Facile preparation of an orthogonally protected, pH-sensitive, bioconjugate linker for therapeutic applications

Article abstract of DOI:10.1021/ol0483347

(Chemical Equation Presented) We describe the facile, three-step synthesis of an orthogonally protected, pH-sensitive linker (8), based on maleic acid, and report its application to the preparation of a pH-sensitive phospholipid (20) for potential use in drug and gene delivery. In addition, we highlight the benefits of our linker over the use of the commercially available cis-aconitic anhydride (4).

Full text of DOI:10.1021/ol0483347

ORGANIC
LETTERS
2004
Vol. 6, No. 23
4245-4248
Facile Preparation of an Orthogonally
Protected, pH-Sensitive, Bioconjugate
Linker for Therapeutic Applications
Steven Fletcher,†,‡ Michael R. Jorgensen,*,§ and Andrew D. Miller*,†,§
Department of Chemistry, Imperial College Genetic Therapies Centre,
Imperial College London, Flowers Building, Armstrong Road, London, SW7 2AZ, UK,
and IC-Vec, Ltd., Flowers Building, Armstrong Road, London, SW7 2AZ, UK
m.jorgensen@icVec.com; a.miller@imperial.ac.uk
Received August 20, 2004
ABSTRACT
We describe the facile, three-step synthesis of an orthogonally protected, pH-sensitive linker (8), based on maleic acid, and report its application
to the preparation of a pH-sensitive phospholipid (20) for potential use in drug and gene delivery. In addition, we highlight the benefits of our
linker over the use of the commercially available cis-aconitic anhydride (4).
Since the first reports in the 1980s of implementing cationic
liposomes as vectors (gene-delivery vehicles) for non-viral
gene therapy,1 a whole array of cationic lipids have been
created to try to improve the efficacy of these early vectors.
An alternative approach has been to attempt to harness the
intrinsic fusogenic2,3 properties of the naturally occurring
lipid dioleoylphosphatidylethanolamine (DOPE, 1), a lipid
that invariably leads to enhanced gene delivery and expres-
sion (transfection).4-7
ing transmembrane aminophospholipid transport and mem-
brane fusion.8 Cyclic, unsaturated anhydrides form pH-sen-
sitive maleamates upon reactions with amines. Stable at high
pH, these compounds recyclize at low pH, causing intramo-
lecular cleavage of the amide bond, thereby regenerating both
the cyclic anhydride and the amine (Scheme 1).9,10
Upon cellular uptake of cationic liposome/DNA complexes
(lipoplexes), there is a concomitant drop in pH, suggesting
the aforesaid modification to DOPE may also be beneficial
for the purpose of gene therapy by masking, and thereby
harnessing, the zwitterionic and fusogenic nature of DOPE.
In 1995, Drummond and Daleke reported the reversible
modification of the amino group of DOPE using a range of
cyclic acid anhydrides, for potential applications in monitor-
(4) Felgner, J. H.; Kumar, R.; Sridhar, C. N.; Wheeler, C. J.; Tsai, Y. J.;
Border, R.; Ramsey, P.; Martin, M.; Felgner, P. L. J. Biol. Chem. 1994,
269, 2550-2561.
† Imperial College London.
‡ Current address: Department of Chemistry, Yale University, 225
Prospect Street, P.O. Box 208107, New Haven, CT 06520-8107.
§ IC-Vec, Ltd.
(5) Farhood, H.; Serbina, N.; Huang, L. Biochim. Biophys. Acta 1995,
1235, 289-295.
(1) Felgner, P. L.; Gadek, T. R.; Holm, M.; Roman, R.; Chan, H. W.;
Wenz, M.; Northrop, J. P.; Ringold, G. M.; Danielsen, M. Proc. Natl. Acad.
Sci. U.S.A. 1987, 84, 7413-7417.
(2) Litzinger, D. C.; Huang, L. Biochim. Biophys. Acta 1992, 1113, 201-
227.
(6) Siegel, D. P.; Epand, R. M. Biophys. J. 1997, 73, 3089-3111.
(7) Keller, M.; Jorgensen, M. R.; Perouzel, E.; Miller, A. D. Biochemistry
2003, 42, 6067-6077.
(8) Drummond, D. C.; Daleke, D. L. Chem. Phys. Lipids 1995, 75, 27-
41.
(3) Gruner, S. M.; Cullis, P. R.; Hope, M. J.; Tilcock, C. P. Annu. ReV.
Biophys. Biophys. Chem. 1985, 28, 211-238.
(9) Dixon, H. B.; Perham, R. N. Biochem. J. 1968, 109, 312-314.
(10) Bender, M. L. J. Am. Chem. Soc. 1957, 79, 1258-1259.
10.1021/ol0483347 CCC: $27.50
© 2004 American Chemical Society
Published on Web 10/20/2004

Scheme 1
Scheme 2
Drummond et al. discovered that reaction with citraconic
anhydride (3) generated the most acid-labile DOPE product.
For gene therapy applications, however, it is necessary to
further stabilize the now anionic DOPE molecule through
the conjugation of a highly hydrophilic group (via an acid-
labile linker) such as a sugar molecule or poly(ethylene-
glycol) (PEG), in order that ion-pairing with cationic lipids,
required to bind the anionic DNA, of the vector formulation
does not lead to self-fusion.11
the desired pH sensitivity of the maleamate products and
thereby prevent liberation of the drug or lipid, may be due
to an unusually low pKa of the methylene group R- to the
free, γ-acid.
In this Letter, we report a de novo and facile, three-step
route to an acid-labile linker moiety (8; Scheme 2), which
Scheme 3
Analogous drug-polymer conjugates have been prepared
in two steps with cis-aconitic anhydride (4). First, the amino
group of the drug is reacted with the anhydride of 4 to form
a cis-R,â-unsaturated amide bond; then, the γ-carboxylic acid
is coupled to the polymer, in the presence of the freed R- or
â-conjugated acid.12 However, such conjugates have been
poorly characterized. As shown in Scheme 1, the importance
of the characterization of amine-4 conjugates is paramount,
since the geometry of the cis-carboxylic acid is a prerequisite
for pH sensitivity. In addition, 4 is known to spontaneously
isomerize to propene-1,2,3-tricarboxylic acid-1,3-anhydride,
leading to undesired trans products upon coupling with
amines.13 Furthermore, Brocchini et al. have reported that
decarboxylation, double-bond isomerization (positional and
geometrical), and hydrolysis reactions have all been observed
during the coupling of an amine to 4.14 The readiness with
which 4 undergoes such isomerizations, which will abolish
(11) Hafez, I. M.; Maurer, N.; Cullis, P. R. Gene Ther. 2001, 8, 1188-
1196.
(12) Shen, W. C.; Ryser, H. J. P. Biochem. Biophys. Res. Commun. 1981,
102, 1048-1054.
(13) Remenyi, J.; Balazs, B.; Toth, S.; Falus, A.; Toth, G.; Hudecz, F.
Biochem. Biophys. Res. Commun. 2003, 303, 556-561.
(14) Dinand, E.; Zloh, M.; Brocchini, S. Aust. J. Chem. 2002, 55, 467-
474.
4246
Org. Lett., Vol. 6, No. 23, 2004

Scheme 4
may have less tendency to undergo cis/trans and positional
conditions with tert-butyl P,P-dimethylphosphonoacetate
afforded almost exclusively (Z)-isomer 8 (as confirmed by
1H NMR and NOE experiments16), in good yield (65%).
Then, removal of the tert-butyl ester of 8 in the presence of
the Boc group was achieved with a 1 M solution of hydrogen
chloride gas in dry EtOAc in excellent yield, giving 9 as its
HCl salt.17
The stabilizing moiety to which 9 would be coupled was
prepared in nine steps (Scheme 3) from methyl R-D-
galactopyranoside (10), a sugar that has a particular tropism
for hepatocytes.18 Such a feature may afford desirable
targeting properties. Briefly, 10 was fully benzylated then
allylated to generate the readily functionalizable alkene 11.19
Hydroboration of 11 with 9-BBN led to the desired anti-
Markovnikov borane, which upon treatment with alkaline
peroxide, followed by oxidation with Jones’s reagent,
furnished acid 1220 in good yield. Next, to improve the
hydrophilic properties of the acyclic portion of 12, glycine
isomerizations, due to an increased spacer between the maleic
anhydride functionality and the linker (acid or amine)
functionality. The application of this moiety to the reversible
modification of an alkynoyl analogue of DOPE (DS(9-yne)-
PE, 2) into an anionic, pH-sensitive, stabilized lipid, suitable
for incorporation into cationic liposomes, is also described.
First, 5 was coupled in excellent yield to (triphenylphos-
phoranylidene)acetonitrile, thereby furnishing cyano keto
phosphorane 6. Subsequent ozonolysis of 6 followed by
quenching of the intermediate vicinal diketo nitrile with
cyanoethanol afforded R-keto cyanoethyl ester 7 in 61%
yield. This methodology, developed by Wasserman et al.,
allows for the simple and swift preparation of R-keto acids,
esters, and amides, compounds that may have important
medical applications as potent inhibitors of proteases.15
Hitherto, ozonolysis of cyano keto phosphoranes followed
by quenching with an alcohol has only been reported with
large excesses of methanol or benzyl alcohol to generate the
R-keto methyl and benzyl esters, respectively. We were able
to perform the reaction with just 1.3 equiv of cyanoethanol,
which therefore did not hinder purification and suggests that
the quenching reaction may be feasible for alcohols in
general. Treatment of 7 under Horner-Wadsworth-Emmons
(16) 1H NMR of alkene region of 8 shows a triplet (4J 1.4 Hz) at δH
5.77 (CDCl3); the less than 1% trans impurity shows a triplet at δH 6.82
(CDCl3). These observations are in agreement with similar functionalities
in the literature: Hosseine Massoudi, H.; Cantacuzene, D.; Wakselman,
C.; Bouthier de la Tour, C. Synthesis 1983, 12, 1010-1012. The NOE
spectrum of 8 is available in Supporting Information.
(17) Gibson, F. S.; Bergmeier, S. C.; Rapoport, H. J. Org. Chem. 1994,
59, 3216-3218.
(18) Kichler, A. J. Gene Med. 2004, 6, S3-S10.
(15) Wasserman, H. H.; Ho, W.-B. J. Org. Chem. 1994, 59, 4364-4366.
Org. Lett., Vol. 6, No. 23, 2004
(19) Giannis, A.; Sandhoff, K. Tet. Lett. 1985, 26, 1479-1482.
4247

tert-butyl ester was coupled, which also installed a desirable
protecting group for the acid functionality. Subsequent
debenzylation gave deprotected C-pyranoside 13 in excellent
yield (94%). Next, treatment of 13 with 2,2-dimethoxypro-
pane (DMP), based on conditions by Liptak et al.,21 afforded
the corresponding 3,4-O-isopropylidene almost exclusively;
then, reaction with FmocCl in pyridine gave the fully
protected sugar 14 in very good yield.22 Finally, removal of
the tert-butyl ester of 14 in TFA/CH2Cl2 also led to rapid
cleavage of the acetal protecting group, but this was easily
remedied by post-treatment with DMP and catalytic PPTS,
furnishing 15 as a white powder in an overall yield of 20%
for nine steps.
Acid-labile linker 9 and sugar 15 were conjugated under
standard EDCI peptide coupling conditions to give fully
protected compound 16 (Scheme 4). Ester 16 was then sub-
jected to a 1:1 mixture of TFA and CH2Cl2 for a prolonged
period (>2 h), to ensure removal of both the tert-butyl
ester and the acetal protecting groups, affording 17 in 95%
yield. Next, a more phase-stable, synthetic analogue of
DOPE, DS(9-yne)PE (2),23 was coupled to acid 17 via acti-
vation with isobutyl chloroformate and N-methyl morpholine
(NMM) (carbodiimides, CDI, and HBTU were unsuccessful
in promoting this reaction, reflecting the deactivated nature
of the conjugated acid) to give 18 in good yield (85%).24
Subsequent deprotection of the base-labile Fmoc and
cyanoethyl groups of 18 with Et3N led to cyclization of the
maleamate derivative, generating imide 19, as detected by
1H NMR and mass spectrometry. Indeed, we discovered that
a range of nonhydrolytic bases such as DBU in CH2Cl2 and
TBAF in DMF all led to imide 19. However, the use of a
40% (w/v) aqueous solution of tetra-n-butylammonium
hydroxide (TBAH) allowed the rapid removal of the protect-
ing groups, followed by in situ cyclization (as monitored by
mass spectrometry), and then reopening of the imide all with
minimal hydrolysis of the oleate esters, giving 20 as a
mixture of equally acid-labile positional isomers in 49%
yield. The apparently inevitable cyclization of maleamate
18 to imide 19 may prove to be serendipitous since these
findings suggest that the pH sensitivity of the acid-labile
maleamic acid moiety of 20 may be “protected” during
synthetic modifications as its corresponding cyclic imide.
In conclusion, building on the work of Wasserman, we
have developed an efficient synthetic route to the preparation
of a fully and orthogonally protected, acid-labile linker (8)
and highlighted its use with reference to the synthesis of a
pH-sensitive lipid (20) for use in drug and gene delivery
applications. The problems of geometrical and positional
isomerizations of the maleamate C-C double-bond func-
tionality upon reaction of 4 with an amine have been
addressed and solved. While the deprotection of 18 led
ultimately to 20 as two positional isomers, these isomers are
positional merely in the alkyl substitution of the maleamate.
The all-important cis-carboxylate remains intact in both
isomers; pH sensitivity has not been compromised upon
undesired cyclic imide formation. Preliminary in vitro
biological data of CDAN:DOPE7 cationic liposomes incor-
porating 20 are encouraging, facilitating the preparation of
temporarily lower-charged (through pH sensitivity), and
therefore more stabilized, lipoplexes that are still competent
at transfection. Finally, the syntheses of other lipids incor-
porating the acid-labile moiety are ongoing in our laboratory.
Supporting Information Available: We thank Mitsubishi
Chemical Corporation and IC-Vec, Ltd., for funding.
(20) Gustafsson, T.; Saxin, M.; Kihlberg, J. J. Org. Chem. 2003, 68,
2506-2509.
(21) Liptak, A.; Nanasi, P.; Neszmelyi, A.; Wagner, H. Carbohydr. Res.
1980, 86, 133-136.
Supporting Information Available: Experimental pro-
cedures and full characterizations for compounds 5-18 and
(22) To the best of our knowledge, this is the first report of a di-Fmoc-
protected pyranoside.
1
(23) A Dialkynoyl Analogue of DOPE Has a Higher LR/HII Phase
Transition TemperaturesImplications for Cationic Liposome-Based Gene
DeliVery; Fletcher, S.; Jorgensen, M. R.; Miller, A. D. Submitted.
(24) If 18 is left in solution for an extended period of time (>8 h),
cyclization to di-Fmoc-protected 19 occurs, as observed by the change in
20, H NMR and m/z for 19, and NOESY NMR spectrum
for 8. This material is available free of charge via the Internet
at http://pubs.acs.org.
1
the H NMR spectrum (vicinal proton: δH 5.91 f 6.20 (CDCl3)).
OL0483347
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Org. Lett., Vol. 6, No. 23, 2004