Note: Descriptions are shown in the official language in which they were submitted.
~6~
MONOSACCHARIDE COMPOUNDS HAVING
IMMUNOSTIMULATING ACTIVITY
Field of the Invention
The present invention relates to novel compounds
which stimulate the activity of immune cells in animals
in a manner similar to that of lipid A, methods o pre-
paring such compounds and methods of treatment using such
compounds.
Description of the Prior Art
Lipid A is a component of the bacterial lipopoly-
saccharide which is a complex amphipathic molecule which
covers the outer surface membrane of Escherichia coll and
other gram-neyative bacteria. Lipid A is a uni~ue hydro-
phobic anchor substance which holds the lipopolysaccharide
molecule in place.
The exact structure of lipid A is still unkn~wn;
however, the components include a ~,1~6-linked glucosamine
disaccharide, 2-3 phosphate groups, 6-7 fatt~ acids, an
aminoarabinose, and ethanolamine. The lipid is structur
ally heterogeneous because the polar aminoarabinose and
phosphorylethanolamine residues occur in only a fraction
of the molecules. Microheterogeneity based on the
estsr-linked fatty acids is also indicated by the
results of a study of the purified monophosphoryl lipid.
The fatty acid composition of lipid A differs from that
of the conventional phospholipids by the presence of
~-hydroxymyristic acid and the near absence o tm-
saturated fatty acids.
There is considerable interest in lipid A, as well
as its precursors and its metabolites because of its
~iological activity. Lipid A is believed to be responsible
for the endotoxic, immunostimulating, tumor cell killing,
and interferon produckion stimulating activities of kh2
lipopolysaccharides. A comprehensive review of the
chemistry and biology of lipid A can be found in the C.
Galanos et al. article which appears in the "Inter~
,
~2~6~
24080-575
-- 2
national P~eview of siochemistry~ Biochemistry of Lipids II",
Volume 14, University Park Press (1977).
Substantial work has been done to determine the molecular
structure of lipid A and to identify the portions of the lipid A
molecule which are responsible for its biological activity.
In an article in the Journal of siological Chemistry, Vol.
254, No. 16 pp. 7837-7844 (1979) Masahiro Nishijima and
Christian R. H. Raetz noted the presence of two unidentiEied
lipids (Y~ and Y) which accumula-ted in certain Escherichia coli
mutan-ts defec-tive in phosphatidylglycerol synthesis, and appeared
"to be metabolites of lipopolysaccharide synthesis, " pp. 7837.
In a la-ter article in Journal of Bacterioloqy~ Vol, 145, No. 1,
pp. 113-121 (1981), Nishijima, Raetz and Christine E. Bulawa
characterized the lips X and Y as "precursors of lipid A bio-
synthesis", p. 118. Still later, Nishijima and Raetz, in an
article in The Journal of Biological Chemistry, Vol. 256, No. 20,
pp. 10690-10696 (1981) purified milligram quantities oE lipids X
and Y from their Escherichia coli mutants defective in
phosphatidylglycerol synthesis, and they speculated that the lipids
X and Y were disaccharides.
It has now been discovered that lipid X is an acylated a-D-
glucosamine l-phosphate containing ~-hydroxymyristoyl groups at
position 2 and 3. It is believed to be a biosynthetic
precursor of lipid A and it possesses a desirable ability to
stimulate the activity of immune cells in a manner similar to
lipid ~.
Summary of the Present Invention
According to the present invention there is provided a
.
, :
.. :
~`:
i
... .
~2~
24080-575
pharmaceutically acceptable composition comprising a compound of
the formula ~II)
A ~
B - ~) ~ JO II
/o~
NH
R
wherein
A and B are the same or different and Rl and R2 are the
same or different and A, B, Rl and R2 each represents H, Cl-C24
alkyl or hydroxyalkyl, C2-C23-alkenyl or a fatty acyl chain of a
C2-C24-alkanoyl or hydroxyalkanoyl or a C3-C24-alkenoyl, a sugar
or a water-solubilizing group and Z represents a water-solubilizing
group, with the provisos that if A, B, Rl and R2 all represent H,
then Z does not represent hydroxyl, phosphate, succinate or a
nucleotide, or if A, s, Rl and R2 all represent methyl or acetyl,
then Z does not represent hydroxyl, phosphate, succinate or a
nucleotide, as an active ingredient, in association with a pharm-
aceutically acceptable diluent or carrier.
According to the present invention there is further
. . provided a compound of formula II
A -~ .
B ~
R~ N H
R~
- 2a -
. .. . .
,~
,: .
; -
:-
~6~
24080-575
wherein A and ~ are the same or different and Rl and R2 are the
same.or different and A, B, Rl and R2 each represent ~, Cl-C24-
alkyl or hydroxyalkyl, C2-C23-alkenyl or a fatty acyl chain of a
C2-C24-alkanoyl or hydroxyalkanoyl or a C3-C24-alkenoyl, a sugar
or a water-solubilizing group and Z represents a water-solubilizing
group,
with the provisos that (a) if A, B, Rl and R2 all represent
H, then Z does not represent hydroxyl, phosphate, succinate or a
nucleotide, (b) if A, B, Rl and R2 all represent methyl or acetyl,
then Z does not represent hydroxyl, phosphate, succinate or a
nucleotide and, (c) if R1 represents hydroxymyristyl and R2 rep-
resents hydroxymyristyl optionally substituted in the hydroxyl
moiety by a palmitate then Z does not represent phosphate.
According to the present invention there is also provided
a process for preparing active ingredients of the pharmaceutical
compositions defined above which proress comprises a process for
preparing a compound of the formula (II)
A ~
~2
~1-0~0
~0~ 1 I
R, NH
R;,
- 2b -
- : .,
: `. ~ , '
:
~fi~ Z
24080-575
wherein
A and B are the same or different and Rl and R2 are the
same or different and A, B, Rl and R2 each represents ~, C -C24
alkyl or hydroxyalkyl, C2-C23-alkenyl or a fatty acyl chain of a
C2-C24-alkanoyl or hydroxyalkanoyl or a C3-C24-alkenoyl, a sugar
or a water-solubilizing group and Z represents a water-solubiliz-
ing group,
with the provisos that if A, s, Rl and R2 all represent H, then
Z does not represent hydroxyl, phosphate, succinate or a nucleo-
tide, or if A, B, Rl and R2 all represent methyl or acetyl, then
Z does not represent hydroxyl, phosphate, succinate or a nucleo-
tide, which process comprises
(a) deprotecting the reaction product of a compound of
formula III
Ph ~` O ~ CH2
O III
C~O N~
Rl R2
wherein
Rl and R2 are as defined above with a compound of the.
formula .
- 2c -
;: ~: : ... ..
~L~6669~;~
24080-575
ZH,
wherein
Z is as defined above,
to yield a compound of formula II and, if required substituting
A or B; or,
(b) deprotecting the reaction pro~uct of a compound of form-
ula IV
Ph ~ O ~ 2
O ~ O
o ~ ,,OH IV
~0
Rl
R2
wherein
Rl and R2 are as defined above
with an electrophilic reagant Z'
to yield a compound of formula II and, if required substituting
A or B.
Based on fast atom bombardment mass spectrometry and .
proton nuclear magnetic resonance studies, we have dis-
- 2d -
,
-3-
covered that lipid X is an acylated monosaccharide derived
from glucosamine 1-phosphate. Lipid X has a Mr of 711.87
as the free acid (C34H66N012P) and contains two ~-hydroxy-
myristate moieties, one attached as an amide at the 2
S position and the other as an ester at the 3 position of
the sugar. It has free hydroxyl groups at the 4 and 6
positions, and the anomeric configuration is alpha. The
structure of lipid X closely resembles the reducing end
subunit of lipid A, and it might represent a very early
precursor in the biosynthesis of lipid A.
The structure of lipid X may be represented as
follows:
H~cH
HO ~ O
CH2 C~O `OH
HC--OH ¢H2
(CH2),0 HC~--OH
CH3 (C~ H2)10
CH3
Lipid X and its immuno stimulating derivatives may
be represented by the following general formula:
A -O ~
CH2
B--O~ o II
0~
NH
36 R,
~ .
.
.
6 ~ /~ J' d
in which the preferred sugar stereochemistry is that of
glucosamine; A and B are the same or different, and are
H, or a hydrocarbon structure (as defined below) or a
fatty acyl chain (as defined below) or another functional
group (as defined below); R1 and R2 are the same or
different, and are H or a hydrocarbon structure or a
fatty acyl chain (preferably ~-hydroxymyristoyl as in the
natural product (I)). When a hydroxylated substituent is
present on A, B, Rl and/or R2 it may be further substi-
tuted with a fatty acyl chain. Substituent Z is a water-
solubilizing group such as a hydroxyl, a phosphate, a
succinate, a sulfate, a sugar residue, or a nucleotide.
A hydrocarbon structure may be an alkyl or a
hydoxyalkyl group of 1-24 carbon atoms, or it may be an
alkenyl group of 2-23 carbons. A fatty acyl chain may be
an alkanoyl or a hydroxyalkanoyl chain of 2-24 carbons,
or it may be an alkenoyl chain of 3-24 carbons. A
functional group may be a sugar or a water solubilizing
group, such as a succinoyl residue, a phosphate, a
2~ sulfate, or a nucleotide.
Combinations are excluded, in which all four of the
substituents (A,B,Rl, and R2) are ~, and Z is a hydroxyl,
a phosphate, a succinate, or a nucleotide; or in which
all four substituents (A,B,Rl,R2) are methyl or acetyl,
or combinations thereof, and Z is a hydroxyl, a phosphate,
a succinate or a nucleotide.
Lipid X may be chemically synthesized or it may be
isolated rom bacterial sources or modified by a combin-
ation of approaches.
The isolation of lipid X from a bacterial source and
verification of its structure are described in the experi-
mental work which follows:
Experimental Procedures
Bacterial Strains and Media - Temperature sensitive
35 E. coli K12 strains MN7 (ATCC No. 39328) was grown in LB
broth, which contains 10 g of NaCl, 10 g of tryptone, and
5 g of yeast extract/liter. Maximum accumulation of lipid
. .
:. .., ~ .
_5_ ~Z~6~
occurred when a log phase culture grown at 30C to an
absorbance at 550 nm of 0.4-0.6 was shifted to 42C for
3 h. This procedure was used whether we grew small
shaker cultures or large cultures (300 liters). At the
time of the harvest, the A550~m was 1.8. In a typical 300
l. fermentation, the cells were harvested by centrifuga-
tion through a continuous flow centrifuge and the cell
paste was stored at -80C. The yield was about 700 g.
of cell paste per 300 l. fermentation.
Growth Conditions for Radiochemical
Labelling of Lipid X
Cells of E. coli strain MN7 were prepared by first
growing them at 30C in 200 ml of LB broth to an absorbance
at 550 nm of 0.5. Then 1 mCi of [1-l4C]-acetate ~60
mCi/mmol or higher) was added to the culture and incubation
was continued at 42C for 4 hr. The cells were harvested
by centrifugation at 5,000 x g for 15 min. These cells
were khe source of the 14C labeled lipid X.
To label lipid X with 32Pi, a 50 ml culture of
MN7 growing on medium was allowed to reach A550 = 0.8
at 30C. The cells were collected by centrifugation and
resuspended in the same volume of medium lacking phos-
phate. Next, the cells were incubated in shaking culture
at 42C, 32Pi (100 ~Ci/ml carrier free) was added, and
the incorporation of label was allowed to take place for
3 hours. Finally, the cells were recovered by centri-
fugation, and the lipid X was obtained by the rapid
radiochemical extraction described below.
Analytical and PreParative Thin Layer Chromatography
(TLC) - TLC was performed either on silica gel H or 60
using either chloroform-methanol-water-concentrated
ammonium hydroxide (50:25:4:2, v/v) (solvent A) or
chloroform-pyridine-formic acid (20:30:7, v/v) (solvent
B).
Extraction and Fractionation of Li~ids - Method I.
Lipopolysaccharide was prepared from 110 g of cell paste
, ~ -
. ; ', ,: ~
.., . ~,
:................. ~ : ' :- '.
~:6~ 2
--6
by the method of Galanos et al. Eur. J. Biochem. 9,
245-249(1969). The yield of crude lipopolysaccharide
(including lipids X and Y) was 337 mg. Next, X and Y
were extracted from this crude material with 90 ml of
chloroform-methanol-water (30:10:1, v/v) to yield 93 mg
of a mixture predominantly consisting of lipids X and Y.
This preparation (59 mg) was subjected to preparative
TLC using 20 x 20 cm silica gel H (500 ~m) plates and
solvent A at a load of 3 mg/plate. The bands were
visualized with I2 vapor and recovered by extracting
the silica gel with chloroform-methanol-water (66:33:4,
v/v). The yield of lipid X was 30.3 mg.
Method II. This method is a modification of the
acidic Bligh-Dyer extraction described in Can. J. Biochem
lS Phvsiol. 37, 911-918(1959), which was designed for more
rapid, large scale purifications. About 35 g of cell
paste was suspended in 950 ml of chloroform-methanol-water
(1:2:0.8, v/v), and the mixture was shaken vigorously in
a 1 1 Erlenmeyer for 60 min. at 30C. The cell debris
was removed by centrifugation at 5,000 x g for lO min.
To the supernatant was added 250 ml each of chloroform
and water to yield a two-phase system. After urther
addition of lO ml. of concentrated HCl and vigorous
shaking in a 2 liter separatory funnel, the layers were
allowed to separate, and the lower phase was washed once
with fresh, acidic pre-equilibrated upper phase. The
washed lower layer was centrifuged to break the emulsion
completely, and it was concentrated by rotary evapor-
ation. The residue was dissolved in 60 ml of chloroform-
methanol-water (2:3:1, v/v) and applied to a 1.5 x 25 cm
column of DEAE cellulose (acetate form). The column was
successively washed with 100 ml of the same solvent and
100 ml of chloroform-methanol-40 mM ammonium acetate,
pH 7.4 (2:3:1, v/v). ~inally lipid X (along with lipid Y,
phosphatidic acid and cardiolipin) was eluted from the
`' '' '-
:
:
i6~
-7-
column with lO0 ml of chloroform-methanol-100 mM ammonium
acetate, pH 7.4 (2:3:1, v/v~.
The eluted material was detected by charring 5
~liter samples of each fraction, and the peak fractions
were pooled (75 ml final volume). Next, enough chloro-
form, methanol and phosphate-buffered-saline were
added in Bligh-Dyer proportions to give an upper phase
volume of 500 ml (approximately 1 ml of upper phase per
100 ~g of lipid X). Under these conditions lipid X
partitioned into the aqueous-methanol phase, while Y and
other phospholipids remained in the chloroform layer.
The purified lipid X was recovered from the upper phase
by adjusting the pH to 1.0 with HCl and adding a fresh
organic phase. This sample was dried by rotary evaporation,
redissolved in 3 ml of chloroform-pyridine-formic acid
(37:30:7, v/v), and finally purified on an 0.8 x 25 cm
silicic acid column equilibrated in this solvent system.
The pyridine and formic acid in samples from the final
silicic acid column were removed by adding methanol and
water in Bligh-Dyer proportions relative to the CHC13.
Additional concentrated HCl was then added until the pH
of the upper phase was l. The upper phase was removed,
and the lower phase was washed twice with pre-
equilibrated acidic upper phase. The washed lower phase
was dried under a stream of ~2 to recover lipid X, pre-
sumably as the free acid. Final recovery was about 30 mg
and the material was stored dessicated at -80~C~
Lipid Y was isolated essentially by the same method
as X. Following DEAE cellulose chromatography and
partitioning at neutral pH (see above), the lower phase
containing Y and some contaminating phospholipids was
dried by rotary evaporation. The residue was redissolved
in 4 ml of chloroform-pyridine-formic acid (60:30:7,
v/v). Final purification was achieved on a silicic acid
column (0.8 x 25 cm) equilibrated with the same solvent.
The pyridine and formic acid was removed as described
' ~' ~ ' .
~, . .
.: ,
~ ~6~
--8--
above for lipid X. Recov~ry of Y in the ree acic~ forrn
was about 12 mg/50 gm cell paste.
Rapid Preparation of RadiochemicallY Labeled Lipid X.
Cells of MN7 labeled with 32Pi or 14C acetate (as
above) are extracted under Bligh-Dyer conditions but
at pH 7 by use of phosphate-buffered saline as the
aqueous component. In this case lipid X is recovered
in the upper aqueous-methanol phase, while phospholipids
and Y are in the lower phase. Relatively pure lipid X
~G can be recovered from the upper phase by adjusting the pH
to 1 with concentrated HCl and adding fresh pre-equili-
brated lower phase. In this way the X is shifted back to
the lower phase, where it can be recovered. Radiochemica].
purity by TLC in solvents A or B is about 95%.
1~ This rapid preparation does not require column
chromatography. If material of greater than 99% purity
is req~lired, this can be achieved by silicic acid chroma~
tography in chloroform-pyridine-formic acid (37:30:7) as
described above. Radiochemical preparation of lipid Y is
not possible with the rapid technique.
Dephosphorylation of Lipid X - About 2-3 mg of
sample was suspended by sonication in 2.0 ml of 0.1 ~
HCl, heated at 100 C for 15 min and cooled. Then 5 ml
of chloroform~methanol (2:1, v/v) was added, mixed, and
allowed to stand for 10 min. The upper aqueous layer was
removed, and the upper aqueous layer of the blank chloro-
form-methanol-water (10:5:6, v/v) mixture was used to
wash the lower organic layer containing the dephosphory-
lated product ("dephospho X~). The organic layer was
filtered and dried with a stream of nitrogen. The extent
of dephosphorylation was 80-90~.
Preparation of Dimethyl Derivative of Lipid X -
~bout 2--3 mg of purified material was dissolved in 1.O ml
of chloroform-methanol (9:1, v/v) and treated with a few
3~ drops of diazomethane in diethyl ether that was sufficient
to give a faint yellow color. This resulted in the
: ~-
9 ~6~
methylation of the phosphate group and the formation of a
phosphate triester ("dimethyl X"). After 30 min at 25~C,
the solution was dried with a stream of nitrogen and
dissolved in 0.3 ml of CDCl3 for NMR spectroscopy.
Preparation of dephospho Y and dimethyl Y was carried
out exactly as for the corresponding X derivatives.
Preparation of li~id Y from lipid Y. Lipid Y was
prepared from Y by hydrolysis in the presence of
triethylamine (TEA). To 3 mg of lipid Y, 3 ml of water
and 100 ~l of TEA were added tO.24 M, pH 12.4~. The
mixture was heated at 100C for 2 hours and evaporated to
dryness under a stream of N2. By this procedure a con-
trolled deacylation was achieved, yielding a partially
deacylated lipid Y (designated Y ) and ~-hydroxymyristate.
The latter was removed from the residue with 2 ml of
acetone. Lipid Y retains the ester-linked palmitate of
lipid Y and has been analyzed further by FAB mass spectro-
metry and NMR analysis.
Preparation of UDP-diacYlglucos~mine and other nucleo-
tide derivatives of liPid X. To 5 mg of lipid X dissolvedin anhydrous pyridine are added 6 mg of UMP-morpholidate.
After an overnight incubation at 37C in a tightly stop-
pered tube, the product is purified by preparative thin
layer chromatography in solvent B, using 500 ~m silica
gel H plates. The nucleotide, which migrates just off the
origin, is eluted and the yield is 70% of theoretical.
FAB mass spectrometry of the nucleotide reveals a molecular
weight of 1018, as predicted for a compound with structure
V. The above method may be used to prepare any deri~ative
of II in which Z is a nucleoside diphosphate, and may be
used to make nucleotide derivatives of Y and Y .
Reaction of UDP-diacylglucosamine (V) with liPid X (I).
Incubation of 0.2 mM V with 0.2 mM I in the presence
of 1 mg per ml of a wild-type E. coli K12 cell-free
extract in 20 mm HEPES buffer at pH 8 results in the
formation of a disaccharide 1-phosphate with the structure
of VI. This disaccharide can be reisolated from the
: .
.' ' ; ~.
.. ..
~26~;~4~
--10--
reaction mixture by the methods described above for the
purification of lipid Y or by preparative TLC.
Chemical Synthesis and Modification of Lipid X and
Related Compounds. Methods for selective acylation or
alkylation of sugar hydroxyl groups are known. Products
of partial acylation or alkylation can be separated from
each other by high performance liquid chromatography or
column chromatography.
Compounds of formula II can be chemically synthesized
as shown in Scheme 1. Suitable known starting materials
would be the allyl 2-acylamido-3-0-acyl-4,6-0-ben~ylidene-
2-deoxy-~-D-glucopyranosides.
"
.
, ... ,~ ,. .. . . .
- - . . : - :
' ,
-11
5SCHEME 1
p~ o CH~ ,~'h~ C~
0~ o~ , a~~ ~
10. C--~O~C~=Ch~ ,
R C o / 2 c
~0 ~o
~O ~ ~H
Z/
Z~ /
~ ~0
Protected Partial ~ \
products ~ o ~
deprotect on c_O ~ Z (Prot)
Deprotection Rz
C~ i) Acylatlon or
2 alkylation
~ ~ ii) Deprotection
c~Z
R, C-O .
7T (/~,~5=~f)
.
: . ,: ,
6~ 2
The allyl glycosides (1) can be isomerized to
l-propenyl glycosides (2) hy treatment with tris(triphenyl~
phosphine) rhodium(I) chloride or with another suitable
isomerization catalyst. Treatment of the 1-propenyl
glycosides (2) with mercuric chloride-mercuric oxide
in an anhydrous medium will yield oxazolines (3). I
water is included in the reaction mixture the products
will be the l~hydroxy compounds (4).
The reaction of the oxazolines (3) with precursors
~ of Z groups, such as diesters of phosphoric acid or with
alcohols or partially protected sugars in the presence
of ferric chloride or other acid catalysts will result
in the attachment of (protected) Z groups to position 1
of the sugar. Removal of the 4,6-0-benzylidene group,
by hydrogenolysis or mild treatment with aqueous acid,
and removal of any protecting groups from the Z-moie-ty,
will give products of structure II, with A and B - H.
Other compounds of the series can be more expeditiously
made by treating the 1-hydroxy compounds (4) with electro-
~0 philic reagents, designated Z' in the scheme, such as acidanhydrides (e.i. succinic anhydride), acid chlorides, or
phosphorochloridates in the presence of suitable bases.
Deprotection will again give }I (A, B = H).
If the products of the reactions of (3) with ZH, and
of (4) wikh Z', are treated so as to selectively remove the
4,6-_-benzylidene group (partial deprotection), then OH-6
and OH-4 can be alXylated or acylated, completely or
selectively, by standard methods. After final depro~
tection, the products would have the structure Il, with A
and B as defined above.
HPLC Fractionation - High Pressure Liquid Chroma-
tography (HPLC) was performed with two ~OOOA solvent
delivery systems a solvent programmer, a universal
liquid chromatograph injector, a variable wavelength
detector and a Radial Compression Module. A 8 mm x 10 cm
Radial pak A cartridge (C18-bonded silica, 10 ~m) was
~- ., . .- :
,i .. :~. , :
-
used. For the analysis of lipid X, a linear gradient of0 to 100% 2-propanol-water (85:15, v/v) in acetonitrile-
water (1:1, v/v) was used over a period of 60 min at a
flow rate of 2 ml/min. Both solvent systems contained
5 mM tetrabutylammonium phosphate. Samples for HFL~
analysis were dissolved in chloroform-methanol (4:1,v/v).
The absorbance was monitored at 210 nm.
Mass Spectral analysis - FAB mass spectrometry was
performed on a MS-50 mass spectrometer at ambient temper~
ature, utilizing a neutral beam of xenon atoms from a
saddle field discharge gun with a translational energy
of 8 Kev and a discharge current of 0.4 mA. Samples
(100 ~g) were dissolved in 100 ~1 of chloroform-methanol
(1:1, v/v) and mixed with an equal volume of either
triethanolamine (for negative mode) or monothioglycerol
(for positive mode). The sample (5 ~liter) was deposited
on the FAB probe tip and the volatile solvents were pumped
away in the insertion lock chamber of the mass spectrometer.
Both negative (M - H) and positive (M ~ cation) ion mass
spectra were obtained by scanning at a rate of 90 atomic
mass units per sec. Measurements were based on a cali-
bration standard glycerol-potassium iodide (1:1, mol/mol)
as cluster ions. Mass assignments were made with an
accuracy of ~1.0 atomic mass units using the DS-55 data
system.
Proton NMR AnalYsis - Spectra were recorded at 200
or 270 MHz, on Nicolet NT-200 and Bruker WH-270 Fourier
transform, superconducting spectrometers interfaced with
Nicolet 1280 and 1180 computers, respectively. In
decoupling experiments the parent spectrum was first
determined with the decoupler power set "off resonance";
then the decoupled spectrum was determined. Difference
spectra were generated by subtracting the parent spectrum
from the decoupled spectra.
Results
Analysis of Purity of LiPid X - Purified lipid X was
examined by analytical TLC using solvents A and B and
.
~' ` `' '"`, , `` ,
. : .,.,, ,:: ::, . .
-14~ ;6~i~2
found to give a single major band which could be visual-
ized with 12 vapor, by charring, or by spraying with an
organic phosphate-specific molybdenum reagent. Lipid X
was readily separable from the incomplete lipid A which
migrated very slowly in the thin layer system with
solvent A.
Chemical analysis of a sample of lipid X purified by
TLC, and containing some silica, showed the following:
total phosphorus, 0.96 ~mol/mg; glucosamine, 1.02
~mol/mg; KD0, 0.04 ~mol/mg. The phosphorus-glucosamine-
KDO molar ratio was 1.00:1.06:0.04. The presence of
glucosamine as the sole amino sugar was confirmed by the
use of the amino acid analyzer on an acid hydrolyzed
sample. These results confirmed the results of a
l.'~ previous chemical analysis of lipid X.
Purified lipid X was analyzed by reverse-phase HPLC
before and after preparative TLC fractionation (Method
I). This analysis showed a single peak of lipid X
eluting at 30 min with an estimated purity by peak height
~0 analysis of at least 95%. There was no evidence for the
presence of a homologous series or further microhetero-
geneity of lipid X. Under these conditions of HPLC, a
purified monophosphoryl lipid A designated TLC-3 from S.
typhimurium and containing 6 fatty acid residues came off
the column at the end of the gradient.
Nature of the Phosphate GrouP in Lipid X - When
[14C]lipid X was treated with 0.1 N HCl at 100C for 15
min, a new product was formed with a Rf value of 0.53 on
TLC in solvent A (unhydrolyzed lipid X had Rf 0.12). The
radioactivity pattern of the TLC separation showed that
almost all of the label in l14C~lipid X was shifted to
the new product after hydrolysis. The time-course of
acid-catalyzed hydrolysis of [14C]lipid X was recorded.
~ver a period of 15 min, there is a rapid decrease of
~14C~lipid X and a corresponding rise in the level of new
product.
., .
.. - : -
` ~ ,
: .
-: ,, :
-15~ 6~
The distribution of the phosphorus content in the
aqueous and organic phases of lipid X before and after 15
min hydrolysis is shown in Table I.
Table I
Distribution of phosphate in the aqueous and organic
phase of control and acid-hydrolyzed lipid X. The sample
was prepared as described. Then 1/3.25 volume of the
aqueous phases and 1/3.75 volume of the organic phases
were analyzed for phosphate content.
,
nmol phosphate
Two-phase system ControlAcid hydrolyzed
Agueous layera 48.4 495.3
lS Organic layer 564.8 97.5
aInorganic phosphate assay (digestion was omitted).
The phosphate content is shifted from the organic
phase before hydrolysis to the aqueous phase after
hydrolysis. By utilizing the data in Table I and
establishing the partition coefficient of lipid X in the
chloroform-methanol-water (20:10:9, v/v) system, the
extent of conversion after 15 min hydrolysis was calcu-
lated to be 78%. Quantitative kinetic analysis of thehydrolysis reaction is complicated by the tendency of
lipid X to aggregate in aqueous solutions, and by the
precipitation of the product (dephospho X). A logarithmic
plot of the time-course data reveals a modest decrease
of the hydrolysis rate constant with time. The evidence
supports the conclusion that lipid X contains a single
type of acid-labile phosphate, attached to the l-position
of the glucosamine.
The results of negative and positive mass spectrometry
establïshed the precise value of Mr for lipid X to be
711 87 as the free acid (C34H66NO12P)
,: , . -
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-16~
lipid X contains one glucosamine, two hydroxymyristate
residues, and one phosphate, the latter linked to
position 1 of the sugar. Since one o the hydroxy~
myristic linkages is stable to mild alkali and the other
is cleaved by mild alkaline treatment, one hydroxymyristate
must be amide-linked (at position 2 of the glucosamine)
and the other ester-linked.
Proton NMR Analysis of Lipid X - Four possible sites
for the ester-linked hydroxymyristate had to be considered,
l() namely positions 3,4, and 6 of the glucosamine residue,
and the ~ position of the amide-linked hydroxymyristoy]
group. The principal purpose of the NMR spectroscopic
analysis was to distinguish between these alternatives.
Initially, spectra were taken of the free acid form of
lipid X dissolved in CDCl3, but these were poorly resolved,
presumably because of the aggregation of the amphipathic
lipid in solution. To obtain satisfactory resolution it
was necessary to esterify the phosphate moiety with
diazomethane, or remove it, and dissolve the resulting
derivatives (dimethyl X and dephospho X, respectively) in
CdCl3 with 1-10 percent dimethyl sulfoxide-d6.
The spectrum of dephospho X (not shown) resembled
that of dimethyl X. Data from decoupling experiments on
dephospho X substantiated the assignments made from
experiments with dimethyl X.
The normal range of the resonances for H-3 and H-4,
and the downshifting of these resonances on acylation at
the respective positions, is well established by obser
vations on numerous glucosamine derivatives, including
synthetic lipid A analogs. Thus, the downfield position
of the H-3 signal in the present case clearly delineates
0-3 o the glucosamine as the site of attachment of the
èstèr-linked hydroxymyristate in lipid X.
Discussion
The complete structure of the ylycolipid, lipid X,
which accumulates in certain phosphatidylglycerol-
deficient mutants of E. _oli (particularly at nonper-
. . -. -
, . : :
-17- ~2666~
missive temperature) has now been established. Lipid X
is 2-deoxy-2- [(R)-3-hydroxytetradecanamido]-3-0-[(R)-3-
hydroxytetradecanoyl]-~-D-glucopyranose l-phosphate and
it bears a striking resemblance to the reducing end subunit
of lipid A. The structure of lipid Y, which is essentially
the same as X but contains an additional esterified
palmitoyl residue on the ~OH of the N-linked hydroxy-
myristate, has also been completed using techniques
essentially identical to those described above for X.
Lipid X might be a very early precursor of lipid A
biosynthesis. Thus radiolabeled lipid X could be useful
as a substrate to study the pathway for the enzymatic
synthesis of lipid A in a cell-free system.
Lipid X might serve as a good model compound to
lS study the relationship between the structure of lipid A
and its numerous biological activities. The attractive
feature of lipid X is the simplicity of its structure and
the ease with which the structure can be modified by
controlled degradation. Preliminary experiments show
that lipid X is relatively nontoxic in the chick embryo
lethality test and that it is a B-cell mitogen.
There appears to be an interaction between
phosphatidylglycerol metabolism and lipid A bio-
synthesis. Mutants like 11-2 or MN7 (pgsA444 ~gsB1)
are isolated by a two stage mutagenesis. They are temp-
erature-sensitive for growth and phosphatidylglycerol-
deficient only when both genetic lesions are present.
Based on subcloning of pgsA and detailed enzymological
studies, it is certain that pgsA represents the structural
gene for phosphatidylglycerolphosphate synthase. The
E~_ may code for an enzyme in the lipid A pathway, for
instance one that converts lipid X to the next inter-
mediate. In this regard, it is relevant that strains
with the genotype pgsA pgsB1 re~ain normal phosphatidyl-
glycerol levels and are not temperature-sensitive, but
they continue to have more than 100 times the lipid X
present in wild-type cells (pgsA pgsB ). Perhaps, the
: ~ ,: .
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-18-
accumulation of lipid X caused by the pgsBl mutation
intereres with phosphatidylglycerophosphate production
when the synthase specified by the pgsA444 allele is
present. Furthermore, phosphatidylglycerol itself might
be required for lipid X utilization, since all components
of phospholipid metabolism and lipid A biosynthesis are
membrane-bound.
The Biological ~CtiVitY of Lipid X
The outer membrane of gram-negative bacteria such as
Escherichia coli and Salmonella tYphimurium consists of
proteins, phospholipids and lipopolysaccharide. The
latter substance is localized almost exclusively on
the outer surface of the outer membrane. It accounts
for many of the immunological and endotoxic properties
of gram-negatlve organisms. Although the complete
structure of lipopolysaccharide is unknown, it consists
of three domains. The outer sugars, which are highly
variable, give rise to the O-antigenic determinants.
The core sugars are relatively conserved and may be
involved in cell penetration by certain bacteriophages.
The lipid A molecule (which is highly conserved between
species) functions as a hydrophobic anchor holding
lipopolysaccharide in place. Since lipopolysaccharide
represents several percent of the dry weight of gram-
negative bacteria, it follows that lipid A (and notphospholipid) must account for most of the outer leaflet
of the outer membrane.
Since lipid X is easy to purify and can be further
manipulated by controlled chemical degradation, we have
examined its ability to activate mouse lymphocytes. We
have found that our lipid X preparations are almost as
active as intact lipopolysaccharide by thi.s criterion,
and that the ester-linked hydroxymyristate at position 3
is crucial for this biological function. The results
suggest that lipid X, like lipid A, is a B cell mitogen.
- ,~
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Methods for Evaluating Mitogenic
Activity of Lipid X
Lipid X was isolated from strain MN7. The mild
alkaline hydrolysis product of lipid X (which retains
the N-linked but not the 0-linked hydroxymyristate
residue) was obtained under standard conditions for
deacylation of phospholipids. Lipid Y was obtained
from lipid Y by treatment with triethylamine ~or 3 hours
at 100C. Dephosphorylated lipid X was generated by
mild acid treatment (~.l M ~Cl at 100C for 60 min). A
lipid A derivative lacking the 1-phosphate moiety at the
reducing end was generated as described in the literature.
Microspheres (polystyrenedivinylbenzene beads, 5.7 + 1.5
~M diameter, were coated with lipopolysaccharide or
dephospho-X by known methods.
Mitogenesis ExPeriments
C57Bl/10 and C3H/HeJ mice were anesthetized with
ether, and the spleens were excised immediately. After
opening the splenic capsule, the cells were suspended
2~ in 10 ml of DMEM, washed twice with the same, and then
resuspended at 5 x 106/ml in DMEM, supplemented with
10% fetal bovine serum, 2mM L-glutamine, 1 mM sodium
pyruvate, 10imM MEM non-essential amino acids, 10 mM
Hepes, 50 ~M 2-mercaptoethanol, 100 units/ml penicillin
G and 100 ~g/ml streptomycin. Multiple wells of a 96 well
Costar microtiter dish were seeded with 0.1 ml of this cell
suspension (5 x 105/well). Next, an additional 0.1 ml of
DMEM supplemented with an appropriate amount of test mitogen
was added. Eollowing addition of mitogen, the dishes were
incubated for 2 days at 37C, 100% humidity and 7.5% C02.
Finally, [methyl- H]-thymidine was added at 1 ~Ci/well, and
the cells were incubated at 37C for an additional six
hours. Cells were washed free of medium and excess
thymidine with 0.9~ NaCl using a Bellco microharves~er.
The washed cells were retained on glass fiber filter
strips, and radioactivity incorporated into the cells was
quantitated by li~uid scintillation counting. Each mitogen
.
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concentration was tested in triplicate wells, and tne
standard deviation of the measurement was approximately
~10%. Induction of plaque forming cells by various mitogen
preparations was quantitated using a monolayer of sheep
S red blood cells as the indicator.
Results
Mitogenic effect of the chloroform-soluble fraction
from mutant MN7. Membrane phospholipids were extracted
under acidic conditions from a strain of Ei. coli which
~0 is wild-type with respect to its membrane lipids or
from mutant MN7 which accumulates lipid X. The crude
lipids were dried under N2 to remove CHCl3, suspended in
1 mM EDTA adjusted to pH 6 with NaOH, and dispersed at a
concentration of 1-2 mg/ml by sonic irradiation for 5 min
J.5 at 25C in bath sonicator. E. coli lipopolysaccharide
was dissolved in 1 mM EDTA in a similar manner. Next,
triplicate sets of mouse lymphocyte cultures were
incubated for 48 hours with increasing amounts of added
phospholipid (0-5 ~g/ml final concentration). Following
~0 this, the cells were labeled for 6 hours with [methYl~-3
H]-thymidine, and incorporation of 3H into DNA was deter--
mined.
The phospholipid dispersions derived from MN7 s-timu-
lated lymphocyte proliferation to a much greater extent
than similar preparations from strains of E. coli with a
wild-type lipid composition. The stimulation of cells by
commercial E. coli lipopolysaccharide was compared. The
results suggest that there is an additional mitogenic
factor in the CHC13-soluble fraction of strain MN7 that
is not present in the wild-type. The main difference
between MN7 and wild-type E. coli lipids has been
attributed to the presence of glycolipids X and Y in
the mutant at levels that are 10,000-fold (or more)
greater than normal.
Mitogenic ActivitY of Purified LiPid X
The predominant glycolipid that accumulates in MN7
is lipid X~ a diacylglucosamine l-phosphate, substituted
. ~ ,
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., '`" , . . :
~; '
-21- ~Z66~
with ~-hydroxymyristate at positions 2 and 3. Lipid ~ is
readily dispersed in 1 mM EDTA (pH 6) at concentrations
of 1-2 mg/ml, especially with mild ultrasonic irradiation.
Dispersions of lipid X (tested in the range of 0-50
~g/ml final concentration) are almost as effective as
commercial lipopolysaccharide in stimulating lymphocyte
proliferation. However, unlike the commercial lipoply~
saccharide, this activity can now be attributed directly
to a highly purified and structurally defined molecule.
Phosphatidic acid, which is structurally similar to lipid
X, has no mitogenic activity. Phosphatidylcholine,
sphingomyelin, phosphatidylinositol, cardiolipin, phos-
phatidylserine, CDP-diglyceride, lysophosphatidylcholine,
lysophosphatidylethanolamine and lysophosphatidic acid
are also ineffective. Polymyxin B (50 ~g/ml), which
inhibits lipopolysaccharide-induced mitogenesis, also
abolishes lymphocyte stimulation by lipid X. This
supports the notion that lipids A and X have similar
mechanisms of action and that the stimulation of the
B lymphocytes is involved.
To further demonstrate that lipid X exerts its
effects by the same mechanism(s) as lipopolysaccharide
(or lipid A), we examined splenic lymphocytes from
C3H/HeJ mice. These are unresponsive to lipopoly-
saccharide, presumably because they lack a membranereceptor (or enzyme) that recognizes this molecule.
The C3H/HeJ lymphocytes also respond poorly (if at all)
to lipid X. However, they are readily activated by the
T cell mitogen concanavalin A, or by PPD-tuberculin,
which function by different mechanisms. T cells do not
seem to be required for lipid X mitogenesis, since splenic
lymphocytes from nude athymic mice respond well to lipid
X and lipopolysaccharide, but not to concanavalin A.
Molecular Requirements for Mitogenicitv
Mild alkaline hydrolysis of lipopolysaccharide
abolishes its mitogenic activity. Since the locations
of the ester-linked fatty acids in lipid A are not
: . '
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-22~ 4~
precisely known, it is unclear which particular ester-
linkage is required. The very fact that lipid X has
strong mitogenic activity implies that the ~-hydrox~my-
ristate esterified at the 3 position of this molecule
must mediate this function. To prove this, we subjected
lipid X to mild alkaline hydrolysis and isolated a
monoacyl glucosamine 1-phosphate derivative, bearing
only the amide-linked ~-hydroxymyristate. The removal of
the esterified hydroxymyristate completely abolishes the B
cell proliferation. Simultaneous addition of equimolar
~-hydroxymyristate and monoacyl glucosamine l-phosphate
or of glucosamine l-phosphate plus ~-hydroxymyristate did
not cause significant proliferation.
In addition to lipid X, mutant MN7 accumulates
lipid Y, especially after 3 hours at 42C. This material
is similar to X but has an additional esterified palmitate
moiety on the ~-hydroxyl group of the N-linked hydroxy-
myristate.
Lipid Y is much less soluble in H2O than lipid X,
but can still be dispersed at l mg/ml by sonic
irradiation at pH 6. The material is also mitogenic,
though somewhat less under these conditions, possibly
because of poor solubility. Selective removal of the
3-O-linXed ~-hydroxymyristate from the glucosamine ring
of lipid Y with triethylamine gives rise to the derivative
Y . This substance retains the esterified palmitate and
therefore is very similar to lipid X in its physical
properties. However, lipid Y is less active as a mitogen,
and in fact may be a useful specific inhibitor.
Mild acid treatment can be used to remove the
1-phosphate moiety from lipid X, leaving the two fatty
acid residues in place. The resulting substance, termed
dephospho lipid X, is very insoluble in H2O and cannot be
dispersed by sonic irradiation. Fine suspensions of
dephospho lipid X or preparations immobilized on hydro-
phobic beads do cause slight cell proliferation (not
shown). The finding that dephospho lipid X retains
- .
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-23- ~fi~
significant biological activity strongly suggests that
the sugar l-phosphate moiety is not obligatory for the
mitogenic response. This conclusion is further supported
by the observation that TLC-3, a lipid A derivative from
S. tvphimurium G30/C21 lacking the l-phosphate residue,
also is fully mitogenic when tested under the same
conditions.
Formation of Plaque-Forming Cells
We evaluated the stimulation of plaque-forming cells
by E. _oli lipopolysaccharide, lipid X, or other prepar-
ations. All derivatives which stimulated radioactive
thymidine incorporation also increased the incidence of
antibody-producing cells significantly. These results
show that lipid X, lipid Y, and perhaps also dephospho
lipid X all stimulate true lymphocyte proliferation and
that the observed increase in [methyl-3H]thymidine
incorporation is not a radiochemical artifact.
Since the complete covalent structure of lipid A is
not known, it has been difficult to elucidate the molecular
mechanisms by which lipid A (or lipopolysaccharide) trig-
gers diverse physiological responses, such as B cell
proliferation or endotoxic shock. The discovery of
biologically active monosaccharide lipid A fragments with
defined structures makes it possible for the first time
to explore lipid A function at a biochemical level. The
lipid X molecule retains most of the properties of intact
lipopolysaccharide with respect to the induction of B
lymphocyte proliferation. In this case the ester-linked
~-hydroxymyristate at position 3 of the glucosamine ring
is critical for function, while the phosphate moiety may
enhance biological activity by increasing the solubility
of the acylated sugar.
Lipid X also possesses other activities normally
associated with lipopolysaccharide. Recently we have
found that sheep respond to intravenous injection of
lipid X in a manner that resembles the pathophysiological
effects of Gram-negative endotoxin including both the
~, '"'
-24-- ~2fi~
characteristic pulmonary hypertension and the increased
lung lymph flow. Further, the limulus lysate assay for
Gram-negative endotoxin is positive with lipid X under
certain conditions, and removal of the ester-linked
hydroxymyristate abolishes clot-forming activit~. On the
other hand, lipid X is relatively nontoxic as judged by
the chick embryo lethality test (CELD50 >lO ~g). It
appears that many of the biological activities of lipid A
are mediated by the esterified hydroxymyristatoyl group
at position 3 o the sugar.
The compound lipid X and its immunostimulating
derivatives may be introduced to the immune cells of an
animal in place of lipid A to stimulate the immune cells
when such stimulation is desired. When thus employed the
compound(s) may be administered in the form of parenteral
solutions containing the selected immunostimulating
compound in a sterile liquid suitable for intravenous
administration. The exact dosage of the active compound
to be administered will vary with the size and weight of
the animal and the desired immunological response.
Generally speaking, the amount administered will be less
than that which will produce an unacceptable endotoxic
response in the animal.
It will be readily apparent to those skilled in the
art that the foregoing description has been for purposes
of illustration and that a number of changes may be made
without departing from the spirit and scope of the
invention. For example, although specific microorganisms
that produce lipid X in recoverable amounts have been
described, it will be apparent that other microorganisms,
including those modified by genetic engineering, may be
used to prepare lipid X. It will also be apparent that
lipid X and some of its useful derivatives may be
chemically synthesized using conventional techniques.
Therefore, it is to be understood that the invention is
not to be limited except by the claims which follow:
. .,
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