Dioxazocinium ortho esters: A class of highly pH-vulnerable amphiphiles

Article abstract of DOI:10.1002/anie.200352589

Making and breaking lipids: A general synthesis of a new class of ortho ester lipids from a dioxazocinium ketene acetal is disclosed. Both single- and dual-chain analogues were prepared, and the most sensitive of these (see structure) was shown to disassemble at 38 °C within 6 h at pH 5.0.

Full text of DOI:10.1002/anie.200352589

Angewandte
Chemie
Lipid Amphiphiles
Dioxazocinium Ortho Esters: A Class of Highly
pH-Vulnerable Amphiphiles**
Kolbot By and Michael H. Nantz*
Micelles and vesicles derived from pH-sensitive amphiphiles
[
1,2]
continue to attract attention for a variety of applications.
Of particular recent interest is the use of cationic, pH-
sensitive liposomes as delivery vehicles for DNA therapeu-
[
3]
tics. Our interest in lipids that disassemble on exposure to
mild acid conditions, such as the pH 5–6 environment within
[
4]
endosomes, led us to engineer lipids that feature the highly
[
5]
pH-vulnerable ortho ester moiety. We disclose herein the
synthesis of an even more sensitive class of pH-vulnerable
lipids from a novel dioxazocinium ketene acetal reagent. The
results of formulation and hydrolysis studies with representa-
tive lipids 1–4 (Scheme 1) and a single-chain analogue are
presented to illustrate the versatility of the synthesis and pH
sensitivity.
[*] K. By, Prof. M. H. Nantz
Department of Chemistry
University of California, Davis, CA 95616 (USA)
Fax: (+1)530-752-8995
E-mail: mhnantz@ucdavis.edu
[
**] This work was partly supported by the Cystic Fibrosis Foundation.
We thank Grete N. Adamson for the transmission electron micro-
graphs and Dara E. Gilbert for technical assistance.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
Angew. Chem. Int. Ed. 2004, 43, 1117 –1117
DOI: 10.1002/anie.200352589
ꢀ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1117

Communications
Scheme 3. Ortho ester synthesis: a) 1,2-ditetradecanoyl-rac-glycerol,
DMF, 69% (1); b) 1,2-dimyristoyloxy-6-hexanol, CH Cl :DMF (3:1),
2
2
8
6% (2); c) 1,3-ditetradecanoyl-rac-glycerol, DMF, 61% (3); d) 1,3-
Scheme 1. Dioxazocinium ortho ester lipids.
dimyristyloxy-2-propanol, CH Cl :DMF (3:1), 43% (4). DMF=N,N-
2
2
dimethylformamide.
[
6]
Lipid synthesis: Exposure of ammonium salt
Scheme 2) to NaOMe gives rise to methyl ortho ester 6
and to ketene acetal 7 as the consequence of a competing
Hofmann-type elimination. We reasoned that the ketene
acetal moiety in 7 could serve as a progenitor of amphiphilic
ortho esters. Hence, we aimed to optimize the yield of 7 from
5
(
of 8 with dual-chain substituted primary and secondary
alcohols gave the ortho esters 1–4 in good to moderate
yields depending on the degree of substitution of the alcohol.
Lipid properties: With the dioxazocinium lipids prepared,
we turned our attention toward liposome formulation and
pH-vulnerability issues. Incorporation of dioloeylphosphati-
dylethanolamine (DOPE) during the hydration and sonica-
tion of the dual-chain analogues 1–4 greatly improved the
[
7]
5. Our initial efforts resulted in a protocol in which sodium
[
11]
ability of these lipids to assemble in water. In separate D O
2
1
experiments, H NMR analysis showed that sonication and
heating of 1–4 at 508C for 1 h did not hydrolyze the ortho
ester. Transmission electron microscopy (TEM) analyses of
representative preparations derived from 1 and 3 revealed
[
12]
multilamellar lipid aggregates with sizes of 95–220 nm.
Light-scattering-particle size measurements also indicated
aggregates with mean diameters of 165 and 159 nm for the
DOPE preparations of 1 and 3, respectively.
To examine the susceptibility of the dioxazocinium ortho
ester linkage toward acid-induced cleavage under different
conditions, we considered the case of the single-chain
derivative 9. Ortho ester hydrolysis is believed to proceed
through a three-stage reaction mechanism that involves, as
the first stage, the generation of a dioxocarbenium ion
Scheme 2. Synthesis of the ketene acetal reagent 8.
triphenylmethoxide and 5 were heated under vacuum in
tetrahydrothiophene 1,1-dioxide (sulfolane). Ketene acetal 7
was collected by direct distillation. Final purification afforded
a 36% yield of pure 7 on a 20-gram scale. Ultimately, we
discovered that simple elution of 5 as a methanol solution
through an anion-exchange resin gave ketene acetal 7 in 89%
yield. Although hydroxide ion exchange resins are often used
to prepare ammonium hydroxide salts, temperatures in excess
of 1008C are typically required to effect subsequent Hofmann
[
13]
intermediate. Though many variables are involved in the
[
14]
hydrolysis, cyclic ortho esters form dioxocarbenium ions
either by endocyclic CÀO or exocyclic CÀO bond cleavage,
with the latter being the preferred mode in small to medium-
[
15]
size rings. In the case of 9, endocyclic CÀO bond cleavage
would give rise to dioxocarbenium species 10 (Scheme 4),
while heterolysis of an exocyclic CÀO bond would give 11
with concomitant formation of myristyl alcohol (12). Subse-
quent interception of these dioxocarbenium species by water
results in products from paths a–c. While 10 may serve as a
precursor to diol 13 (path a), the monoacetate 15 (path b),
and the accompanying myristyl hydrolysates 14 and 12,
respectively, the cyclic species 11 can form only monoacetate
15. This mechanistic interpretation suggests that the appear-
ance of the myristyl hydrolysis products could be used as a
convenient probe for monitoring the progress of the hydrol-
ysis.
[
8]
eliminations. Thus the formation of 7 under these mild
conditions represents a convenient but unusual ketene acetal
[
9]
synthesis.
Ortho ester formation with 7 proved to be sluggish
without the assistance of an acid catalyst and low yielding
when only catalytic amounts of acid were employed. How-
ever, quaternization of 7 cleanly afforded 8 (Scheme 2). In
contrast to 7, ammonium salt 8 can be stored without
[
10]
decomposition
and readily converted into ortho esters
(
Scheme 3). The synthesis is general. For example, under
Incubation of 9 in HOAc/NaOAc buffer solutions at
pH 5.0, 5.5, and 6.0 with monitoring by GC/MS to quantify the
appearance of 12 and 14 revealed that myristyl acetate was
the predominant by-product, thereby implicating path a as
HBF catalysis, reaction of 8 with myristyl alcohol provided
ortho ester 9. Other catalysts were examined (trifluorome-
thanesulfonic acid, methanesulfonic acid, anhydrous HCl in
diethyl ether), but none were as effective as HBF . Reaction
4
[
12]
the principal mode of hydrolysis. Only trace amounts of
4
1
118
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Angewandte
Chemie
(approximately 10–50%) hydrolysis. While the hydrolysis
rate certainly is influenced by differences in the aggregation
state (that is, micellar with 9, lamellar with 1–4), substituent
effects also contribute. Lipid 3 is the slowest to hydrolyze, a
fact which is consistent with the expected electron-withdraw-
ing influences of the b-acyloxy and b-alkoxy groups. Lipid 2,
which closely approximates the alkyl side-chain structure of 9,
is the most sensitive of the dual-chain amphiphiles. In
competition hydrolysis experiments of 9 mixed with each
dual-chain lipid we found that 9 was clearly more vulnerable
except when mixed with 2, where we observed essentially no
difference in the rate of hydrolysis between 2 and 9. The ortho
ester moiety in 2 undergoes complete hydrolysis at pH 5.0
within 6 h. These results show that subtle structural differ-
ences can be used to adjust the pH sensitivity of a dioxazo-
cinium ortho ester. This facet may be useful in tailoring
amphiphiles of this type to cleave at a targeted, mild
pH value.
In summary, we have reported an efficient synthesis of a
dioxazocinium ketene acetal that can serve as a reagent for
the transformation of hydrophobic primary or secondary
alcohols into pH-vulnerable ortho ester amphiphiles. Hydrol-
ysis studies revealed unprecedented pH-sensitivity among the
cationic lipids and provided preliminary indications that
hydrolysis of dioxazocinium ortho esters occurs through the
endocyclic CÀO bond cleavage mode.
Scheme 4. Hydrolysis of the ortho esters.
myristyl alcohol (12, < 5%) were detected in these experi-
ments. The data (Figure 1) suggest an apparent zero-order
rate law consistent with the acid-catalyzed dissociative
mechanism reported for ortho ester hydrolysis. The rate
of hydrolysis of 9 is faster than that of other cationic lipids—9
[
13]
was completely hydrolyzed at pH 5.0 within six hours (t1/2
=
1
20 Æ 1 min). Extrapolation of the linear plot data for the
pH 5.5 and 6.0 conditions gave t1 values of 250 Æ 17 and
/2
4
07 Æ 15 min, respectively.
Received: August 7, 2003[Z52589]
Keywords: amphiphiles · cleavage reactions · hydrolysis · lipids ·
.
ortho esters
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), and pH 6.0 (*). Hydrolyses were
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[
[
[
[
[
12] See Supporting Information for TEM images and representative
GC chromatographs.
Angew. Chem. Int. Ed. 2004, 43, 1117 –1117
www.angewandte.org
ꢀ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1119

Communications
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