Versatile synthesis of head group functionalized phospholipids via oxime bond formation

Article abstract of DOI:10.1021/ol8019346

(Equation Presented) A method for introduction of various head groups on phospholipid frameworks via oxime bond formation has been developed for the synthesis of cyclen-Cu(II), pyrene, naphthalene, and other headgroup functionalized phospholipids that can cleave the membrane protein, hemagglutinin.

Full text of DOI:10.1021/ol8019346

ORGANIC
LETTERS
2008
Vol. 10, No. 21
4847-4850
Versatile Synthesis of Head Group
Functionalized Phospholipids via Oxime
Bond Formation†
Takumi Furuta,*,‡ Masayoshi Mochizuki,§ Mai Ito,§ Tadanobu Takahashi,§
Takashi Suzuki,§ and Toshiyuki Kan*,§
Institute for Chemical Research, Kyoto UniVersity, Uji, Kyoto 611-0011, Japan, and
School of Pharmaceutical Sciences, UniVersity of Shizuoka and Global COE Program,
52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan
furuta@fos.kuicr.kyoto-u.ac.jp; kant@u-shizuoka-ken.ac.jp
Received August 21, 2008
ABSTRACT
A method for introduction of various head groups on phospholipid frameworks via oxime bond formation has been developed for the synthesis
of cyclen-Cu(II), pyrene, naphthalene, and other headgroup functionalized phospholipids that can cleave the membrane protein, hemagglutinin.
Artificial phospholipids have been employed as valuable
molecular probes to investigate membrane related biosys-
tems.1 Furthermore, they have occupied a central position
on supramolecular chemistry as useful building blocks to
construct a nanoscale structure such as polymerizable lipo-
some.2 Among such artificial lipids, headgroup functionalized
derivatives have been paid attention to as key molecules for
membrane surface engineering,3 target specific MRI,4 siRNA
delivery,5 and protein crystallization.6 Natural product,7
peptide,8 as well as photoactivatable functionality9-phospho-
lipid conjugates at the headgroup have also been prepared.
Although utility of headgroup-functionalized phospholip-
ids has been recognized, a mild and efficient method to
incorporate a functionalized headgroup onto a phospholipid
framework has not yet been well exploited.
During the course of a previous investigation, we synthe-
sized a phospholipid possessing EDTA-Fe(III) chelating
headgroup and demonstrated its ability to cleave the influenza
† In memory of Professor Kiyoshi Tanaka, who passed away December
8, 2004.
‡ Kyoto University.
(5) Utku, Y.; Dehan, E.; Ouerfelli, O.; Piano, F.; Zuckermann, R. N.;
Pagano, M.; Kirshenbaum, K. Mol. BioSyst. 2006, 2, 312.
(6) (a) Drakopoulou, E.; Tsivgoulis, G. M.; Mukhopadhyay, A.; Brisson,
A. Tetrahedron Lett. 2000, 41, 4131. (b) Thompson, D. H.; Zhou, M.; Grey,
J.; Kim, H. Chem. Lett. 2007, 36, 956.
§ University of Shizuoka.
(1) For examples, see: (a) Prestwich, G. D. Acc. Chem. Res. 1996, 29,
503. (b) Regen, S. L. Curr. Opin. Chem. Biol. 2002, 6, 729.
(2) Ringsdorf, H.; Schlarb, B.; Venzmer, J. Angew. Chem., Int. Ed. 1988,
27, 113.
(7) Matsuoka, S.; Matsumori, N.; Murata, M. Org. Biomol. Chem. 2003,
1, 3882.
(3) (a) Grogan, M. J.; Kaizuka, Y.; Conrad, R. M.; Groves, J. T.;
Bertozzi, C. R. J. Am. Chem. Soc. 2005, 127, 14383. (b) Rabuka, D.;
Forstner, M. B.; Groves, J. T.; Bertozzi, C. R. J. Am. Chem. Soc. 2008,
130, 5947.
(8) Cavalli, S.; Handgraaf, J.-W.; Tellers, E. E.; Popescu, D. C.;
Overhand, M.; Kjaer, K.; Vaiser, V.; Sommerdijk, N. A. J. M.; Rapaport,
H.; Kros, A. J. Am. Chem. Soc. 2006, 128, 13959.
(4) Frias, J. C.; Williams, K. J.; Fisher, E. A.; Fayad, Z. A. J. Am. Chem.
Soc. 2004, 126, 16316.
(9) Wang, P.; Blank, D. H.; Spencer, T. A. J. Org. Chem. 2004, 69,
2693. See also, ref 1a.
10.1021/ol8019346 CCC: $40.75
Published on Web 09/30/2008
2008 American Chemical Society

virus membrane protein, hemagglutinin (HA).10 This work
suggested that phospholipids could be a valuable platform
for pursuing the affinity cleavage of integral membrane
proteins. However, due to the requirement of headgroup
introduction at an early stage of the synthesis, the synthetic
route was not suitable for further derivative preparation.
Chemical cleavage of proteins is a valuable technology
for investigating protein structures,11 protein-protein inter-
actions,12 and also for degrading specific proteins and
abrogating their functions.13 However, such chemical cleav-
age has not yet expanded into the field of integral membrane
protein research, although the methods to investigate the
roles, functions, and structures of integral membrane proteins
still require development.
Scheme 2. Preparation of the Oxyamino-Phospholipids
This background motivated us to develop a general and
convenient method to incorporate a functional group onto a
phospholipid framework for preparing a series of derivatives,
which would be beneficial for screening for more useful
protein cleavable molecules. Here, we report the versatile
synthesis of protein cleavable phospholipids 2, by efficient
introduction of protein cleavable head groups on 1 via oxime
bond formation (oxime ligation)14 at the latter stage of the
synthetic route via the pivotal oxyamino functional group
(Scheme 1). Additionally, we observed that these synthesized
alcohol 4,17 which had a protected oxyamino group,
proceeded smoothly in the presence of 1H-tetrazole, and
successive oxidation of the phosphite intermediate by
TBHP afforded the phosphotriesters 5a and 5b in 85 and
69% yields, respectively.
Removal of the phthalimide group of 5a, by treatment with
methyl hydrazine, yielded the desired O-benzyl-protected
phosphodiester derivative 1a in 91% yield. The same
deprotection reaction afforded the O-cyanoethyl protected
1b without any elimination of the cyanoethyl-protecting
group (Scheme 2). Both of these protected phospholipids 1a
and 1b worked well as key intermediates, in which the benzyl
and cyanoethyl groups could be removed under catalytic
hydrogenation and basic conditions, respectively, after
introduction of the headgroup, via the formation of an oxime
bond as shown in Table 1.
Scheme 1
.
Strategy for Synthesizing Artificial Phospholipids
with Protein Cleavable Head Groups
At first, we tested the introduction of imidazole groups,
which have been used as catalytic centers for peptide bond
hydrolysis18 (Table 1, entries 1 and 2). Oxime bond
formation between 1a and 2-imidazole carboxyaldehyde (6a)
proceeded smoothly in the mixed solvent system of
CHCl3-MeOH to give 7a in 77% yield under neutral
conditions without addition of any additive and/or catalyst
(Table 1, entry 1). The isomeric 4-imidazole moiety was also
incorporated into the cyanoethyl protected 1b in excellent
yield (Table 1, entry 2). The pyrene moiety, which has been
reported to be a protein photocleavable group,19 was also
phospholipids had protein cleavage activities within proteo-
liposomes.
First, phospholipid derivatives containing the oxyamino
headgroup were prepared (Scheme 2). The amidite cou-
pling between the phosphoroamidite 3a15 or 3b16 and the
(10) Furuta, T.; Sakai, M.; Hayashi, H.; Asakawa, T.; Kataoka, F.; Fujii,
S.; Suzuki, T.; Suzuki, Y.; Tanaka, K.; Fishkin, N.; Nakanishi, K. Chem.
Commun. 2005, 4575.
(11) For example, see: Ermacora, M. R.; Ledman, D. W.; Fox, R. O.
Nat. Struct. Biol. 1996, 3, 59.
(12) Datwyler, S. A.; Meares, C. F. Trends Biochem. Sci. 2000, 25, 408.
(13) (a) Jeon, J. W.; Son, S. J.; Yoo, C. E.; Hong, I. S.; Song, J. B.;
Suh, J. Org. Lett. 2002, 4, 4155. (b) Chae, P. S.; Kim, M.; Jeung, C.-S.;
Lee, S. D.; Park, H.; Lee, S.; Suh, J. J. Am. Chem. Soc. 2005, 127, 2396.
(c) Suzuki, A.; Tsumura, K.; Tsuzuki, T.; Matsumura, S.; Toshima, K. Chem.
Commun. 2007, 4260. (d) Suh, J.; Yoo, S. H.; Kim, M. G.; Jeong, K.; Ahn,
J. Y.; Kim, M.; Chae, P. S.; Lee, T. Y.; Lee, J.; Lee, J.; Jang, Y. A.; Ko,
E. H. Angew. Chem., Int. Ed. 2007, 46, 7064.
(16) Xu, Y.; Lee, S. A.; Kutateladze, T. G.; Sbrissa, D.; Shisheva, A.;
Prestwich, G. D. J. Am. Chem. Soc. 2006, 128, 885.
(17) Wey, S.-J.; Augustyniak, M. E.; Cochran, E. D.; Ellis, J. L.; Fang,
X.; Garvey, D. S.; Janero, D. R.; Letts, L. G.; Martino, A. M.; Melim,
T. L.; Murty, M. G.; Richardson, S. K.; Schroeder, J. D.; Selig, W. M.;
Trocha, A. M.; Wexler, R. S.; Young, D. V.; Zemtseva, I. S.; Zifcak, B. M.
J. Med. Chem. 2007, 50, 6367.
(18) Suh, J.; Oh, S. J. Org. Chem. 2000, 65, 7534.
(14) The oxime bond formation (oxime ligation) has been used to
conjugate functional groups, such as fluorescent label, to biomolecules. See
the following reference and references cited therein: Dirksen, A.; Hackeng,
T. M.; Dawson, P. E. Angew. Chem., Int. Ed. 2006, 45, 7581.
(15) Fukase, K.; Yoshimura, T.; Kotani, S.; Kusumoto, S. Bull. Chem.
Soc. Jpn. 1994, 67, 473.
(19) (a) Kumar, C. V.; Buranaprapuk, A. Angew. Chem., Int. Ed. 1997,
36, 2085. (b) Kumar, C. V.; Buranaprapuk, A.; Opiteck, G. J.; Moyer, M. B.;
Jockusch, S.; Turro, N. J. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 10361.
(c) Kumar, C. V.; Buranaprapuk, A. J. Am. Chem. Soc. 1999, 121, 4262.
(d) Buranaprapuk, A.; Kumar, C. V.; Jockusch, S.; Turro, N. J. Tetrahedron
2000, 56, 7019.
4848
Org. Lett., Vol. 10, No. 21, 2008

able side reactions including reductive cleavage of the oxime
bond. This headgroup introduction-deprotection sequence
was readily applied to other artificial phospholipids such as
the photoactivatable benzophenone derivative 2g, which
could be employed for photoaffinity labeling studies,9 and
the aryl bromide derivative 2h, which could be used for
further chemical modifications (Table 1, entries 7 and 8).
Next, the phenanthroline and cyclen modified phospho-
lipids 2e and 2f were converted to their Cu-chelated forms22
(Scheme 3). Treatment of 2e with CuCl2 in the presence of
Table 1. Introduction of Functionalized Head Groups and
Subsequent Deprotections
Scheme 3. Preparation of Cu(II) Chelated Phospholipids
i-Pr2NEt gave the desired Cu-chelated phenanthroline deriva-
tive 8e. Removal of the Boc groups of the cyclen derivative
2f with TFA treatment and successive Cu(II) chelation in
the presence of i-Pr2NEt gave the desired Cu-chelated cyclen
phospholipid 8f (Scheme 3).
After producing these artificial phospholipids, we turned
our attention to evaluating the ability of 2a-2d, 8e, and 8f
to cleave hemagglutinin (HA).23
These prepared phospholipids were relatively hydrophobic,
so simple incubation of these phospholipids with influenza
virus (A/WSN/33, H1N1) in MOPS buffer (pH 7.0) was
ineffective for cleaving HA due to their insolubility. To
overcome this difficulty, we reconstituted a membrane with
the artificial phospholipids to place the phospholipids in close
proximity to HA. The artificial phospholipids and HA were
dissolved in a 5% octylglucoside solution, which was
dialyzed against an external buffer solution to afford
precipitates which included HA24 when the phospholipids
2a, 2c, 2d, and 8f were used. A TEM image of the precipitate
with 8f clearly shows a liposome structure (50-150 nm in
diameter), in which all of the surface is coated with
cyclen-Cu(II) moieties (Figure 1a).
a Obtained as an E/Z mixture (E:Z ) 2:1) of the oxime bond. b Obtained
as a t-butylamine salt.
introduced (Table 1, entry 3). Besides 2-hydroxy-1-naph-
thaldehyde (6d), which is both a photocleavable protein and
a metal-chelating group (Table 1, entry 4), the nitrogen-
containing metal-chelating groups such as 1,10-phenanthro-
line (6e)20 and the cyclen derivative 6f21 were also easily
introduced, giving 7e and 7f, respectively, in excellent yields
(Table 1, entries 5 and 6).
Subsequent deprotection of both the benzyl and cyanoethyl
groups of these derivatives by catalytic hydrogenation and
basic conditions, respectively, proceeded smoothly to yield
the corresponding phospholipids 2a-2f without any observ-
(22) For protein cleavage by the 1,10-phenanthroline-Cu(II) complex,
see: (a) Wu, J.; Perrin, D. M.; Sigman, D. S.; Kaback, H. R. Proc. Natl.
Acad. Sci. U.S.A. 1995, 92, 9186. (b) Galasco, A.; Crouch, R. K.; Knapp,
D. R. Biochemistry 2000, 39, 4907. For protein cleavage by the cyclen-Cu(II)
complex, see the following references and references cited therein: Jang,
S. W.; Suh, J. Org. Lett. 2008, 10, 481.
(23) Wilson, I. A.; Skehel, J. J.; Wiley, D. C. Nature 1981, 289, 366.
(24) Determined by running SDS-PAGE on the precipitates from 2a,
2c, 2d, and 8f and visualizing bands for HA1 and HA2, both of which
correspond to HA. We had difficulty applying the phenanthroline phos-
pholipids 8e to membrane reconstitution due to its insolubility in the 5%
octylglucoside solution. See the Supporting Information.
(20) Zaid, A.; Sun, J.-S.; Nguyen, C.-H.; Bisagni, E.; Garestier, T.;
Grierson, D. S.; Zain, R. ChemBioChem 2004, 5, 1550.
(21) For details of how the cyclen 6f was prepared, see the Supporting
Information.
Org. Lett., Vol. 10, No. 21, 2008
4849

that HA was being cleaved.26,27 It is noteworthy that no
cleavage of HA was observed when the cyclen-Cu(II)
complex was added to the proteoliposomes made with
phosphatidylcholine under the same reaction conditions
(Figure 1b, lane 2).
Although the imidazole-tethered phospholipid 2a did not
cleave HA, the pyrene and 2-hydroxynaphthalene modified
phospholipids 2c and 2d did exhibit cleavage activities under
photoirradiation conditions with UV light at 365 nm and
degraded HA within 5 and 30 min, respectively.28,29 The
efficient cleavage by 8f, 2c, and 2d clearly indicates that
these proteoliposomes provide excellent reaction environ-
ments for integral membrane protein cleavage, in which the
active site of the cleavage reactions is in close proximity to
the proteins. It also indicates that the phospholipid structure
is a promising platform for the development of further
protein-specific scissor molecules.
In conclusion, we have developed a mild and convenient
way to access a variety of headgroup functionalized phos-
pholipids. We are currently modifying the phospholipid
structure further toward site-selective as well as molecule-
specific cleavage of integral membrane proteins.
Figure 1. (a) TEM image of a proteoliposome with 8f. (b) SDS-
PAGE of the cleavage reactions with 8f (coomassie-blue staining):
gel shows proteoliposomes construced with phosphatidylcholine
(lane 1, control) or with phosphatidylcholine incubated with
Cu(II)-cyclen for 4 h (lane 2) or the reaction mixture immediately
after initiation, and 1 h, 2 h, and 4 h later (lanes 3-6, respectively)
with the proteoliposome 8f.
The HA cleavage reactions were performed with these
proteoliposomes. In the case of the proteoliposome 8f, the
hydrolytic cleavage of HA was initiated by changing the pH
of the buffer from 7.0 (MOPS buffer) to 9.0 (boric acid
buffer). The reaction was monitored by SDS-PAGE over 4 h
(Figure 1b). SDS-PAGE of the liposomes immediately after
cleavage initiation shows the HA1 and HA2 bands, both of
which correspond to HA (Figure 1b, lane 3).25 After a 2 h
incubation at 50 °C, a decrease in the HA bands (HA1 and
HA2) was observed (Figure 1b, lane 5). Moreover, after a
4 h incubation, they had almost completely disappeared
(Figure 1b, lane 6). These band decreases clearly indicated
Acknowledgment. We thank Prof. Koji Nakanishi and
Drs. Nathan Fishkin and Craig Parish (Columbia University)
for their valuable discussions. Financial support was provided
by JSPS and a Grant-in Aid for Young Scientists (B)
17710187.
Supporting Information Available: Detailed experimen-
tal procedures and spectroscopic data (PDF). This material
is available free of charge via the Internet at http://pubs.acs.org.
OL8019346
(25) Selimova, L. M.; Zaides, V. M.; Zhdanov, V. M. J. Virol. 1982,
44, 450.
(27) The oxidative conditions were extremely powerful for HA cleavage.
HA was almost completely cleaved within 3 min at 0 °C after addition of
H2O2 and sodium ascorbate. See Supporting Information.
(28) For the cleavage of HA with 2c and 2d, see Supporting
Information.
(29) Pyrene and 2-hydroxynaphthalene themselves did not show cleavage
activity. See Supporting Information.
(26) Staining with biotin-ConA also decreased the HA1 band. Cleaved
HA fragments were visualized as smears by immunostaining with anti-P50
(H1N2) rabbit polyclonal antibody. See Supporting Information. For anti-
P50, see: Suzuki, T.; Takahashi, T.; Guo, C.-T.; Hidari, K. I.-P. J.;
Miyamoto, D.; Goto, H.; Kawaoka, Y.; Suzuki, Y. J. Virol. 2005, 79, 11705.
4850
Org. Lett., Vol. 10, No. 21, 2008