Lipase and Phospholipase Protocols

354 Pages·1999·22.872 MB·English

by  Mark Doolittle

1
-
Phospholipase A2 and Phosphatidylinositol-
Specific Phospholipase C Assays by HPLC
and TLC with Fluorescent Substrate
H. Stewart Hendrickson
1. Introduction
Llpolytic enzymes have traditlonally been assayed by radlometrlc and t&-l-
metric methods (I). RadIometrIc methods are quite sensitive but require ex-
pensive radlolabeled substrates and tedious separation of labeled substrate and
products. In addition, the safe use of radioactive materials 1so f mcreasmg con-
cern. Tltrlmetrtc assaysa re contmuous and quite straightforward and use natu-
ral substrates but suffer from low sensltlvlty and are subject to condltlons that
may alter the amount of free hydrogen Ions released Fluorescence-based assays
have sensltlvltles that approach those of radtometrlc methods; although they
require synthetic fluorescent-labeled substrates, they are often more conve-
nient and rapid. For a recent review, see Hendrickson (2).
We first used dansyl-labeled glycerol ether analogs of phosphatidylcholme
as substrates for the assay of enzymes of the platelet-actlvatmg factor (PAF)
cycle m peritoneal polymorphonuclear leukocytes (3). This became a general
method for the assay of phosphollpase A2 (PLA,) (see Fig. 1; refs. 4 and 5).
This method can be modified to assay other enzymes of the PAF cycle such as
lyso-PAF acyltransferase, lyso-PAF acetyltransferase, and PAF acetylhydro-
lase (3) Smce the probe remains attached to the glycerol backbone, simulta-
neous assay of all of these enzymes IS possible.
The method described here for the assay of PLA2 uses thin-layer chroma-
tography (TLC) to separate products from substrate, and quantltatlon by fluo-
rescent scanning. The use of high-pressure hquld chromatography (HPLC) with
fluorescence detection IS included as an alternative to TLC. The assay IS spe-
From Methods m Molecular &o/ogy, Vol 109 Lipase and Phospholipase Protocols
Edlted by M H Doohttle and K Reue 0 Humana Press Inc , Totowa, NJ
I
Hendrickson
H20POjCH2CH2N(CH3)3
+
S02NHH 0 --CH2
HO 1 -H
+
H2OP03CH$IH2N(CH3)3
+
Fig 1 Reactrons chemef or the assayo f PLA, with dansyl-PC.
cific for PLA2 since the substrate, with an ether lmkage at the sn-1 position of
glycerol, ts not hydrolyzed by PLA,. Hydrolysis of the substrate by phosphoh-
pases C or D present m crude enzyme preparattons will be apparent as addi-
tional products that will be seen by TLC or HPLC
Phosphattdylmosttol-specific phospholipase C (PI-PLC) from Bacdlus
cerexs catalyzes the hydrolysis of PI to a diglycertde and 1 o-myo-inositol- 1,2-
(cychc)phosphate (6) The latter is subsequently slowly hydrolyzed by the same
enzyme to 1o -myo-mosttol- l-phosphate. This enzyme also catalyzes the release
of a number of enzymes linked to glycosylphosphatidylmosttol membrane an-
chors (7)
Several years ago we synthestzed 4-(l-pyreno)butylphosphoryl-l-myo-
mosttol (pyrene-PI) as a substrate for the assay of PI-PLC from B. cereu~ (see
Fig. 2; refs. 8 and 9) The method described here uses reverse-phase HPLC
wtth fluorescence detectton to separate and quantitate the product released.
The methods described here are well suited to the assay of crude enzyme
preparattons since the presence of other phospholtpase acttvmes will be appar-
ent. The methods are independent of the specific condtttons for the enzyme
reaction, so a variety of detergents can be used. These assays can also be auto-
mated by the use of an autosampler for HPLC and larger plates wtth multiple
Fluorescence-Based Phospholipase Assays
H&
po3=
,
PI-PLC
I
H,CH2CH&H20H
Fig 2 Reactlon scheme for the assay of PI-PLC wrth pyrene-PI
lanes for TLC; they can thus be used to screen many enzyme samples and
potential mhibltors. The TLC-based assays can done m a qualitative manner
by simply vlsuahzing the plates under an ultraviolet (UV) lamp
2. Materials
2.1. PL A2 Assay
1 PLA, buffer. 0.395 M NaCl, 66 nuW Tns, 13.2 mi!4 CaCl,, pH 7 0 (adjust with
HCl)
2 PLA, substrate dansyl-PC, 1 n-J4 In CHCl, Dissolve 1 mg of dansyl-PC (cat
no D-3765, Molecular Probes, Inc , Eugene, OR) m 1 23 mL of chloroform (see
Note 1) Store m a brown bottle at -20°C (see Note 2)
3 Trlton X-100 solutlon, 10 mM Triton X-100 (cat no. T9284, Sigma, St. LOUIS,
MO) m water
4 PLA, stock assay solution. place 100 pL of PLA, substrate (1 mMdansyl-PC) m
a small test tube (10 x 75 mm) and dry under a stream of mtrogen and then under
high vacuum for 10-15 mm to remove all traces of solvent Add 20 pL of Trlton
X-100 solution (see Note 3) and 380 pL of PLAz buffer Vortex and somcate
(bath-type somcator) repeatedly until the lipid 1s completely dissolved and the
solution is optlcally clear.
4 TLC solvent* CHC13/CH30H/conc ammoma/water (90.54 5.5:2 [v/v])
5 TLC plates. 10 x 10 cm, HPTLC plates, (cat. no. 60077, Analtech, Newark, DE).
6. Fluorescence scanner: densitometer (model CS9000, Shlmadzu) with fluorescence
accessory, or other sultable Instrument for fluorescence scanning of TLC plates.
7 Quenching solvent* hexane/lsopropanol/acetlc acid (6:8: 1.6)
8. PLA, HPLC solvent hexane/lsopropanol/water (6 8.1.6)
4 Hendrickson
2.2. PI-PLC Assay
1. PI-PLC buffer. 50 mM2-(N-morpholmo)ethane sulfomc acid (MES, Sigma), pH
7 0 (adjust with NaOH)
2. PI-PLC substrate 1 tipyrene-PI m CHCls/CH,OH (2.1). Dissolve 0 5 mg of
racemic 4-( 1- pyreno)butylphosphoryl- 1 -myo-mositol (cat no P-3764, Molecu-
lar Probes) m 1 25 mL of solvent Store m a brown bottle at -20°C (see Note 2)
3. PI-PLC stock assay solution place 250 pL of PI-PLC substrate (1 mM pyrene-
PI) m a small test tube (10 x 75 mm) and dry under a stream of nitrogen and then
under high vacuum for 10-15 min to remove all traces of solvent Add 200 yL of
PI-PLC buffer. Vortex and sonicate (bath-type somcator) repeatedly until the
lipid is completely dissolved and the solution is clear
4. PI-PLC HPLC solvent 5 mM tetrabutylammonmm dihydrogenphosphate (cat
no 26,8 10-0, Aldrich, Milwaukee, WI) in acetomtrile/methanol/water (70 10.20)
2.3. HPLC Analysis
1 HPLC column for PLA, assay 15 cm x 4.6 mm, 5 pm spherical silica gel (cat
no 85774, Waters, Milford, MAtprotect with a guard column.
2. HPLC column for PI-PLC assay’ 25 cm x 4.6 mm 5 pm Spherisorb ODS (cat. no.
583 12, Supelco, Bellefonte, PA)--protect with a guard column (see Note 4).
3. Fluorescence detector Kratos model 980 or other suitable detector
4. HPLC mstrument with autosampler (optional) and mtegrator/recorder
3. Methods
3.1. PLAl Assay
1 MIX 40 pL of PLA, stock assay solution with 60 pL of enzyme (containing the
equivalent of about 2 ng of pure snake venom PLA2 (cat no 525 150, Calbiochem,
La Jolla, CA) (see Note 5) m a 500~PL microcentrifuge tube (vortex) Incubate at
room temperature. The final concentrations are 0.15 MNaCI, 0.1 &substrate,
0 2 rnA4 Triton X-100, 5 mMCaC12, 25 mMTris-HCl, pH 7 0
2 At various times over a period of 30-60 mm, remove 5-pL aliquots Spot directly
on a TLC plate for TLC analysis, or add to 90 pL of quenchmg solvent m a 500~pL
microcentrifuge tube for HPLC analysis (vortex)
3 For TLC analysis. dry the spots and develop the TLC plates m TLC solvent After
the solvent has evaporated, scan the plates with a fluorescence densitometer (set
excitation at 256 nm; measure emission above 400 nm [cutoff filter]). The Rr val-
ues for dansyl-PAF and lyso-dansyl-PAF are 0 35 and 0 15, respectively
4 For HPLC analysis centrifuge the quenched samples at top speed m a rmcrocenmfuge for
several minutes to remove any precipitated protein Equilibrate the s&a gel HPLC col-
umn in PLAZ HPLC solvent at a rate of 1 ml/r&n Set the fluorescenced etector at excna-
hOn 256 nrn and emission >4 18 nm (cutoff filter). InJect 20 pL of sample onto the column,
Elution times for dansyl-PC and lyso-dansyl-PC are about 6 and 19 min, respectively
5 Calculation of acttvtty. the mol fraction of product released is determined by
dividmg the area of the lyso-dansyl-PC peak by the sum of the areas of the dansyl-
Fluorescence-Based Phospholipase Assays 5
PC and lyso-dansyl-PC peaks Thts value times the mmal amount of substrate
present in the assay (0 01 pmol) equals the amount of product released Plot the
pmol of product released vs time to determine the mitral lmear rate (acttvtty,
umol/min)
3.2. Pi-PLC Assay
1 Add 5 pL of PI-PLC (contammg the equivalent of 0 2-4 ng of pure enzyme (cat
no P-6466, Molecular Probes) (see Notes 5 and 6) to 20 pL of PI-PLC stock
assay solutton m a 500~uL microcentrifuge tube (vortex). Incubate at room tem-
perature Fmal concentratton of substrate, 1 mM
2 At vartous times over a period of l&30 mm remove 5-uL altquots and dilute
with 95 uL of PI-PLC HPLC solvent in 500~pL microcentrifuge tubes (vortex)
Centrifuge these samples at top speed in a microcentrtfuge for several minutes to
remove any prectpttated protein (see Note 7)
3 Equilibrate the ODS reverse-phase HPLC column m HPLC solvent at a rate of
1 mL/mm (see Note 4) Set the fluorescence detector at excitation 343 nm and
emission >370 nm (cutoff filter) Inject 20 PL of sample onto the column. Elu-
tion times for pyrene-PI and pyrenebutanol are 2 4 and 6.2 mm, respectively
4 Calculatton of activity the mol fraction of product released is determmed by
dtvtdmg the area of the pyrenebutanol peak by the sum of the areas of the pyrene-
PI and pyrenebutanol peaks. This value ttmes the untial amount of substrate
present m the assay (0 025 pmol) equals the amount of product released Plot the
pmol of product released vs time to determme the mttial linear rate (activity,
pmol/min)
4. Notes
Dertvatives of dansyl-PC are useful in the assay of other enzymes of lipid
metaboltsm Lyso-dansyl-PC and dansyl-PAF (cat. no D-3766 and D-3767,
respectively, Molecular Probes) can be used as substrates m assays of lyso-PAF
acyltransferase, lyso-PAF acetyltransferase, and PAF acetylhydrolase (3)
This solution 1s stable for a year or more tf protected from moisture and stored m
a brown bottle at -20°C Before use, warm to room temperature and make sure
the lipid ts completely dissolved
Snake venom PLA, IS quite active in the presence of Trtton X- 100, but other
PLA2s (parttcularly pancreattc PLA2) may be less active with this detergent. The
pancreatic enzyme is best assayed using sodium cholate as a detergent Hexa-
decylphosphocholme (cat no H6722, Stgma) IS also a good detergent for other
phosphohpases. The concentration of substrate and the ratto of substrate to deter-
gent may be varied as destred The spectfic activity of pure snake venom PLA, in
this assay with dansyl-PC is about 13 ymol/mm/mg
Do not leave the reverse-phase ODS column in PI-PLC HPLC solvent (wtth
tetrabutylammonmm dthydrogen phosphate) for any length of time without sol-
vent running through; if so, the column will become plugged After use, tmmedt-
ately wash the column with CHsOH/H,O (80.20)
6 Hendrickson
5 Phospholipases readily adsorb onto glass or plastic surfaces Dilute soluttons of
PLA, and PI-PLC (<I mg/mL) should be stabthzed by the presence of 1% (w/v)
bovme serum albumm
6 With pure B cereus PI-PLC, the specific activity m this assay wtth pyrene-PI is
about 60 pmol/mm/mg, about 4 % hydrolysis IS observed m 1 mm wtth 4 ng of
pure enzyme
7 R, values for TLC of pyrenebutanol and pyrene-PI on silica gel plates 0.75 and
0 0, respectively, m CHCl,/CH,OH (95 5), 1 0 and 0 25, respectively m CHCl,/
CH,OHIH,O (65 35 3)
References
1. Reynolds, L J., Washburn, W N., Deems, R. A., and Dennis, E A (199 1) Assay
strategies and methods for phospholtpases Methods Enzymol 197,3-23
2 Hendrickson, H S (1994) Fluorescence-based assays of hpases, phospholtpases,
and other ltpolytx enzymes Anal Blochem 219, l-8
3. Schmdler, P W , Walter, R., and Hendrickson, H S (1988) Fluorophore-labeled
ether lipids Substrates for enzymes of the PAF cycle m peritoneal polymorpho-
nuclear leukocytes Anal Blochem 174,477-484
4 Hendrickson, H S , Kotz, K J , and Hendrtckson, E K (1990) Evaluation of
fluorescent and colored phosphatidylcholme analogs as substrates for the assay of
phosphohpase A, Anal Bzochem 185,80-83
5 Hendrickson, H. S. (1991) Phosphohpase A, assays with fluorophore-labeled lipid
substrates Methods Enzymol 197,9&94
6 Bruzik, K S and Tsar, M -D (1994) Toward the mechamsm of phosphomosttide-
spectfic phospholtpase C Bloorg Med Chem 2,49-72
7 Low, M G and Saltiel, A R (1988) Structural and functional roles for
glycosylphosphattdylmositol m membranes. Sczence 239,268-275
8 Hendrickson, E K , Johnson, J L., and Hendrickson, H S (1991) A fluorescent
substrate for the assay of phosphattdylmosnol-specific phosphohpase C 4-( l-
pyreno)butylphosphoryl- 1-myo-inositol Bloorg Med Chem Lett 1,6 19-622
9 Hendrickson, H S , Hendrickson, E K , Johnson, J L , Khan, T H , and Chlal, H
J (1992) Kinetics ofB cereus phosphatldylmositol-specific phospholtpase C with
thiophosphate and fluorescent analogs of phosphatidylmosnol Bzochemlstry 3 1,
12,169-12,172
2
Fluorometric Phospholipase Assays Based
on Polymerized Liposome Substrates
Wonhwa Cho, Shih-Kwang Wu, Edward Yoon.
and Lenka Lichtenbergova
1. Introduction
Phospholtpases are a drverse group of ltpolyttc enzymes wrth specrflcr-
ties for dtfferent sites on the phosphohptd molecule (2) Based on the site
of then hydrolytrc attack, they can be classrfted into several groups, among
whtch phospholtpase A, (PLA,), phospholipase C (PLC), and phosphoh-
pase D (PLD) have been the most extensrvely studied. Because of then
crltlcal mvolvement n-r many extra- and intracellular processes, a senstttve
contmuous kmetrc assay for phospholrpase IS an essentral tool for many
blologtcal studtes
Among the vartous assays used for the different classes of phospholt-
pases (2), those based on fluorometry (3-7) serve as the most sensttrve type
of contmuous assay. However, a maJority of current fluorometrtc methods
rely on the use of synthetic phospholiptds containmg a fluorophore at the
~2-2 posrtion of the phospholtptd that restricts then use to the assay of
PLA*. In order to measure the activity of many types of phospholtpases, we
have recently developed a novel fluorometrrc assay utlhzmg polymertzed
mixed lrposomes (7).
Polymerized mixed hposomes are composed of a pyrene-labeled phospho-
hptd monomer Inserted mto a polymertzed phospholtptd matrix, the pyrene-
labeled phospholiprd constitutes only a small mole percentage of the total
polymerized mixed hposome substrate. In this system, the polymerized matrix
is essentially inert, and only the monomer (unpolymerrzed) inserts are selec-
tively hydrolyzed by the phosphohpase (8). For example, the PLA2 actrvtty
From Methods m Molecular Biology, Vol 109 Lfpase and Phospholfpase Protocols
Edlted by M H Dooltttle and K Reue D Humana Press Inc , Totowa, NJ
7
Cho et a/.
Fig 1 SchematIc lllustratlons of fluolometrlc assays usmg polymerized mixed
hposomes for PLA, (A) and PLC and PLD (B). F indicates a fluorescent label, such as
pyrene, attached to either the acyl cham (A) or the polar head group (B)
assay utlhzes a polymerized mixed hposome substrate that IS composed of l-
hexadecanoyl-2-( I-pyrenedecanoyl)-srt-glycero-3-phosphoglycerol (pyrene-PG)
inserted into a polymerized matrix of 1,2-bls[ 12-(hpoyloxy) dodecanoyll-sn-
glycero-3-phosphoglycerol (BLPG). The pyrene-PG Insert contams a
pyrenedecanoyl fatty acid at the sn-2 posltlon, with the fluorescent pyrene
molecule located at the methyl-termmal end of the fatty acid (see Fig. 1A);
the fluorescence emtsslon Intensity of the pyrene moiety ts largely
quenched by the lipolc acid moieties of the BLPG molecules. Using this
substrate, PLA2 hydrolysis can be contmuously monitored by measuring an
increase m pyrene fluorescence, since the hydrolyzed pyrene labeled-fatty
acids are no longer quenched as they are removed from the llposome sur-
face by serum albumin m the reaction mixture (see Fig. 1A). Alternatlvely,
for measurement of PLC and PLD hydrolysis, a new Insert IS used where
the phosphollpld monomer contams a pyrene-labeled head group; agam,
quenchmg of the pyrene moiety by the polymerized phosphollpld matrix IS
lost as the hydrolyzed pyrene moiety readily diffuses away from the llpo-
some surfaces (see Fig. 1B). Thus, polymerized mixed llposomes serve as
an extremely versatile assay system for different phosphollpases, since one
can readily modify the structure of the polymerized matrix, and the pyrene-
labeled phospholipid inserts, to create an ideal combmatlon of llposome
surfaces and hydrolyzable substrates for a specific phosphollpase. Herein,
we describe the synthesis of polymerlzable phosphollplds and pyrene-
labeled phospholiplds, the preparation of polymerized mixed llposomes
from these synthesized products, and the use of the prepared llposomes as
substrates to fluorometrlcally monitor phosphollpase hydrolysrs
Polymerized Liposome Substrates 9
2. Materials
2.1. Synthesis of the Polymerized Phospholipid Matrix,
1,2-bis[lZ-(lipoyloxy)-dodecanoy/l-sn-Glycero-
3-Phosphoglyceroi (BL PG)
1 12-hydroxydodecanotc acid, 3,4-dihydro-2H-pyran, p-toluenesulfonic actd
monohydrate, dtcyclohexylcarbodnmtde (DCC), 4-(dtmethylammo)pyrtdme
(DMAP), glycerol (Aldrtch, Mtlwaukee, WI)
2 L-a-glyceropl~ospl~orylchohne, 1. I cadmmm chloride adduct (Sigma, St Louts, MO)
3 Cabbage PLD IS prepared as follows The Inner ltght-green leaves of a Savoy
cabbage (1 Kg) IS homogemzed wtth 200 mL of cold water for 5 mm The homo-
genate IS filtered and the filtrate IS centrifuged at 20,OOOg for 30 mm The super-
natant IS kept at 55 “C for 5 mm and placed on tee for 5 mm The prectpttate 1s
removed by centrtfugatton (20,OOOg for 30 min), the supernatant 1s cooled to 0°C
and 2 vol of acetone cooled to -15°C is added to the supernatant After 10 mm,
the prectpttate containmg the active PLD IS collected by centrifugatton (20,OOOg
for 30 mm) and used for transphosphattdylatton. The acetone power can be stored
at -20°C for several months
4. PLD reaction buffer 0 1 M sodium acetate buffer, pH 5 6, 0 1 A4CaC12
2.2. Synthesis of Pyrene-Labeled Phospholipid Monomers (In-
serts) Specific for the Measurement of Cytosolic PLA2 Activity
1 lo-(1-Pyreno)decanotc actd (a k a l-pyrenedecanotc actd) (Molecular Probes,
Eugene, OR)
2 Ltthtum alummum hydrtde, pyridme, mesyl chloride, sodtum hydrtde, 2,3-
tsopropyltdene-sn-glycerol, phosphorus oxychlortde, trtethylamme (Aldrrch,
Mtlwaukee, WI)
3 Cholme tosylate, arachtdomc actd (Sigma, St Louis, MO).
2.3. Preparation of the Polymerized Mixed Liposome Substrate
1 Macro extruder Ltposofast (Avestm, Ottawa, Canada) with 0.1 -mm polycarbon-
ate filter (M&pore, Bedford, MA).
2 Polymenzatton buffer, 10 mMTris-HCI, pH 8.4,O 16 M KC1
3 Sephadex G-SO (Pharmacta, Uppsala, Sweden).
4. Mobile phase for Sephadex G-50, 10 mM HEPES buffer, pH 7.4 , 0.16 KC1
2.4. Kinetic Measurements
1 Spectrofluorometer equtpped with a thermostated cell holder and a magnettc stnrer.
2. 1-Hexadecanoyl-2-( 1- pyrenedecanoyl)-sn-glycero-3-phosphocholme (pyrene-PC),
-phosphoethanolamme (pyrene-PE),-phosphoglycerol (pyrene-PG) (Molecular
Probes)
3. N-( I-pyrenesulfonyl)-egg phosphattdyl ethanolamme (N-pyrene-PE) (Avanti,
Alabaster, AL)
4 Fatty acid-free bovine serum albumin (BSA) (Bayer, Kankakee, IL)
70 Cho et al
5 Secretory PLA2 assay buffer 10 mMHEPES, pH 7 4,0 16 M KCI, 10 mMCaCl,,
and 10 mMBSA
6 Cytosohc PLAz assay buffer 10 mMTns-HCI, pH 8.0,1 mA4CaCl,, and 10 mMBSA
7. PLC assay buffer 10 mMMES buffer, pH 6 0,O 1 n&‘ZrCl,, and 10 mA4CaClz
8 PLD assay buffer 10 mA4HEPES, pH 7 4,0 16 M KCl, and 10 mM CaClz
3. Methods
3.1. Synthesis of the Polymerized Phospholipici Matrix, 1,2-bis[12-
(lipoyloxy)=doctecanoyl;l-sn -Glycero-5 Phosphoglycerol(6 L PG)
BLPG IS prepared from L-u-glycerophosphorylchohne by a four-step syn-
thesis according to the procedure by Sadowmk et al. (9) with slight modlfica-
tions (7,s).
3.7.1. Synthews of 12-(Tetrahydropvranyloxy)Dodecanoic Acid (TCA)
1 Suspend 10 mmol (2 16 g) of 12-hydroxydodecanolc acid m 20 mL of dry
tetrahydrofuran
2 Add 17 mm01 (1 39 g) of 3,4-dihydro-2H-pyran and stir the mixture for 10 mm at
room temperature
3 Add 0 1 mm01 (20 mg) of crystallme p-toluenesulfomc acid monohydrate and
stir the mixture for an addItIona 2 h at room temperature
4 Evaporate the solvent and purify the product by slllca gel column chromatogra-
phy usmg chloroform/methanol (20 1, v/v) as eluent. Check the product by thm-
layer chromatography (TLC) (R,= 0 5 with the same solvent) Completely remove
the solvent zn vacua
3.72. Synthesis of 1,2-Bis(lZ-Hydroxydodecanoyl)-sn-
Glycero-3-Phosphocholine
1 Dissolve the purified 12-(tetrahydropyranyloxy)dodecanolc acid (TCA) m dry
dtchloromethane (15 mL) and add DCC (2.6 mmoU4 mm01 of TCA) dissolved m
dry duzhloromethane
2 Stir the reactlon mixture at room temperature until it gets cloudy (approx 5 mm),
then add L-a-glycero-phosphorylcholme (1 mmol/4 mmol of TCA) and DMAP
(2 6 mmol/4 mm01 of TCA) Stir the mixture at room temperature overnlght
3 Filter the reactlon mixture to remove dlcyclohexylurea, evaporate the solvent
and purify the product by slhca gel column chromatography using chloroform/
methanol (5 1) first and then chloroform/methanol/water (65 25.4) Check the
product by TLC Rf= 0 35 with chloroform/ methanol/water (65 25:4)
4 Dtvlde the product mto about 200 mg fractions, dissolve each fraction m metha-
nol (2 mL) and add equlmolecular crystallme p-toluenesulfomc acid monohy-
drate Stir the mixture at room temperature for approx 1 5 h while checking the
progress of the reactlon every 15 mm by TLC usmg chloroform/methanol/water
(4 5 1) as developmg solvent (Rf = 0 3 for the product) The reaction must be
completed within 2-2 5 h, otherwlse the hydrolysis of acyl groups of the phos-