Synthesis of VIP-lipopeptide using a new linker to modify liposomes: Towards the development of a drug delivery system for active targeting

Article abstract of DOI:10.1248/cpb.c13-00518

A new component for the solid phase peptide synthesis of lipopeptide, 2-[(4R,5R)-5-({[(9H-fluoren-9-yl)- methoxy]carbonylaminomethyl}-2,2-dimethyl-1, 3-dioxolan-4-yl)methoxy]acetic acid (2), was designed and synthesized from (-)-2,3-O-isopropylidene-D-threitol (3) in 4 steps. The key step was the selective alkylation of 3 with benzyl bromoacetate in the presence of Cs ₂CO₃. Vasoactive intestinal peptide (VIP)-lipopeptide (1) incorporating this linker was synthesized by solid phase peptide synthesis.

Full text of DOI:10.1248/cpb.c13-00518

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Note
Chem. Pharm. Bull. 61(11) 1184–1187 (2013)
Vol. 61, No. 11
Synthesis of VIP-Lipopeptide Using a New Linker to Modify Liposomes:
Towards the Development of a Drug Delivery System for Active
Targeting
Toru Masaka,a,b Takuya Matsuda,a Yingpeng Li,a Yuki Koide,a Akira Takami,a Kenji Yano,a
Ryosuke Imai,a Risa Ichihara,a Nobuhiro Yagi,a Hideharu Suzuki,a Hidemasa Hikawa,a
,a
Katsuhide Terada,a and Yuusaku Yokoyama*
a School of Pharmaceutical Sciences, Toho University; 2–2–1 Miyama, Funabashi, Chiba 274–8510, Japan: and
b Sakura Medical Center, Toho University; 564–1 Shimoshizu, Sakura, Chiba 285–8741, Japan.
Received June 27, 2013; accepted August 9, 2013; advance publication released online August 23, 2013
A new component for the solid phase peptide synthesis of lipopeptide, 2-[(4R,5R)-5-({[(9H-fluoren-9-yl)-
methoxy]carbonylaminomethyl}-2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]acetic acid (2), was designed and
synthesized from (ꢀ)-2,3-O-isopropylidene-D-threitol (3) in 4 steps. The key step was the selective alkylation
of 3 with benzyl bromoacetate in the presence of Cs2CO3. Vasoactive intestinal peptide (VIP)-lipopeptide (1)
incorporating this linker was synthesized by solid phase peptide synthesis.
Key words drug delivery system; linker; isopropylidene-D-threitol; lipopeptide; peptide synthesizer
Surface modification of liposomes is essential for the devel- mimic domain (structurally equivalent to the glycerol in phos-
opment of drug delivery systems (DDS),1,2) especially for the pholipids), a hydrophilic domain (structurally equivalent to the
active targeting of tumor cells.3,4) Peptides are generally used phosphate group in phospholipids), and a linker domain (to
to modify liposomes in order to recognize the target receptor prevent steric hindrance in the liposome).
on the cell membrane.5–9) It is easiest to modify the liposome
We intend to explore this technology for generating active
surface when the functional groups of a pre-formed lipo- targeted DDS for tumor cells. It has been reported1,13) that the
some are reacted with peptides in an aqueous environment.10) receptor for vasoactive intestinal peptide (VIP-R) is overex-
However, such methods have several limitations5,6): 1) steric pressed in some tumor cells. We believed that a peptide-lipid
hindrance of the liposome leads to low yield, 2) purification of analog conjugate (VIP-lipopeptide) could easily be synthesized
the liposomes is very difficult due to by-products and unreact- and modified on the liposome surface. Further, encapsulation
ed reagents, and 3) the amino acid sequence of the peptide can of an anti-tumor drug, doxorubicin (DOX), in the liposome
hinder reactivity. Although a promising approach to solving could prove effective as an active targeted anti-tumor DDS.
this problem is to produce a lipid-protein/peptide conjugate
The purification of this lipopeptide and its modification of
molecule by total synthesis and use it to make liposomes, the liposomes proved difficult due to the unexpected amphiphilic
modification of the phospholipid requires many protection and characteristics of the compound. These characteristics arise
deprotection steps of the phosphate group, resulting in poor from the combination of a lipophilic alkyl chain and the hy-
yields due to hydrolysis in aqueous media.7,8)
drophilic peptide. A new, more hydrophilic linker domain
We therefore designed11,12) a novel lipid analog mimicking was therefore designed and incorporated into the lipopeptide,
the structural features of phospholipids that is easily synthe- providing better orientation of the peptide on the aqueous
sized using a peptide synthesizer (Fig. 1). The lipid moiety surface of the lipid bilayer. The new linker domain contained
was composed of the following four domains: the anchor two hydroxyl groups (lipopeptide 1, Fig. 2). Incorporation of
domain (anchoring the molecule into the liposome), a glycero this linker into the lipopeptide using a peptide synthesizer re-
Fig. 1. General Structure of the Novel Lipid Analogue (Lipopeptide)
The authors declare no conflict of interest.
© 2013 The Pharmaceutical Society of Japan
*To whom correspondence should be addressed. e-mail: yokoyama@phar.toho-u.ac.jp

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Fig. 2. Structure of VIP-Lipopeptide (1) with the New Linker Domain
Table 1. Results of Alkylation
Run
Base (eq)
NaH (1.8)
Solvent
BrCH2COOBn (eq)
Yield of 4 (%)
1
2
3
4
THF
Toluene
DMF
2
—
—
11
34
NaOH (1.5)
K2CO3 (1.1)
Cs2CO3 (1.8)
1.5
1.5
1.6
DMF
quired a 9-fluorenylmethoxycarbonyl (Fmoc) protected amino bromoacetate and the hydroxyl anion of 3, resulting in side
acid with an acetonide-protected hydroxyl group (2). The syn- reactions that provided a complex product mixture. Indeed,
thesis of linker 2 and lipopeptide 1 using a solid phase peptide selective mono-alkylation of more acidic poly-phenolic com-
synthesizer is described below.
(−)-2,3-O-Isopropylidene-D-threitol (3) was selected as the the desired product in high yield under similar conditions.18,19)
starting material. Due to our experience developing unpro- Although the yield was not satisfactory, we carried out the
pounds with benzyl bromoacetate smoothly proceeded to give
tected synthesis strategies for highly polar compounds such as next step. Tosylation of 4 smoothly proceeded to give 5 (73%
free amino acids,14–17) we attempted the direct introduction of yield), which was reacted with azide anion to give the desired
benzyl acetate moiety into unprotected diol 3.
azide 6 in 84% yield (Chart 2).
The reaction of benzyl bromoacetate with diol 3 provided
Non-selective reduction of 6 to reduce the azide group and
a complex mixture in non-polar aprotic solvent [tetrahydro- remove the benzyl group by H2/Pd–C provided amino acid 7,
furan (THF), toluene] with strong base (NaH, NaOH) (Chart which was reacted with N-(9-fluorenylmethoxycarbonyloxy)-
1, Table 1, runs 1, 2). The desired product 4 was isolated in succinimide (Fmoc-OSu) without purification to give the final
low yield (11%) when a weaker base, K2CO3 in dimethylfor- product 2 in 77% yield (Chart 3).
mamide (DMF), was used, and no residual starting material
VIP-Lipopeptide (1) with new linker 2 was synthesized
was detected (run 3). By changing the base, reaction tempera- on TGS-RAM resin by Fmoc solid-phase synthesis using
ture, and molar ratio, the yield was improved to 34% using an automated peptide synthesizer (Chart 4). Fmoc-protected
Cs2CO3 as a base (run 4). The low yield could have been due amino acids were coupled sequentially to synthesize VIP
to the simultaneous formation of the carbanion of benzyl using
benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium
hexafluorophosphate (PyBOP) and N-hydroxybenzotriazole
(HOBt) as condensation reagents (8→9). Fmoc-tryptophan
(probe for fluorescence marker), Fmoc-Asp(OH)-OtBu and
Fmoc-Dap(Fmoc)-OH were coupled sequentially on the syn-
thesizer (9→10→11→12). Since the last condensation reac-
tion, with palmitic acid (anchor domain), did not proceed
satisfactorily due to its insolubility in DMF, palmitic acid was
Chart 1. Alkylation with Benzyl Bromoacetate
Chart 2. Conversion from Alcohol 4 to Azide 6
Chart 3. One-Pot Synthesis of Linker Component 2

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Vol. 61, No. 11
Chart 4. Solid Phase Synthesis of VIP-Lipopeptide (1) with the New Linker
Fig. 3. TOF-MS Spectrum of 1: (A) before Purification, (B) after Purification
Molecular weight was 4351.16 (C203H330N50O53S).
manually reacted with O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-
We synthesized Fmoc-protected amino acid 2 in 4 steps in
tetramethyluronium hexafluorophosphate (HATU) in the pres- 16% overall yield. Compound 2 can be used as a component
ence of HOBt and N-methylmorfoline (NMM) (12→13). After of the linker domain during the synthesis of VIP-lipopeptide
de-protection and cleavage from the resin, crude VIP-lipopep- (1) using a peptide synthesizer. The key aspects of this syn-
tide (1) was obtained as a yellow solid, which was purified by thetic route are selective alkylation of diol 3 by benzyl bro-
preparative HPLC to give pure 1 in 28% yield. The purity and moacetate, as shown in Chart 2, and one-pot reaction of 6
structure of 1 were confirmed by time-of-flight mass (TOF- (reduction and protection of Fmoc group) to provide the final
MS) spectra and analytical HPLC (Figs. 3, 4).
product 2, as shown in Chart 3. The synthesis and straight-

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Fig. 4. HPLC Profile of 1: (A) before Purification, (B) after Purification
HPLC column [Capcell pak Phenyl UG120 (Shiseido) 5µm, 4.6mmϕ×250mm]; detector, UV (280nm); flow rate, 1.0mL/min; gradient, H2O (0.1%TFA)–CH3CN (0.1%
TFA): 40:60.
Schraa A. J., Janssen A. P. C. A., Kok R. J., Molema G., Storm G.,
J. Control. Release, 91, 115–122 (2003).
6) Pastorino F., Brignole C., Marimpietri D., Cilli M., Gambini C.,
Ribatti D., Longhi R., Allen T. M., Corti A., Ponzoni M., Cancer
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7) Maeda N., Takeuchi Y., Takada M., Sadzuka Y., Namba Y., Oku N.,
forward purification of VIP-lipopeptide (1) was accomplished
using solid phase peptide synthesis and preparative HPLC.
Modification of liposomes with 1 and their evaluation as ac-
tive targeting DDS for cancer therapy will be published in the
near future.
J. Control. Release, 100, 41–52 (2004).
Experimental
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General chemistry methods, synthesis procedures, spectral
data, and bioassay methods are given in Supplemental Infor-
mation.
10) Gregoriadis G., Florence A., Patel H., “Liposome in Drug Deliv-
ery,” Harwood Academic Publishers, NJ, 1993, pp. 227–238.
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12) Yagi N., Yano Y., Hatanaka K., Yokoyama Y., Okuno H., Bioorg.
Med. Chem. Lett., 17, 2590–2593 (2007).
Acknowledgments This work was supported by a Grant-
in-Aid for Scientific Research [(C)-No. 13672237] from the
Ministry of Education, Culture, Sports, Science and Technol-
ogy of Japan. We are also grateful to Toho University for sup-
porting the Toho University Joint Research Fund.
13) Pan C. Q., Hamren S., Roczniak S., Tom I., DeRome M., Peptides,
29, 479–486 (2008).
14) Hikawa H., Yokoyama Y., Org. Biomol. Chem., 9, 4044–4050
(2011).
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