-
2
b
dioxalane; IIIb, 2-ethyl-2-iso-propoxy-4-methyl-1,3-dioxalane. Esti-
mated by extrapolating the kinetic curves.
spectroscopy. Figure 2 shows the representative time-dependent
1
H NMR spectra of ortho ester Ia hydrolyzed at pH 7.5. The
hydrolysis degree was estimated by monitoring the change in
integration intensity of peaks at ∼2.3 ppm (proton b′) using the
peak at ∼4.8 ppm (HDO) as a standard. Compound Ia is very
sensitive to the mildly acidic environment, showing a half-life
time of 14 min even though in the neutral medium. By the same
method, hydrolysis kinetics of the other ortho esters was also
evaluated (Figures S10−S13), and the half-life times at different
pHs are summarized in Table 2. As expected, all of these ortho
esters were hydrolyzed faster with the decrease of pH. When the
exocyclic alkyloxy group was identical, at the same pH and
temperature, hydrolysis rate decreased in the order of Ia > IIa >
IIIa, and Ib > IIb > IIIb.
For the other two pairs of reactions, EDO with glycol versus
EDL with 1,3-propanediol (A4 vs B2) or EDL with 1,2-
propanediol versus EMD with glycol (B3 vs C3), a similar trend
was obtained. Furthermore, when the reaction was carried out in
1
CDCl as in situ monitored by H NMR spectroscopy, the
3
similar phenomenon was observed (data not shown). These
results demonstrate that the proportion of the two competing
cyclic ortho esters is thermodynamically controlled, mainly
depending on their stability (Scheme 5).
pH-Dependent Hydrolysis of Various Cyclic Ortho
Esters. Three pairs of ortho esters, which are different in the
number of the cyclic atoms or in the substituent on the rings,
were studied to demonstrate their pH-dependent hydrolysis
behaviors (Table 2). The experiments were carried out in the
For acid-triggered hydrolysis of ortho esters, it is generally
46
accepted that there are three reaction stages. The first stage,
that is, generation of the dialkoxycarbocation intermediate,
which consists of the probable concerted proton transfer and
1
deuterated aqueous buffer, as monitored by H NMR
1
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1
Figure 2. H NMR (300 MHz) spectra of ortho ester Ia (20 mg/mL) at different hydrolysis times at 15 °C in deuterated PB (10 mM, pH 7.5). The
relative amount of the remaining ortho ester (Ia) is calculated by the formula: 1 − (I − I )/(I − I ), where I , I , and I , denote the peak intensity
t
0
∞
0
0
t
∞
of proton b′ (∼2.4 ppm) at 0 min (zero), t min, and of the completely hydrolyzed sample, respectively. The spectrum at 0 min is measured in the
buffer of pH 8.5, and # denotes the spectrum of the completely hydrolyzed sample. The proton peak of HDO (∼4.8 ppm) is used as the internal
standard, and * denotes the proton signals of impurities.
47,48
C−O bond cleavage,
is the rate-determining step in the
49
mildly acidic or neutral media. The faster hydrolysis rate of
the six-membered cyclic ortho ester (Ia or Ib) as compared to
the five-membered counterpart (IIa or IIb) can be explained by
the well-known anomeric (i.e., stereoelectronic) effect. For Ia
or Ib, the molecule adopts an almost ideal chair conformation,
which makes the exocyclic oxygen highly basic and is helpful for
C−O breaking and leaving of the protonated exocyclic alkoxy
2
6,50
group.
Regarding the hydrolysis of ortho esters with five-
and six-membered rings, Jones et al. reported an opposite
trend: the five-membered ortho ester hydrolyzed faster than
51
the six-membered one. This discrepancy might be due to the
difference in substituents on the rings. In Jones’ molecules, the
amide group might affect conformation of the ortho esters via
intramolecular hydrogen bonding, which would influence the
hydrolysis.
Modification of PHEA and PVA by CKAs. PHEA and
PVA were used as the polyols not only for their good
biocompatibility, but also for their different hydroxyl groups.
PHEA has the primary hydroxyl groups while PVA possesses
the secondary hydroxyl groups in the form of the 1,3-diol
structure. PHEA was first modified in anhydrous DMSO by
EDL with various feed ratios. As shown in Figure 3, the degree
of substitution (DS) of the ortho ester can be simply tuned by
changing the feed ratio of EDL to hydroxyl group. However,
DS was generally lower than the corresponding feed ratio,
which is mainly attributed to the partial hydrolysis of the ortho
ester during the postreaction purification procedure. By
increasing the feed ratio of EDL to hydroxyl group and
adopting a modified purification protocol, DS can be increased
up to 95% (Figure S16). Following a similar procedure, PHEA
was also modified by EDO and EMD (Figure S17). To further
clarify the polymer structure, EDO-modified PHEA was
1
Figure 3. H NMR (300 MHz) spectra of PHEA in D O and the
2
EDL-modified PHEAs in CDCl . Molar feed ratios of EDL to hydroxyl
group of PHEA are 0.6 (EDL-PHEA-1), 0.8 (EDL-PHEA-2), and 1.0
EDL-PHEA-3), respectively. The degrees of substitution (DS) in the
final polymers are estimated by comparing the intensity of peak d (Id)
and peak f + f′ (I ), being 44, 58, and 74%, respectively (DS = I /I ).
3
(
f+f′
d
f+f′
of PHEA and 1,3-propanediol monopropionate were detected,
indicating that PHEA reacted with EDO through the simple
addition of a hydroxyl group to the carbon−carbon double
bond as ethanol did (Figure S18).
completely hydrolyzed in acidic D O. Only the proton signals
2
1
77
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Article
Figure 4. Hydrolysis kinetics of the CKA-modified PHEAs.
By contrast, when PVA was modified by EMD with a feed
ratio of 1:1 in anhydrous DMSO, the isomerized structure of a
six-membered cyclic ortho ester on the polymer backbone was
observed, besides the pendent five-membered cyclic ortho
ester, which was formed by the simple addition of a hydroxyl
group to EMD (Figure S19). These results are very consistent
with those for the model reactions of CKAs with the corre-
sponding small monohydroxyl alcohol and 1,3-diols.
Cytotoxicity of two EMD-modified polymers, EMD-PVA
and EMD-PHEA, was evaluated by a MTT assay in Hela cells,
using poly(ethylene glycol) methyl ether (mPEG 5k) and
branched poly(ethylenimine) (PEI 25k) as negative and
positive controls, respectively (Figure S20). Compared to
PEI 25k, the two acid-labile polymers showed much lower
cytotoxocity. However, the cell viabilities for both EMD-
PVA and EMD-PHEA were lower than that for mPEG 5k at
high polymer concentration, which might be attributed to
their hydrophobic character.
neutral medium. By contrast, the other two polymers, EDO-
PHEA and EDL-PHEA, showed a significant hydrolysis, even at
pH 8.5. This demonstrates that EMD may be a better candidate
than the other two CKAs for the preparation of ortho ester-
containing polymers that are stable enough in weakly basic
media but sensitive enough to mildly acidic environments.
CONCLUSION
■
The reaction of CKAs with different alcohols and the hydrolysis
of their ortho ester products were studied in detail. Less steri-
cally hindered primary alcohol is much more active than the
secondary one. When 1,2- or 1,3-diols react with CKAs, the
isomerized cyclic ortho esters are formed; the ratio of the
competing ortho esters is mainly determined by their stability.
Hydrolysis of the cyclic ortho esters is pH-dependent and
greatly affected by their structure. Facile synthesis of the acid-
labile polymers with pendent cyclic ortho esters has been
achieved by modifying the biocompatible polyols with CKAs.
The chemical structure and hydrolysis kinetics of these acid-
labile polymers can be easily manipulated by using different
polyols and CKAs or by changing the feed ratio. The mild and
efficient reaction condition, structural diversity of the polyols
and CKAs, and ease of obtaining the starting materials make
the present approach very potential for the preparation of bio-
compatible and acid-sensitive polymeric drug carriers.
Hydrolysis of CKA-Modified PHEAs. Three CKA-modified
PHEAs with the similar degree of substitution (∼44%, Figure S17),
that is, EDO-PHEA, EDL-PHEA, and EMD-PHEA, were studied
on their pH-dependent hydrolysis in a mixed solvent of
deuterated phosphate buffer and acetone (Figure 4). As expected,
all the polymers showed significant pH-dependent hydrolysis
behaviors: the lower the pH, the faster the hydrolysis rate. At pH
8.5, the hydrolysis rate followed the order of EDO-PHEA > EDL-
PHEA > EMD-PHEA, which is consistent with the results of their
corresponding small model compounds (Table 2).
ASSOCIATED CONTENT
■
However, at the same pH, the polymers hydrolyzed much
slower than their small model compounds. This can be attrib-
uted to the hydrophobic aggregation of the polymers in
*
S
Supporting Information
NMR spectra of the CKAs, ortho esters, polymers, and their
hydrolysis kinetics; results of cell viability evaluation; and more
experimental details. This material is available free of charge via
the Internet at http://pubs.acs.org.
1
6,52,53
aqueous buffers.
Interestingly, EMD-PHEA was quite
stable at pH 8.5 but showed a moderate hydrolysis rate in the
1
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Biomacromolecules
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29) Masson, C.; Garinot, M.; Mignet, N.; Wetzer, B.; Mailhe, P.;
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Corresponding Author
E-mail: zcli@pku.edu.cn (Z.L.); fsdu@pku.edu.cn (F.D.).
(
1
(
*
ACKNOWLEDGMENTS
■
3
(
This work was supported by the National Natural Science
Foundation of China (21174002, 50973002, and 21090351).
We thank Prof. Wenlin Huang (Institute of Microbiology,
CAS) for his kind help regarding cytotoxicity evaluation.
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