Synthesis of a C-disaccharide analog of the Thomsen-Friedenreich (T) epitope

Article abstract of DOI:10.1055/s-2001-9698

The 2,3,4,6-tetra-O-[(tert-butyl)dimethylsilyl]-C-β -D-galactopyranosylformaldehyde (2,6-anhydro-1,3,4,6-tetra-O-[(tert-butyl)dimethylsilyl] -D-glycero-L-manno-heptose, 2) was condensed with isolevoglucosenone (3) in the presence of Et₂AlI to give 1,6-anhydro-3-C-{(1R)-2,6-anhydro-3,4,5,7-tetra-O-[(tert-butyl)dimethylsilyl] -D-glycero-L-manno-heptitol-1-C-yl}-2,3-dideoxy- β-D-glycero-hex-2-enopyranos-4-ulose (8). Diastereoselective conjugate addition of BnONHBn to 8, followed by diastereoselective ketone reduction with LiBH₄ and treatment with Me₃SiSPh/ZnI₂ provided phenyl 3-C-{(1R)-2,6-anhydro-3,4,5,7-tetra-O[(tert-butyl)dimethylsilyl]-D-glycero-L- manno-heptitol-1-C-yl}-2-[(N-benzoxyl-N-benzyl)amino]-2,3 -dideoxy-1-thio-β-D-galacto-hexopyranoside (14).

Full text of DOI:10.1055/s-2001-9698

LETTER
79
Synthesis of a C-Disaccharide Analog of the Thomsen-Friedenreich (T)
Epitope
Yao-Hua Zhu, Pierre Vogel*
Section de Chimie de l'Université, BCH, CH-1015 Lausanne-Dorigny, Switzerland
Fax +41 21 692 3975; E-mail: pierre.vogel@ico.unil.ch
Received 22 September 2000
vaccine. We report here our efforts toward the synthesis of
a C-glycoside analog of epitope T disaccharide. The syn-
thesis relies on our recently developed methodology for
the synthesis of C(1Æ3)-linked disaccharides based on a
Abstract: The 2,3,4,6-tetra-O-[(tert-butyl)dimethylsilyl]-C-b-D-
galactopyranosylformaldehyde (2,6-anhydro-1,3,4,6-tetra-O-[(tert-
butyl)dimethylsilyl]-D-glycero-L-manno-heptose, 2) was con-
densed with isolevoglucosenone (3) in the presenc e of Et2AlI
to give 1,6-anhydro-3-C-{(1R)-2,6-anhydro-3,4,5,7-tetra-O-[(tert- Baylis-Hillmann type of condensation5 between the D-ga-
butyl)dimethylsilyl]-D-glycero-L-manno-heptitol-1-C-yl}-2,3-
dideoxy-b-D-glycero-hex-2-enopyranos-4-ulose (8). Diastereose-
lective conjugate addition of BnONHBn to 8, followed by diastere-
lactose derived carbaldehyde 2 and isolevoglucosenone
(3) induced with a dialkyl aluminum salt.
oselective ketone reduction with LiBH4 and treatment with
Me3SiSPh/ZnI2 provided phenyl 3-C-{(1R)-2,6-anhydro-3,4,5,7-
OR
RO
RO
O
tetra-O[(tert-butyl)dimethylsilyl]-D-glycero-L-manno-heptitol-1-
C-yl}-2-[(N-benzoxyl-N-benzyl)amino]-2,3-dideoxy-1-thio-b-D-
galacto-hexopyranoside (14).
O
CHO
O
O
OR
Key words: Baylis-Hillman reaction with Et2AlI, C-disaccharide,
epitope T, C-b-D-galactopyranosylformaldehyde, isolevoglucose-
none, non-hydrolyzable epitope analog
2 R = (t-Bu)Me2Si
4 R = Bn
3
OAc
OR
O
AcO
AcO
RO
3
SiMe3
1
O3
O
8
9
7
2
OAc
6
-78°C
BF3·Et2O
Me3SiOTf
RO
Malignancy is often associated with profound alterations
in cell surface bound carbohydrate components of glyco-
conjugates.1 Such structural changes are due to incom-
plete glycosylation or novel glycosylation by tumor cells.
Among the tumor associated carbohydrate antigens the
Thomsen-Friedenreich antigen (T antigen) is found in car-
5
4
OAc
OR
6 R = Ac (92%)
7 R = (t-Bu)Me2Si (85%)
5
TBDMSOTf
pyr, DMAP
Scheme 1
cinoma-associated mucins; it is
a
disaccharide,
The required C-b-D-galactopyranosylformaldehyde 2 was
prepared following a procedure similar to that described
by Bednarski and co-workers6 for the preparation of 4,
starting from D-galactose pentaacetate (5).7 Trimethylsilyl
triflate (2 equivalents), then BF3◊OEt2 (3 equivalents)
were added dropwise to a solution of 5 and propargyl tri-
methylsilane (3 equivalents) in MeCN at 0 °C. After 2 h
at 0 °C acidic work-up (1 N HCl) and flash chromatogra-
phy on silica gel gave 6 in 92% yield. Zemplén methanol-
ysis (MeONa/MeOH) of 6 followed by silylation with
(t-Bu)Me2SiOSO2CF3/pyridine and 4-dimethylamino-
pyridine afforded 7 in 85% yield.8 Ozonolysis (2% O3 in
O2, CH2Cl2, -78 °C) provided aldehyde 2 (Scheme 1), an
unstable compound that must be used directly in the next
operation (Scheme 2).
Galb1Æ3GalNAcaÆO linked to serine or threonine. The
T antigens have been prepared and their immunogenicity
in conjugate vaccines has been confirmed.2 The clustered
antigen motifs such as 1 prepared by Danishefsky and co-
workers3 have demonstrated the potential for antitumor
vaccines4 based on T antigen conjugates.
OH
HO
OH
HO
OH
OH
O
HO
HO
HO
OH
O
O
O
O
O
AcHN
NHAc
OH
HO
O
O
O
O
H
N
H
N
H
N
AcHN
OH
N
SH
H H
O
O
O
AcHN
O
HO
O
O
A 1 M solution of Et2AlI in toluene was added to a 1:1.5
mixture of crude 2 and isolevoglucosenone (3)9 in CH2Cl2
at -78 °C. After 2 h at -78 °C and acidic work-up, enone
8 was obtained as the major product of condensation in
61% yield.10 The diastereoselectivity of the reaction was
not established unambiguously. Nevertheless, by analogy
with all the other cases of aldol condensations involving
aluminum enolates derived from isolevoglucosenone
HO
OH
OH
OH
1
Disaccharide conjugates are relatively short-lived in the
blood stream because of their hydrolysis catalyzed by
ubiquitous glycosidases. Disaccharide mimetics such as
C-linked disaccharide analogs offer an improved stability
towards hydrolysis as required for a disaccharide-based
(Zimmerman-Traxler model,11 steric
factors), the
Synlett 2001, No. 1, 79–81 ISSN 0936-5214 © Thieme Stuttgart · New York

80
Y.-H. Zhu, P. Vogel
LETTER
hydroxymethano linker must have the (R) configuration.5
BnONH2
8
1
20 °C
The H NMR spectrum of 8 confirmed the configuration
3
of the C-glycosidic linkage, with J(H-2’,H-3’) = 7.0 Hz
OR
OR
RO
RO
RO
O
NOBn
HO
HO
and 3J(H-3’,H-4’) = 8.6 Hz.
O
O
+
O
O
RO
When a neat mixture of enone 8 and O-benzyl hydroxy-
lamine was stirred at 20 °C, oxime 10 was obtained and
isolated in 70% yield. Control experiments indicated that
the initial conjugate addition of BnONH2 to 8 giving ad-
duct 9 is somewhat faster than the oxime formation, al-
though the latter reaction could not be avoided (Scheme
3). We thus looked for a nitrogen nucleophilic reagent that
would not undergo 1,2-addition to enone 8 or to its prod-
uct of 1,4-addition. We found that N,O-dibenzyl hydrox-
ylamine adds to enone 8 between -78 °C and -15 °C in
the presence of one equivalent of Me2AlCl. This gave rise
to pure 11, the product of conjugate addition, in 41%
yield.12 Attempts to reduce the carbonyl group of 11 with
NaBH4 induced fast 1,4-elimination of BnONHBn. Using
(i-Bu)2AlH led to decomposition. Finally, ketone 11 was
reduced with LiBH4 in THF into the desired D-galac-
tosamine derivative 12 with high diastereoselectivity and
good yield (70%). The structure of 12 was deduced from
its 1H NMR and 2D 1H-NOESY spectra. It was confirmed
by the structures of the derivatives 13 and 14 (see below).
OR
OR
10 (70%)
BnONH
BnONH
O
O
9
OR
RO 7'
5'
O
6
H
HO
BnONHBn
Me2AlCl
-78 °C
LiBH4
THF
6'
O
O
2
RO
1'
8
3'
OR2'
4'
3
1
-78 to -15 °C
H
BnN
(41%)
O
(70%)
BnO
11
OR
RO
RO
OH
H
HO
O
Me3SiSPh
ZnI2
O
H
OR
H
BnN
BnO
O
12
OR
O
OR'
O
RO
RO
OH
OH
5
4
SPh
2
3
OR
BnONBn
R = (t-Bu)Me2Si
H
13 R' = (CH3)3Si (53%)
K2CO3/MeOH
(85%)
14 R' = H (85% from 12)
Scheme 3
O
OR
RO 7'
6
Et2AlI
-78 °C
(61%)
4
OH
1'
O
2 + 3
O
O
RO
3'
OR2'
1
4'
3
2
Acknowledgement
R = (t-Bu)Me2Si
8
We thank the Swiss National Science Foundation, the Fonds Her-
bette (Lausanne) and the "Office Fédéral de l'Education et de la Sci-
ence" (European program COST D13/0001/999) for financial
support. We are grateful also to Mr. Martial Rey and Franscisco
Sepúlveda for their technical help.
Scheme 2
Since our C-disaccharide analog of the antigen T sugar
must be conjugated with serine and threonine we convert-
ed the anhydro moiety of 12 into a more flexible glycoside
donor.13 With this goal in mind we obtained a single dias-
tereomer of the phenyl thiogalactopyranoside 13, on treat-
ment with Me3SiSPh and ZnI2,14 in 53% yield after
column chromatography on silica gel. This compound
was not stable in the presence of water, losing its trimeth-
ylsilyl moiety rapidly at room temperature. It was thus
treated with MeOH in the presence of K2CO3 to provide
triol 14 in 85% yield. The latter product was obtained in
85% yield directly from 12 when omitting the purification
of the intermediate product 13. The 1H NMR spectra of 13
and 14 confirmed their structures.15 In particular the b-C-
D-galactopyranosyl configuration of the 2-aminohexose
moiety was established by the vicinal coupling constants
References and Notes
(1) See e.g.: Springer, G. F. Science 1984, 224, 1198; Taylor-
Papadimitriou, J.; Epenetos, A. A. Trends Biotechnol. 1994,
12, 227; Kim, Y. J.; Varki, A. Glycoconj. J. 1997, 14, 569;
Hakomori, S. Acta Anat. 1998, 161, 79; Orntoft, T. F.;
Vestergaard, E. M. Electrophoresis 1999, 20, 362.
(2) MacLean, G. D.; Reddish, M. A.; Bowen-Yacyshyn, M. B.;
Poppema, S.; Longenecker, B. M. Cancer Invest. 1994, 12,
46; Jennings, H.J.; Ashton, F. E.; Gamian, A.; Michon, F.
Roy, R. Can. Prog. Biotechnol. 1987, 3, 149; Springer, G. F.
Crit. Rev. Oncog. 1995, 6, 57.
(3) Kuduk, S. D.; Schwarz, J. B.; Chen, X.-T.; Glunz, P. W.;
Sames, D.; Ragupathi, G.; Livingston, P. O.; Danishefsky, S.
J. J. Am. Chem. Soc. 1998, 120, 12474.
(4) Hakamori, S.; Zhang, Y. Chem. Biol. 1997, 4, 97; Ragupathi,
G. Cancer Immunol. Immunother. 1996, 43, 152; Lo-Man, R.;
Bay, S.; Vichier-Guerre, S.; Deriaud, E.; Cantacuzene, D.;
Leclerc, C. Cancer Res. 1999, 59, 1520; Allen, J. R.;
Danishefsky, S. J. J. Am. Chem. Soc. 1999, 121, 10875;
Kuberan, B.; Linhardt, R. J. Curr. Org. Chem. 2000, 4, 653;
see also: Bousquet, E.; Spadaro, A.; Pappalordo, M. S.;
Bernardini, R.; Romeo, R.; Panza, L.; Ronsisvalle, G. J.
Carbohydr. Chem. 2000, 19, 527.
3J(H-1,H-2) = 9.5 Hz, J(H-2,H-3) = 11.5 Hz, J(H-3,H-
3
3
3
1
4) and J(H-4,H-5) <3 Hz and by the 2D-NOESY H
NMR spectrum of 14.
This letter describes a short and stereoselective approach
to a partially protected C-disaccharide analog of the oli-
gosaccharide portion of the T-antigen. Our work estab-
lishes another strategy for the preparation of C(1Æ3)
disaccharides that are C-glycosides of aminosugars.16
Synlett 2001, No. 1, 79–81 ISSN 0936-5214 © Thieme Stuttgart · New York

LETTER
Synthesis of a C-Disaccharide Analog of the Thomsen-Friedenreich (T) Epitope
81
(5) Zhu, Y.-H.; Vogel, P. Tetrahedron Lett. 1998, 39, 31; Zhu,
Y.-H.; Demange, R.; Vogel, P. Tetrahedron: Asymmetry
2000, 11, 263.
(6) Koberts, W. R.; Bertozzi, C. R.; Bednarski, M. D.
Tetrahedron Lett. 1992, 33, 737; see also: Gaertzen, O.;
Misske, A. M.; Wolbers, P.; Hoffmann, H. M. R. Synlett 1999,
1041.
(7) For other galactopyranosylcarbaldehyde derivatives, see:
Petrusova, M.; BeMiller, J. N.; Krihova, A.; Petrus, L.
Carbohydr. Res. 1996, 295, 57; Dondoni, A.; Kleban, M.;
Zuurmond, H.; Marra, A. Tetrahedron Lett. 1998, 39, 7991;
Dent, B. R.; Furneaux, R. H.; Gainsford, G. J.; Lynch, G. P.
Tetrahedron 1999, 55, 6977.
3J(H-4’,H-5’) <2 Hz, H-5'), 3.87 (ddd, 1H, 3J(H-2',H-3') = 9.2
Hz, 3J(H-1',H-2') = 7.0 Hz, 4J(H-2',H-4') = 1.7 Hz, H-2'), 3.82
(d, 1H, 3J(H-5',H-6') = 3.9 Hz, H-6'), 3.81 (d, 1H, 2J = 7.6 Hz,
Hendo-6), 3.75 (dd, 1H, 2J = 7.6 Hz, 3J(H-5,Hexo-6) = 5.8 Hz,
H
exo-6), 3.72 (dd, 1H, 3J(H-3',H-4') = 11.9 Hz, 4J(H-2',H-
4') = 1.7 Hz, H-4'), 3.64 & 3.27 (2m, 2H, CH2(BnN)), 2.64
(dd, 1H, 3J(H-2,H-3) = 11.0 Hz, 3J(H-1',H-3) = 2.5 Hz, H-3),
0.97, 0.97, 0.91, 0.88 (4s, 36H, 4 t-Bu), 0.15, 0.14, 0.14, 0.12,
0.08, 0.07, 0.015, 0.01 (8s, 24H, 4 Me2Si); 13C NMR (100.6
MHz, CDCl3): dc 214.9, 136.7, 130.1, 129.3, 128.2, 100.3,
79.4, 77.3, 73.3, 70.2, 67.3, 67.0, 66.5, 65.2, 58.5, 58.4, 46.1,
26.0, 18.4, 18.3, 18.1, 18.0, -4.4, -4.5, -4.6, -4.8, -4.9, -5.0, -
5.3, -5.3, -5.5.
(8) Data for 7: colorless oil; 1H NMR (400 MHz, CDCl3): dH 5.27
(q, 1H, 4J(H-7,H-9a) = 4J(H-7,H-9b) = 3J(H-6,H-7) = 7.0 Hz,
H-7), 4.80 (ddd, 1H, 2J = 10.8 Hz, 4J(H-7,H-9a) = 6.7 Hz,
5J(H-6,H-9a) = 1.9 Hz, H-9a), 4.76 (ddd, 1H, 2J = 10.8 Hz,
4J(H-7,H-9b) = 6.7 Hz, 5J(H-6,H-9b) = 1.9 Hz, H-9b), 4.42
(d, 1H, 3J(H-5,H-6) = 7.0 Hz, H-6), 4.22 (m, 1H, H-3), 4.13
(m, 1H, H-5), 3.91 (m, 1H, H-4), 3.74 (m, 2H, H-1), 3.63 (m,
1H, H-2), 0.93, 0.92, 0.91, 0.89 (4s, 36H, 4 t-Bu), 0.10, 0.095,
0.09, 0.085, 0.08, 0.07, 0.05, 0.04 (8s, 24H, 4 Me2Si).
(9) Horton, D.; Roski, J. P.; Norris, P. J. Org. Chem. 1996, 61,
3783.
(13) See e.g.: Pozsgay, V.; Jennings, H. J. J. Org. Chem. 1988, 53,
4042; Sato, S.; Mori, M.; Ito, Y.; Ogawa, T. Carbohydr. Res.
1986, 155, C6; Mallet, J.-M.; Meyer, G.; Yvelin, F.; Jutand,
A.; Amatore, C.; Sinaÿ, P. Carbohydr. Res. 1993, 244, 237;
Geurtsen, R.; Holmes, D. S.; Boons, G.-J. J. Org. Chem. 1997,
62, 8145; Kahne, D.; Walker, S.; Cheng, Y.; Van Engen, D.
J. Am. Chem. Soc. 1989, 111, 6881; Taylor, C. M.
Tetrahedron 1998, 54, 11317.
(14) Wang, L.-X.; Sakairi, N.; Kuzuhara, H. J. Chem. Soc., Perkin
Trans 1 1990, 1677.
(15) Data for 14: colorless oil; 1H NMR (400 MHz, CDCl3): dH
7.66- 7.57 (m, 4H), 7.42-7.30 (m, 6H), 7.25-7.20 (m, 3H),
7.02-6.96 (m, 2H), 4.90 (d, 1H, 3J(H-1,H-2) = 9.5 Hz, H-1),
4.47 & 4.04 (2d, 2H, 2J = 9.5 Hz, CH2(BnN)), 4.46 (m, 1H, H-
4), 4.44 & 4.22 (2d, 2H, 2J = 8.5 Hz, CH2(BnO)), 4.27 (dd,
1H, 3J(H-5',H-6') = 7.5 Hz, 3J(H-6',H-7'b) = 2.2 Hz, H-6'),
4.26 (m, 1H, H-3'), 4.13 (m, 1H, H-2'), 4.09 (m, 1H, H-2), 4.03
(m, 1H, H-1'), 3.97 (dd, 1H, 2J = 12.0 Hz, 3J(H-5,Ha-6) = 6.5
Hz, Ha-6), 3.91 (d, 1H, 3J(H-5',H-6') = 7.5 Hz, H-5'), 3.86 (d,
1H, 2J = 4.2 Hz, Ha-7'), 3.82 (dd, 1H, 3J(H-3',H-4') = 12.5 Hz,
4J(H-2',H-4') = 1.5 Hz, H-4'), 3.78 (dd, 1H, 2J = 4.2 Hz, 3J(H-
6',Hb-7') = 2.2 Hz, Hb-7'), 3.74 (dd, 1H, 2J = 12.0 Hz, 3J(H-
5,Hb-6) = 2.5 Hz, Hb-6), 3.39 (br. dd, 1H, 3J(H-5,Ha-6) = 6.5
Hz, 3J(H-5,Hb-6) = 2.5 Hz, H-5), 2.42 (br. d, 1H, 3J(H-2,H-
3) = 11.5 Hz, H-3), 0.93 & 0.90 (2s, 36H, 4 t-Bu), 0.11-0.05
(m, 24H, 4 Me2Si); 13C NMR (100.6 MHz, CDCl3): dc 137.9,
132.0, 130.0, 129.6, 128.8, 128.4, 128.3, 128.1, 127.4, 127.2,
85.8, 80.0, 73.2, 70.4, 69.1, 67.8, 67.3, 65.9, 63.6, 60.3, 58.6,
41.6, 26.1, 26.0, 25.9, 25.9, 25.7, 18.4, 18.1, 18.0, 17.8, -4.5,
-4.5, -4.6, -4.8, -4.9, -5.0, -5.4; CI-MS (NH3) m/z 1100 (13,
[M+H]+), 1099 (76, M•+), 991 (11, [M+H-PhS]+•), 990 (11,
[M-PhS]+), 91 (100).
(10) Data for 8: colorless oil; 1H NMR (400 MHz, CDCl3): dH 7.39
(dd, 1H, 3J(H-1,H-2) = 3.5 Hz, 4J(H-2,H-1’) = 1.0 Hz, H-2);
5.83 (d, 1H, 3J(H-1,H-2) = 3.5 Hz, H-1), 4.75 (dd, 1H, 3J(H-
5,H-6) = 6.4 Hz, 3J(H-5,H-6) = 1.3 Hz, H-5), 4.45 (dd, 1H,
3J(H-3’,H-4’) = 8.6 Hz, 3J(H-2’,H-3’) = 7.0 Hz, H-3’), 4.26 (d,
1H, 2J = 12.4 Hz, H-7’a), 4.23 (ddd, 1H, 3J(H-1’,H-2’) = 7.0
Hz, 3J(H-1’,OH) = 6.7 Hz, 4J(H-2,H-1’) = 1.0 Hz, H-1’), 4.06
(dd, 1H, 2J = 8.3 Hz, 3J(H-5, Hexo-6) = 6.4 Hz, Hexo-6), 3.99
(br.d, 1H, 3J(H-3’,H-4’) = 8.6 Hz, 3J(H-4’,H-5’) <2 Hz, H-4'),
3.96 (br.d, 1H, 3J(H-5',H-6') = 4.1 Hz, H-5'), 3.91 (t, 1H, 3J(H-
1',H-2') = 3J(H-2',H-3') = 7.0 Hz, H-2'), 3.80 (dd, 1H, 3J(H-
5',H-6') = 4.1 Hz, 3J(H-6',H-7'b) = 1.9 Hz, H-6'), 3.75 (dd, 1H,
2J = 12.4 Hz, 3J(H-6', H-7'b) = 1.9 Hz, H-7'b), 3.65 (dd, 1H,
2J = 8.3 Hz, 3J(H-5,Hendo-6) = 1.3 Hz, Hendo-6), 2.94 (d, 1H,
3J(H-1',OH) = 6.7 Hz, OH), 0.94, 0.93, 0.91, 0.90 (4s, 36H, 4
t-Bu), 0.14, 0.12, 0.10, 0.09, 0.08, 0.07, 0.06, 0.05 (8s, 24H, 4
Me2Si). Anal. Calcd for C37H74O9Si4: C 57.32, H 9.62; found:
C 57.37, H 9.58.
(11) Zimmerman, H. E.; Traxler, M. D. J. Am. Chem. Soc. 1957,
59, 1920.
(12) Data for 11: colorless oil; 1H NMR (400 MHz, CDCl3): dH
7.47-7.41 (m, 2H), 7.36-7.30 (m, 3H), 7.28-7.25 (m, 3H),
7.21-7.14 (m, 2H), 6.12 (s, 1H, H-1), 4.50 (dd, 1H, 3J(H-5,H-
6) = 5.6 Hz, 4J(H-3,H-5) = 0.9 Hz, H-5), 4.43 & 4.18 (2d, 2H,
2J = 10.2 Hz, CH2(BnO)), 4.26 (dd, 1H, 3J(H-1', H-2') = 7.0
Hz, 3J(H-3,H-1') = 2.5 Hz, H-1'), 4.24 (d, 1H, 3J(H-2,H-
3) = 11.0 Hz, H-2), 4.22 & 3.92 (2d, 2H, 2J = 13.0 Hz,
H2C(7')), 4.18 (dd, 1H, 3J(H-3',H-4') = 11.9 Hz, 3J(H-2',H-
3') = 9.2 Hz, H-3'), 3.98 (br.d, 1H, 3J(H-5',H-6') = 3.9 Hz,
(16) For the synthesis of a-D-Manp (1Æ3)CH2-D-GalNAc and a-
D-Manp (1Æ3)CH2-D-TalNAc, see: Pasquarello, C.; Picasso,
S.; Demange, R.; Malissard, M.; Berger, E. G.; Vogel, P. J.
Org. Chem. 2000, 65, 4251.
Article Identifier:
1437-2096,E;2001,0,01,0079,0081,ftx,en;G20300ST.pdf
Synlett 2001, No. 1, 79–81 ISSN 0936-5214 © Thieme Stuttgart · New York