Towards the development of antitumor vaccines: A synthetic conjugate of a tumor-associated MUC1 glycopeptide antigen and a tetanus toxin epitope

Article abstract of DOI:10.1002/1521-3773(20010119)40:2<366::AID-ANIE366>3.0.CO;2-J

Proliferation of cytotoxic T-cells, a prerequisite for the development of antitumor vaccines, was induced by 1, but not by its partial structures A and B. The conjugate 1 containing a tumor-associated Sialyl- T_N- MUC-1 glycopeptide antigen A an

Full text of DOI:10.1002/1521-3773(20010119)40:2<366::AID-ANIE366>3.0.CO;2-J

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[29] The 1H (300 MHz) and 13C (75 MHz) NMR spectra of 1 were re-
measured in CDCl3. For the 1H NMR shift of 1, see also M. Jonathan,
S. Gordon, B. P. Dailey, J. Chem. Phys. 1962, 36, 2443 ± 2448.
[30] P. W. Fowler, E. Steiner, R. Zanasi, B. Cadioli, Mol. Phys. 1999, 96,
1099 ± 1108.
[31] J. M. Schulman, R. L. Disch, J. Comput. Chem. 1998, 19, 189 ± 194.
[32] L. T. Scott, P.-C. Cheng, M. M. Hashemi, M. S. Bracher, D. T. Meyer,
H. B. Warren, J. Am. Chem. Soc. 1997, 119, 10963 ± 10968, and
references therein.
[33] A. Ayalon, M. Rabinovitz, P.-C. Cheng, L. T. Scott, Angew. Chem.
1992, 104, 169 ± 170; Angew. Chem. Int. Ed. Engl. 1992, 31, 1636 ± 1637.
[34] A. Pasquarello, M. Schlüter, R. C. Haddon, Phys. Rev. A 1993, 47,
1783 ± 1789.
[35] P. Lazzeretti, R. Zanasi, SYSMO package (University of Modena),
1980. Some additional routines for the evaluation and plotting of
current density were written in Exeter (E. Steiner, P. W. Fowler,
unpublished results).
mice. A monoclonal antibody (82-A6) obtained by hybridoma
techniques was selectively directed against the T-antigen
glycopeptide. It exhibited affinity to epithelial tumor cells in
enzyme-linked immunosorbant assays (ELISAs), but did not
show sufficient tumor selectivity. Further characterizations of
this antibody disclosed no affinity for glycophorin, but binding
to asialoglycophorins was observed,[5, 6] which confirms spe-
cificity towards the T-antigen structure. Interestingly, the
antibody proved to be distinctly more reactive to asialogly-
cophorins of the M blood group than those of the N blood
group. The former contains an N-terminal peptide sequence
identical to the one in the synthetic T-antigen glycopeptide.
Thus, the antibody differentiated between the two glycopro-
teins which both carry numerous identical T-antigen saccha-
rides but are slightly different in their peptide sequences. This
observation implies that the specificity of an antibody
directed against a carbohydrate epitope is distinctly modu-
lated by the peptide sequence in the linkage region.[4±6] We
conclude that a tumor-selectitve antigen not only requires a
tumor-associated saccharide epitope but also a tumor-selec-
tive peptide structure.
[36] R. D. Amos, J. E. Rice, The Cambridge Analytical Derivatives Pack-
age, Issue 4.0, 1987.
[37] J. C. Hanson, C. E. Nordman, Acta Crystallogr. Sect. B 1976, 32, 1147 ±
1153.
During recent years, amino acid sequences of tumor-
associated mucins have been elucidated.[7] Our syntheses are
now aimed at glycopeptides of the tumor-associated mucin
MUC1.[8] MUC1 is overexpressed up to tenfold on epithelial
tumors and is incompletely glycosylated because of a pre-
maturely occurring sialylation. The low glycosylation rate of
the polymorphic epithelial mucin (PEM) MUC1 influences its
conformation. Peptide epitopes located in its tandem repeat
region become accessible to the immune system. They should
constitute tumor-selective peptide-structure information.
Therefore, we have synthesized TN-,[9] T-,[10] and sialyl ± TN ±
antigen glycopeptides[11] from the tandem repeat region of
MUC1.[12] As only one of these glycopeptides exhibited a
proliferating effect on peripheric blood lymphocytes, we have
developed a novel concept for the antigen construction of
antitumor vaccines. According to this strategy, the tumor-
associated MUC1 glycopeptide antigen is combined with a
T-cell epitope of tetanus toxin using a flexible spacer to give a
conjugate 1 (Figure 1).
Towards the Development of Antitumor
Vaccines: A Synthetic Conjugate of a Tumor-
Associated MUC1 Glycopeptide Antigen and
a Tetanus Toxin Epitope**
Stefanie Keil, Christine Claus, Wolfgang Dippold, and
Horst Kunz*
Since it was recognized that tumor cells and normal cells are
distinctly different in the glycoprotein profile of their outer
cell membranes,[1] numerous efforts have been made to
develop tumor-selective antigens for vaccinations against
tumors.[2] First investigations focused on glycolipids.[3] Since
1977, Springer et al.[1b] have described glycoproteins carrying
the Thomsen ± Friedenreich antigen (T- or TF-antigen:
Galb(1,3)-GalNAc-O-Ser/Thr) as being tumor-associated sur-
face structures on epithelial cells. A close structural relation
between T-antigen glycoproteins and asialoglycophorins was
supposed because of cross reactivities of antibodies induced
by these components. Based on these results, we synthesized
TN- and T-antigen glycopeptides 15 years ago in order to
obtain antigens for vaccinations against tumors.[4] To this end,
synthetic T-antigen glycopeptides with the N-terminal se-
quence of the M blood group glycophorin were coupled to
bovine serum albumin (BSA) and used for vaccination of
Figure 1.
[*] Prof. Dr. H. Kunz, Dipl.-Chem. S. Keil
Institut für Organische Chemie der Universität Mainz
Duesbergweg 10 ± 14, 55099 Mainz (Germany)
Fax : (‡ 49)6131-392-4786
The peptide motif PDTRPAP was incorporated into the
target structure as the immunodominant domain[13]. The
neuraminic acid was protected as its benzyl ester,[14] and the
whole construct 1 was formed from two large portions in a
solid-phase fragment condensation. The sialyl ± TN building
block was synthesized from a TN ± threonine conjugate and a
neuraminic acid donor.[11] The O-acetylated sialic acid benzyl
ester 2[14] was treated with acetyl chloride to give the glycosyl
chloride which, with potassium O-ethylxanthogenate, was
E-mail: hokunz@mail.uni-mainz.de
Dr. C. Claus, Prof. Dr. W. Dippold[‡]
Naturwissenschaftlich-Medizinisches Forschungszentrum
der Universität Mainz
‡
[ ] Additional address: St. Vincenz-Hospital Mainz
[**] This work was supported by the Deutsche Forschungsgemeinschaft
and by the Stiftung Rheinland-Pfalz für Innovation. S.K. is grateful for
Â
a Kekule-Stipendium from the Fonds der Chemischen Industrie.
366
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converted into the sialyl xanthogenate 3;[15] this had already
been demonstrated as a favorable glycosyl donor in the case of
the sialic acid methyl ester.[11] Reaction of 3 with the partially
protected TN ± threonine precursor 4[11] regioselectively fur-
nished the desired STN ± threonine conjugate 5 (STN ˆ Sialyl-
TN). Low temperature and intermediate acetonitrile conju-
gates favor the formation of the a-sialoside (Scheme 1).
After O-acetylation of 5, the tert-butyl ester was removed
from 6 by careful acidolysis. The obtained STN ± threonine
building block 7[16] was used in the solid-phase synthesis of the
glycododecapeptide sequence of the MUC1 tandem repeat
region.
ester, 13 was coupled to the HYCRON linker.[9] The Fmoc-
Val ± spacer± HYCRON conjugate 14 was coupled to the
polymer (!15) and then the tetanus toxin peptide ± spacer
conjugate was assembled according to the Fmoc strategy. In
the first chain extension, a dipeptide was coupled[18] to furnish
16 in order to avoid formation of a diketopiperazine. Finally,
the Fmoc group was exchanged for an acetyl group
(Scheme 3).
The synthesis of the STN ± glycododecapeptide started with
Fmoc-proline linked to the aminomethylpolystyrene (AMPS)
resin 8 through the allylic HYCRON anchor[9] and b-alanine.
In order to avoid the formation of a diketopiperazine, a
change to the Boc strategy was chosen for the second amino
acid (Ala). This is possible because of the stability of the
allylic anchor to both acids and bases.[9] The further synthesis
of the resin-bound STN ± glycododecapeptide 9 was per-
formed according to the Fmoc strategy (Scheme 2).
Parallel to this, tert-butyl 12-hydroxy-4,7,10-trioxa-dodeca-
noate[17] (10) was converted through the 12-azido derivative
11 into the 12-amino derivative 12, which was then condensed
with Fmoc-valine to give 13; after cleavage of the tert-butyl
Scheme 3. a) 1. MesCl, NEt3, CH2Cl2, 208C, 3 h, 2. NaN3, DMF,
608C, 18 h; b) Raney-Ni, H2, 2-propanol, 208C, 12 h, 73%;
c) Fmoc-Val-OH, EEDQ, CH2Cl2, 208C, 12 h, 70%; d) 1. TFA,
208C, 2 h, 2. Br-HYCRON-OtBu (ref. [9]), NBu4Br, NaHCO3/
CH2Cl2, 208C, 12 h, 75%; e) TFA, 208C, 2 h, 95%; f) H-AMPS,
TBTU, HOBT, NMM, DMF, 208C, 18 h 74%; g) morpholine/
DMF (1/1); h) Fmoc-Pro-Ser(tBu)-OH, TBTU, HOBT, NMM,
DMF; i) 4 times [1. coupling: Fmoc-AS-OH, TBTU, HOBT,
NMM, DMF; 2. capping: Ac2O/pyridine (1/3); 3. Fmoc-removal:
morpholine/DMF (1/1)]; j) Ac2O/pyridine (1/3); k) [Pd(PPh3)4],
morpholine, DMF/DMSO (1/1) 44% based on 15. Mesˆbmor-
pholinylethanesulfonic acid, EEDQ ˆ 2-ethoxy-N-ethoxycarbon-
yl-1,2-didydroquinoline.
Scheme 1. a) AcCl, 208C, 3 d, quant.; b) KCS2OEt, EtOH, 208C, 4 h, 73%;
c) CH3SBr/AgOTf, CH3CN/CH2Cl2 (2/1), ꢀ628C, 3 h, 60%; d) Ac2O, pyridine, 08C,
4 h, 84%; e) TFA/CH2Cl2 (1/1), anisole, 208C, 6 h, quant. Ac ˆ acetyl, Tf ˆ trifluor-
omethanesulfonyl, TFA ˆ trifluoroacetic acid.
The TTX ± T cell epitope ± spacer conjugate 17 was
obtained by Pd0-catalyzed cleavage of the allylic
anchor.[9] This fragment was coupled to the solid-
phase-bound MUC1 glycododecapeptide after car-
boxy-group activation using HATU/HOAT[19] to fur-
nish the T-cell epitope ± spacer± MUC1 antigen con-
jugate 18 (Scheme 4).
After Pd0-catalyzed cleavage of the HYCRON
anchor and purification by means of preparative
HPLC, the protected T cell epitope ± spacer± MUC1
antigen conjugate 19 was isolated in pure form (as
judged by NMR spectroscopy).[20] Hydrogenolysis of
the sialic acid benzyl ester, acidolysis of the side-chain
protecting groups, and mild methanolysis of the
O-acetyl groups gave the pure conjugate 20 (as
analyzed by HPLC and NMR spectroscopy)[21] in
47% yield. Compound 20 corresponds to the delineat-
ed construct 1 containing the tetanus toxin epitope, a
spacer, and the tumor-associated MUC1 glycopeptide
antigen.
Scheme 2. a) Morpholine/DMF (1/1); b) Boc-Ala-OH, TBTU, HOBT, NMM, DMF;
c) 1. CH2Cl2/TFA (1/1) 2. DIPEA, CH2Cl2; d) ten times [1. 2.±8., 10., and 11. coupling:
Fmoc-AS-OH, TBTU, HOBT, NMM, DMF; 9. coupling: 7; 2. capping: Ac2O/pyridine
(1/3); 3. Fmoc-removal: morpholine/DMF (1/1)]. Boc ˆ tert-butoxycarbonyl,
HOBTˆ hydroxybenzotriazole, TBTU ˆ O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetra-
methyluronium tetrafluoroborate, NMM ˆ N-methylmorpholine, DIPEA ˆ diiso-
propylethylamine, Pmc ˆ 2,2,5,7,8-pentamethylchroman-6-sulfonyl, Fmoc ˆ 9-fluore-
nylmethoxycarbonyl.
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(FACS analysis). The analyses showed that
conjugate 20 induces proliferation of up to
100% for CD3-positive T-cells, and amongst
those for 53% of CD8-positive T-cells. The other
substances also induced proliferation of CD3-
positive T-cells, but a much lower extent of
proliferation for the desired cytotoxic CD8-
positive T-cells. These preliminary results justify
the anticipation that conjugates of T cell epito-
pes and tumor-associated MUC glycopeptide
antigens such as 20 can induce cytotoxic T-cell
response[22] and that on this basis efficient
synthetic antitumor vaccines can be developed.
Received: August 9, 2000 [Z15608]
[1] a) T. Feizi, Nature 1985, 314, 53; b) G. F. Springer,
Science 1984, 224, 1198.
[2] Reviews: a) S. J. Danishefsky, J. R. Allen, Angew.
Chem. 2000, 39, 882; Angew. Chem. Int. Ed. 2000, 112,
836; b) R. R. Koganty, M.-A. Reddish, B. M. Longe-
necker in Glycopeptides and Related Compounds
(Eds.: D. G. Large, C. D. Warren), Marcel Dekker,
New York, 1997, p. 707; c) H. Kunz, P. Wernig, M.
Schilling, J. März, C. Unverzagt, S. Birnbach, U. Lang,
H. Waldmann, Environ. Health Perspect. 1990, 88, 247.
[3] Review: S.-I. Hakomori, Y. Zhang, Chem. Biol. 1997,
4, 97.
[4] H. Kunz, S. Birnbach, Angew. Chem. 1986, 98, 354;
Angew. Chem. Int. Ed. Engl. 1986, 25, 360. TN anti-
gen ˆ Ser/Thr (aGalNAc).
Scheme 4. a) 17, HOAT/HATU/NMM, DMF, 208C, 2 d, 42%; b) Ac2O/pyridine (1/3);
c) [Pd(PPh3)4], morpholine, DMF/DMSO (1/1) 20% based on 8; d) 1. Pd/C (10%), H2,
MeOH, 208C, 1 h 2. CH2Cl2/TFA/thioanisole/ethanedithiol (10/10/1/1), 208C, 2 h 3. NaOMe,
MeOH, pH 8.5, 208C, 18 h, 47% over 3 steps. HOATˆ 7-aza-1-hydroxy-1H-benzotriazole,
HATU ˆ N-[(dimethylamino)-1H-1,2,3-triazol[4,5-b]-pyridin-1-ylmethylene]-N-methylme-
thanaminium hexafluorophosphate.
[5] W. Dippold, A. Steinborn, K.-H. Meyer zum Bü-
schenfelde, Environ. Health Perspect. 1990, 88, 255.
[6] W. Dippold, A. Steinborn, S. Birnbach, H. Kunz,
unpublished results; A. Steinborn, PhD Thesis, Uni-
versity Mainz, 1990.
[7] Review: P. L. Nguyen, G. A. Niehans, D. L. Cherwitz,
Y. S. Kim, S. B. Ho, Tumor Biol. 1996, 17, 176.
[8] S. J. Gendler, C. A. Lancaster, J. Taylor-Papadimitriu, T. Duhig, N.
Peat, J. Burchell, L. Pemberton, E. N. Lalani, D. Wilson, J. Biol. Chem.
1990, 265, 15286.
[9] O. Seitz, H. Kunz, Angew. Chem. 1995, 107, 901; Angew. Chem. Int. Ed.
Engl. 1995, 34, 803.
[10] a) M. Leuck, H. Kunz, J. Prakt. Chem. 1997, 339, 332; b) P. Braun,
G. M. Davies, M. R. Price, P. M. Williams, S. B. J. Tendler, H. Kunz,
Bioorg. Med. Chem. 1998, 6, 1531.
For immunological evaluation, conjugate 20 and, for
comparison, the MUC1 dodecapeptide, the MUC1 STN
glycododecapeptide of idenical sequence, the STN threonine
conjugate, and the tetanus toxin ± spacer conjugate (all in
completely deblocked form) were applied to approximately
105 peripheric blood lymphocytes (PBL) from four healthy
donors in concentrations of 0.1 mg and 1 mg per200 mL. Since a
primary stimulation of the cells did not occur, restimulation
with the same antigen was carried out after seven days until
centers of proliferation appeared. According to an ELISA
with murine antihuman IFNg antibodies, cells from three of
the four donors produced interferon-g after the second
restimulation with all these substances and before the T-cell
growth factor interleukin 2 was added. After the fourth
restimulation, the proliferation of the PBLs was measured
by [3H]thymidine incorporation. Proliferation was found to a
different extent for the cells from each donor with all of the
five substances, and also in a medium free of fetal calf serum.
However, the proliferation only proceeded in the presence of
antigen-presenting cells (APCs) and was not found for
purified T-cells. This is considered as distinct proof of an
antigen-specific reactivity. The proliferating lymphocytes
were characterized using monoclonal antibodies directed
against surface antigens in a fluorescence-activated cell sorter
[11] B. Liebe, H. Kunz, Angew. Chem. 1997, 109, 629; Angew. Chem. Int.
Ed. Engl. 1997, 36, 618.
[12] Other groups have also synthesized mucin glycopeptides with TN ± ,
T± and STN ± antigen saccharide portions, see, for example, a) E.
Meinjohanns, M. Meldal, A. Schleyer, H. Paulsen, K. Bock, J. Chem.
Soc. Perkin Trans. 1 1996, 985; b) S. D. Kuduk, J. B. Schwarz, X.-T.
Chen, P. W. Glunz, D. Sames, G. Ragupanti, P. O. Livingston, S. J.
Danishefsky, J. Am. Chem Soc. 1998, 120, 12474; c) J. B. Schwarz,
S. D. Kuduk, X.-T. Chen, D. Sames, P. W. Glunz, S. J. Danishefsky, J.
Am. Chem Soc. 1999, 121, 2662.
[13] a) J. Burchell, J. Taylor-Papadimitriu, M. Boshell, S. Gendler, T.
Duhig, Int. J. Cancer 1989, 44, 691; b) M. R. Price, F. Hudecz, C.
OꢁSullivan, R. W. Baldwin, P. M. Edwards, S. B. J. Tendler, Mol.
Immunol. 1990, 62, 795.
[14] K. Furuhata, K. Kimio, H. Ogura, T. Hata, Chem. Pharm. Bull. 1991,
39, 255.
[15] A. Marra, P. SinayÈ, Carbohydr. Res. 1989, 187, 36.
[16] Trihydrate of 7: [a]2D2 ˆ 29.1 (c ˆ 1.0, MeOH). MALDI-MS (MeOH,
‡
‡
‡
dihydroxybenzoic acid (DHB)): m/z: 1239.1 [M ꢀ H ‡K ‡Na ],
1223.3 [M ꢀ H ‡2Na ], 1217.0 [M‡K ], 1201.5 [M‡Na ]; 1HNMR
‡
‡
‡
‡
(1H-1H COSY) (400 MHz, CD3OD, TMS): d ˆ 4.99 (dd, 1H, J2,3
ˆ
368
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Angew. Chem. Int. Ed. 2001, 40, No. 2

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11.7 Hz, J3,4 ˆ 3.2 Hz, H-3), 4.90 ± 4.84 (m, 2H, H-1, H-4'), 4.69 (dd,
1H, J ˆ 6.2, 10.9 Hz, OCH2Fmoc), 4.51 (dd, 1H, J ˆ 5.9, 10.9 Hz,
OCH2Fmoc), 4.10 (dd, 1H, J ˆ 5.3, 12.3 Hz, H-9'b), 3.84 (dd, 1H,
J5,6a ˆ 7.5 Hz, J6a,6b' ˆ 10.3 Hz, H-6a), 3.15 (dd, 1H, J5,6b ˆ 4.4 Hz,
Synthesis and Membrane-Binding Properties of
a Characteristic Lipopeptide from the
Membrane-Anchoring Domain of Influenza
Virus A Hemagglutinin**
J6a,6b ˆ 10.3 Hz, H-6b), 2.69 (dd, 1H, J3'e,3'a
ˆ 12.6, J3'e,4' ˆ 4.4 Hz,
H-3'e), 1.22 (d, 3H, J ˆ 6.5 Hz, Tg); 13CNMR (DEPT) (100.6 MHz,
CD3OD): d ˆ 100.77 (C-1), 99.93 (C-2'), 38.83 (C-3'), 19.32 (Tg).
[17] M. Gewehr, H. Kunz, Synthesis 1997, 1499.
Frank Eisele, Jürgen Kuhlmann, and
Herbert Waldmann*
[18] J. Habermann, H. Kunz, Tetrahedron Lett. 1998, 39, 265.
[19] L. A. Carpino, A. S. El-Faham, C. Minor, F. Albericio, J. Chem. Soc.
Chem. Commun. 1994, 210.
Key events in the establishment and progression of viral
infection are attachment and fusion of the virus with the cell
and budding of new virus particles from the infected cell. This
complex multistep process is decisively influenced and
determined by posttranslationally modified proteins embed-
ded in the viral lipid bilayer. For instance, hemagglutinin from
influenza virus A is glycosylated in the extracellular domain[1, 2]
and S-palmitoylated next to the transmembrane region (Fig-
ure 1).[3, 4] The glycoprotein part is responsible for initiation of
viral infection through selective binding to sialic acid recep-
tors on the surface of the host cell. The lipid residues are
required for the interaction between the cell membrane and
the free capsid during budding of viral offspring[5, 6] and are
thought to mediate protein ± protein and protein ± lipid inter-
actions in the viruses.[7] In addition, the lipidated regions may
play an important role in fusion processes of the viral
membrane with the endosome after entry of the viral particle
into the cell.[8, 9] However, this proposal is controversial, since
other investigations indicated that the lipidated cytoplasmic
tail of the complex viral lipoglycoproteins is not essential for
its membrane fusion activity.[10]
For the study of these and related processes in precise
molecular detail, lipidated peptides which represent the
characteristic linkage region between the protein backbone
and the lipid groups and which additionally carry a marker by
which they can be traced in biological systems may be
employed as efficient molecular probes.[11] However, their
synthesis is complicated by the pronounced base lability of the
palmitic acid thioesters which hydrolyse spontaneously at
pH > 7.[12] For the synthesis of such labile peptide conjugates
enzymatic methods may open up viable alternatives to
classical chemical techniques.[11] In this paper we report on
the development of the p-phenylacetoxybenzyl (PAOB) ester,
a new enzyme-labile protecting group for carboxyl function-
[20] 19: [a]2D2 ˆ ꢀ20.8 (c ˆ 1.0, CH3CN). FAB-MS (nitrobenzyl alcohol
(nba), positive ion): m/z (%): 3754.4 (62.3) [M(2 Â 13C) ꢀ 4 Â (tert-
‡
butyl)‡2  Na ], calcd: 3753.74, 3755.1 (100) [M(3  13C) ꢀ 3  (tert-
‡
butyl)‡2  Na ], 3755.8 (82.7) [M(4  13C) ꢀ 4  (tert-butyl)‡2 Â
‡
‡
1
Na ], 3757.2 (49.1) [M(5  13C) ꢀ 4  (tert-butyl) ‡ 2  Na ]; HNMR
(400 MHz, [D6]DMSO, TMS): d ˆ 8.48 ± 7.41 (m, 18H, NH), 7.38 (mc
5H, HAr-Bn), 7.28 ± 7.16 (m, 5H, HAr-F), 7.11 (d, 2H, J ˆ 7.9 Hz), 7.03
(d, 2H, J ˆ 7.6 Hz, 2  (H2-Y, H6-Y)), 6.82 ± 6.76 (m, 4H, 2  (H3-Y,
H5-Y)), 6.70, 6.55 (Sbr, RNH(e,x,h)), 5.22 ± 5.14 (m, 5H, H-4, H-7', H-8',
CH2Bn), 4.92 ± 4.85 (m, 2H, H-1, H-3); 4.78 ± 469 (m, H-4'), 4.65 ± 3.24
a
a
a
a
b
ˆ
(m, 58H, F , 2 Â Y , 2 Â V , 3 Â S , 3 Â S , CH2CH2(OCH2CH2CH2)2
CH2CH2, Ra, Da, 2 Â Aa, 4 Â Pa, 2 Â Ta, Ga, Va, H-2, H-5, H-6', H-9',
2 Â Tb, 4 Â Pd, H-6a, H-5'), 3.22 ± 2.60 (m, 10H, Fb, 2 Â Yb, H-6b, Rd,
H-3'e), 2.57 ± 2.38 (m, 4H, Db, CH2CH2Pmc), 2.38 ± 2.35 (m, 2H,
CH2CONH-G-V), 2.49, 2.46 (s, 6H, o,o'-CH3Pmc), 2.18 ± 1.58 (m,
18H, 4 Â Pb, 4 Â Pg, CH2CH2Pmc), 2.03, 2.00, 1.99, 1.93, 1.91, 1.85, 1.82,
1.75, 1.71, 1.66 (10 Â s, 30H, m-CH3Pmc, 6 Â OAc, 3 Â NHAc), 1.53 ±
1.02 (m, 9H, 2 Â Vb, Rg, Rb, H3'a, 2 Â Ab), 1.35, 1.23, 1.20, 1.15, 1.11,
1.08, 1.06, 1.05, (s, 69H, 7 Â C(CH3)3, C(CH3)2Pmc), 0.98 ± 0.90 (m,
3H, T1g), 0.89 ± 0.83 (m, 3H, T2g), 0.82 ± 0.78 (m, 12H, Vg); 13CNMR
(DEPT) (100.6 MHz, [D6]DMSO): d ˆ 98.40 (C-1), 98.01 (C-2'), 41.18,
40.97, 38.40, 36.95, 35.87 (2 Â Yb, Fb, C-3', Db, Rd, CH2CONH), 32.12
(CH2CH2Pmc), 19.05, 18.98, 18.75, 18.75, 18.16, 18.07, 17.95 (2 Â Vg,
g
g
T1 , o,o'-CH3Pmc), 16.77 16.72, 16.00 (T2 , 2 Â Ab), 11.82 (m-CH3Pmc).
[21] 20: [a]2D2 ˆ ꢀ76.9 (c ˆ 1.0, H2O); FAB-MS (nba ‡ LiBr, positive ion):
m/z (%): 2770.7 (0.62) [M‡Li ], calcd: 2770.30; 1HNMR (1H-1H
‡
COSY) (400 MHz, D2O): d ˆ 7.37 ± 7.24 (m, 3H), 7.23, 7.16 (m, 2H,
HAr-F), 7.08 ± 6.97 (m, 4H, 2 Â (H2-Y, H6-Y)), 6.81 ± 6.72 (m, 4H, 2 Â
(H3-Y, H5-Y)), 4.90 (mc, 1H, H-1), 4.72 ± 4.66 (m, 1H, Da), 4.64 ± 4.59
(m, 2H, Fa, Ra), 4.57 ± 4.50 (m, 1H, A1a), 4.49 ± 4.42 (m, 6H, A2a, 2 Â
Sa, 2 Â Ya), 4.41 ± 4.33 (m, 6H, 4 Â Pa, S3a, T1a), 4.32 ± 4.29 (m, 2H, V1
,
a
a
T2 ), 4.28 ± 4.23 (m, 1H, T1b), 4.22 ± 4.17 (m, 1H, T2b), 4.12 ± 4.03 (m,
3H, H-2, V2a, S1b), 4.01 ± 3.27 (m, 40H, H-3, H-4, H-5, H-6, H-4', H-5',
H-6', H-7', H-8', H-9', NH(CH2CH2O)3CH2CH2CONH, 2 Â Sb, Ga,
4 Â Pd), 3.17 (mc, 2H, Rd), 3.10 ± 2.99 (m, 1H, Fba), 2.90 ± 2.77 (m, 7H,
2 Â Yb, Db, Fbb), 2.67 (dd, 1H, H-3'e), 2.60 ± 2.54 (m, 2H, CH2CONH-
G-V-), 2.33 ± 2.18 (m, 4H 4 Â Pba), 2.11 ± 1.77 (m, 15H, 2 Â Vb, 4 Â Pbb
,
4 Â Py, Rba), 2.03, 2.01, 1.93 (s, 9H, 3 Â NHAc), 1.76 ± 1.59 (m, 4H,
H-3'a, Rbb, Rg), 1.34 (m, 6H, J ˆ 6.5 Hz, 2  Ab), 1.30 ± 1.21 (m, 3H,
T1g), 1.15 (d, 3H, J ˆ 6.2 Hz, T2 ), 0.97 ± 0.85 (m, 12H, 2  Vg);
g
13CNMR (DEPT) (100.6 Mhz, D2O): d ˆ 99.62 (C-1), 99.20 (C-2'),
22.14, 21.94, 21.49, (3 Â NHAc), 18.73, 18.39, 18.22, 17.54 (2 Â Tg, 2 Â
Vg), 15.11, 14.92 (2 Â Ab).
[*] Prof. Dr. H. Waldmann
[22] For the importance of this key property, see Section 2 in ref. [2a],
p. 885.
Max-Planck-Institut für molekulare Physiologie
Abteilung Chemische Biologie
and Universität Dortmund
Institut für Organische Chemie
Otto-Hahn-Strasse 11, 44227 Dortmund (Germany)
Fax: (‡49)231-133-2499
E-mail: herbert.waldmann@mpi-dortmund.mpg.de
Dr. J. Kuhlmann
Max-Planck-Institut für molekulare Physiologie
Abteilung Strukturelle Biologie
Otto-Hahn-Strasse 11, 44227 Dortmund (Germany)
Dr. F. Eisele
Universität Karlsruhe
Institut für Organische Chemie
Richard-Willstätter-Allee 2, 76128 Karlsruhe (Germany)
[**] This research was supported by the Deutsche Forschungsgemeinschaft
and the Fonds der Chemischen Industrie.
Angew. Chem. Int. Ed. 2001, 40, No. 2
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
1433-7851/01/4002-0369 $ 17.50+.50/0
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