Note: Descriptions are shown in the official language in which they were submitted.
r,
134os~s
.. .
Membrane anchor/active compound conjugate, its prepara-
tion and its use
The invention relates to membrane anchor/active compound
conjugates having at least one active compound cov.alently
bonded to the membrane anchor compound(s), to a process
for their preparation and to their use.
Membrane anchor compounds are compounds which can pene-
trate into biological and synthetic membranes.
For example, these membrane anchor compounds can be
natural membrane lipoproteins as have already been iso-
lated from the outer membrane of Escherichia coli and have
now also been synthesized. The E. coli membrane anchor
compound is composed in the N-terminal region of three
fatty acids which are bonded to S-glyceryl-L-cysteine
(G. Jung et al. in "Peptides, Structure and Function",
V.J. Hruby and D.H. Rich, pages 179 to 182, Pierce Chem.
Co. Rockford, Illinois, 1983).
Moreover, conformation-stabilized alpha-helical polypep-
tides have already been described for the investigation
of biological membranes by means of models, see inter
alia alamethicin, an alpha-helical amphiphilic eicosapep-
tide antibiotic which forms voltage-dependent ionically
conducting systems in lipid membranes (Boheim, G., Hanke,
W., Jung, G., Biophys. Struct. Mech. _9, pages 181 to 191
(1983); Schmitt, H. and Jung, G., Liebigs Ann. Chem.
pages 321 to 344 and 345 to 364 (1985)).
There is a description in European Patent A1-330 of the
immunopotentiating action of lipopeptides which are ana-
logs of the lipoprotein from E. coli Which has been known
since 1973. Another European patent application, A2-114787,
deals with the ability of lipopeptides of this type to acti-
vate alveolar macrophages of rats and mice in vitro so that,
134~~5~
_ 2 _
after incubation with the substance for 24 hours, the
macrophages are able to eliminate tumor cells and, in'
particular, they significantly increase the production of
antibodies, for example against porcine serum albumin.
It is proposed in European Patent A2-114787 to use these
lipoprotein derivatives as adjuvants for immunization,
that is to say to employ the lipoprotein derivatives of
the E. coli membrane protein mixed with antigens to
improve the immune response.
There is a great need for substances which stimulate and
potentiate the immune response, in particular because
purified antigens can often be obtained in only minuscule
amounts; furthermore, when new batches of antigens are
employed there is always the possibility of new contami-
nants or decomposition products.
It is furthermore desirable not to have to inoculate an
experimental animal frequently but, where possible, to
obtain the desired immune response by a single dose of the
immunogenic material.
Hence it is an object of the present invention to increase
the formation of antibodies against antigens or haptens
and thus to obtain a specific immunopotentiating action.
The object is achieved according to the invention by the
new membrane anchor/active compound conjugate having at
least one membrane anchor compound and at least one active
compound covalently bonded to the membrane anchor com-
pound(s).
According to the invention, a process for the preparation
of membrane anchor compounds is also proposed, which
process comprises synthesis of the peptide, which is pro-
tected with protective groups in a manner known per se on
the functional groups at which no reaction is to take
~
r
134afi5~
- 3 -
place, by means of known coupling processes on a solid or
soluble carrier, such as a polymer (for example Merrifield
resin); covalent bonding of the carrier-bound peptides,
which have been synthesized in this way, via N-termini or
side-groups of the peptide to the membrane anchor com-
pound; isolation of the polymer/peptide conjugate, which
has been prepared in this way, by cleavage of the protec-
tive groups and the peptide/carrier bond in a manner known
per se, and thus the membrane anchor/peptide or the mem-
brave anchor/active compound conjugate being obtained.
The invention also relates to the use of the compounds for
the preparation of conventional and monoclonal antibodies -
in vivo and in vitro; however, it is also possible, in
an advantageous manner, to use the compounds according to
the invention in genetic engineering to facilitate cell
fusion, for the preparation of synthetic vaccines, for the
preparation of cell markers with fluorescence labels, spin
labels, radioactive labels or the like, for affinity chro-
matography, in particular for affinity columns; for lipo-
some preparations; as additive to human foodstuffs or
animal feeds, and as additTVe to culture media for micro-
organisms and, generally, for cell cultures. This may
entail, where appropriate, the compounds according to the
invention.being used, together with vehicles known per se,
in solution, ointments, adsorbed onto solid carriers, in
emulsions or sprays, for purposes in human or veterinary
medicine.
The membrane anchor compound is preferably a compound of
one of the following general formulae:
R - CO-0-CH2 R - O-CH2 R - 0-CO-CH2
I [
R'- CO-0-CH* R'- O-CH* R'- 0-CO-CH*
i i
(CH2)n (CH2)n (CH2)n
i
A A A
i i
(CH2)m (CH2)m (CH2)m
I
R"-CC-NH-CH*-CO-X R"-CO-VH-CH*-CO-X R"-CO-NH-CH*-CO-X
I. II. III.
- 4 -
130656
R -NH-CO-CH2 R -CO-NH-CH2
I
R'-NH-CO-CH* R -CO-NH-CH*
I I
(CH2)n (CHZ)n B
A A
i
(CH2)m (CH2)m (CH2)m
R"-CO-NH-CH*-CO-X R"-CO-NH-CH*-CO-?( R-NH-CO-CH*-CO-K
IV. V. VI.
R~-CH2
I
R2-CH*
- I
(CH2)n
A
I
(CHZ)m
R-CO-NH-CH*-CO-X
VII.
it being possible for A to be sulfur, oxygen, disulfide
(-S-S-), methylene (-CH2-) or -NH-;
n being 0 to 5; m being 1 or 2;
B can be each of the substituted s-alkyl radicals
formulated in I to V;
C* being an asymmetric
carbon atom with R or S configuration; R, R' and R" being
identical or different and being an alkyl, alkenyl or
alkynyl group having 7 to 25 carbon atoms or hydrogen,
which can optionally be substituted by hydroxyl, amino,
oxo, acyl, alkyl or cycloalkyl groups, and R1 and R2 being
identical or different and being defined as R, R' and R"
or possibly being -OR, -OCOR, -COOR, -NHCOR or -CONHR, and
X being an active compound or a spacer-active compound
group.
It is also possible, in an advantageous manner, to use a
membrane anchor/active compound conjugate according to the
invention of the following general formula:
i3~as~~
-5-
i
R3 - NH - CH - CO - X
VT_iI.
R3 being an alpha-acyl-fatty acid residue having between
7 and 25 carbon atoms; preferably between 10 and 20 car-
bon atoms and very particularly preferably having between
14 and 18 carbon atoms; an alpha-alkyl-beta-hydroxy-fatty
acid residue or its beta-hydroxy ester, the ester group
being preferably straight-chain or branched chain and
having more than 8 carbon atoms, preferably between about
and 20 and very particularly preferably between 14 and
10 18 carbon atoms; it is possible and preferable for for- '
mula VIII to be an active compound conjugate with the
following membrane anchor compounds: N,N'-diacyllysine;
N,N'-diacylornithine; di(monoalkyl)amide or ester of
glutamic acid, di(monoalkyl)amide or ester of aspartic
acid, N,0-diacyl derivative of serine, homoserine or
threonine and N,S-diacyl derivatives of cysteine or homo-
cysteine; serine and homoserine; R4 being a side chain of
an amino acid pr hydrogen; and X being hydrogen or a
spacer-active compound group, it being possible when R3 is
a side chain of lysine, ornithine, glutamic acid, aspartic
acid or their derivatives for the latter to be bonded,
both in the manner of an ester and in the manner of an
amide in the same molecule, in alpha or omega positions
to R4.
25. It is particularly preferred for the membrane anchor/
active compound conjugates to be prepared by synthesis of
the peptide, which is protected with protective groups in
a manner known per se on the functional groups at which
no reaction is to take place, by means of known coupling
processes on a solid or soluble carrier, such as a polymer
(for example Merrifield resin); covalent bonding of the
carrier-bound peptides, which have been synthesized in
this way, via N-termini or side-groups of the peptide to
the membrane anchor compound; isolation of the peptide
conjugate, which has been prepared in this way, by
134065
- 6 -
cleavage of the protective groups and the peptide/
carrier bond in a manner known per se, and thus the mem-
brane anchor peptide or the membrane anchor/active com-
pound conjugate being obtained.
The linkage between the peptide and the membrane anchor
compound can be produced by condensation, addition, sub-
stitution or oxidation (for example disulfide formation).
It is possible to use, in an advantageous manner, con-
formation-stabilizing alpha-alkylamino acid helices
with an alternating amino acid sequence as the membrane
anchor, it not being permissible for the alpha-helix to
be destabilized by the other amino acids, such as of the
type X-(Ala-Aib-Ala-Aib-Alan-Y, n being 2 or 4, and X and
Y being protective groups which are known per se or -H,
-OH or -NH2.
It may be advantageous for the active compound to be co-
valently linked to two membrane anchor compounds which
are, where appropriate, different.
In addition, it is also possible for the active compound
to be covalently linked to an adjuvant which is known per
se for immunization purposes, such as, for example, mura-
myldipeptide and/or to a lipopolysaccharide.
Examples of active compounds which we propose are: an
antigen such as, for example, a low molecular weight par-
tial sequence of a protein or conjugated protein, for
example of a glycoprotein, of a viral coat protein, of a
bacterial cell wall protein or of a protein of protozoa
(antigenic determinant, epitope), an intact protein, an
antibiotic, constituents of bacterial membranes, such as
muramyldipeptide, lipopolysaccharide, a natural or syn-
thetic hapten, an antibiotic, hormones such as, for exam-
ple, steroids, a nucleoside, a nucleotide, a nucleic acid,
an enzyme, enzyme substrate, an enzyme inhibitor, biotin,
avidin, polyethylene glycol, a peptidic active compound
such as, for example, tuftsin, polylysine, a fluorescence
134a65G
marker (for example FITC, RITC, dansyl, luminol or cou-
marin), a bioluminescence marker, a spin label, an alka-
loid, a steroid, biogenic amine, vitamin or even a toxin
such as, for example, digoxin, phalloidin, amanitin,
tetrodoxin or the like, a complex-forming agent or a
drug.
The nature of the active compound determines the completely
novel areas of use which emerge for the substances accord-
ing to the invention.
It may also be beneficial for several membrane anchor/
active compound conjugate compounds to be crosslinked '
together in the lipid part and/or active compound part.
The membrane anchor compounds and the active compound can
also be connected together via a crosslinker, which
results in the active compound becoming more remote from
the membrane to which it is attached by the membrane
anchor.
Examples of suitable crosslinkers are a dicarboxylic acid
or a dicarboxylic acid derivative, diols, diamines, poly-
ethylene glycol, epoxides, malefic acid derivatives or the
like.
According to the invention, an unambiguously defined, low
molecular weight conjugate which is suitable, inter alia,
for immunization and which covalently links together the
carrier/antigen/adjuvant principles is prepared. The carrier
and adjuvant can be not only a lipopeptide having mitogenic
activity, such as, for example, tripalmitoyl-S-glyceryl-
cysteine (Pam3Cys) and its analogs, but also lipophilic
conformation-stabilized alpha-helices and combinations
thereof, such as Pam3Cys-antigen-helix, alpha-helix-
antigen-helix or even merely Pam3Cys-antigen or antigen-
Pam3Cys (N- or C-terminal linkage), and antigen-helix or
helix-antigen (N- or C-terminal, or incorporated in the
helix on side chains of Glu, Lys and the like). Thus, the
1340~~~
_8_
new compounds differ in essential aspects from all the
high molecular weight conjugates of antigens with high
molecular weight carrier substances which have hitherto
been used, for example proteins, such as serum albumins,
globulins or polylysine or, in general, high molecular
weight linear or crosslinked polymers.
In particular, however, the new compounds also differ from
all hitherto known adjuvants which are merely admixed
and, accordingly, do not bring about specific presentation
of the antigen on the cell surface. The adjuvants hither-
to known have frequently required multiple immunizations
and have also resulted in inflammatory reactions in animal
experiments. A particular advantage according to the
invention is the possibility of reproducible preparation
of pyrogen-free, pure, unambiguously chemically defined
compounds, and this - in contrast to conventional com-
pounds or mixtures of various substances - also results
in an improvement in the reproducibility of antibody for-
mation. Hence, a particular area of use of the com-
pounds according to the invention is viewed as being the
area of antibody production, genetic engineering, the
preparation of synthetic vaccines, diagnostic methods and
therapy in veterinary and human medicine, since the new
conjugates have for the first time an action which speci-
fically stimulates the immune response, whereas the adju-
vants hitherto used have merely stimulated the immune
response non-specifically. Surprisingly, it is even
possible with the compounds according to the invention to
convert weakly immunogenic compounds into highly immuno-
genic compounds. Thus, a particular importance of the
invention derives from the possibility of dispensing with
animal experiments and costs' for the preparation of anti-
bodies, since the new immunogens are also highly active
in vitro. Moreover, because the immunization method is
not inflammatory, an animal can be used several times for
obtaining different antibodies.
Finally, it might also be possible to use the new immunogens
134as~s
- 9 -
to prepare polyvalent vaccines, i.e. for example a mem-
brane anchor to whose side chains several antigens or
haptens have been linked so that several different active
antibodies can be prepared by means of one immunization.
One example of a water-soluble, mitogenic lipid anchor
group is Pam3Cys-Ser(Lys)n-OH, which is particularly suit-
able for the preparation of the new immunogens as well as
for the preparation of fluorescent, radioactive and bio-
logically active cell markers. A particularly desirable
property of the membrane anchor/active compound conjugates
according to the invention is their amphiphilicity, i.e.
a partial water-solubility, since in this case it is con- '
siderably more straightforward to carry out biological
tests on animals and investigations with living cells.
Moreover, the artificial lipid bilayer membranes, lipo-
somes and vesicles which are required for some experiments
can be prepared, and are stable, only in an aqueous medium. -
An example of a suitable amphiphilic, biologically active
membrane anchor is Pam3Cys-Ser(Lys)n-OH. The serine resi-
due coupled to Pam3Cys favors immunogenic properties,
whereas the~polar, protonated epsilon-amino groups of the
lysine residues represent the hydrophilic part of the
molecule. Because of its multiple charges, this type of
compound has further interesting properties. Owing to
induction of interaction between cells, it can be used as
a fusion activator in the preparation of hybridoma cells,
especially when the lysine chain is relatively long, when
coupling to polyethylene glycol, or on covalent incorpora-
tion of the biotin/avidin system.
Furthermore, in an advantageous manner, it is possible to
use the compounds according to the invention for the pre-
paraticvn of novel liposomes by crosslinking, it being
possible for this to take place either in the fatty acid
moiety or in the peptide moiety.
The membrane anchor (Pam3Cys and analogs, and the helices)
i3~ass~
_ 10 - s
are also suitable for potentiating the cell/cell inter-
action when, for example, they are covalently combined
with the biotin/avidin system. Other advantageous proper-
ties of the compounds according to the invention are that
S they may facilitate cell fusion as is required, for
example, for work in genetic engineering. Furthermore,
the new immunogens can also be used in ELISA, RIA and bio-
luminescence assays.
Various Pam3Cys derivatives are lipid- and water-soluble
and have potent mitogenic activity in vivo and in vitro.
They are also very suitable for labeling of cells with
FITC and other markers such as RITC, dansyl and coumarin.
In particular, they can also be used for fluorescence
microscopy and fluorescence activated cell sorting (FACS).
A reasonably priced membrane anchor having an analogous
action to Pam3Cys is S-(1,2-dioctadecyloxycarbonyl-
ethyl)cysteine, whose preparation is described in detail
in the experimental.part. .
Specific coupling of the mitogenically active lipid
anchors to antigens can also be effected by crosslinkers,
such as, for example, with dicarboxylic acid monohydrazide
derivatives of the general formula:
X-NH-NH-CO-A-CO-B-Y
or
X-NH-NH-CO-A-COOH
where A and B are amino acid or (CH2)n, and X and Y are
protective groups known per se.
It is also possible to use every other suitable cross-
linker ~r spacer for the preparation of the new substances,
a particularly preferred embodiment of the invention
always being represented by the principle (low molecular
weight carrier and adjuvant)-(antigen) as long as it con-
tains lipopeptide structures with lipid membrane anchor
~~~as5s
- 11 -
functions and/or conformation-stabilized helices.
Particularly advantageous effects can be found by use of
the compounds according to the invention in affinity chro-
matography, for which purpose lipopeptide-antigen(hapten)
conjugates are particularly suitable. The latter are out-
standingly suitable for loading conventional reversed
phase HPLC columns (or preparation RP columns), this
entailing, for example, anchoring of a tripalmitoyl com-
pound, which has been applied in organic aqueous systems,
in the apolar alkyl layer. The presentation of the anti-
gen to the mobile aqueous phase remains the same as on
cell surfaces, and thus it invites adsorption of the
antibodies. Hence, it is possible to enrich or isolate
antibodies, which specifically react with the relevant
antigen, from dilute serum directly on an affinity column
of this type. The elution of the antibodies is effected
as with other affinity columns, for example by adjusting
the pH.
The intention now is to illustrate the invention in
detail below by means of examples, but first the abbre-
viations used in them are listed:
Aib - 2-methylalanine
TFA - trifluoroacetic acid
EGF R - epidermal growth factor receptor
Pam = palmitoyl radical
OCC - dicyclohexylcarbodiimide
DMF - dimethylformamide
FITC - fluorescein isothiocyanate
Fmoc - fluorenylmethoxycarbonyl
But - tert.-butyl radical
PS - DVB = styrene/divinylbenzene copolymer with 4-
(hydrox~methyl)phenoxymethyl anchor groups
HOBt - i-hydroxybenzotriazole
RITC - rhodamine isothiocyanate
Hu IFN-(Ly) 11-20 = antigenic determinant of human inter-
feron
DCH = Dicyclohexylurea
EE = Ethylacetate
134t~65~
- 12 -
The figures which are attached to illustrate the inven-
tion show:
Fig. 1 the scheme for the preparation of Pam-Cys(C1g)2-
Ser-Ser-Asn-Ala-OH
Fig. 2 the table of the 13C NMR spectra
Fig. 3 the 13C NMR spectrum of Pam3Cys-Ser-(Lys)4-OH x
3TFA in CDCl3
Fig. 4 the 13C NMR spectrum of Pam-Cys(Pam)-OBut in
CDCl3
Fig. 5 the 13C NMR spectrum of Pam-Cys(Pam)-OH in
CDCl3/CD30D 1:1
Fig. 6 the 13C NMR spectrum (J-modulated spin-echo
spectrum) of Pam(a-Pam)Cys-OBut
Fig. 7 the 13C NMR (100 MHz) of the alpha-helix
Fig. 8 the CD spectrum of the alpha-helix of HuIFN-(a-
Ly)-11-20
Fig. 9 the obtaining of antibodies using Pam3Cys-Ser-
EGF-R (516 to 529)
Fig. 10 an in vivo immunization experiment
Fig. 11 a comparison of the in vivo and in vitro immuni-
zation experiments and
Fig. 12 the mitogenic activation of Balb/c mouse spleen
cells using Pam3Cys-Ser-(Lys)4FITC.
First some preparation processes for substances according
to the invention and their precursors are now described
below:
I. Preparation of Pam~Cys-EGF-R (516 - 529)
After the customary stepwise synthesis (Merrifield synthe-
sis protecting with N a-Fmoc/CBut), with DCC/HOBt and
symmetric anhydrides) of the EGF-R segment (526-529), the
final e:rtWno acid attached was Fmoc-Ser(8ut)-OH. After
elimination of the Fmoc group with piperidine/DMF (1:1,
15 min), the resin-bound pentadecapeptide of EGF-R H-Ser-
(But>-Asn-Leu-Leu-Glu-(OBut)-Gly-Glu(OBut)-Pro-Arg(H+)-Glu-
(OBut)-Phe-Val-Glu(OBut)-Asn-Ser(But)-0-p-alkoxybenzyl-
134os5s
- 13 -
Copoly(divinylbenzene/styrene) (1 g, loading 0.5 mmol/g)
was linked with Pam-Cys(CH2-CH(OPam)CH2(OPam) (2 mmol, in
DMF/CH2Cl2 (1:1)) and DCC/HOBt (2 mmol, preactivated at
0oC for 20 min) (16h), followed by a second coupling
(4 h). The lipohexadecapeptide was cleaved off with tri-
fluoroacetic acid (5 ml) with the addition of thioanisole
(0.25 ml> within 2 h.
Yield:
960 mg - (76%) Pam-Cys(CH2-CH(OPam)CH2(OPam))Ser-Asn-Leu-
leu-Glu-Gly-Glu-Pro-Arg-Glu-Phe-Val-Glu-Asn-Ser-OH x
CF3COOH (correct amino acid analysis, no racemization).
II. Preparation of S-(1,2-dioctadecyloxycarbonylethyl)-N-
palmitoyl-L(or D)cysteine tert.-butyl ester
Dioctadecyl maleate can be obtained by the general pro-
cedure for esterifications of malefic acid (H. Klostergaard,
J. Org. Chem. 23 (1958), 108).
13C NMR spectrum:
see Fig. 2.
1.2 mmol (500 mg) of N-palmitoyl-L-cysteine tert.-butyl
ester and 1.2 mmol (745 mg) of dioctadecyl maleate are
dissolved in 20 ml of THF. After addition of 20 mmol
(3 ml) of N,N,N',N'-tetramethylethylenediamine, the mix-
ture is stirred under nitrogen with a reflux condenser
for 12 h. After addition of 100 ml of methanol and
5 ml of water, the colorless precipitate is filtered off
with suction, washed with water and methanol and dried in
vacuo over P205.
Yield:
1 g (83%>;
Melting point:
51 degrees Celsius
1340656
- 14 -
Thin-layer chromatography:
RF - 0.80; (mobile phase: CHCl3/ethyl acetate - 14:1)
13C NMR:
see Fig. 2.
Molecular weight:
C63H113N07S (1035.7)
Elemental analysis:
Calculated C 72.99 H 11.76 N 1.35 S 3.09
Found C 73.08 H 11.92 N 1.27 S 3.27
III. Preparation of S-(1,2-dioctadecyloxycarbonylethyl)-
N-palmitoylcysteine
0.48 mmol (500 mg) of the t-butyl ester described under
II is stirred in 65.3 mmol (7.45 g, 5 ml) of trifluoro-
acetic acid in a closed vessel at room temperature for
1 h. The mixture is evaporated in a rotary evaporator
under high vacuum, the residue is taken up in 1 ml of
chloroform, 50 ml of petroleum ether is added to precipi-
tate at -20 degrees C, and the product is dried in vacuo
over P205.
Yield:
420 mg (89%);
Melting point:
64 degrees Celsius
Thin-layer chromatography on silica gel plates:
RF - 0.73; (mobile phase: CHCl3/MeOH/H20 - 65:25:4)
13C NMR:
see Tab. 1.
Molecular weight:
C59H113N07S (980.6)
134os~s
- 15 -
Elemental analysis:
Calculated C 72.27 H 11.62 N 1.43 S 3.27
Found C 72.46 H 11.75 N 1.36 S 3.50
The new cysteine derivative and its t-butyl ester can be
separated into the diastereomers on silica gel and RP
chromatography. It is thus possible to prepare the two
pairs of diastereomers of the L- and D-cysteine derivative.
IV. Preparation of S-(1,2-dioctadecyloxycarbonylethyl)-
' N-palmitoyl-Cys-Ser(But)-Ser(But)-Asn-Ala-OBut
0.2 mmol (196 mg) of S-(1,2-dioctadecyloxycarbonylethyl)-
N-palmitoylcysteine is dissolved in 5 ml of dichloromethane,
and preactivation is carried out with 0.2 mmol (27 mg) of
HOBt in 0.5 ml of DMF and 0.2 mmol (41 mg) of DCC by
stirring at 0 degrees C for 30 min.
After addition of 0.2 mmol (109 mg> of H-Ser(But)-Ser(But>-
Asn-Ala-OBut in 3 ml of dichloromethane, the mixture is
stirred at room temperature for 12 hours. Without further
working up 40 ml of methanol are added to the reaction
mixture. The colorless product can be filtered off with
suction after 3 h. It is taken up in a little dichloro-
methane and again precipitated with methanol. After
washing with methanol, it is dried in vacuo over P205.
Yield:
260 mg (86%)
Melting point:
194 degrees Celsius
Thin-layer chromatography:
RF - 0.95; (mobile phase: CHCl3/MeOH/H20 - 65:25:4)
RF - 0.70; (mobile phase: CHCl3/MeOH/glacial acetic
acid - 90:10:1)
- 16 - 134x656
13C NMR:
see F ig. 2
Molecular weight:
C84H158N6014S ( 1508.3')
Elemental analysis:
Calculated C 66.89 H 10.56 N 5.57
Found C 67.10 H 10.41 N 5.52
V. Preparation of S-(1,2-dioctadecyloxycarbonylethyl)-N-
palmitoyl-Cys-Ser-Ser-Asn-Ala
53 umol (80 mg) of protected lipopeptide (IV) are stirred
with 13 mmol (1.5 g; 1 ml> of trifluoroacetic acid in a
closed vessel at room temperature for 1 h. After evapora-
tion under high vacuum, the residue is taken up twice with
10 ml of dichloromethane each time and evaporated each
time in a rotary evaporator. The residue is taken up in
3 ml of chloroform and precipitated with 5 ml of methanol
at 4 degrees Celsius in 12 h. The product is filtered
off with suction, washed with methanol and dried in a
desiccator over P205.
~ Yield:
63 mg (87%)
Melting point:
208 degrees Celsius (decomposition)
Thin-layer chromatography:
RF - 0.63; (mobile phase: CHCl3/MeOH/glacial acetic acid/
H20 = 64:25:3:4)
RF - 0.55; (mobile phase: CHCl3/MeOH/H20 = 64:25:4)
RF - 0.06; (mobile phase: CHCl3/MeOH/glacial acetic
acid - 90:10:1)
1340656
- 17 -
Amino acid analysis:
Cysteic acid 0.6; aspartic acid 0.93; serine 1.8;
alanine 1.0
Molecular weight:
C72H134N6014S (1340)
VI. Preparation of Pam~Cys-Ser-(Lys)4-OH:
Pam3Cys-Ser(Lys)4-OH was synthesized by the solid-phase
method (MERRIFIELD) on a p-alkoxybenzyl alcohol/PS-DVB
(1%) copolymer with N-Fmoc-amino acids and acid-labile
side-chain protection (but for serine and 8oc for lysine).
The symmetric anhydrides of the Fmoc-amino acids were used.
The coupling to Pam3Cys-OH was carried out by the DCC/HOBt
method and repeated in order to achieve as near quantit-
ative conversion as possible. In order to cleave the
lipopeptide off the carrier resin and to remove the side-
chain protection, the resin was treated twice with tri-
fluoroacetic acid for 1.5 h and the acid was then removed
in a rotary evaporator under high vacuum. The product was
recrystallized from acetone.
The elemental analysis and the 13C spectrum indicate thar
the lipopeptide is in the form of the trifluoroacetate.
Assuming that Pam3Cys-Ser-(Lys)4-OH is in the form of a
zwitterion, there are still three e-amino groups remain-
ing which can be protonated by three trifluoroacetic acid
molecules.
The 13C NMR spectrum of Pam3Cys-Ser-(Lys)4-OH x 3 TFA
shows that the compound is in the form of the trifluoro-
acetate. (Quartet of the CF3 group at 110-120 ppm, and
carbonyl signals at 161-162 ppm). Owing to the aggregation
of the polar part of the molecule, .the lines for the Lys
and Ser carbon atoms are greatly broadened. The carbonyl
signal at 206.9 ppm is produced by acetone which was used
for the recrystallization and which is still adherent.
1340656
- 18 -
Molecular weight:
1510.4
Elemental analysis:
Calculated C 56.40 H 8.70 N 7.56 S 1.73
Found C 55.58 H 9.33 N 6.54 S 2.61
Amino acid analysis:
The amino acid analysis showed that the ratio of
serine to lysine is 1:4.2. The characteristic decompo-
sition products of S-glycerylcysteine produced during the
hydrolysis (6 N HCI, 110°C, 18 h) were present (com-
parison with known standards). The peptide content was
calculated to be 83%. 3 TFA molecules per lipopeptide
correspond to a peptide content of 80.2%, which agrees
well with the analysis.
VII. Preparation of Pam~Cys-Ser-(Lys)4-OH x 3 TFA
VII.1. Coupling of Fmoc-Lys(Boc)-OH to the carrier resin
Fmoc-Lys(Boc)-OH (4.5 g, 9.6 mmol) in 15 to 20 ml of OMF/
CH2Cl2 1:1 (v/v) at 0 degrees C is mixed with DCC
(0.99 g, 4.8 mmol). After 30 min, the precipitated urea
is removed by filtration directly into a shaker vessel
which contains p-benzyloxybenzyl alcohol resin (2.5 g,
1.6 mmol of OH groups). After addition of pyridine
(0.39 ml, 4.8 mmol), the mixture is shaken at room tempera-
ture for 18 h. The solvent is removed by filtration with
suction, and the resin is washed 3 x each with 20 ml of
DMF/CH2Cl2 and DMF for each time. The resin is added
to 20 ml of CH2Cl2 and then mixed first with pyridine
(28.8 mmol, 6 equivalents) and then with benzoyl chloride
(28.8 mmol, 6 equivalents). The mixture is shaken at room
temperature for 1 h. The solvent is removed by filtration
with suction, and the resin is washed 3 x each with 20 ml
of CH2Cl2, OMF, isopropanol and PE 30/50.
- ,9 - 134065
11II.2. Symmetric Fmoc-amino acid anhydride
Fmoc-Lys(Boc)-OH (4.5 g, 9.6 mmol, 3 equivalents) is dis-
solved in 15 ml of CH2Cl2/DMF and, at 0 degrees Celsius,
DCC (4.8 mmol, 1.5 equivalents) is added. After 30 min
at 0 degrees Celsius, the urea is removed by filtration
directly into the reactor, and the process is continued
as indicated in the table below.
The following procedure applies to 1/5 of the amount of
resin used at the start (0.5 g, 0.32 mmol of OH groups>.
Fmoc-0-butyl-serine dissolved
(0.74 in
g, 1.91 '
mmol)
is
4 ml CH2/Cl2/DMF ius, (0.96 mmol)
of and, DCC
at
0
degrees
Cels
is adde d.
.Table: Sequential using
synthesis
of
the
peptide
symmetric s
Fmoc-amino
acid
anhydride
Operati on Reagent Time Number of
Cminl times
1 CH2Cl2 2 2
2 DMF 2 2
3 55% piperidine/DMF (v/v) 5 1
4 55% piperidine/DMF Cv/v) 10 1
5 DMF ~ 2 3
6 isopropanol 5 2
7 DMF 2 3
8 CH2Cl2 2 3
9 DMF 2 2
10 Coupling with 3 eq. of
symmetric Fmoc-amino acid
anhydride in DMF/CH2Cl2 1:1
(v: v); after 15 min addition
of 3 eq. of NMM
11 DMF 2 3
12 CH2Cl2 2 3
1340fi56
Operation Reagent Time Number
CminJ of
times
13 Completeness of coupling
checked by the Kaiser test;
steps 10-12 repeated if
necessary
14 Acetylation: 2 eq. of 15 1
Ac20 and 0.5 eq. of NMM in
20 ml of CH2Cl2
10 15 CH2Cl2 2 3
16 isopropanol 2 3
17 CH2Cl2 2 3
After 30 min, the urea is removed by filtration at
0 degrees C directly into the reactor, and the procedure
15 is continued as usually.
VII.3. Coupling to Pam;Cys-OH
Pam3Cys-OH (0.58 g, 0.64 mmol) is dissolved in 5 ml of
CH2Cl2/DMF 1:1 (v: v) and, at OoC, is mixed with H08t
(93 mg, 0.64 mmol) and DCC (0.64 mmol). After 30 min at
20 0°C, the mixture is poured directly into the reactor.
After shaking for 16 h, a second coupling is carried out,
with the same molar ratios as above, for 4 h. The solvent
is removed by filtration with suction, and the resin is
washed 3 x each with 20 ml of DMF/CH2Cl2 and DMF.
VII.4. Cleavage of the hexapeptide from the polymer
The Boc-protected peptide/polymer resin compound (about
1 g) from VII.3 is thoroughly Washed with CH2Cl2 and
shaken 2 x 1.5 h with a mixture of 5 ml of TFA and 0.5 ml
of anisole. The filtrate is evaporated in vacuo, and the
residue is taken up in 5 ml of CHCl3. Pam3Cys-Ser-(Lys)4-
OH x 3TFA crystallises out after addition of 50 ml of
acetone at -20 degrees Celsius, is removed by centrifuga-
tion and is dried under high vacuum.
13406~~
- 21 -
Yield:
0.41 g (85%)
Melting point:
205 degrees Celsius (decomposition)
Thin-layer chromatography on silica gel plates:
RF - 0.42;
(mobile phase: n-BuOH/pyridine/H20/glacial acetic acid -
4:1:1:2)
RF - 0.82
(mobile phase: n-BuOH/MeOH/H20/glacial acetic acid -
10:4:10:6)
Amino acid analysis:
Ser 0.95 (1); Lys 4 (4)
Molecular weight:
Cg7H159N10019SF9 (1852.6)
Elemental analysis:
Calculated C 56.40 H 8.70 N 7.56 S 1.73
Found C 55.58 H 9.33 N 6.94 S 2.61
VIII. Preparation of Pam~Cys-Ser-(Lys)4-OH-FITC x 2 TFA
Fluoresceine isothiocyanate (3.9 mg, 10 micromol) is
dissolved in 2 ml of chloroform and added to a solution
of Pam3Cys-Ser-(Lys)4-OH x 3TFA (18.5 mg, 10 micromol)
in 2 ml of chloroform. After addition of 4-methylmorpho-
line (10 microliters, 10 micromol), the mixture is stirred
for 1 h and the solvent is then removed in a rotary
evaporator. The residue is dissolved in 10 ml of chloro-
form/acetone 1:1. The yellow product forms a voluminous
precipitate at -20 degrees Celsius and is removed by
centrifugation and dried under high vacuum.
_ 22 -
Yield:
16 mg after purification on Sephadex LH 20
The product is in the form of the trifluoroacetate and
fluoresces very strongly on excitation with UV light of
wavelength 366 nm. Compared with the starting material,
a -amino group is covalently linked with FITC. This
results in the molecular formula Pam3Cys-Ser-(Lys)4-OH-
FITC x 2TFA, assuming the zwitterionic structure is
retained.
Molecular weight:
C106H169N11022S2F6 (2127.68)
Thin-layer chromatography on silica gel plates:
RF - 0.72
(mobile phase: n-butanol/pyridine/water/glacial acetic
acid = 4:1:1:2)
RF - 0.73
(mobile phase: n-butanol/formic acid/water - 7:4:2)
Amino acid analysis:
Ser 1.11 (1.00) Lys 4.00 (4.00)
The hydrolysis products of glycerylcysteine are present.
IX. Preparation of Pam~Cys-Ser-(Lys)'-OH x 3HCl
Pam3Cys-Ser-(Lys)4-OH x 3 TFA (185.2 mg, 0.1 mmol) is
just dissolved in a little chloroform, and approximately
the same volume of ethereal HCl solution is added. The
mixture is thoroughly shaken, whereupon there is some
precipitation but the major part remains in solution. The
mixture is evaporated to dryness in a rotary evaporator
and ether/HCl is added once more. After this procedure
has been repeated several times, the residue is dissolved
in a little chloroform, and acetone is added until the
solution becomes cloudy. The product crystallizes as a
colorless powder at -20 degrees C and is filtered off
with suction and dried under high vacuum.
- 23 - 134065fi
Yield:
153 mg
Molecular weight:
C81H159N10013SC13 (1619, 63)
Elemental analysis:
Calculated C 60.07 H 9.89 N 8.65
Found C 57.64 H 11.20 N 8.39
Excess HCl is still adherent to the product.
Field-desorption mass spectrometry:
The M+ peak appears at m/e 1510, together with M++1 and
M++2. The protonated fragments Pam3Cys-NH (908.5) at
m/e 909, 910, 911 and 912 are characteristic.
X. N,S-Dipalmitoylcysteine tert.-butyl ester
Palmitic acid (2.5 g, 9.6 mmol), dimethylaminopyridine
(130 mg, 0.9 mmol) and dicyclohexylcarbodiimide (9.6 mmol)
are dissolved in 100 ml of chloroform. The solution is
stirred for half an hour and N-palmitoylcysteine tert.-
butyl ester (2 g, 4.8 mmol), which has previously been
dissolved in 50 ml of chloroform, is added dropwise to the
other solution. After 1 1/2 hours, the solvent is removed
in a rotary evaporator, and the residue is taken up in
100 ml of chloroform/methanol 1:5. The product forms a
voluminous precipitate at -20 degrees C. It is filtered
off with suction and dried under high vacuum.
Yield:
2.3 g (73%)
Molecular weight: (mass spectrometer)
C39H75N~4S (655.20)
Elemental analysis:
Calculated: C 71.48 H 11.71 N 2.13 S 4.89
Found: C 71.72 H 12.14 N 2.12 S 4.77
1340656
- 24 -
Thin-layer chromatography on silica gel plates:
RF - 0.67 (mobile phase: chloroform/ethyl acetate 95:5)
RF - 0.73 (mobile phase: chloroform/cyclohexane/MeOH
10:7:1)
_13C NMR:
se ure 4
XI. N-(a-Tetradecyl-B-hydroxyoctadecanoyl)cysteine tert.-
butyl ester
N-(a-Palmitoylpalmitoyl)cysteine tert.-butyl ester (1.5 g,
2.3 mmol) is dissolved in 10 ml of i-propanol, and 1.5
times the molar amount of sodium borohydride is added.
The mixture is stirred for two hours, and after completion
of the reaction nitrogen-saturated 1 N hydrochloric acid
is added until there is no further evolution of hydrogen.
- This results in a voluminous precipitate of the product.
It is filtered off with suction, washed several times with
nitrogen-saturated water and dried under high vacuum.
Yield:
1.4 g (93X>
Molecular weight: (determined from the mass spectrum)
C39H77N04S - 656.11
Thin-layer chromatography on silica gel plates:
RF - 0.84 (mobile phase: chloroform/ethyl acetate 95:5>
Elemental analysis:
Calculated: C 71.39 H 11.83 N 2.13 S 4.89
Found: C 71.32 H 12.39 N 2.04 S 5.33
XII. N-(a-Tetradecyl-B-hydroxyoctadecanoyl)cysteine
N-(a-Tetradecyl-B-hydroxyoctadecanoyl)cysteine tert.-butyl
ester (1 g, 1.5 mmol) is treated with anhydrous trifluoro-
acetic ac id for 1/2 hour, and the latter is then removed
1340656
- 25 -
in a rotary evaporator under high vacuum. The residue is
dissolved in tert.-butanol and is freeze-dried.
Yield:
0.7 g (78%)
Molecular weight: (determined from the mass spectrum)
C35H69N04S 600.0
Elemental analysis:
Calculated: C 70.06 H 10.92 N 2.33 S 5.34
Found: C 70.36 H 10.44 N 2.45 S 5.01
Thin-layer chromatography on silica gel plates:
RF - 0.43 (mobile phase: chloroform/methanol/water
65:25:4)
XIII. N,S-Dipalmitoylcysteine
N,S-Dipalmitoylcysteine tert.-butyl ester (1 g, 1.5 mmol)
is treated with anhydrous trifluoroacetic acid for 1 h.
The latter is then removed in a rotary evaporator under
high vacuum, and the residue is taken up in tert.-butanol
and freeze-dried.
Yield:
0.8 g (89%)
Molecular weight: (determined from the mass spectrum)
C35H67N04S (598.00)
Elemental analysis:
Calculated: C 70.18 H 11.44 N 2.34 S 5.34
Found: C 69.97 H 11.31 N 2.50 S 5.17
Thin-layer chromatography:
RF - 0.30
(mobile phase: chloroform/methanol/glacial acetic acid
90:10:1)
134656
- 26 -
RF - 0.75
(mobile phase: chloroform/methanol/water 65:25:4)
RF - 0.81
(mobile phase: chloroform/methanol/ammonia (25%)/water
65:25:3:4)
13C NMR:
see F ig. 5
XIV. N-(a-Palmitoylpalmitoyl)-N'-palmitoylcysteine di-
tert.-butyl ester
Palmitoyl chloride (8 g, 30 mmol) is dissolved in 40 ml
of nitrogen-saturated dimethylformamide, and triethyl-
amine (60 ml, 60 mmol) is added. The mixture is stirred
under reflux in a stream of nitrogen for three hours,
during which the triethylammonium chloride which is pro-
duced in the formation of the tetradecylketene dimer pre-
cipitates out as a colorless salt. The reflux condenser
is then replaced by a dropping funnel, and a solution of
cysteine di-tert.-butyl ester (4.9 g, 15 mmol) in 20 ml of
dimethylformamide is slowly added dropwise. After 6 hours,
the solvent is removed in a rotary evaporator, and the
residue is taken up in chloroform and washed twice with
100 ml of 5% strength potassium bisulfate solution each
time and once with 1,200 ml of water. The organic phase
is dried over anhydrous sodium sulfate, and the solvent
is removed once more. At -20 degrees Celsius a mixture
of N-(a-palmitoylpalmitoyl)-N'-palmitoylcysteine tert.-
butyl ester and N,N'-dipalmitoylcysteine di-tert.-butyl
ester crystallizes out and the products are separated by
gel filtration on Sephadex~LH-20 in chloroform/methanol
1:1.
Yield:
6.4 g (40%)
Molecular weight: (mass spectrum)
C62H118N207S (1067.76)
deno+es ~~~l~rrYJa r /l
1340656
_ 27 _
Thin-layer chromatography on silica gel plates:
RF - 0.69 (mobile phase: chloroform/ethyl acetate 91:5>
XV. N-(a-Palmitoylpalmitoyl)cysteine tert.-butyl ester
N-(a-Palmitoylpalmitoyl)-N'-palmitoylcysteine di-tert.-
butyl ester (3.2 g, 3 mmol> is dissolved in a little
methylene chloride, and 100 ml of 9.1 N methanolic hydro-
chloric acid are added. The solution is transferred into
an electrolysis cell with a silver electrode as anode and
mercury as cathode, and is reduced at a constant voltage
of -1.1 V. The current falls from about 200 mA to almost
zero at the end of the electrochemical reduction. The
solvent is then removed in a rotary evaporator, and the
mixture of products comprising N-(a-palmitoylpalmitoyl)-
cysteine tert.-butyl ester and N-palmitoylcysteine tert.-
butyl ester is precipitated from methanol at -20 degrees
Celsius. These two compounds are separated by gel fil-
tration on Sephadex LH-20 in chloroform/methanol 1:1.
Yield:
1.5 g (76%)
Molecular weight: (determined from the mass spectrum)
C3gH75N045 654.09
Thin-layer chromatography on silica gel plates:
RF - 0.75 (mobile phase: chloroform/ethyl acetate 95:5)
Elemental analysis:
Calculated: C 71.48 H 11.71 N 2.13 S 4.89
Found: C 71.16 H 11.31 N 2.00 S 4.65
XVI. Preparation of an antigen conjugate with a confor-
mation-stabilized a-helical membrane anchor
Synthesis of HuIFN-a(Ly)(11-20)-(L-Ala-Aib-Ala-Aib-Ala)2-
OMe, a 20-peptide which has on the N-terminal end an anti-
genic determinant of human interferon (a(Ly » .
- 28 - 134x656
The synthesis of the lipophilic membrane anchor with a
functional amino group at the end, H-(Ala-Aib-Ala-Aib-
Ala)2-OMe, can be applied to other conjugates. The alpha-
helix can also be extended once or twice by the Ala-Aib-
Ala-Aib-Ala unit. It is advantageous for this purpose to
start from the pentapeptide Boc-Ala-Aib-L-Ala-Aib-L-Ala-
OMe. (R. Oekonomopulos, G. Jung, Liebigs Ann. Chem.
1979, 1151; H. Schmitt, W. Winter, R. 8osch, G. Jung,
Liebigs Ann. Chem. 1982, 1304).
XVII. Preparation of Boc-Asn-Arg(NO?)-Arg(N0~)-OH
XVII.1. Boc-Arg(NO?)-OMe
Boc-Arg(N02)-OMe (15.97 g, SO mmol) and HOet (6.67 g,
50 mmol) in DMF (100 ml) were added at -10 degrees Celsius
to HCl x H-Arg(N02)-OMe (13.49 g, 50 mmol) and NMM (5.5 ml,
50 mmol) in CH2Cl2 (12 mol) and the mixture was stirred
at -10 degrees Celsius for 30 min, at 0 degrees Celsius
for 1 hour and at room temperature for 3 hours. The reac-
tion was then stopped with a few drops of glacial acetic
acid. The precipitated DCU was removed by filtration, and
the solvent was removed under high vacuum. The oily resi-
due was dissolved in ethyl acetate with the addition of
a little n-butanol. After the organic phase had been
washed with 5% KHS04 solution, 5% KHC03 solution and
saturated NaCI solution, it was dried over Na2S04, and
petroleum ether (30-50) was added.and the mixture was
cooled to precipitate.
Yield:
20.30 g (76%);
Melting point:
130 degrees Celsius (decomposition);
Thin-layer chromatography:
RF(I) - 0.69,
RF(II) - 0.87,
l3~oss~
- 29 -
RF(III) - 0.81,
RF(IV) - 0.32,
RF(V) - 0.42.
Molecular weight determination
C1gH54N1009 (534.5)
Elemental analysis:
Calculated C 40.45 H 6.41 N 26.20
Found C 40.39 H 6.55 N 26.11
XVII.2. HCl x H-Arg(NO )-Arg(NO )-OMe
Boc-Arg-(N02)-Arg(N02)-OMe (20.00 g, 37.42 mmol) was
mixed with 1.2 N HCl/acetic acid (110 ml) and, after
30 min, the mixture was poured into stirred ether (600 ml).
This resulted in precipitation of HCl x H-Arg(N02)-Arg-
(N02)-OMe which was pure by thin-layer chromatography.
Yield:
17.3 g (98X);
Thin-layer chromatography on silica gel plates:
RF(I) - 0.37,
RF(II) - 0.29,
RF(III) - 0.44,
RF(IV) - 0.07,
RF(V) - 0.10.
XVII.3. 8oc-Asn-Arg(NO )-Arg(NO )-OMe
Boc-Asn-OH (8.39 g, 36.10 mmol) and HOBt (4.89 g,
36.10 mmol) in DMF (75 ml> were added at -10°C to HCl x
H-Arg(N02)-Arg(N02)-OMe (17.00 g, 36.10 mmol) and NMM
(3.98 mmol> in DMF (75 ml). After addition of OCC (7.53 g,
36.50 mmol) in CH2Cl2 (10 ml), the mixture was stirred
at -10 degrees Celsius for 30 min, at 0 degrees Celsius
for 1 hour and at room temperature for 3 hours. After the
reaction had been stopped with a few drops of glacial
1340fi5fi
- 30 -
acetic acid, the solvent was removed by evaporation in
vacuo, and the residue was taken up in a little methanol.
This solution was added dropwise to stirred dry ether.
The residue was removed by filtration and taken up in
methanol. The pure product precipitated out in th'e cold.
Yield:
18.25 g (78%)
Melting point:
170 degrees Celsius
Thin-Layer chromatography -
RF(I) - 0.59,
RF(II) - 0.67,
RF(III) - 0.66,
RF(IV) - 0.45,
RFCV) - 0.65.
Amino acid analysis:
Asx 1.00 (1), Arg 1.85 (2)
Molecular weight determination:
C22H40N12011 (648.6)
Elemental analysis:
Calculated C 40.74 H 6.22 N 25.91
Found: C 40.70 H 6.40 N 25.79
XVII.4. Boc-Asn-Arg(NO )-Arg(NO )-OH
8oc-Asn-Arg(N02)-Arg(N02)-OMe (18.00 g, 27.75 mmol) in
methanol (180 ml) was hydrolyzed with 1 N NaOH (80 ml> at
room temperature. After 2 h, the mixture was neutralized
with dilute HCI, and the methanol was removed by evapora-
tion in vacuo. Exhaustive extraction with ethyl acetate
was carried out at pH 3. The organic phases were washed
with a little saturated NaCI solution, dried over Na2S04
and the product was crystallized from a methanolic solu-
1340656
- 31 -
tion at -20 degrees Celsius.
Yield:
15.84 g (90%)
Melting point:
228 degrees Celsius (decomposition)
Thin-layer chromatography
RF(I) - 0.49,
RF(II) - 0.21
RF(III) - 0.26
RF(IV) - 0.05, -
RF(V) - 0.21.
XVIII. Boc-Ala-Leu-Ile-Leu-Leu-Ala-Gln-(Ala-Aib-Ala-Aib-
Ala)?-OMe
XVIII.1 Boc-Ala-Aib-Ala-Aib-Ala-OH
Boc-Ala-Aib-Ala-Aib-Ala-OMe (10.03 g, 20 mmol> in MeOH
<150 ml) was hydrolyzed with 1 N NaOH (40 ml, 40 mmol).
After 2.5 hours, the mixture was neutralized with 1 N HCI,
evaporated in vacuo and partitioned between EA/SX KHC03
(1:1; 1,000 ml). The aqueous phase was acidified to pH 4
with 5% KHS04 and was extracted five times with EA/1-
butanol (5:1). The organic phase was dried over Na2S04,
PE (30-50) was added, and the pentapeptide acid was pre-
cipitated in the cold.
Yield:
6.54 g (65%)
Melting point:
195 degrees Celsius (decomposition)
Thin-layer chromatography
RF(I> - 0.72,
RF(II) - 0.80,
134x656
- 32 -
RF(III) - 0.87,
RF(IV) - 0.95,
RF(V) - 0.80.
Amino acid analysis:
Ala 3.08 (3), Aib 1.98 (2>
Molecular weight:
~22H39N508 (501.6)
Elemental analysis:
Calculated C 52.68 H 7.84 N 13.96
Found C 52.70 H 7.90 N 13.89 '
XVIII.2. Boc-(Ala-Aib-Ala-Aib-Ala)~-OMe
Boc-Ala-Aib-Ala-Aib-Ala-OH (1.75 g, 3.48 mmol) and HO8t
(470 mg, 3.48 mmol) in DMF (10 ml) were added at -10
degrees Celsius to HCl x H-Ala-Aib-Ala-Aib-Ala-OMe (1.57 g,
3.48 mmol) and NMM (384 art, 3.48 mmol) in DMF (8 ml).
After addition of DCC (825 mg, 4.00 mmol) in CH2Cl2 (3 ml)
at -10 degrees Celsius, the mixture was stirred for 15 h
allowing it slowly to warm up spontaneously. After the
reaction had been stopped with a few drops of glacial
acetic acid, the DCU which~had precipitated out was
removed by centrifugation, the residue was washed twice
with cold DMF, and the solvent was removed by evaporation
in vacuo. The residue was taken up in 10 ml of CHCl3/MeOH
1:1 and chromatographed on Sephadex LH 20 in CHCl3/MeOH 1:1.
Yield:
2.246 g (72%),
Melting point:
160 degrees Celsius,
Thin-layer chromatography
RF(I) - 0.61,
RF(II) - 0.76,
1340656
- 33 -
RF(III) - 0.83,
RF(IV) 0.95,
-
RF(V) 0.81.
-
Molecular weight determination:
C40H7pN10013 (899.1)
Elemental analysis:
Calculated C 53.44 H 7.85 N 15.58
Found ~ C 53.42 H 7.90 N 15.40
XVIII.3. HCl x H-(Ala-Aib-Ala-Aib-Ala)2-OMe
8oc-(Ala-Aib-Ala-Aib-Ala)2-OMe (2.046 g, 2.276 mmol) was
mixed with 1.2 N HCl/AcOH (10 ml). After stirring for
30 min, the hydrochloride was precipitated with ether,
filtered off and dried over KOH under oil pump vacuum.
Yield:
1.805 g (95%)
Thin-layer chromatography:
RF(I) - 0.50, '
RF(II) - 0.38,
RF(III) - 0.71,
RF(IV) - 0.48,
RF(V) - 0.53.
XVIII.4. Boc-Gln-(Ala-Aib-Ala-Aib-Ala)~-OMe
Boc-Gln-OH (997 mg, 4.05 mmol) and HOBt (547 mg, 4.05
mmol> in DMF (10 ml) were added at -10 degrees Celsius to
HCl x H-(Ala-Aib-Ala-Aib-Ala)2-OMe (2.250 g, 2.70 mmol)
and NMM (298 Nl, 2.70 mmol) in DMF (13 ml). After addi-
tion of DCC (846 mg, 4.10 mmol) in CH2Cl2 (2 ml) at
-10 degrees Celsius, the mixture was stirred for 15 h
allowing it slowly to warm up spontaneously. After the
reaction had been stopped with a few drops of glacial
acetic acid, the precipitated DCU was removed by centri-
34 - 1340656
fugation, the residue was washed twice with a little cold
DMF, and the solvent was removed under oil pump vacuum.
The residue was taken up in 10 ml of CHCl3/MeOH 1:1 and
chromatographed on Sephadex LH 20 in CHCl3/MeOH (1:1).
Yield:
2.60 g (94%)
Melting point
223 degrees Celsius (decomposition)
Thin-layer chromatography:
RF(I) - 0.66, -
RF(II) - 0.73,
RF(III) - 0.79,
RF(IV) - 0.94,
RF(V) - 0.80.
Molecular weight determination:
C45H78N12015 (1027.2)
Elemental analysis:
Calculated C 52.62 H 7.65 N 16.36
Found C 52.65 H 7.68 N 16.32
XVIII.S. HCl x H-Gln-(Ala-Aib-Ala-Aib-Ala) -OMe
8oc-Gln-(Ala-Aib-Ala-Aib-Ala>2-OMe (2.60 g, 2.701 mmol)
was mixed with 1.2 N HCl/AcOH (15 ml). After 40 min, the
hydrochloride was precipitated with ether while stirring,
removed by filtration and dried over KOH under oil pump
vacuum.
Yield:
2.209 g (85%);
Thin-layer chromatography:
RF(I) - 0.48, -
RF(II) - 0.25,
- 35 - 134os~s
RF(III) - 0.54,
RF(IV) - 0.24,
RF(V) - 0.35.
XVIII.6. Boc-Ala-Leu-Ile-Leu-Ala-Gln-(Ala-Aib-Ala-Aib-
Ala)Z-OMe
Boc-Ala-Leu-Ile-Leu-Leu-Ala-OH (760 mg, 1.07 mmol) and
HOBt (145 mg, 1.07 mmol) in DMF (12 ml) were added at
room temperature to HCl x H-Gln-(Ala-Aib-Ala-Aib-Ala)2-OMe
(818 mg, 0.85 mmol) and NMM (94 ~rl, 0.85 mmol) in DMF
(10 ml). After addition of DCC (227 mg, 1.10 mmol) in
CH2Cl2 (1.5 ml), the mixture was stirred for 64 hours.
After the reaction had been stopped with a few drops of
glacial acetic acid, the precipitated DCU was removed by
centrifugation, the residue was washed twice with a little
cold DMF, and the solvent was removed under oil pump
vacuum. The residue was taken up in 8 mt of CHCl3/MeOH
(1s1) and chromatographed on Sephadex LH-20 in CHCl3/MeOH
(1:1>.
Yield:
774 mg (57%);
Melting point:
260 degrees Celsius (decomposition>;
Thin-layer chromatography:
RF(I) - 0.80,
RF(II) - 0.86,
RF(III) - 0.91,
RF(IV) - 0.77
RF(V) - 0.78.
Amino acid analysis:
Glx 1.00 (1), Ile 0.89 (1), Leu 3.10 (3), Aib 4.08 (4),
Ala 7.95 (8).
1340656
- 36 -
Molecular weight determination
C75H132N18021 (1622.0)
Elemental analysis
Calculated C 55.54 H 8.20 N 15.54
Found C 55.58 H 8.31 N 15.52
XIX. Preparation of Boc-Asn-Arg(N02_)-Arg(N02)-Ala-Leu-
Ile-Leu-Ala-Gln-(Ala-Aib-Ala-Aib-Ala)2-
XIX.1. HCl x H-Ala-Leu-Ile-Leu-Leu-Ala-Gln-(Ala-Aib-Ala-
Aib-Ala) )-OMe
8oc-Ala-Leu-Ile-Leu-Leu-Ala-Gln-(Ala-Aib-Ala-Aib-Ala)2-OMe
(754 mg, 0.465 mmol) was mixed with 1.2 N HCl/AcOH (10 ml).
After 50 min, the mixture was partly evaporated under oil
pump vacuum and, after addition of water (10 ml), freeze-
dried.
. Yield:
690 mg (95%>;
Thin-layer chromatography:
RF(I) - 0.71,
RF(II) - 0.52,
RF(III) - 0.78,
RF(IV) - 0.56,
RF(V) - 0.54.
XIX.2. 8os-Asn-Arg(N02)-Arg(N0~)-Ala-Leu-Ile-Leu-Leu-Ala
Gln-(Ala-Aib-Ala-Aib-Ala)~-OMe
Boc-Asn-Arg(N02)-Arg(N02)-OH (634 mg, 0.995 mmol) and HOBt
(135 mg, 1.13 mmol) in DMF (5 ml) were added at -5 degrees
Celsius to HCL x H-Ala-Leu-Ile-Leu-Leu-Ala-Gln-(Ala-Aib-
Ala-Aib-Ala)2-OMe (20 mg, 0.398 mmol) and NMM (44 micro-
liters, 400 micromole) in DMF (7 ml). After addition of
OCC (217 mg, 1.05 mmol) in CH2Cl2 (1.5 ml) at -5 degrees
Celsius, the mixture was stirred for 48 h allowing it to
1340656
- 37 -
warm up spontaneously. After the reaction had been
stopped with 3 drops of glacial acetic acid, the precipi-
tated DCU was removed by centrifugation. The working up
and purification by chromatography were carried out as
described previously.
Yield:
630 mg (74%);
Melting point:
195 degrees Celsius (decomposition);
Thin-layer chromatography:
RF(I) - 0.70,
RF(II) - 0.51,
RF(III) - 0.56,
RF(IV) - 0.45,
- 15 RF(V) - 0.68.
Amino acid analysis:
Asx 0.94 (1), Glx 1.00 (1), Ile 0.89 (1), Leu 3.16 (3),
Arg 1.95 (2).
Molecular weight determination:
Cg1H160N30029 (2138.5)
Elemental analysis:
Calculated C 51.11 H 7.54 N 19.65
Found C 51.14 H 7.60 N 19.66
XX. Preparation of the free eicosapeptide
XX.1. 8oc-Asn-Arg-Arg-Ala-Leu-Ile-Leu-Leu-Ala-Gln-(Ala-
Aib-Ala-Aib-Ala) -OMe x 2HCl
8oc-Asn-Arg(N02)-Arg(N02)-Ala-Leu-Ile-Leu-Leu-Ala-Gln-
(Ala-Aib-Ala-Aib-Ala)2-OMe (350 mg, 0.164 mmol) in 3 ml
of anhydrous methanol was mixed with 35 mg of Pd/active
charcoal and 12 ~l (0.075 mmol> of 6 N HCI. Hydrogen was
134656
- 38 -
passed through the solution while stirring at room tempera-
ture. After 20 min 8 ~l (49 micromole), and after 35 min
7 girl (42 micromole), of 6 N HCl were added. After a
hydrogenation time of about 50 min the cleavage off, as
checked by TLC, was quantitative. The catalyst was
removed by filtration and washed several times with a
little methanol. The solvent was rapidly removed by dis-
tillation in a rotary evaporator (oil pump vacuum, bath
temperature 25 degrees Celsius>, and the residue was taken
up in a little water and freeze-dried.
Yield:
332 mg (95%>;
Thin-layer chromatography:
RF(I) - 0.16,
RF(II) - 0,11,
RF(III) - 0.21,
RF(III) - 0.10.
XX.2. H-Asn-Arg-Arg-Ala-Leu-Ile-Leu-Leu-Ala-Gln-(Ala-
Aib-Ala-Aib-Ala) -OMe x 3HCl
Boc-Asn-Arg-Arg-Ala-1.eu-Ile-Leu-Leu-Ala-Gln-(Ala-Aib-Ala-
Aib-Ala>2-OMe x 2HCl (600 mg, 0.283 mmol> was mixed with
1.2 N HCl/AcOH (5 ml). After 30 min, the mixture was
partly evaporated in a rotary evaporator, and the residue
was mixed with water (10 ml> and freeze-dried.
Yield:
564 mg (97%);
Melting point:
245 degrees Celsius (decomposition)
Thin-layer chromatography:
RF(I) - 0.11
- 39 - 1340656
Molecular weight determination:
C86H157N28023C13 (2057.7)
Elemental analysis:
Calculated C 50.20 H 7.69 N 19.06 Cl 5.17
Found C 50.31 H 7.78 N 18.95 Cl 5.28
Materials and methods for the experiments
Chemicals
Analytical grade solvents were obtained from Merck, while
other solvents were dried and distilled by customary
methods. N-Methylmorpholine (Merck) was distilled over '
ninhydrin to remove sec. amines. 1-Hydroxybenzotriazole
and dicyclohexylcarbodiimide likewise originated from
Merck. All L-amino acid derivatives were obtained from
Sachem. Boc-Aib-OH and H-Aib-OMe x HCl were synthesized
by literature methods.
Thin-layer chromatography
Precoated silica gel 60 F254 plates (supplied by Merck)
and the following mobile phases were used:
(I) 1-8utanol/glacial acetic acid/water 3:1:1
(II) Chloroform/methanol/glacial acetic acid/water
65:25:3:4
(III) Chloroform/methanol/concentrated ammonia/water
65:35:3:4
(IV> Chloroform/methanol/water 65:25:4
(V) Chloroform/methanol 1:1
The following spray reagents were used: ninhydrin re-
agent, chlorine/4,4'-bis(dimethylamino)diphenylmethane
(TDM reagent) and Sakaguchi reagent. The reference used
was dicyclohexylurea with the following values:
RF(I) 0.91, RF(II) 0.82, RF(III) 0.92, RF(IV) 0.81,
RF(V) 0.83.
130656
- 40 -
Amino acid analyses
To establish the identity of the intermediates approxi-
mately 200 microgram samples of each of the protected
peptides were hydrolyzed in 6 N HCl at 110 degrees Celsius
for 24 hours. The intermediates and the target sequence
of the hexapeptide segment which contains the Leu-Leu bond
were hydrolyzed for 72 hours under conditions which were
otherwise identical. The amino acid analyses were carried
out with a Biotronic LC 6000 E amino acid analyzer using
the standard program.
Racemate determination
The hydrolyzed amino acids were derivatized as the n- '
propyl esters of the pentafluoropropionylamino acid and
the enantiomers were separated by gas chromatography on
glass capillary columns with Chirasil-Val. The reported
percentages of D-amino acids have not been corrected for
racemization caused by the hydrolysis.
Elemental analyses
Single C, H and N-determinations were carried out using
a model 1104 (Carlo Erba, Milan) elemental analyzer.
Melting points
Melting points were determined~according to Tottoli and
are uncorrected.
Recording of the spectra
13C NMR spectra: 30 mg of the protected eicosapeptide
were dissolved in 400 microliters of 12C2HC13/12C2H302H
(1:1) (supplied by Merck) and measured in a WM 400 8ruker
NMR spectrometer at 30oC for 12 h. Circular dichroism
spectra: solutions of the free eicosapeptide (c - 1-1.7 mg/
ml) in ethanol, trifluoroethanol, methanol, 1,1,1,3,3,3-
hexafluoro-2-propanol, water and ethanol/water mixtures
were measured in a Dichrograph II (Jouhan-Roussel).
Purification by chromatography
The protected peptide intermediates were, after termina-
1340656
- 41 -
tion of the coupling reaction and removal of the solvent
under oil pump vacuum, dissolved by addition of the same
volume of CHCl3/MeOH 1:1, the dicyclohexylurea was
removed by centrifugation, and the product was chromato-
graphed on Sephadex LH 20: column 3 x 115 cm; eluting
agent CHCl3/MeOH 1:1; amount applied 35 ml; flow rate
8.40 ml/10 min. The 3 ml fractions were examined by thin-
layer chromatography in system II (TDM reagent). The
peptides appeared in the elution volume 165-190 ml. The
fractions were combined, the solvent was removed in vacuo,
and the residue was dried over P205. Amino acid analysis
produced the expected values and a peptide content of
92-96%. .
Immunization tests
We have for the first time covalently linked a B-cell
mitogen, which is simultaneously an outstanding carrier
and a highly active adjuvant, to synthetic antigenic
determinants. For this we have used, inter alia, the syn-
thetic lipopeptide S-(2,3-bis(palmitoyloxy)propyl)-N-pal-
mitoylcysteinylserine (Pam3Cys-Ser) which represents the
N-terminal end of the lipoprotein from the outer membrane
of Escherichia coli. The amphiphilic properties, which
are particularly pronounced when covalently bonded to an
antigen, ensure, on the one hand, stable anchoring of the
S-glyceryl compound,, which carries three fatty acid
residues, in the lipid layer of the cell membrane. On
the other hand, this means that the antigen (or hapten),
which is usually more polar, is presented in the outer
hydrophilic layer of the membrane. Since the activating
effect of the lipoprotein is determined entirely by its N-
terminal part, the immunostimulant effect of Pam3Cys-Ser,
or analogs, is retained in all the conjugates which carry
it.
As an example, we detail the use of the concept for the
generation of specific antibodies against epidermal growth
factor receptor (EGF-R) Fig. 9. For this purpose, a
computer-assisted search for epitopes led to selection of
134os5s
42
the extracytoplasmic region 516-529, which was constructed
by Merrifield synthesis and finally Fmoc-Ser(But)-OH and
then Pam3Cys-OH were attached. The conjugate, which was
found to be homogeneous by analysis, was cleaved off from
the resin and then administered i.p., without further
additives, in a single dose to mice. After only 2 weeks
high titers of specific antibodies against the tetradeca-
peptide were found by ELISAs. An essential point is that
no antibody titers were obtained with the tetradecapeptide,
which is by itself obviously a weak immunogen, in control
experiments.
Since Pam3Cys conjugates are likewise highly immunogenic '
in cell cultures, it is possible in a rapid and elegant
manner to obtain conventional and monoclonal antibodies,
even against weakly immunogenic compounds, by in vitro
immunization.
The advantages of our concept in association with cell
cultures are: straightforward preparation of a chemically
unambiguously defined antigen-adjuvant conjugate in any
desired amount, in contrast to other conjugates a single
administration without multiple "boosters", and high
efficiency in vivo and in vitro. The considerable saving
in experimental animals, and frequently even dispensing
completely with in vivo immunization and a drastic saving
in time, especially in genetic engineering procedures, are
obvious. The experiments can also be carried out with
human cell culture systems.
Example of an in vivo immunization:
6- to 10-week old Balb/c mice were immunized by a single
i.p. administration of 50 micrograms and 500 micrograms
(0.2 ml of a 10 1 to 10 2 molar solution of adjuvant co-
valently coupled to antigen) of Pam3Cys-Ser-(EGF-R 515-529).
The controls used were antigen, adjuvant and a mixture of
antigen and adjuvant, in each case in comparable molar
amounts, and medium. Two weeks after the injection, blood
was taken -from the retroorbital venous plexus of the mice
i34os5s
- 43 -
to obtain serum, and the antibody titer was determined by
ELISA.
Analogous immunizations can also be obtained by other
administrations, for example i.v., oral,~rectal, i.m.
and s.c.
The formation of specific antibodies without Freund's
adjuvant against the tetradecapeptide EGF-R 516-529 after
in vivo immunization was examined.
8alb/c mice were immunized once i.p. with 0.2 micromol of
the conjugate -
I. Conjugate Pam3Cys-Ser (EGF-R 516-526)
II. Free tetradecapeptide EGF-R 516-529 alone
III. Pam3Cys-Ser alone
IV. Free tetradecapeptide (EGF-R 516-529) mixed together
with Pam3Cys-Ser
as shown in Fig. 10. The antibody titer was determined by
ELISA. (Ordinate OD at 405 nm) (Fig. 10).
14 days after the immunization the mice were bled from the
ophthalmic vein and the serum which was obtained was used
in ELISA. The values emerge from the mean (3-5 mice) of
the difference between the ELISA values of PEP 14 - BSA
conjugate and BSA (Fig. 10).
It is evident that only when the membrane anchor/active
compound conjugate according to the invention is used are
drastically elevated antibody concentrations, which exceed
the activity of those with previous processes by a multi-
ple, found.
Example of an in vitro immunization
Samples of mouse spleen cells were cultivated for 5 days
in the presence of the conjugate Pam3Cys-Ser-(EGF-R
515-529), of the adjuvant Pam3Cys-Ser, of the tetradeca-
l~4as~s
- 44 -
peptide EGF-R 516-529, of a mixture of the antigen and
adjuvant, and of medium.
The lymphocytes were cultivated at a cell density of
2.5 x 106/ml in 0.2 ml aliquots in RPMI-1640 medium
enriched with 10% heat-inactivated FCS, glutamine (2 mM>,
penicillin (100 U/ml), streptomycin (100 rg/ml) and 2-
mercaptoethanol (5 x 10 5 M>, for 48 h.
The supernatants were obtained for examination for speci-
fic antibodies by ELISA.
Mitogenic activation of mouse spleen cells '
The mitogenic activation of Balb/c spleen cells by Pam3-
Cys-Ser-(Lys)4-FITC (circles), Pam3Cys-Ser-(Lys)4-OH x
3HCl (triangles) and Pam3Cys-Ser-(Lys)4-OH x 2 TFA (squares)
is shown in Fig. 12. The cell cultivation conditions have
been described (Z. Immunforsch. 153, 1977, pp. 11 et seq.
and Eur. J. Biochem. 115, 1981). In the figure, the
stimulation index for the incorporation of 3H-thymidine
into the DNA <cpm for incorporation/cpm for the control
without mitogen) is plotted as the ordinate against the
concentration of active compound used.
In vivo/in vitro comparison
In Fig. 11 the in vivo experiment detailed above is com-
pared with an in vitro experiment:
In vitro experiment in microtiter plates: cell density:
2.5 x 106 cells/ml; substance concentration: 5 x 10 7
millimolar; incubation conditions: 37oC, 5% C02, 5 days.
Conj.. conjugate Pam3Cys-Ser-(EGF-R 515-529)
Pep . tetradecapeptide EGF-R 516-529
Adj. . Pam3Cys-Ser
Mix . mixture of free tetradecapeptide EGF-R 516-529
and Pam3Cys-Ser.
The drastic rise in the antibody concentration also emerges
in vitro, and this considerably extends the utilizability
-. 1340656
- 45 -
of cell cultures, in particular for the preparation of
antibodies.
