Note: Descriptions are shown in the official language in which they were submitted.
~l~6~
- 1 - 16777IL
This is a divisional of Canadian Application
S.N. 466,742, filed October 31, 1984.
RELATIONSHIP TO THE PRIOR ART
The concept of usiny conjugates of leuko-
trienes in a radioimmunoassay was described, by L.
Levine, R.A. Morgan, R.A. Lewis, K.F. Austin, D.A.
Clark, A. Marfat, and E.J. Corey, Proceeding of the
National Academy of Sciences, U~S~Ao~ Vol. 78, No. 12
7692 (1981). This method uses direct coupling through
an activated acid derivative to the protein. This
method is much less effective than the present in-
vention.
Bifunctional cross-linking reagents useful
in preparation of protein-hapten conjugates have also
been prepared, see Kitagawa, J. Biochem. 79, 233-236;
and Kitagawa, Chem. Pharm. Bull. 29(4), 1130-1135;
/
/ : :
::
: ~
.
~6~t35~
2298P/0832A - 2 - 16777IB
describing maleimido-succinimide derivatives. The
present invention relates to conjugates of
leukotrienes C4, B4, D4 or E4 (preferably
C4 and B4) with a protein selected from hemo-
cyanine from giant keyhole limpets (KLH), bovineserum albumin (BSA), human serum albumin, tetanus
antigen, diphtheriae toxoid, or CRM 197 (a diphtheriae
toxoid produced by a mutant of Corynebacterium
diphtheriae), through the coupling agents
1,5-difluoro-2,4-dinitrobenzene or 6-N-maleimido-
alkanoic acid chloride,-preferably 6-N-maleimido-
hexanoic acid chloride, wherein the alkanoic moiety
has 2 to 8 carbon atoms. The conjugates are useful
in a sensitive and specific immunoassay and are also
useful immunotherapeutic agents in the treatment of
various allergic and chronic inflammatory diseases of
the skin, lung and airways, including asthma,
allergic rhinitis, rheumatoid arthritis,~and skin
diseases such as psoriasis and eczema.~ The present
invention also relates to useful reagents for
preparing such conjugates. ~ ~
Leukotriene C4 (LTC4) has the~following
structure: ~
~ COOH
S ~ COOH
: J~
NH2
: : ~ ~ :
~ ` ~
COOH ~ 4- ; ;
SH
::: :
: :
.
356
2298P/0832A ~ 3 - 16777IB
Leukoteiene B4 (LTB4) has the following
structure:
H` ? \C02H
~ LTB4_
Leukotriene D4 (LTD4) has the following structure:
~1
S ~ N ~ CO H
~ ~H2 2
~ CO2N
LTD4_
:
Leukotriene E4 (LTE4) has~the following structure:
2H
~ NH2
O2H~
TE
`:
35~
- 4 - 16777Is
The present invention also relates to the
following compounds which are useful in preparing the
conjugates (especially the conjugates of LTB4):
1)
HO ~ CONH(CH2~nNH2
~-/~/
wherein n is O to lOj preferably O or 2 to lO, more
preferabIy O or 3.
2)
HO ~ ~ CoNH~cH2)nNH ~ ~
02N N02
:
wherein n is O ~o lO, preferably O or 2 to lO, more
preferably O or 3.
3)
~V 1'
: :: : : :
2298P/0832A - 5 - 16777IB
wherein n is 0 to 10, preferably
0 or 2 to 10, more preferably 0 or 3.
In the above three compounds, the compounds
where n is 1 are likely to be less stable than the
other compounds having the same generic formula.
The preparation of the conjugates of the
present invention may be illustrated by reference to
conjugates of LTC4 and LTB4.
For LTC4, coupling procedures were
selected so that the reactions took place on the free
amino group of the glutamyl residue, thus retaining
the most important parts of the LTC4 molecule
unchanged.
The general conjugation procedure utilized a
stepwise method with well characterized
intermedlates. The strong W absorption of the
triene chromophore in LTC4 ~ = 40,000~at 280 nm)
was used as a~probe for determining coupling
efficiencies and for monitoring the state of the
LTC4 molecules throughout the procedures.
Coupling ratios~in~th~e ranges~of 5 to 15
equivalents~of LTC4 per 100,000 daltons~of prot~in
were desired.
Con~ugates of LTC4
Conjugations using 1,5-difluoro-2,4-
; dinitrobenzene as~coupl~ing ~
~Th~e reagent, 1,~5~-d~lfluoro-2,~4-dinitrob;enzene
(DFDNB) reacts~quite specifically with amino
functions~, allowing clean~stepw~ise replacement~of the
two ~fluorine atoms (the second fluorine being~
:
~:
5~i
2298P/0832A - 6 - 16777IB
replaced at a much slower rate). In addition, the
strong and characteristic W absorptions of the
reagent, and its mono- and diamino substituted
derivatives allows one to follow the course of the
coupling procedure and to quantitate the final
adducts by W spectroscopy.
LTC4 was found to react essentially
quantitatively with excess DFDNB in pH 7.2 buffered
aqueous methanol within 30 minutes. The
intermediates thus formed could be characterized by
HPLC analysis, by the appearance of a strong W band
at 345 nm characteristic of 1-amino-5-fluoro-2,4-
dinitrobenzenes. After removal of methanol from the
reaction the excess DFDNB could be removed by ether
extraction. The intermediatés could be further
purified by HPLC but this was found to not offer any
advantage and, in general, the cr~ude reaction mixture
was then allowed to react with protein in pH 8.5
bufer for two days in the~dark. Final separation of
the conjugates from unreacted LTC4 or reagents was
achieved by filtration on Sephadex G-50~ The derived
coupled products now showed~W absorptions at 342 and
420 nm characteristic of 1,5-diaminodinitrobenzenes
as well as the characteristic absorptions of the
triene system at 271, 282, and 291 nm in the case of
the LTC4 conjugates. In this manner, S-p-chloro- `
phenacylglutathione when reacted in 10:1 molar ratio
with BSA gave a~con~ugat;e wlth~about 6~moles of
hapten per mole of BSA.
~ ~ Simila~rly,~LTC4~1n 30 fold molar excess
gave a conjugate with BSA with~9-10 moles LTC4 per
mole BSA, and LTC4~in ca. 30 fold molar excess
:
.
8~
2298P/0832A - 7 - 16777IB
(calculated per 100,000 daltons protein), gave a
conjugate with KLH with 11-12 equivalents LTC4 per
100,000 daltons KLH.
Conjugation using 6-N-maleimidohexanoic acid
chloride as c upling agent
Since this invention provides a second
LTC4 protein conjugate using a~different spacer
group, a number of potential coupling methods were
examined. A direct coupling using a reagent such as
DCC or ECDI (6) was considered but quickly rejected
due to the expectation that a heterogeneous mixture
of adducts would be formed. Also, preliminary
experiments indicated that the efficiency of such a~
coupling would be low. The known agents, toluene
diisocyanate and m-maleimidobenzoyl-N-hydroxy-
succinimide ester~were not used due to the
possibility of immunological~cross reactivity with~ ~
respect to the spacer un~its between the two ~ ;
conjugatesO `~ ~ ~
The coupling agent 6-N-maleimidohexanoic
acid ch;loride provide~s rapld,~selective~
functionalizati~on of~the~gl~utamyl~amino~group~of
LTC4, as well as high coupling~eficiency.~
The agent chosen w~as 6-N-maleimidohexanoic
acid~ch~loride~which was readlly~pre~pared~from~
6-amino:hexanolc~acld.~ 0ther~analogous~rea~g~ents ;~
having~from 2-~8~carbon atoms~in~ the;chain can~be
used`, e.g~.~, 2-aminoace~tic ac~ up~to 8-amino octanoic ;
acid.
The 6-N-maleimidohmxanoic~acid~amide~of
~LTC4 wa~s~prepared~by~reac~ti~ng~à~methanolic solution
:
~6~8~;
2298P/0832A - 8 - 16777IB
of LTC4 tripotassium salt with the reagent (1.5
equivalents in dry THF) in the presence of excess
Et3N~ HPLC analysis showed essentially complete
conversion to the amide (eluting before LTC4 on
RP-HPLC). A portion of this adduct, isolated from
HPLC, had W characteristics essentially unchanged
from those of LTC4. For subsequent coupling with
thiolated protein (KLH) the crude mixture (in pH 7.2
borate buffer) was used as such.
The thiolated protein used, in this case
derived from KLH, was prepared by reaction with
S-acetylmercaptosuccinic anhydride. As no report of
thiolation of XLH could be found in the literature,
trials were done to determine conditions for
obtaining KLH with about 20 S-acetyl groups per
100,000 daltons protein [thio] content, after
hydrolysis of the acetyl groups, was determined by
Elleman's method. The S-acetylmercaptosuccinyl
derivatized KLH was highly unstable to oxygen until
further reacted with N-ethyl maleimide (NEM).
However, once any free SH groups were thus reacted,
the material could be handled and purified by
Sephadex~G-50 filtration.
Concentration of the resulting purified
protein was accomplished by dialysis agalnst a
packing of anhydrous Sephadex~G-200 resin. Just
prior to coupling with derivatized LTC4, the thiol
groaps were liberated by hydrolysis of the rigorously
deoxygenated ~solution at pH 11.5 followed by
reduction of the pH to 7.2.
This mixture was then reacted with the
deoxygenated solution of the 6-N-maleimidohexanoic
,
,: :
ra.~ s6
2298P/0832A - 9 - 16777IB
acid amide of LTC4 in a ratio o~ 80 equivalents
LTC4 per 100,000 daltons RLH. After stabilization
with NEM and purification by Sephadex~G-50, the
protein conjugate showed 7-10 moles LTC4 per
100,000 daltons KLH by W analysis.
The protein solution has proven to be very
stable during several months storage frozen at -78C.
More detailed examples follow. It is noted
that IR spectra were recorded on a Perkin-Elmer 267
Grating Spectrophotometer. PMR spectra were recorded
on a Varian EM-390 spectrometer. UV spectra were
recorded on a Cary 210 spectrophotometer. Spectra
were recorded in water unless otherwise indicated.
Sephadex~G-50 (medium grade) was obtained from
Pharmacia Fine Chemicals.
Bov1ne Serum Albumin was obtained as
crystallized and lyophilized grade from Sigma
Chemical Co. and Hemocyanin (Keyhole Limpet) was
obtained as lyophilized powder~from Calbiochem
Behring Co~p. Lèukotriene C4 was synthetic material
prepared in our laboratories using known procedures,
Rokach et al~, Tet~. Lett., 21, 1485 tlg8o).
Preparation o~LTB4 conjugates is
illustrated by the following reaction schemes:
:
.
:
:;::
::
2298P/0832A - 10 - 16777IB
Scheme 1
HO_~ CO 2 Et
H~ O~Ph
II
H 2N/V\NH 2 ¢~
H
15 H = ~CON~\ H 2
2 0
1 ~ ~N~IIO~
HQ
~\CON~V\NH : ~ F
~/ ; ~(
H2N-BSA ~:
::
, .
~6~8~
2298P/0832A ~ 16777IB
r H~CON~M~NH~BSA
L v 02N~\N0
This method makes use of the immediate
synthetic precursor to LTB4, Ethyl 5(S)benzoyloxy-
12(R)-hydroxy-6,14(2)-8,10(E)-eicosatetraenoate
(II). We reasoned that reaction of II with a
volatile diamine such as l,3-diaminopropane would at
the same time remove the benzoate protecting group
and convert the ethyl e~ster to the ~-aminopropyl-
amide, all under mild weakly basic conditions. The
solvents could then be removed under vacuum leaving
only a mixture of the product (IIIj and N-~-amino-
propylbenzamide. In model studies, using ethyl
5-(4-octylphenyl)-5-benzoyloxypentanoate this
reaction was found to~be extremely sluggish,~even in
neat 1,3-diaminopropane. ~However, when a oatalytic
amount of 2-hydroxypyridine was~added to~the reaction
mixture~,~the~diester;was~smoothly converted ~o the
desired~aminoamide.~Wh~en~appl~ied~to the~ protec~ed ~;
LTB4 lII) a similar smooth con~version to III was
effected~ could~be ~reacted~directly~in~the nex~
step, after~removal~of the~volatile components. The
aminoamide~ was~réacted~with;excess~1,5-difluoro-
~ 2,4~-dinitr~obenzene ~n~th~e~presen~e of t~rlethylamine
30 ~ to~ prov~ide the ~: ~.dduct~ IV~in hlgh yield. ~ This ~product
was~purif~ied~by~reverse~pha~se~HPLC;and was~fully
characte~rized~by W and~PMR~spectroscopyO~ Finally,~
,
~6~8~
2298P/0832A - 12 - 16777IB
IV reacted smoothly with bovine serum albumin (BSA)
(mole ratio-12:1) in a mixture of dimethylformamide
and pH 8.5 borate buffer to provide the conjugate V
which was puriPied by chromatography on Sephadex
G-50. UV spectral analysis indicated that the triene
chromophore was unchanged and allowed the estimation
that 5.5-8.3 moles of LTB4 were coupled per mole of
BSA. (45-70% coupling efficiency).
The aminoamide III could also be prepared by
direct reaction of LTB4 -lactone with
1,3-diaminopropane at room temperature. This
provided III in quantitative yield free of side
products.
: : : : : : `
: . :
: : : ~ : :: ::
~,
:: ~ : : :
'
.
.
2298P/0832A - 13 - 16777IB
Sc}~eme 2
~\C00 K ~, COOH
H~ CH ~ H'
R ~ T . ~ ~3~ H0
Tl*,R. U~ 1 0
VI VII
0
~1, Et311
: ~
,, ~ :
-
~6~8~
- 14 - 16777IB
Another type of LTB4 conjugate could be pre-
pared as illustrated in Scheme 2. Lactone VI reacted
cleanly with hydrazine to provide the hydrazide (VII)
in quantitative yield. VII was reacted further with
6-N-maleimidohexanoic acid chloride to give the diacyl
hydrazide (VIII). This material could be purified by
reverse phase HPLC to remove the excess reagent by-
products. However, attempts to concentrate the product
in order to obtain a PMR spectrum led to partial de-
composition apparently due to hydration or methanolysisof the maleimide system. It was found however, that
the crude reaction product could be used in the subse-
~uent coupling reaction. VIII was reacted with thiol-
ated KLH in a ratio of 50 moles of VIII per 100,000
daltons KLH, to provide the desired conjugate IX which
was purified by filtration through Sephadex~ G-50. UV
analysis indicated that 12 equivalents of LTB4 were
bound per 100,000 daltons of KLH.
EXAMPLE 1
Conjugates of LTC4 Using 1,5-Di~f~luoro-2,4-dinltro-
benzene as Coupling Agent
A. Conjugation of S-p-Chlorophenacylglutathlone
and Bovine Serum Albumin (BSAj
1,5-Difluoro-2~,4-dinltrobenzene (120 mg, 0.59
mmol) in methanol (6 ml) was added~to a solution of
S-p-chlorophenacylglutathione (88 mg, 0.19 mmol) in 9
mL of phosphate buffer (pH 7.2, 0 lN). Af;te~r stirring
12 hours at room temperature the me-thanol was~removed
ln vacuo and~the~resulting aqueous solution was washed
~ith ether ~ Ihe q F~
,_
~ `: :
::
: ~
::
2298P/0832A - 15 - 16777IB
was chromatographed on C-18 Silica Gel (eluting with
~ethanol:water (1:1) to provide the pure adduct
intermediate (105 mg). W: ~max ~) 260 (24,000),
347 nm (19,000). PMR (D2O): ~ 8.62 (lH, d, J = 7.5
Hz), 7.6 (2H, d, J = 9Hz, A of AB), 7.1 (2H, d, J =
9Hz, B of AB), 6.7 (lH, d, J = 15Hz), 3.9 (2H, s,
phenacyl CH2).
The adduct (1.05 mg, 1.63 X 10 6 mol) in
water (0.1 mL) was added to a solution of BSA (10 mg,
1.49 X 10 7 mol) in borate buffer (pH 8.5, 0.2 N, 1
mL). After standing in the dark at room temperature
for 71 hours the solution was centrifuged and
filtered on Sephadex~G-50 (1.5 X 75 cm) eluting with
water. Fractions (10.5 mL) eluting after the void
volume (55 mL) contained protein and were~analyzed by
W. A sample of this solution diluted 5 times had a
W spectrum (in H2O) ~max (Absorbance) 342 (0.359),
425 nm (0.133). Assuming 8 my of protein were
recovered and assuming for the l,5-diamino-2,4-
dinitrobenzene chromophor of ca. 27,000 (3) at 342 nm
the W indicated 6 moles of S-~-chlorophenacyl-
glutathione were conjugated per mole o~ BSA.
B. Conjug~ation of 1eukotriene~C4 and
Bovine Serum Albumin~ ~
_ _ .
Leukotriene C4 (tripotassium salt)~ (2.5 mg) was
dissolved in l mL of phosphate~buffer (pH 7.2, 0.1
N). 1,5-Difluoro-2,4-dinitrobenzene (l mgj~ in~
methanol ~0.6 m~) was;added and the mixture wa5 left
3t min. at~room~temper~ature~.~ The methanol waa
removed under a~stream~of~ N2~and then in vacuo to
: : .
~26~
2298P/0832A - 16 - 16777IB
remove final traces followed by extraction with ether
(3 x 2 mL) to remove unreacted reagent. ~he last
traces of ether were removed under N2 and in
vacuo. To this mixture was added bovine serum
albumin (BSA) (10 mg) in borate buffer (0.2M, pH 8.5,
1 mL), and the mixture was left to stand at room
temperature in the dark for two days. The reac~ion
mixture was filtered on a column of Sephadex G-50
(1.5 x 75 cm) eluting with water and the yellow
protein eluting in 18 mL, after the void volume, of
ca. 55 mL, was collected. At a~bout the 140 mL dead
volume a peak considered to contain unreacted LTC4
eluted. Direct W analysis on the protein fractions
(combined) gave a spectrum ~max (A) 271 (sh), 282
(3.57), 291, 342 (1.835) and 420 nm (0.91). Assuming
about 9 mg of protein were recovered, and assuming
for the 1,5-diamino 2,4-dinitrobenzene of about
27,000 at 340 nm and for~LTC4 at 280 nm of
40,000, calculations based on the 282 nm absorption
about 10.0 mole of LTC4 per mole BSA while
calculations based on the absorption of 342~nm
indicated 9.1 moles LTC4 per mole BSA.
C. Conjugation of Leukotriene C4 and
Hemocyanin ~from Giant Keyhole Limpets
(KLH)~
~ Leukotrl~ene C4 (tripotassium salt) (~2.1
mg), and~l,5~-di~luoro-2,4-dinitrobenzene (8 mg)~ were
reacted as in react~ion A. ~To the r~sultant~adduct
was~added~KLN~;(15~mg~ borate buffer (pH a;.5, 0.2M,
0.8~3 mL)~-and~the~m1~xture~;was ~allowed to stand at room
temperature 60 hours. At~this time a precipitate of
:
:
:
~fi~
- 17 - 16777IB
denatured KLH had formed which was removed by centrif-
ugation ~6 mg, dry weight). The supernatant was
filtered on Sephadex G-50 as before yielding a yellow
protein fraction eluting in 17 mL following the void
volume which by UV analysis indicated 11-12 equivalents
of LTC4 per 100,000 daltons of KLH.
D. Conjugation of 2,4(E),6,9(7)-Pentadecatetraen-
l-ol with BSA _ __
A solution of DFDNB (2.04 g, 10 mmol) in
dioxane (20 mL) was added to L-proline (0.58 g, 5 mmol)
in phosphate buffer (pH 7.5, 0.1 N, 5 mL) and the
mixture was stirred 2 hours at room temperature. The
mixture was reduced to dryness and the residue was
chromatographed on silica gel (eluting with chloro-
form:methanol (9:1) to yield W-2,4-dinitro-5-fluoro-
phenylproline as a foam (1.1 g).
PMR (CDC13): &9.43 (lH, broad, exchanged by
D2O, COOH),` 8.55 (lH, d, JH F = 7~.5 Hz, H-3 of phenyl),
6.62 (lH, d, JH F = 15 Hz, H-6 of phenyl), 4,5 (lH,
broad t, J = 6Hz, proline methyne), 3.7-3.1 (2H, m),
2.7-1.9 ppm (4H, m).
To a mixture of 2,4(E),6,9(~)-pentadeca-
tetraen-l-ol (123 mg, 0.56 mmol) and the proline deriv-
ative above (170 mg, 0.5~7~mmol) in methylene chloride,
at -10 C, were added successively,~l-cyclohexyl-3-
(2-morpholinoethyl) carbodiimide methyl-p-toluene-
sulphonate (266 mg, 0.63 mmol) and~pyrrolidlnopyridine
(9 mg, 0.06 mmo1). The solution was~stirred under W2
at room temperature for 7 hours. ~The mixture was
filtered and the flltrate was washed with water,~5
NaHCO3, brine and drled~over ; ;~
, ~ ~
22~8P/0832A - 18 - 16777IB
Na2SO4. The residue after concentration was
chromatographed on silica gel teluting with
chloroform:ethano] ~99.25:0.75)] to yield the pure
adduct as an oil
PMR (CDC13): ~ 8.57 (lH, d, J = 7.5 Hz),
6.55 (lH, d, J = 15 Hz), 6.7-5.2 ~8H, m, olefinic),
4,65 (2H, d, J = 6Hz, -CO~C~2-), 4.47 (1~, t~ J a
6Hz, proline methyne), 3.45 (2H, m), 2.95 (2H, m)
2.7-1.8 (6H, m), 1.5-1.2 (6H, m), 0.88 (3H, t).
W (dioxane: ~ max (~) 275 (48 "00), 347nm (18,450).
Anal. calcd for C26H32N3O6F: C, 62.26; H, 6.43;
N, 8.38; F, 3.79. Found: C, 61.88; H, 6.72; N,
8.48; F, 3.47.
A suspension of the adduct (5 mg, 1 X 10 5
mol) and BSA (10 mg, 1.5 X 10 7 mol) in dioxane (1
mL) and borate buffer (pH 8.5, 0.2M, 3 mL) was slowly
stirred at room temperature for 4 days in the dark.
The mixture~was centrifuged:and the suspernatant was
filtered on Sephadex~G-50 (1.5 X 7~5 cm),:eluting with
water. The protein fraction eluti:ng in 7 ml after
the void volume analyzed by W~for approximately 4
mol~s hapte~n per mole~of BSA.~ : ;
~ ~ ~ : EXAMPLE 2~
Conjugates of LTC4 Using 6~-N-Maleimidohexanoic
Acid Chloride as CouPling:~Aqent
A. Prepara:tion of 6;-N-Maleimidohexanoic ::; :
Acid~Chloride
~ 6-Amin~ohexanoic~acid (2 g, 0.02 mol) and
maleic:anhydride ~(2 9,~ 0.~02:mol) were ref~lùxed
togethe~r in~xyl~ene~:(20~mL~)~under a Dean-:Stark~wat~r~ :~
separator such that the internal temperature reaohed
:
.
63L1551i;
2298P/0832A - 19 - 16777IB
ca. 165C. The mixture was cooled, diluted with
chloroform-methanol and washed with lN hydrochloric
acid. The organic layers were washed with water,
dried, and reduced to dryness to yield a residue (1
g) which after chromatography on silica gel (eluting
with 5% methanol-chloroform) provided pure
6-N-maleimidohexanoic acid, m.p. 84-85C.
IR(KBr): 3300-2500 (COOH), 1700 cm 1 (maleimide and
COOH). PMR (CDC13): ~ 11.10 (lH, s, exchanged by
D2O, COOH), 6.72 (2H, s, maleimide CH), 3.53 (2H,
t, J = 7Hz), 2.34 (2H, t, J = 7Hz), 1.6 ppm (6H, m).
Mass spectrum: m/e 211 (M ).
Anal. Calcd for CloH13NO4: C, 56.87; H, 6.20;
N, 6.63. Found: C, 56.87; H, 6.24; N, 6.62.
6-N-Malelmidohexanoic acid (50 mg, 0.23
mmol) and a,~-dichloromethyl methyl ether (150 ~1,
1.5 mmol) were refluxed~together in anhydrous
dichloromethane (1 mL) overnight. ;The mixture was
reduced to dryness and the resultant highly
hygroscopic solid~ (6-N-malelmldohexanolc acld
chloride t54 mg) was used,~freshly prepared~, in the
coupling reactions.
IR (fil~m):~1795 (COC~lj, 1700~cm l (maleimide).
PMR (CDC13): ~ 6.60 ~(2H~, s, maleimlde CH),~3.53
(2H, t, J = 7Hz), 2.90 (2H, t, J`= 7Hz), 1.6~ppm (6H,
m).
~ : :
B. Reaction of~6-N-Ma~leimidohexanoic Acid
Chloride with Leukotriene C~
- 4
~LTC4~tr;ipotas~sium~salt~5 mg)~ was~
~dlssolve~d~i~n;~anhydrous~methanol~(l;mL) and~
triethylamine (80 ~L) under~nitrogen and the acid
. ~ :
~26~
2298P/0832A - 20 - 16777IB
chloride (25 ~L of a solution of 10 mg acid chloride
in 100 ~L anhydrous THF) was added. The reaction was
stirred at room temperature and was followed by HPLC
(Whatman Partisil M9 10/25 ODS, eluting with
MeOH:H2O:HOAc; 70:30:0.01, 4 mL/min). The adduct
eluted at 4.8 min. and LTC4 eluted at 6.6 min.
After 10 and 30 min. about 15% of unreacted LTC4
remained. More of the acid chloride solution (5 ~L)
was added and after a further 10 min. 5% unreacted
10 LTC4 remained. The reaction mixture was
concentrated to 0.2 mL under a stream of N2,
diluted with borate buffer (pH 7.2, 0.IM, 0.5 mL) and
the residual methanol was removed ln vacuo. This
solution had UV spectrum essentially unchanged from
LTC4 itself, and was used as such in reaction with
thiolated KLH (see following).
C. Reaction of KLH with S-Acetylmercapt
succinic Anhvdride
KLH (60 mg) was dissolved in borate buffer
(0.2M, pH 8, 1.5 mL) and centrifuged to remove
denatured protein. The resultant solut~ion analyzed
for 24.6 mg/mL by UV;[E278(mg/mL) = 1.36]. The
solution was deoxygenated (by three purges
alternating high vacuum and pure N2 flush) then
treated under N2 with S-acetylmercaptosuccinic
anhydride (45 mg~added in~5 mg portions over~one
hour). The pH was maintained at 8 by addition of lN
~NaOH (total 400 ~L). Af~ter standing one ~our more,
N-ethylmaleimide~(20 mg in 0.1 mL MeOH) w s added to
bind any free thiol groups~and stabilize the;solution
to air.~ After stand;ing l.5 hours more the solution
: :
:
~: :
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~6~35~
2298P/0832A 21 - 16777IB
was centrifuged and applied to a column Sephadex G-50
tl.5 x 75 cm) eluting with 0.1N saline buffered with
0.01N pH 6.2 phospha~e buffer. Two fractions (7 mL)
eluting after the void volume contained the bulk of
the protein (2.4 mg/mL). An aliquot analyzed for
thiol content, after hydrolysis at pH 11.5 for one
hour, indicated 18 thiol groups per 100,000 daltons
protein.
D. Coupling of 6-N-Maleimidohexanoic Acid
Amide of LTC4 and Thiolated KLH
A solution of S-acetylmercaptosuccinate
derivative of KLH (from reaction C) (10.8 mg, in 4.5
mL 0.1N salinej buffered to pH 6.2 with 0.01N
phosphate) was rigorously deoxygenated and then the
pH was raised to 11.5 with lN NaOH (150 ~L) under
N2 and the mixture was left at room temperature for
one hour. The pH was then reduced to 7.2 by addition
o~ deoxygenated lN HCl (150 ~L) and the~solution of
the 6-N-maleimldohexanoic acid amide derivat~lve of
LTC4 from reaction B~was added. After standing 2
hours at room temperature, N-ethylmaleimide (1 mg in
10 ~L methanol) was added and the mixture~was left
one hour more at room temperature. This so~lution was
appli~d to a Sephadex~G-50 column~(l.5~x 7~,5~cm)
elu~ing with 0.lN saline~buffered~to pH 6 with 0.01N
phosphate. The protein fraction eluted with 85% in
ll;mL after~he vold~volume. Unreacted r~agents
eluted at the dead volume (150 mL). The protein
i 30 ~solution~was ad~usted to pH 7.2 with lN NaOH ~:~fqr ::
storage. ~
:
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~:
~26~35~
2298P/0832A - 22 - 16777IB
Analysis of the protein solution by W
indicated 7-10 equivalents of LTC4 were coupled per
100,000 daltons protein.
The conjugates of LTC4 with the proteins
BSA and KLH have been used to raise antibodies with
rabbits, at a dose of 200 ~g/rabbit, approximately
weighing 1 kg; the antibodies specifically recognize
Leukotrienes C4, D4, and E4. A detailed
description of the antibody production, specificity,
and the use of these conjugates in an immunoassay for
the leukotrienes follows.
In addition to LTC4 and the specific
proteins used, it will be appreciated that other
leukotrienes, such as LTD4 and LTE4 can be
conjugated with other antigenic proteins such as
tetanus antigen, human se~um albumin (HSA), as well
as diphtheriae toxide, tetanus antigen,~ and CRM 197
(from coryne bacterium diphtheriae) and other similar
antigenic materials. ``
EXAMPLE 3
Immunization Usin~ LTC~g~lg~g~
The following ~iS~ thè~immunization reglme
used employing KLH-maleimi;do-LTC4~as the immunogen.
Three 4 month old~New~Zealand White rabbits
each received sub-cataneoas~injectlons at~mult1ple
sites of 200 ~g KLH-LTC4 in complete Freunds~ ~
adjuvant~fôllowed~in~thre~e weeks by sub-cutaneous
injections~at mult~iple~sites;with~lOO ~g~LKH-LTC4
3 ~ in~lncomplete~Freunds~adjuvant; . The~rabbits were
bled lO~dàys aft~er~the second injection and every~
three~ weeks~thereafter. When~a~significant decline
:
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2298P/0832A - 23 - 16777IB
in the level of antibody was observed, the animals
were boosted with 200 ~g KLH-LTC4 in incomplete
Freunds adjuvant and the animals bled again on the
same schedule.
The antigen BSA-DNP-LTC4 was employed in a
solid-phase-immuno-radioassay (SPIRA) in order to be
used for the dectectîon of leukotrienes.
Polyvinyl chloride - 96 well microtiter
plates (Dynatech Laboratories) were coated with
antigen (BSA-DNP-LTC4) by incubating 100 ~1
aliquotes of the antigen at 0.1 mg protein/ml in PBS
for 18 h at 4C. The wells were washed three times
with 200 ~1 PBS and then unreacted sites in the wells
were blocked by incubating a 200 ~1 aliqout of 10%
horse serum in PBS in the wells for 2 h at 22C. The
wells were then washed three times with 200 ~1 of
PBS-1.5 H.S.(1.5% horse serum in PBS). One hundred
(100)~1 of a reaction m`ixture containing a diiution
of the immune or pre-immune rabbit serum was added to
the wells and the pl~ates 1ncubated for~4 h~;at 2~2C.
The 100 ~I reaction employed for the ti~ration of
rabbit serum consisted of~50 ~1 of dilutions~of the
sera in PBS-1.5~H.S. and~50~ 1 bf~P~S-l.5~H.S.~;For
competition~analysis~this~reaction~mixture consisted
of 50 ~1 of a dilution of~immune serum in PBS-1.5
H.S. which contained a limiting amount~of leukotriene~
specific~antibody and;50 ~1~ of PB5~ .5~. H.S.~
containing various~c;oncent;rations of~leukotrienes or
chemically~relat~ed~compounds.~ This 100 ~1 reaction
mixture~was prei;ncubated l~h at 22C be~ore it wa.s
added~to~the~well of;the~microtlter plate.~
:
: ~
2298P/0832A - 24 - 16777IB
The wells of the microtiter plate were then
washed three times with 200 ~1 PBS-1.5 H.S. and then
100 ~1 of 125I-labeled rabbit anti-mouse
[F (ab)2 fragments of rabbit anti-mouse IgG (H +
L)] in PBS containing 10% horse serum was added to
the wells and the plates incubated 4 h at 22C.
Approximately 2 X 104 cpm of the iodinated reagent
was added to each well. After the incubation period,
the wells were washed five times with 200 ~1 PBS-1.5
H.S. and once with 200 ~1 PBS. The wells were then
cut from the plate and the radioactivity in each well
was determined in a gamma counter.
The advantage of this assay is that,
although the rabbits were immunized with
KLH-maleimido-LTC4, therefore antibodies are
present in these animals against KLH, against the
maleimido linker and against the hapten-LTC4,
however, antibodies directed against the KLH and the
maleimido linker do not cross react or bind to the
BSA or DNP linker of the~material coated onto the the
surface of~ the wells. Therefore the only antibodies
that bind to the;material coated on the;wells~
~LTC4-DNP-BSA) are~directed against the LTC4.
These rabbit LTC4~antibodies bind~to;the
LTC4 portion of the conjugate and~they~in turn are
detected by adding a second species of antibody ~ `~
(125I-labeled goa~t ant~i rabbit ant~ibodiesj.~ Thqre~
a`ntibodies are radio-labeled with iodine and wilI
bind~to the rabbit~antibodies wh~ich in turn are bound
3~. to LTC4.~ The net~result is~ the~more antibodies
directed against~LTC4,~the~;more~radloactivity
associated~wlth the wel~
~::
~ ~ ,
.
. ' .
- 25 - 16777IB
In order to determine if free LTC4 is in a
biological sample, an aliquot is added to the plastic
well. Some of this free LTC4 will bind to the rabbit
anti LTC4 displacing if from the antigen coating which
is bound to the surface. This results in a decrease in
the number of counts (125I) bound to the surface of the
well and by comparing this decrease to the decrease in
a standard curve where known amounts of free LTC4 are
added, the amount of LTC4 in the sample can be deter-
mined.
The other compounds described in Examples land 2 can be similarly used in an assay system, as
reagents.
The antisera produced in rabbits by immuni-
zation with these conjugates can also be used in con-
junction with radio-labelled leukotrienes C4, D4, or E4
as the basis of a radioimmunoassay for Leukotrienes C4,
D4, and E4.
These conjugates are useful as chemical
immunotherapeutic agents in the treatment various
allergic and chronic inflammatory diseases of the~skin,
lung, and airways,~ including asthma, allergic rhinitis,
rheumatoid arthritis, and skin diseases such as pso-
riasis and eczema.
~ LTC4 ANTIBODY ASSAY
In a standard Guinea Pig Ileum assay, 4
t~issues were set up ln 10~ml~baths~ of Kreb'~s~buffer
with atropine and pyrilamine both~at 10 6M.
Standard~contractions were observed;using l0
~l of 2.7 x l0 6M LTC4~solution in a 10 ml~bath, for a
final eoneen~ra=l~r of~2.'~x l~0 9M LTC4~
:
:
~ ~ ,q ~.
s~
2298P/0832A - 26 - 16777IB
The standard response tension was 1.1-2. 0
grams.
20 ~1 of stock LTC4 solution was mixed
with a varying amount (10 ~1, 40 ~1, 100 ~1 and 400
~1) of anti-LTC4 serum (rabbit).
(1 ml of serum contained 7.9 x 10 9 M of
specific anti-LTC4 antibody).
The serum was incubated on ice (in the dark)
for 1/2 hour before use.
Control samples were run using similar~
amounts of normal rabbit serum.
The mixed samples (15 ~1, 30 ~1, 60 ~1 and
210 ~1 respecitvely) were added to baths and the
response recorded.
~ RESULTS
Volume o~ Antlbody_Serum ~ % of Control Response
205;~ 100.0
20 ~ 72.7
50 ~ 92.3
200~ yl ` ~ ; 64.7
Volume`of Normal Serum
2Q ~ 85.7
~ 50~ 102.~6
30 ~ 200~ ?' ~ :114.~3
,
.
.
85~
2298P/0832A - 27 - 16777IB
From the above results, clearly the
anti-LTC4 serum diminished the effect of LTC4 in
each sample. Thus the conjugates can be used to
raise antibodies to LTC4, LTD4, LTE4, in humans
(in a manner similar to that employed herein in
rabbits). The resulting circulating levels of
antibodies would serve to diminish plasma levels of
LTC4, and LTD4 and LTE4 released during an
asthmatic anaphylactic re~ponse and thus serve to
alleviate the symptoms. Since the antibodies would
be present during long periods of time, this would
represent a long term asthma therapy.
EXAMPLE 4_- Conju~ates of LTB4
Materials and Cond_tions
PMR spectra were recorded on a Varian EM-390
or Bruker WM-4Q0 spectrometer. W spectra were
recorded on a Cary 210 spectrophotometer. Optical
rotations were measured using a Perkin Elmer Model
241 Polarimeter. Sephadex G-S0~tmedium grade) was
obtained from Pharmacia Fine Chemicals.
Bovine serum albumin was obtained as ;
crystallized~and lyophilized~grade from Sigma
Chemical Co. and Hemocyanin (Keyhole Limpet)~ was
obtained as lyophilized powder from Calbiochem
Behring Corp. ;
1. Conjugatlon of LTB4 with Bovine Serum Albumin
A. 5(S),12(R)-dihydroxy-6,14(Z)-8,10(Ej-eicosatetra-
eonic acid S;-1actone (LTB4 -lactone) (VI): ~ ~
~ 5(S),12(R)-dihydroxy-6,14(Z)-8,10(E)-eico
satetraenoa~te (12 mg) was stirred under nitrogen in
::
: :~: :: :
:
2298P/0832A - 28 - 16777IB
methanol (1.5 mL) and water (0.4 mL) with postassium
carbonate (22 mg) for 2.5 days at ambient
temperature. Most of the methanol was removed under
a stream of nitrogen (to leave about 0.4 mL volume)
and the mixture was diluted with O.lN pH 6.2
phosphate buffer (2.5 mL). The mixture was extracted
with ether (5 x 2 mL) and the combined ether extracts
were dried (Na2SO4) and reduced to dryness. UV
analysis of the resulting oil indicated that 8 mg of
LTB4 free acid was thus obtained. The oil was
dissolved in anhydrous ether (5 mL) and treated with
dicyclohexylcarbodiimide (DCC) (20 mg) at 0 under
nitrogen for 24 hours. TLC analysis (ethyl acetate:
hexane 2:3) indicated about 50~ conversion of LTB4
to the ~-lactone (Rf LTB4 = 0, Rf LTB4
lactone = 0.6). More DCC (30 mg) was added and after
2 days at 0 TLC indicated essentially complete
conversion to the ~-lactone. The mixture was
concentrated to 1 mL under N2, filtered, reduced to
dryness, taken up in ethyl acetate:hexane (2:3) (1
mL) and chromatographed on silica gel column (10 g)
eluting with the same solvent ot provide the
~-lactone contaminated with a small amount of
dicyclohexylurea. This material was further purified
by HPLC (Wa~ers 10~, ~-porasil; ethylacetate:hexane;
(1:2); 4 mL~min) to provide~the pure LTb4r-lactone
~VI) eluting at 5.7 min (603 mg, 77~ yield~from II).
The lactone crystallized as fine needles from
ether:hexàne~, mp 50.0~_50.5 ~]RT~= ~231.0
(C=0.3,~CHC13) W: ~max (e) (MeOH) 260 (37,20Q), 270
(5Q,Q00), 280 nm (39~400). S PMR (400 MHz) (CDC13):
0.87 (3H, t), 1~.2-1.4 (6H, m), 1.65 (2H, m), 1.93
: :~: :
:
:: : :~ :
:
.
5~i
2298P/0832A - 29 - 16777IB
(2H, m), 2.03 (2H, q, CH2, C-16), 2.32 ~2H, m, CH,
C-13), 2.48 (lH, dt, J=17.5, 7 Hz, one of CH2,
C-2), 2.62 (lH, dt, J=18, 5 Hz, one of CH2, C-2),
4.22 (lH, m, methine, C-12), 5.23 (lH, dt, J=10~5,
2Hz, methine, C-5), 5.35 (lH, dd), 5.45 (lH, t),
5.58 (lH, dd), 5.81 (lH, dd), 6.15 (lH, t), 6.29 (2H,
m), 6.41 (lH, dd).
B. N-(3-aminopropyl)-5-(S),12(R)-dihydroxy-6,14(Z)-
8,10(Ej-eicosatetraenoic acid amide (III):
Method 1. LTB4 ~-lactone (VI) 1.75 mg)
was dissolued in redistilled 1,3-diaminopropane (0.5
mL) and the mixture was left at room temperature for
18 hours. The excess diaminopropane was removed
under high vacuum to give the amide III, quantitative
yield, la~RT = -2 (C=0.17, CHC13).
W: ~max (~)~(MeOH) 259.5 (29,800) 269.5 (46,500),
280 (36,500). o PMR (400 MHz): ~ 2.03 (2H, q,~CH2
C-16), 2.21 (2H, t, -CH2-CONH-), 2~.31~(2H,-m,
CH2, C-13) r~ 2.76 (2H,~t, -CH2-NH2)~ 3.33 (2H~,~
q, -CONH-CH2-),~4~20 (lH, q,~ methine, C-12)~, 4.58
(lH, q, methine, C-5j,~5.3-5.43 (2H, m),~5.5S (lH,
dd)`, 5.78 (~lH, dd)~, 6.05~(1H, ~t~,~6.18-6.31 ~(2H, m),
6.36 (lH, broad NH, amide), 6.47 (lH,-~dd).
~ Method 2. Ethyl 5(5)~-be~n20yloxy-12(R)-
hydroxy-6,1g~(Z)-8~,10(E)-eicosatetraenoate (2.5~mg)
and~2-hydroxypyrid~ine~(1.5~ mg)~were dissolved in
1,~3-diaminopropane (0.~5 mL)~and~the~mixture~was left ~ ~
30 àt room;~temperature,~under~nitrogen, for 3~days. The ~;
` excess~diaminopropane was~removed~under hlgh~vacuum ~ ~
: `
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:: :: :
:
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85il~
2298P/0832A - 30 - 16777IB
at room temperature to provide crude III which was
used as such in the next reaction tw: ~ max 227,
260, 270, 280, 298 mm).
C. N-(3-[2,4-dinitro-5-fluorophenyl]aminopropyl)-
5(S), 12(R~-dihydroxy-6,14 (Z)-8,10(E)-
eicosatetraenoic acid amide (IV)-
, _ . . .......... . _.. _ __ .. .
The crude amino amide (III) from Step B,
Method 2 (2 mg) in anhydrous methanol (400 ~) and
triethylamine (8 ~1) was treated with 1,5-difluoro-
2,4-dinitrobenzene (4 mg) in methanol 200 ~1) at room
temperature for 15 minutes at which time reverse
phase TLC (RPTLC) (acetonitrile:water, 85:15:)
indicated complete~reaction of III (Rf=0.1) and the
appearance of a new yellow product (Rf=0.7). The
mixture was chromatographed on RPHPLC (Waters, 10 ~
~bondapak, C-18, acetonitrile:water, 70:30, l mL/min)
to provide the product IV~(1.8 mg) [a]RDT -18~.9
(C=0.37, MeOH).
W: ~ max (MeOH) 260, 270~ 280, 335, 380 (sh). ~PMR
(400 MHz) (acetone-d6:~ r 3.34 (2H,~q,~-CONH~-CH2
3.61 (2H, m, -CH~2-)~, 3.61 ~(2H, m, -CH2-NH-Ar), ~
3.84 (2H, m, 2-OH), 4.14 (lH, m, methine,~C-12) 4.58
(lH, m,~methine, C 5)~, 5.42~(3H,; m), 5.78 ~(~lH, dd,
J=14, 6Hz, H-ll), 6.00 (lH,~t, J=llHz,~H-7j, 6.21
(lH, dd, J=14, llHz, H-10), 6.30~(1H, dd~,~J=14, llHz,
H-9), 6.57 (lH, dd,;J-`14, llHz, H-8)~,~7.15~(1H,~d,~
JHF=15Hz), 7.27 tlH, broad,~ NH~ amide), 9.00 (lH,
d, JH, F=8Hz),~9~.~15 (1H, broad,~NH, amine).
,
.
~: .
85~
2298P/0832A - 31 - 16777I3
D. Coupling of compound IV with Bovine Serum
Albumin (BSA)
A solution of compound IV (from step C) (1.5
mg) in dimethylformamide (0.5 mL) was added to a
solution of BSA (15 mg) in 0.2N pH 8.5 borate buffer
(0.75 mL) and the mixture was allowed to stand in the
dark under nitrogen and at room temperature for 4
days. The mixture was centrifuged and the clear
supernatant was applied to a column of Sephadex~G-50
(1.5 x 75 cm) eluting with water. The yellow protein
fraction eluted cleanly in 20 mL, after the void
volume of about 55 mL. At about the 140 mL dead
volume a peak containing unreacted IV and byproducts
eluted. W analysis of the protein frac~ions gave a
spectrum max 266, (sH), 273, 283, 336, 420 nn.
Assuming 100~ recovery of BSA from the column,
calculations based on the peak at 273 nm, corrqcting
for contributions due to BSA and to the
dinitrobenzene chromophore, indicated that 5~.5 moles
of L?B4 were coupled per mole of BSA. The
absorption at 336 nm (assuming~ for~the ~
1,5-diamin~-2,4-dinitrobenzene~chromophor o~ about
27,000) indicated that~8.3 moles of LT84 were
coupled per mole BSA.
2. Con~ugation of LTB4 with Hemocyanin from~
Kevhole Limpets;(KLH) ;~
LT84 ~-la~tone (~I) (4 mg) was disso~lved
in a mixture of THF (l mL) and 99~ hydrazine~hydrate
~0c5 mL) and the mixtui~ was~stirred vigorously under
nitrogen~at room~-temperature~for 0.5 hours.~The
mixture was~estra~cted with ether ~3 x 2 m1)~ and the
:: : : : ~ : : ~ : :
:: : : :~ :: : :
359C~
2298P/0832A - 32 - 16777IB
combined organic layers were dried (Na2SO4) and
evaporated to dryness under a stream of nitrogen and
then ln vacuo to provide the hydrazide VII ~4.2 mg).
~]D =8.9 (C=0.28, MeOH). UV ~max (~)=260
(37,000) 269.5 (50,000), 280 (39,000). PMR (400 MHz)
acetone-d6): ~ 2.1 (2H, t), 2.27 (2H, m), 3.82 (lH,
m, NH2), 3.99 (lH, broad NH2), 4.14 (lH, m,
methine, C-12), 4.56 (lH, m, methine, C-5), 5.3-5.5
(3H, m), 5.77 (lH, dd, J=14, 6Hz, H-ll), 6.00 (lH, t,
J=llHz), 6.22 (lH, dd, J=14, llHz, H-10), 6.31 ~lH,
dd, J=14, llHz, H-9), 6.56 (lH, dd, J=14, 11 Hz,
H-8), 8.22 (lH, broad, -CO-NH-).
B. Reaction of LTB4 hydrazide (VII) with 6-N-
Maleimidohexanoic acid chloride-
LTB4 hydrazine (VII) 2.5 mg, 7 x 10
moles), in anhydrous methanol (l mL) and
triethylamine (20 ~LI was treated with a solution of
6-N-maleimidohexanoic a~id~chloride (8j (3.3 mg, 1.4
x 10 5 moles) in~ anhydrous~ THF~(10~0 ~L) under
nitrogen at room~temper~ature. TLC analysis
(chloroform~methanol~, 8~5~:15)~ indicated~complete
conversion~to~a less~polar product,~ The mixture~was
reduced to~dryness,~and the~residue was taken up in
deoxygénated methanol~(l.2~mL)~and used as such in
the next reac~tion. Th~e prodùct;could~be purified if ;
desired~by~reverse~phase HPLC ~ (Waters~10~, ~-Bondapak~
C-18; methanol:water; 75:25,~2mL/min), to give the ~
pure addu~ct VIII~elu~ting at~4.~5 min. W ~max ~(MeOH)
~ 260~(36,300),~270 ~(50,~000)~, 280.5 nm~(39,400).
On~concent~rat;ion~to~obtain PMR;spectra some
decompositlon~was~;noted;by~TLC. NoweVer~the spectrum
:
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:
2298P/0832A - 33 - 16777IB
(400 MHz) (acetone d6) contained a weak signal at
6.82 ppm indicating that the malemide unit was
present although partially reacted.
C. Coupling of Compound VIII with Thiolated KLH:
S-Acetylmercaptosuccinylated KLH was
prepared as previously described (8~. The
derivatized protein (KLHSAc) (10 mg) in 0.1 N Saline
buffered with 0.01 N pH 6.2 phosphate buffer (PBS) (5
mL) was rigorously deoxygenated; then the pH was
raised to llo S by addition of 0. lN NaOH. After
standing 1 hour nitrogen at room temperature, the pH
was reduced to 7.~2 by addition of 0.1N HCl. The
adduct VIII in methanol (1.2 mL) from reaction B
above, was added~and the mixture was stirred sIowly
under nitrogen for 18 hours. N-ethylmalemide (5 mg)
in methanol (0.1 mL) was added and the mixture was
stirred 1 hour more~ The methanol was~removed under
a stream of~nitrogen during l hour, the~mixtu~re was~
centrifuged and the sapernatant was fil~ered on ~
Sephadex G-50 eluting with pH 6.2 PBS. The~protein
eluted with 95% in 19 ml after the void~volume and
gave a~ W spectrum: ~max ~264 (sH),~273.5,~283.5 nm.
Assuming 9 mg of protein was recovered from the
25 - column and correcting the absorption at 273.5 nm for
contributions due to coupled per 100,000 daltons KLH.
`
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:i : : :
