Design and synthesis of artificial phospholipid for selective cleavage of integral membrane protein

Article abstract of DOI:10.1039/b507917a

An artificial phospholipid, possessing saturated alkyl chains as a membrane anchor and protein recognition site as well as an Fe(III)-EDTA moiety as a protein cleavable polar head group, was designed and synthesized based on the amidite method for the pur

Full text of DOI:10.1039/b507917a

View Article Online / Journal Homepage / Table of Contents for this issue
COMMUNICATION
www.rsc.org/chemcomm | ChemComm
Design and synthesis of artificial phospholipid for selective cleavage of
integral membrane protein{
Takumi Furuta,*a Minatsu Sakai,a Hiroyasu Hayashi,a Tomohiro Asakawa,a Fumi Kataoka,b Satoshi Fujii,b
Takashi Suzuki,c Yasuo Suzuki,c Kiyoshi Tanaka,{a Nathan Fishkind and Koji Nakanishi*d
Received (in Cambridge, UK) 6th June 2005, Accepted 29th July 2005
First published as an Advance Article on the web 17th August 2005
DOI: 10.1039/b507917a
It has been known that integral membrane proteins are
embedded in lipid bilayer membrane, where their lipophilic
a-helical transmembrane domains interact with the hydrocarbon
chains of a variety of phospholipids such as phosphatidylcholine
and phosphatidylethanolamine.9 This affinity between lipids and
integral membrane proteins led us to design ‘‘scissor’’ phospholipid
(R,S)-1, bearing saturated alkyl chains as a membrane anchor
and recognition site for the transmembrane domain, and an
Fe(III)–EDTA complex as a protein cleavable polar head group of
phospholipid (Fig. 1).
An artificial phospholipid, possessing saturated alkyl chains as
a membrane anchor and protein recognition site as well as an
Fe(III)–EDTA moiety as a protein cleavable polar head group,
was designed and synthesized based on the amidite method for
the purpose of examination of cleavage of integral membrane
proteins.
Integral membrane proteins, such as G-protein coupled receptors,
ion channels and transporters, play important roles in a variety of
fundamental biological processes and have attracted much
attention as potential targets for drug discovery. Therefore,
detailed structural and functional investigations of integral
membrane proteins have been much required.
The synthetic pathway for (R,S)-1, using a coupling reaction
between a phenolic head group and an amidite derivative of a tail
group as a key step, is depicted in Scheme 1.
Reduction of the amide group of O-benzyl-L-tyrosinamide (S)-2
with BH3?THF followed by alkylation with tert-butyl bromoace-
tate afforded the benzyl protected EDTA derivative (S)-3 in 44%
yield for two steps. Removal of the benzyl protecting group of
(S)-3 under catalytic hydrogenation conditions gave desired
phenolic derivative (S)-4. Coupling of (S)-4 with phosphoroami-
dite 510 in the presence of 1H-tetrazole afforded phosphite (R,S)-6
in 64% yield. The characteristic phosphorus resonance in (R,S)-6
was observed at 134 ppm by 31P NMR analysis. Oxidation with
MCPBA afforded phosphate (R,S)-7 in 84% yield.11 Deprotection
of the benzyl group under catalytic hydrogenation conditions gave
(R,S)-8. The EDTA modified phospholipid (R,S)-9 was obtained
quantitatively as a colorless amorphous solid after the removal of
tert-butyl groups by treatment with TFA. Finally, the CHCl3–
MeOH (2 : 1) solution of (R,S)-9 was treated with FeCl3 in the
The non-enzymatic chemical cleavage of proteins, which has
been accomplished by the use of transition metal chelates such as
Fe–EDTA,1 Cu–phenanthroline2 and Cu–cyclen3 complexes, is
recognized as a powerful investigative tool both for determination
of the three-dimensional structure of proteins and mapping
protein–nucleic acid or protein–protein interactions. However,
such cleavage methods have been mainly applied to the water-
soluble proteins and the examples of cleavage reactions of integral
membrane proteins are quite limited.4
While metal-chelating lipids have attracted attention as a key
element for sensing of metal ions,5 protein immobilization6 and
magnetic resonance imaging (MRI),7 the application of such
artificial lipids for cleavage of integral membrane proteins has not
been demonstrated so far.8
Herein, we report the synthesis of Fe(III)-chelating phospholipid
(R,S)-1 as a ‘‘scissor’’ molecule for integral membrane proteins and
preliminary investigation of its cleavage activity toward influenza
virus membrane protein, hemagglutinin.
i
presence of Pr2NEt to give desired phospholipid (R,S)-1. The
Fe(III) chelated structure of (R,S)-1 was confirmed by FAB-MS
spectra, which exhibited a peak at m/z 1083, corresponding to the
molecular ion [(M 2 2) + Fe]+ (Scheme 1).
Next, we turned our attention to the evaluation of the cleavage
activity of (R,S)-1 toward an integral membrane protein,
hemagglutinin (HA).12 The influenza virus (A/WSN/33, H1N1)
in MOPS buffer (pH 7.0) was incubated with (R,S)-1 (0.58 mM
final conc.) at 30 uC for 1 h to incorporate (R,S)-1 into the viral
membrane. After removal of excess (R,S)-1 from incubated
aDepartment of Synthetic Organic & Medicinal Chemistry, School of
Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka,
422-8526, Japan. E-mail: furuta@u-shizuoka-ken.ac.jp;
Fax: +81-54-264-5745; Tel: +81-54-264-5742
bDepartment of Physical Biochemistry, School of Pharmaceutical
Sciences, University of Shizuoka, 52-1 Yada, Shizuoka, 422-8526, Japan
cDepartment of Biochemistry, School of Pharmaceutical Sciences,
University of Shizuoka, CREST, JST, COE Program in the 21st
Century, 52-1 Yada, Shizuoka, 422-8526, Japan
dDepartment of Chemistry, Columbia University, New York, NY 10027,
USA. E-mail: kn5@columbia.edu; Fax: +1-212-932-8273;
Tel: +1-212-854-2169
{ Electronic supplementary information (ESI) available: experimental
procedures for chemical synthesis of (R,S)-1 and cleavage reactions. See
http://dx.doi.org/10.1039/b507917a
{ Deceased December 8, 2004. This paper is dedicated to the memory of
the late Professor Kiyoshi Tanaka.
Fig. 1 Structure of (R,S)-1.
This journal is ß The Royal Society of Chemistry 2005
Chem. Commun., 2005, 4575–4577 | 4575

View Article Online
(NP, 56 kDa), which exist inside of the virus virion (Fig. 2a), were
completely protected from the cleavage reaction even after 2 h
incubation (lanes 4 and 5), suggesting a selective cleavage reaction
at the outer leaflet of the virion without disruption of the virus
bilayer membrane. In contrast, no cleavage of HA was observed
by the simple Fe(III)–EDTA complex, which has been used as a
diffusible protein cleavable reagent,15 over the period of 2 h under
the same reaction conditions for (R,S)-1 (lanes 7 and 8).
For further examination of the cleavage activity with (R,S)-1
and Fe(III)–EDTA complex employing the same reaction condi-
tions, cleavage of bovine fetuin,16 a water-soluble glycoprotein
(50 kDa), has been carried out (Fig. 3). In both cases, decrease of
the density of bands corresponding to fetuin has been observed
over the period of 1 h. These results suggested cleavage potential of
both (R,S)-1 and Fe(III)–EDTA toward water-soluble proteins.
Taking the results from these experiments (Figs. 2 and 3) into
account, the lipophilic hydrocarbon chains of (R,S)-1 are essential
structural elements for cleavage of HA, which resists cleavage
with the simple Fe(III)–EDTA complex.17 It can therefore be
presumed that (R,S)-1 is incorporated into the membrane so as to
interact with HA via its hydrocarbon chains, then the selective
cleavage reactions of the Fe(III)–EDTA moiety can occur in
proximity to HA.
In conclusion, the synthesis and the cleavage activity of the
designed ‘scissor’ phospholipid (R,S)-1 toward viral integral
membrane protein, HA, have been demonstrated. The synthetic
strategy described here might be readily applicable to preparation
of artificial phospholipids possessing other protein cleavable
head groups such as Cu–phenanthroline2 and Cu–cyclen3 systems
instead of Fe(III)–EDTA.
Scheme 1 Reagents and conditions: (a) BH3?THF, reflux, 7 h; (b) t-butyl
bromoacetate, Proton sponge, NaI, CH3CN, 44% (2 steps); (c) 10% Pd–C,
H2, MeOH, rt, 2 h, 78%; (d) 1H-tetrazole, CH2Cl2, rt, 2 h, 64%; (e)
MCPBA, CH2Cl2, 0 uC, 1 h, 84%; (f) 10% Pd–C, H2, MeOH, rt, 4.5 h,
83%; (g) TFA, rt, 10 h, quant.; (h) FeCl3, iPr2NEt, CHCl3–MeOH (2 : 1).
The extramembrane domains of integral membrane proteins are
functionally important for ligand–protein as well as protein–
protein interactions in many biological processes.18 Since the active
site for cleavage reaction (Fe(III)–EDTA moiety) of (R,S)-1 could
be fixed just on the membrane surface, the separation of extra-
membrane domains from transmembrane domains by cleavage
and successive analysis of cleaved peptide fragments might be
feasible. Improvement of site-selectivity of cleavage reactions using
a variety of conditions, including cleavage on the surface of
proteoliposomes reconstituted with (R,S)-1 and HA, is currently
under investigation in our laboratory.
mixture by repeated centrifugation and washing, the cleavage
reaction was initiated by the sequential addition of sodium
ascorbate (4.0 mM final conc.) and H2O2 (4.0 mM final conc.).
The reaction mixture was incubated at 30 uC and the cleavage of
HA was monitored by SDS-PAGE with Coomassie-blue staining
(Fig. 2b). As can be seen from the figure, disappearance of the
bands corresponding to HA1 and HA2, derived from disulfide
bond cleavage of HA by 2-mercaptoethanol,13 was observed after
1 h (lane 4). In spite of no detection of peptide fragments cleaved
on SDS-PAGE, this result indicated scission of HA.14 It is note-
worthy that both matrix protein (M, 28 kDa) and nucleoprotein
We thank Dr Yasuhiro Itagaki (Department of Chemistry,
Columbia University) for helpful discussions. We also thank the
Fig. 2 (a) Schematic view of influenza virus; (b) SDS-PAGE of cleavage
reaction mixtures: Lanes: 1, influenza virus (control); 2, influenza virus
incubated with (R,S)-1; 3, reaction mixture after 30 s with (R,S)-1; 4,
reaction mixture after 1 h with (R,S)-1; 5, reaction mixture after 2 h with
(R,S)-1; 6, reaction mixture after 30 s with Fe(III)–EDTA; 7, reaction
mixture after 1 h with Fe(III)–EDTA; 8, reaction mixture after 2 h with
Fe(III)–EDTA.
Fig. 3 Cleavage of bovine fetuin with (R,S)-1 and Fe(III)–EDTA. Lanes:
1 and 9, fetuin (control); 2 and 10, reaction mixture after 30 s; 3 and 11,
reaction mixture after 10 min; 4 and 12, reaction mixture after 20 min;
5 and 13, reaction mixture after 30 min; 6 and 14, reaction mixture after
40 min; 7 and 15, reaction mixture after 50 min; 8 and 16, reaction mixture
after 1 h.
4576 | Chem. Commun., 2005, 4575–4577
This journal is ß The Royal Society of Chemistry 2005

View Article Online
10 Phosphoroamidite 5 was prepared according to the reported procedure.
See, K. Fukase, T. Yoshimura, S. Kotani and S. Kusumoto, Bull. Chem.
Soc. Jpn., 1994, 67, 473.
Japan Health Sciences Foundation for financial support (Research
on Health Sciences focusing on Drug Innovation KH12072).
11 The observation of a phosphorus resonance at 25.65 ppm suggested the
oxidation occurred selectively at the phosphorus atom.
Notes and references
12 Because of the abundance (ca. 20% of total viral proteins) and ease of
detection by SDS-PAGE gel electrophoresis without any troublesome
purification, HA has been selected as a protein substrate for this
experiment. For the X-ray structure of HA, see, I. A. Wilson, J. J. Skehel
and D. C. Wiley, Nature, 1981, 289, 366.
1 S. A. Datwyler and C. F. Meares, Trends Biochem. Sci., 2000, 25, 408;
A. Schepartz and B. Cuenoud, J. Am. Chem. Soc., 1990, 112, 3247;
D. Hoyer, H. Cho and P. G. Schultz, J. Am. Chem. Soc., 1990, 112,
3249; M. R. Ermacora, D. W. Ledman and R. O. Fox, Nat. Struct.
Biol., 1996, 3, 59.
2 J. Gallagher, O. Zelenko, A. D. Walts and D. S. Sigman, Biochemistry,
1998, 37, 2096.
3 J. Suh, Acc. Chem. Res., 2003, 36, 562.
4 Only intramolecular cleavage reactions of integral membrane proteins,
using Fe(III)–EDTA and Cu–phenanthroline complexes conjugated
to cysteine thiols, have been reported. See, J. Wu, D. M. Perrin,
D. S. Sigman and H. R. Kaback, Proc. Natl. Acad. Sci. USA, 1995, 92,
9186; A. Gelasco, R. K. Crouch and D. R. Knapp, Biochemistry, 2000,
39, 4907; J. B. Ghaim, D. P. Greiner, C. F. Meares and R. B. Gennis,
Biochemistry, 1995, 34, 11311.
5 B. C. Roy, B. Chandra, D. Hromas and S. Mallik, Org. Lett., 2003, 5, 11.
6 E. Drakopoulou, G. M. Tsivgoulis, A. Mukhopadhyay and A. Brisson,
Tetrahedron Lett., 2000, 41, 4131.
13 L. M. Selimova, V. M. Zaides and V. M. Zhdanov, J. Virol., 1982, 44,
450.
14 Although LC/MS analysis of the cleavage reaction mixture was
performed, no peaks corresponding to cleaved peptide fragments were
detected on HPLC analysis. This suggests that the cleavage reaction
occurred non site-specifically and HA was cleaved into peptide
fragments that are too small to allow for SDS-PAGE as well as
LC/MS analysis. This non-specific cleavage is presumably due to
simultaneous occurrence of the following different mechanisms:
hydrolytic cleavage by Fe(II)–hydroperoxo complex and oxidative
cleavage by hydroxyl radical, in which the active species were generated
by reduction of Fe(III) with sodium ascorbate and successive reaction of
reduced Fe(II) with H2O2. Both these mechanisms of amide bond
cleavage have been reported. See, T. M. Rana and C. F. Meares, Proc.
Natl. Acad. Sci. USA, 1991, 88, 10578; I. E. Platis, M. R. Ermacora and
R. O. Fox, Biochemistry, 1993, 32, 12761.
15 For example, see, D. P. Greiner, K. A. Hughes, A. H. Gunasekera and
C. F. Meares, Proc. Natl. Acad. Sci. USA, 1996, 93, 71.
16 E. D. Green, G. Adelt, J. U. Baenziger, S. Wilson and H. Van Halbeek,
J. Biol. Chem., 1988, 263, 18253.
17 It might be assumed that both the sugar side chains modifying the
extramembrane domain of HA and the viral membrane protect HA
from the cleavage reaction by inhibition of the approach of Fe(III)–
EDTA complex.
18 For example, see, N. Kunishima, Y. Shimada, Y. Tsuji, T. Sato,
M. Yamamoto, T. Kumasaka, S. Nakanishi, H. Jingami and
K. Morikawa, Nature, 2000, 407, 971.
7 R. W. Storrs, F. D. Tropper, H. Y. Li, C. K. Song, J. K. Kuniyoshi,
D. A. Sipkins, K. C. P. Li and M. D. Bednarski, J. Am. Chem. Soc.,
1995, 117, 7301.
8 Artificial phospholipids possessing a photoactivatable group and a
disulfide moiety have been prepared for the investigation of protein–
lipid and lipid–lipid interactions, respectively. For example, see,
G. D. Prestwich, Acc. Chem. Res., 1996, 29, 503; Y. Ogawa,
W. Hahn, P. Garnier, N. Higashi, D. Massotte, M.-H. Metz-
Boutigue, B. Rousseau, M. Kodaka, J. Sunamoto, G. Ourisson and
Y. Nakatani, Chem. Eur. J., 2002, 8, 1843; M. Sugahara, M. Uragami
and S. L. Regen, J. Am. Chem. Soc., 2003, 125, 13040.
9 For example, see, M. Beck, F. Siebert and T. P. Sakmar, FEBS Lett.,
1998, 436, 304.
This journal is ß The Royal Society of Chemistry 2005
Chem. Commun., 2005, 4575–4577 | 4577