column using MeOH–CH2Cl2 (1 : 1) to provide compound 24
(12 mg, 85%). dH (300 MHz, CD3OD–CDCl3): 5.10 (1 H, m),
4.17 − 3.83 (5 H, m), 3.72–3.63 (3 H, m), 3.58–3.51 (3 H, m),
3.22 (1 H, t, J 9.7), 3.10 (1 H, m), 2.90 (2 H, m), 2.82 (1 H, dd),
2.25–2.27 (4 H, m), 1.70 − 1.20 (45 H, m), 0.87 (3H, t); MS
(negative-ion ESMS, M − H−) calcd for C33H63O13NP 712.4037,
found 712.4123. The compound 24 (12 mg, 0.0163 mmol) was
dissolved in anhydrous DMF (2 mL) containing triethylamine
(0.05 mL). This was treated with N-hydroxysuccinimidyl-
6-[7-nitrobenz-2-oxa-1,3-diazolobenz-4-yl)amino]-hexanoate
(NBD-amino-caproic NHS ester, 10 mg, 25.6 lmol) and the
reaction was stirred at rt for 4 h, when negative-ion ESMS
showed completion of the reaction. The solvents were removed
under a reduced pressure and residual DMF was removed by
repetitive evaporation with toluene. The residue was washed
with hexane–ethylacetate (1 : 1) until the washed solvent
showed no fluorescent active (NBD-aminocaproic acid) or
UV-active (N-hydroxysuccinimide) byproducts. The fluorescent
product was purified by preparative TLC (n-butanol–ethanol–
ammonia–water; 4 : 4 : 1 : 1), which provided the desired
NBD-labeled phosphatidylinositol compound 1 (7.7 mg, 55%).
The purity of the compound was further established by HPLC
on a RP-18 column using CH3CN–H2O gradient system and
photodiode array detector. TLC (n-BuOH–EtOH–NH3–H2O,
4 : 4 : 1 : 1): Rf = 0.45. dH (300 MHz, CDCl3): 8.42 (1 H, d,
J 8.4, NBD), 6.21 (1 H, d, J 8.4, NBD), 5.28 (1 H, m), 4,42 −
3.80 (8 H, m), 3.48 (2 H, m), 3.36 (2 H, m), 2.74 (2 H, t,
J 6.8), 2.30–2.37 (6 H, m), 1.40 − 1.20 (42 H, m, aliphatic),
0.87 (3 H, t, CH3); dP (100 MHz, CD3OD–CDCl3): 0.90 ppm;
MS (negative-ion ESMS, M − H−) calcd for C45H75O17N5P
988.4896, found 988.4875.
1D-myo-Inositol 1-O-myristoyl-2-O-[6-[(7-nitro-2-oxa-1,3-di-
azolobenz-4-yl)-amino-hexano yl]-sn-glycer-3-yl]-phosphate (5).
This fluorescent PI was prepared by same method as used for the
synthesis of 1, from D-inositol intermediate 11 and natural lipid
H-phosphonate donor 27. The fluorescent product was purified
by preparative TLC (n-butanol–ethanol–ammonia–water; 4 :
4 : 1 : 1, by volume), which provided the desired NBD-labeled
phosphatidylinositol 5. The purity of the compound was further
established by HPLC on a RP-18 column using CH3CN–H2O
gradient system and photodiode array detector: Rf = 0.46. dH
(300 MHz, CDCl3): 8.44 (1 H, d, J 8.4, NBD), 6.20 (1 H, d,
J 8.4, NBD), 5.28 (1 H, m), 4.42 − 4.08 (6 H, m), 3.99 (1 H, m),
3.66 (1 H, m), 3.49 (2 H, m), 3.37 (2 H, m), 2.74 (2 H, t, J 6.7),
2.30–2.40 (6 H, m), 1.40–1.25 (42 H, m, aliphatic), 0.86 (3 H, t,
CH3); dP (100 MHz, CD3OD–CDCl3): 0.94 ppm; MS (negative-
ion ESMS, M − H−) calcd for C35H56O16N4P 819.3429, found
819.3455.
1D-myo-Inositol 3-O-myristoyl-2-O-[6-[(7-nitro-2-oxa-1,3-di-
azolobenz-4-yl)amino-hexano yl]-sn-glycer-1-yl]-phosphate (6).
This fluorescent PI was prepared by same method as used for the
synthesis of 1, from D-inositol intermediate 11 and unnatural
lipid H-phosphonate donor 30. The fluorescent product was
purified by preparative TLC (n-butanol–ethanol–ammonia–
water; 4 : 4 : 1 : 1), which provided the desired NBD-labeled
PI 6. The purity of the compound was established by HPLC
on a RP-18 column using CH3CN–H2O gradient system and
photodiode array detector: Rf = 0.46. dH (300 MHz, CDCl3):
8.42 (1 H, d, J = 8.4 Hz, NBD), 6.23 (1 H, d, J 8.4, NBD), 5.29
(1 H, m), 4,41 − 4.00 (7 H, m), 3.66 (2 H, m), 3.36 (2 H, m),
2.75 (2 H, m), 2.30–2.38 (6 H, m, CH2CO), 1.40 − 1.22 (42 H,
m, aliphatic), 0.86 (3 H, t, CH3); dP (100 MHz, CD3OD–
CDCl3): 0.90 ppm; MS (negative-ion ESMS, M − H−) calcd
for C35H56O16N4P 819.3429, found 819.3410.
1D-myo-Inositol 3-O-[1-[6ꢀ[[6-[(7-nitro-2-oxa-1,3-diazolobenz-
4-yl)amino]hexanoyl] amino]-hexanoyl]-2-O-stearoyl-sn-glycer-
1-yl]-phosphate (2). This fluorescent PI was prepared by the
same reaction sequence as used for the synthesis of 1, from D-
inositol intermediate 11 and unnatural lipid H-phoshphonate
donor 22. The spectral data of compound 2: dH (300 MHz,
CDCl3): 8.43 (1 H, d, J 8.4, NBD), 6.22 (1 H, d, J 8.4, NBD),
5.30 (1 H, m), 4,43 − 4.40 (7 H, m), 3.67 (1 H, m), 3.45 (2 H, m),
3.38 (2 H, m), 2.76 (2 H, t, J 7), 2.30–2.38 (6 H, m), 1.40 − 1.20
(42 H, m, aliphatic), 0.87 (3 H, t, CH3); dP (100 MHz, CD3OD–
CDCl3): 0.96 ppm; MS (negative-ion ESMS, M − H−) calcd for
C45H75O17N5P 988.4896, found 988.4879.
Assays of flippase activity
Reconstitution of liposomes and proteoliposomes. Proteoli-
posomes were reconstituted as described previously9–11,25,30 from
1 ml mixtures of TE, ePC (4.5 lmol) and ∼0.3 mol% of fluo-
rescent acyl-NBD–phospholipids solubilized in 10 mM Hepes–
NaOH, pH 7.5, 100 mM NaCl and 1% (w/v) Triton X-100. The
amount of TE used was varied as required to prepare prote-
oliposomes with different protein–phospholipid ratios (PPR’s).
Liposomes were prepared in parallel from identical ingredients
except that TE was omitted. The PPR of proteoliposome
samples was determined as described.31–34 Recovery of protein
and phospholipid after reconstitution was ∼70%.9,11,12 Triton
X-100 in the vesicle preparation was undetectable indicating
that >99.9% of the detergent was removed by the Bio-Bead
treatment.9,11,25
1L-myo-Inositol 1-O-[1-[6ꢀ[[6-[(7-nitro-2-oxa-1,3-diazolobenz-
4-yl)amino]hexanoyl] amino]-hexanoyl]-2-O-stearoyl-sn-glycer-
3-yl]-phosphate (3). This fluorescent PI was prepared by same
reaction sequence as used for the synthesis of 1, from unnatural
L-inositol intermediate 12 and natural lipid H-phoshphonate
donor 17. The spectral data of compound 3: dH (300 MHz,
CDCl3): 8.43 (1 H, d, J 8.45, NBD), 6.19 (1 H, d, J 8.5, NBD),
5.27 (1 H, m), 4.44 − 4.05 (6 H, m), 3.99 (1 H, m), 3.67 (1 H,
m), 3.50 (2 H, m), 3.36 (2 H, m), 2.75 (2 H, t, J 6.8), 2.31–2.38
(6 H, m), 1.41 − 1.22 (42 H, m), 0.86 (3 H, t); dP (100 MHz,
CD3OD–CDCl3): 0.93 ppm; MS (negative-ion ESMS, M − H−)
calcd for C45H75O17N5P 988.4896, found 988.4875.
Flippase assay in a reconstituted system. Flippase activity in
freshly prepared proteoliposomes was measured using dithionite
reduction of the NBD fluorophore or BSA back-extraction as
described.11,35 Assays were conducted at 23 ◦C.
Stopped-flow analysis of NBD–phospholipid flipping in SWER
vesicles. SWER membranes were thawed rapidly and adjusted
to a phospholipid concentration of 1 mM with 10 mM Hepes–
NaOH, pH 7.5 and 0.25 M sucrose. Aliquots were incubated at rt
with NBD-labeled phospholipid analogs (added from stock so-
lutions of 200 lM in 10 mM Hepes–NaOH, pH 7.5) such that the
final amount of the NBD–phospholipid analog was ∼0.5 mol%
of total phospholipid. All fluorescence measurements were
performed with an Aminco Bowman Series 2 spectrofluorom-
eter (SLM Instruments, Rochester, NY, USA) equipped with
a stopped-flow accessory (RX 1000, Applied Photophysics,
1L-myo-Inositol 3-O-[1-[6ꢀ[[6-[(7-nitro-2-oxa-1,3-diazolobenz-
4-yl)amino]hexanoyl] amino]-hexanoyl]-2-O-stearoyl-sn-glycer-
yl]-phosphate (4). This fluorescent PI was prepared by same
reaction sequence as used for the synthesis of 1, from unnatural
L-inositol intermediate 12 and unnatural lipid H-phoshphonate
donor 22. The spectral data of compound 4: dH (300 MHz,
CDCl3): 8.41 (1 H, d, J 8.4, NBD), 6.22 (1 H, d, J 8.4, NBD),
5.29 (1 H, m), 4,43 − 4.03 (6 H, m), 3.99 (1 H, m), 3.67 (1 H,
m), 3.48 (2 H, m), 3.36 (2 H, m), 2.73 (2 H, t, J 6.7), 2.30–2.37
(6 H, m), 1.42 − 1.23 (42 H, m), 0.87 (3 H, t); dP (100 MHz,
CD3OD–CDCl3): 0.89 ppm; MS (negative-ion ESMS, M − H−)
calcd for C45H75O17N5P 988.4896, found 988.4895.
◦
Leatherhead, UK). Measurements were done at 20 C (unless
stated otherwise) using quartz microcuvettes. Incorporation of
NBD–phospholipids into SWER vesicles was determined by
monitoring the time course of fluorescence emission at 540 nm
1 2 8 2
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 2 7 5 – 1 2 8 3