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Mycoloylpeptides and Other Lipopeptide Adjuvants from Higher Aldoketene Dimers.

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cycloheptatrienylacetylenesand the I-chloro-l-cycloheptatrienyl-2-cyclopentadienylideneethylenes.Compounds l a c (Table 1 ) were obtained by treatment of the mixture with
excess triethylamine in ether. They are fairly stable in the
solid state and give correct M @ peaks in their mass spectra.
No dimer formation was observed and the compounds
gradually decompose in solution.
Table I. Yields and melting points of the products l a - c and 4a-c.
Compound
Yield [%]
M.p. ["C]
la
90
61
62
57
69
68
183-184 (dec.)
Ib
lc
4a
4b
4c
198-199 (dec.)
147-150 (dec.)
182- I84
198-200
121-123
Mycoloylpeptides and Other Lipopeptide Adjuvants
from Higher Aldoketene Dimem**
The stretching vibrations due to the cumulene bonds (in
KBr; 6 [cm-'I = 2036 m (la), 2040 m (lb), 2028 m (lc))
resemble those of tri- and tetraarylbutatrienes, whose absorptions were reportedc7'at about 2032 cm- I. Compounds
l a - c exhibit n o absorptions in the acetylenic region, however. These values indicate that the small contribution of
the resonance structure 1' is influenced by the substituents
on the aryl groups at the 3- and 4-positions of the cyclopentadienylidene ring: the - I effect of C1 in l b increases
the contribution of the resonance structure corresponding
to 1'; the + M effect of OMe in l c decreases it.
The lowest-field I3C-NMR signals of l a - c were observed around 6= 143 (in CDC13). The high-field shift of
la-c compared with that of tetraphenylbutatriene
(S= I52)I8' indicates a weak acetylenic character of the central bond of l a - c , corresponding to a small contribution
of the resonance structure 1'.
The electronic spectra of l a - c showed three different
absorption bands: I,,, [nm] (E): l a , 280 (27500, sh), 360
(4000, sh), and 538 (13200); lb, 287 (29500), 360 (SOOO),
and 547 (37 100); lc, 280 (25 700), 360 (7250), and 545
(19900). The longest-wavelength bands are very broad.[']
An interesting feature of the cumulenes is their emission
spectra. When l a - c were excited by 360-nm light, emission was observed around 450 nm. Since no emission was
observed by excitation of the longest-wavelength absorption band at I = 530-560 nm, the fluorescence spectra
should be attributed to the emission from the S2 state.["l
Received: October 22, 1986;
revised: January 2, 1987 [Z 1962 IE]
German version: Angew. Chem. 99 (1987) 367
[ I J Reviews: H. Fischer in S . Patai (Ed.): The Chemistry ofAlkenes, Wiley,
New York 1964, Chap. 13; S. Patai (Ed.): The Chemistry of Ketenes, Allenes. and Related Compounds, Wiley, New York 1980, Part I and 11.
(21 Ried reported the synthesis of tetrabenzo derivatives of this system.
However, no properties were described: W. Ried, W. Schlegemilch, S.
Piesch, Chem. Ber. 96 (1963) 1221. Iyoda et al. and Yoshida reported the
synthesis of the isoelectronic compounds containing the bisdehydro[l3]annulene group as a terminal group. Also, Gompper reported the
synthesis of butatrienes containing different push-pull functional
groups: M. lyoda, S. Tanake, K. Nishioka, M. Oda, Tetrahedron Lett. 24
(1983) 2861; Z. Yoshida, PureAppl. Chem. 54 (1982) 1059; R. Gompper,
U. Wolf, Tetrahedron Lett. 1970. 4263.
[3] T. Toda, N. Shimazaki, T. Mukai, Chem. Lett. 1979. 171; T. Toda, N.
Shimazaki, T. Mukai, C. Kabuto, Tetrahedron Lett. 21 (1980) 4001.
[4] R. M. Hoskinson, Aust. J. Chem. 23 (1970) 399.
[ S ] The cyclopentadienones were prepared by Johnson's method from dibenzylketone and the corresponding benzil: J. R. Johnson, 0. Grummitt,
Org. Synth., Collect Yo/. 3 (1955) 805.
161 A11 of the mixtures show weak stretching bands around V=2210 and
1950 c m - ' in their IR spectra.
336
0 VCH Verlagsgesellschafr mbH. 0-6940 Weinheim, 1987
[7] R. Kuhn, H. Fischer, Chem. Ber. 92 (1959) 1849.
(81 J. P.,C. M. van Dongen, M. J. A. de Bie, R. Stern, Tetrahedron Lett.
1973. 1371.
191 Redshift of the first absorption bands was observed when the electronic
spectra of l a - c were measured in more polar solvents such as acetonitrile, acetone, anisole, indicating that the cumulenes l a - c are less polar
in the ground state and more polar in the excited state.
[I01 Emission from the S2 state in solution is rather rare. For examples see:
M. Beer, H. C. Longuet-Higgins, J. Chem. Phys. 23 (1955) 1390; S . Murata, C. Iwanaga, T. Toda, H. Kokubun, Ber. Bunsenges. Phys. Chem. 76
(1972) 1176. See also the following reviews: N. J. Turro: Modern Molecular Photochemrstry, Benjamin/Cummings, Menlo Park, CA, USA 1978,
p. 147-148, 184-185; N. J. Turro, V. Ramamurthy, W. Cherry, W. Franeth, Chem. Rev. 78 (1978) 125.
By Jorg Metzger and Giinther Jung*
Dedicated to Professor Ernst Buyer
on the occasion of his 60th birthduy
During the development of carrierladjuvant systems
based on lipopeptides such as tripalmitoyl-S-glycerylcysteine,"] we encountered an interesting side product, which
was formed during the acylation of L-cystine bis(tert-butyl)
ester with palmitoyl chloride (Pam-CI) and which made
the purification of larger batches of the symmetric cystine
derivative'Ih1 more difficult: N-palmitoyl(a-palmitoy1)-N'palmitoyl-L-cystine bis(tert-butyl) ester. The formation of
the a-alkyl-8-ketoacyl moiety Pam(a-Pam) could be explained by the intermediary formation of a 4-alkylidene3-alkyl-2-oxetanone, 1, which was experimentally confirmed. Such diketenes, formed from higher fatty acid halides,['] are known to react with amines 2 to give fl-ketoamides 3l3l ( R = long-chain alkyl groups, R'= alkyl, aryl).
We have optimized this side reaction in order to develop
a simple method for the preparation of lipopeptides, particularly mycolic acid derivatives (N-(a-alkyl-fi-hydroxyacyi)
amino-acid derivatives 6),which are also valuable as adjuvants. Mycolic
are found in naturally occurring
lipids and, like the muramyldipeptide (MDP), are active
components of Freund's adjuvant, a mixture of killed tubercle bacteria in paraffin oil that is used for the induction
of antibodies.
An example is the toxic trehalose-6,6'-dimycolate
(C[&i366017, cord factor), isolated from Gram-positive
m y ~ o b a c t e r i a . ~0-Acylated
~"~
@-hydroxyacyl moieties are
also present in lipid A, the toxic component of the endotoxin'4h1 of Gram-negative bacteria. Finally, active analogues of M D P containing mycoloyl moieties are
Previous syntheses of mycoloyl amino acids employed mycolic acids isolated from bacteria (semisyntheses).[51
[*I Prof. Dr. G. Jung, DipLChem. J. Metzger
Institut fur Organische Chemie der Universitat
Auf der Morgenstelle 18, D-7400 Tiibingen (FRG)
[**I J. M. thanks the Studienstiftung des Deutschen Volkes for a Promotionsstipendium. We thank Prof. Dr. W. G. Bessler, lnstitut fur Immunbiologie der Universitat Freiburg, for carrying out mitogenicity tests.
0570-0833/87/0404-0336 $ 02.50/0
Angew. Chem. In!. Ed. Engl. 26 (1987) No. 4
The aldoketene dimers 1 required for our synthesis are
readily obtainable from fatty acid halides by reaction with
tertiary aminesI2]and are stable upon storage. They react
with the free amino group of amino acid esters 4 in anhydrous pyridine in the presence of catalytic amounts of 4dimethylaminopyridine (DMAP) within 2-4 h to give the
a-alkyl-f3-ketoacyl compounds 5 . In solvents such as dimethylformamide, CH2C12,or CHCI, and with bases such as
triethylamine or N-methylmorpholine, however, mainly
the simple acylation product, e.g., the N-palmitoyl derivative, is isolated, even when the reaction is carried out for
long reaction times and at high temperatures.
Table 2. N-(a-Hexadecyl-B-hydroxyicosanoyl)
amino acid terf-butyl esters 6
(R =n-C16H,&
R+ value [b] in
Amino acid ester
in 6
Yield
[Yo1
M.p.
["C] [a]
CHCI?
EA [cl
Gly-OIBu
Ala-Of Bu
Ser(fBu)-OtBu
Lys( Boc)-OfBu
(Cys-Of Buh
Asp(rBu)-OtBu
Tyr(1Bu)-OtBu
Phe-OfBu
92
92
72
75
91
74
92
77
91
84
55
76
75
65
78
75
0.19/0.28
0.17-0.2410.29-0.35
0.2I-0.2610.29-0.35
0.05/0.07
0.09-0.24
0.22-0.32
0.20-0.27
0.33-0.40
0.82
0.81
0.81
0.81
0.82-0.87
0.8I
0.82
0.8I
[a-c] See Table I.
+
R-CH+
'
R
DMAP
I
4
R"
t BU
I
I
5
two-chain mycoloyl compounds 6 can be transformed to
three-chain derivatives by esterification of the OH group.
The latter compounds are, in general, stronger mitogens
than the non-0-acylated compounds 6. Four-chain compounds are accessible via N-(a-alkyl-a-hydroxyacy1)-Sglycerylcysteine (Fig. 1) or N",N"-bis[Pam(a-Pam)]lysine.
Several of the prepared lipopeptides and amino acids are
effective B-cell mitogens.
C5H5N. 50%
R-CH2-CO-CH-CO-NH-CH-CO-0
R
>-
H2N-CH-CO-OtBu
NaBH,
>
Me2CH-OH
R''
R-CH2-CH
I
-CH-CO-NH-CH-CO-0
I
I
t Bu
0
m-
R''
OH R
6
-PamIn-Peml-Pam~Cys-Ale-Gly-OH
= -Pemla-Paml-Cya-OH
0
0
The reaction of aldoketene dimers 1 from higher fatty
acids with amino acid esters 4 is thus a simple and economical route to novel lipoarnino acids. The resulting aalkyl-P-ketoacyl derivatives 5 (Table I), after removal of
the carboxyl protecting group, are mitogenic compounds
(Fig. I), which, for example, can be covalently linked to
the free N-terminus of amino-acid or peptide sequences to
give lipopeptides that are also biologically active (Fig. 1).
Compounds 5 can be quickly reduced to give, in high
0
-Psmla-Paml-Als-Ale-Gly-OH
-Pem$yS-Aia-Gly-OH
'D-
O
Y
Table I . N-(a-Hexadecyl-~-oxoicosanoyl)
amino acid fert-butyl esters 5
(R = n-C,,Hd.
Amino acid ester 4
H-Gly-OIBu. HCI
H-Ala-OfBu- HCI
H-Ser(f Bu)-Or Bu
H- Lys( Boc)-OtBu .HCI
(H-Cys-OfB U ) ~
H-Asp(fBu)-OfBu.HCI
H-Tyr(fBu)-OIBu. HCI
H-Phe-OIBu-HCI
Reaction
time [hl
Yield
[%I
M.p.
["Cl [a1
RF value [b] in
EA [c]
CHCI,
72
76
52
63
50
62
51
62
72
64
41
60
76
48
62
66
0.47
0.53
0.46
0.14
0.41
0.44
0.45
0.57
0.81
0.81
0.80
0.80
0.83
0.79
0.80
0.82
[a] Melting point of the mixture of diastereomers, uncorrected. [b] Silica gel
plates 60 FZ1+,10 x 20 cm (Merck), preequilibrated once in eluent; detection
with iodine. The mixtures of diastereomers in Table 2 produce one or two
long spots, the upper and lower edges of which are given. [c] EA = ethyl
acetate.
yield, the mycoloyl compounds 6 (Table 2). For example,
the dimeric tetradecylketene 1 (R=n-C,,H,,), obtained
from Pam-CI, reacts with alanine tert-butyl ester to give a
product that can be reduced to a derivative of corynomycolic acid, a C32fatty acid found in the lipids of Corynebacterium diphtheriae. The mixture of diastereomers formed in
the reduction can, for example in the case of the alanine
derivative, be separated on silica gel. However, it is also
possible to use the mixture of diastereomers directly as a
adjuvant o r mitogen in animal experiments. Several of the
Angew. Chem. I n r . Ed. Engl. 26 (1987) No. 4
,
I,
q
O l o ~ " " ' " ""';"o
;
I
' "'1'00'
Mitogen concentration [pg/muFig. I . Mitogenic effect of the two-cham lipids Pam(a-Pam)-L-Cys-OH and
Pamfa-Pam)-L-Ala-L-Ala-Gly-OH,
prepared from 5, in comparison to the
very potent, three-chain adjuvant S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]N-palmitoyl-(R)-cysteinyl-(S)-alanylglycine (Pam,Cys-Ala-Gly-OH; J.-K.
Wiesmiiller et al., unpublished results) and the appreciably less active, fourchain derivative S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-N-palmitoyl(a-palmitoy1)-(R)-cysteinyl-(S)-alanylglycine(Pam(a-Pam)-PamZCys-Ala-Gly-OH).
The dose-response curves were obtained by incorporation of ['Hlthymidine
in spleen cells of C3H/HeJ mice 161.The three-chain 0-acylated lipopeptides
prepared from the mycoloyl amino acid esters 6 were also tested in vivo and
in vitro 171.cpm = counts per minute.
For the synthesis of immunogenic peptides and synthetic vaccines, it is important that the a-alkyLf3-ketoacyI
can be inmoiety, like tripalmitoyl-S-glycerylcysteine,['~'l
troduced at the end of a Merrifield synthesis (Fmoc/tBu
strategy). Since the lipid moiety is stable in trifluoroacetic
acid, the resulting lipopeptide can be cleaved from the carrier with simultaneous removal of all protecting groups.
0 VCH VerlagsgesellschafimbH, 0-6940 Weinheim. 1987
0570-0833/87/0404-0337 $ 02.50/0
337
Experimental Procedure
N-(a-Alkyl-8-ketoacyl) amino acid tert-butyl ester 5: The fatty acid diketene
1 ( I mmol) was added to a stirred solution of the amino acid ester 4
( I mmol) in 3 mL of anhydrous pyridine. DMAP (0.1 mmol) was added at
50°C. During the reaction, the color of the solution turned from yellow to a
cognaclike color and, after 2-4 h, thin-layer chromatography indicated the
consumption of all the diketene. The pyridine was removed in vacuo and the
crude product was precipitated from a small amount of chloroform by addition of methanol to the cooled solution. Impurities due to excess diketene
could be precipitated from ethyl acetate or diisopropyl ether at O"C, the
usually still yellow or orange product remaining in solution. Final purification of 5 was achieved by flash chromatography on a silica gel column with
chloroform.
Mycoloyl amino acid tert-butyl ester 6 : The N-(a-alkyl-B-ketoacyl) amino
acid tert-butyl ester 5 (1 mmol) was dissolved in 15 m L of 2-propanol by
heating and treated with solid sodium tetrahydridoborate ( I mmol). The reduction product slowly precipitated. After 3 h stirring, the precipitation was
driven to completion by neutralization with 0.1 N HCI. The colorless mycoloyl compound 6 was filtered off, washed several times with water, and dried
over P 2 0 5 .
this type of bonding is expected (in solution) when the arene ligands are linked."] Optimal conditions and thus the
greatest tendency of formation and stability should exist,
according to this concept, for doubly linked rings, such as
those present in cyclophanes. However, a11 attempts to introduce G a + , I n + , or T1+ into the cavity between the benzene rings of [2.2]- and [3.3]paracyclophane~,".~~
as well as
between the arene rings of the related naphthalene derivatives,l5I have failed so far. The metal atoms in the resulting
adducts are always coordinated to the benzene rings of the
cyclophanes from the outside. Nevertheless, the centric q6
coordination, present in B, is still found for the arene ligands, which are bent toward each other (e.g., in C).[41
y
I
Received: October 7, 1986;
revised: February 5 , 1987 [Z 1950 IE]
German version: Angew. Chem. 99 (1987) 343
-_-
A
[I] a) G. Jung, K.-H. Wiesmuller, G. Becker, H.-J. Buhring, W. G . Bessler,
Angew. Chem. 97 (1985) 883; Angew. Chem. Int. Ed. Engl. 24 (1985) 872;
b) K.-H. Wiesmiiller, W. G. Bessler, G. Jung, Hoppe-Seylerk 2. Physiol.
Chem. 364 (1983) 593; c) G. Jung: Synthesis and Perspectiues of New Adjuvants and Carrier Systems for Potential Applicution for Synthetic Vaccines (Proc. lnt. Symp. Synth. Antigens. Siena, Italy October 29/30, 1984);
Ann. Sclauo 1984, 191; d) W. G. Bessler, B. Suhr, H.-J. Biihring, C . P.
Muller, K.-H. Wiesmiiller, G. Becker, G. Jung, lmmunobrology (Stuttgart)
170 (1985) 239.
[2] a) J. C. Sauer, J . A m . Chem. SOC.69 (1947) 2444; b) E. S. Rothman, J.
Org. Chem. 32 (1967) 1683.
[3] a) S. Piekarski, C.R . Hebd. Shames Acad. Sci. 38 (1954) 1241; b) J. Rech.
C. N. R . S . 40 (1957) 197.
141 a) E. Lederer, Angew. Chem. 72 (1960) 372; b) J. Y. Homma, S. Kanegasaki, 0. Liideritz, T. Shiba, 0. Westphal (Eds.): Bacterial Endotoxin. Chemical, Biologicul and Clinical Aspects, Verlag Chemie, Weinheim 1984; c) T.
Shiba, S. Kusomoto, M. Inage, S. Sawaki in E. Gross, J. Meienhofer
(Eds.): Peptides: Structure and Biological Function (Proc. 6th Am. P e p .
Symp.]. Pierce, Rockford, IL 1979, p. 425-430.
[S] a) Y. Yamamura, A. Tanaka, M. Kato, J. Biochem. (Tokyo) 4 7 ( 1960) 5 0 5 ;
b) A. Tanaka, K. Tanaka, T. Tsubone, Y. Kuroda, K. Sugiyama, Int. Arch.
Allergy Appl. Immunol. 28 (1965) 340.
[6] A. Lex, K.-H. Wiesmiiller, G. Jung, W. G. Bessler, J. Immunol. 137 (1986)
2676, and literature cited therein.
[71 G. Jung, J. Metzger, W. G. Bessler, unpublished results.
C
The knowledge of the preferred contact distances and
modes of coordination has now been used by us to achieve
centric coordination of the cation G a + in the cavity of
[2.2.2]paracyclophane. Indeed, it was surprisingly easy to
achieve this novel coordination experimentally. Crystalline
Ga[GaBr,], whose structure was recently elucidatedJ6] dissolves in benzene as the dimeric tetraarene complex
[(C6H6)2Ga.GaBr4],.~71
Upon addition of the cyclophane,
such solutions afford crystalline precipitates having the
analytical composition of the I : 1 complex 1.
Arene Complexes with Weak Interactions:
A Macrocyclic Tris(arene) Complex of Galliurn(1)
with 9
'
' Coordination**
By Hubert Schmidbaur, * Rudolf Hager, Brigitte Huber.
and Gerhard Miiller
Main-group metals with an ns2 configuration form centrically $-bonded complexes with aromatic hydrocarbons;
however, the long contact distances between the metal and
the ring center indicate relatively weak interactions."-31 In
addition to I : 1 complexes (A), 1 :2 complexes (B), in
which the rings are bent toward each other, are known for
gallium, indium, and thallium in the oxidation state 1.
Owing to the entropy effect, considerable stabilization of
+
[*] Prof. Dr. H. Schmidbaur, R. Hager, Dipl.-Chem. B. Huber [+I,
Dr. G. Muller ['I
Anorganisch-chemisches Institut der Technischen Universitat Miinchen
Lichtenbergstrasse 4, D-8046 Garching (FRG)
['I X-ray structure analysis.
[**I This work was supported by the Deutsche Forschungsgemeinschaft
(Leibniz program), the Fonds der Chemischen Industrie, Hoechst AG,
Siemens AG, and Preussag AG. We thank P. Kiprof for preparing the
SCHAKAL drawing on the cover.
338
0 VCH Verlagsgesellschafr mbH. 0-6940 Weinheim, 1987
1
Complex 1 forms colorless, only slightly air-sensitive
crystals, which are sparingly soluble in aromatic hydrocarbons and afford solutions exhibiting no electrical conductivity. The latter finding indicates that 1 has pronounced
ion-pair character.
The X-ray crystal structure analysis revealed[*] that the
metal atom in 1 is indeed located in the interior of the cyclophane macrocycle and is involved in identical centric
($) bonding to all three benzene rings (Fig. la). The gallium atom is thus equidistant, within narrow limits, from
all 18 carbon atoms of the arenes. The distances correspond to values found earlier for similar bonds.") Instead
of lying completely in the plane defined by the three ring
centers ( D l , D2, D3), however, the metal lies 0.43 A above
it. Taking into consideration the position of the anion, it
becomes immediately clear that this displacement is d u e t o
the proximity of a bromine atom (Ga2-Br2 3.388(2)A),
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Angew. Chem. Int. Ed. Engl. 26 (1987) No. 4
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