Synthesis of a tumor-associated 2,3-sialyl-T glycododecapeptide antigen from the tandem repeat region of the mucin MUC1

Article abstract of DOI:10.1055/s-2003-41009

As a cell surface antigen for the development of selective antitumor vaccines, a tumor-associated glycododecapeptide from epithelial MUC1 carrying the 2,3-sialyl-T antigen was synthesized. The 2,3-ST building block was assembled from a T_N-threo

Full text of DOI:10.1055/s-2003-41009

LETTER
1623
Synthesis of a Tumor-Associated 2,3-Sialyl-T Glycododecapeptide Antigen
from the Tandem Repeat Region of the Mucin MUC1
Synthesis of
a
e
2,3-Sialyl-
b
a
capeptide
f
s
rom MU
C
t
1
ian Dziadek, Horst Kunz*
Institut für Organische Chemie, Johannes Gutenberg-Universität Mainz, Duesbergweg 10-14, 55128 Mainz, Germany
Fax +49(6131)3924786; E-mail: hokunz@uni-mainz.de
Received 18 June 2003
Dedicated to Professor Wolfgang Steglich on the occasion of his 70th birthday.
We here describe a novel synthetic approach toward the
tumor-associated 2,3-sialyl-T antigen building block and
its incorporation into a glycododecapeptide of the tandem
repeat domain of the MUC1 mucin. In contrast to other
strategies,6 the complex sialic acid was introduced at a late
stage of the synthesis in a regio- and stereoselective re-
action. Starting from the readily available glycosylated
amino acid Fmoc-Thr(aGalNAc)-O-t-Bu7 1, the T disac-
charide was synthesized first and subsequently converted
into the a-2,3-sialyl T antigen (Scheme 1).
Abstract: As a cell surface antigen for the development of selective
antitumor vaccines, a tumor-associated glycododecapeptide from
epithelial MUC1 carrying the 2,3-sialyl-T antigen was synthesized.
The 2,3-ST building block was assembled from a TN-threonine con-
jugate by stepwise saccharide chain extension and utilized for the
solid-phase glycopeptide synthesis on the fluoride-labile PTMSEL
linker.
Key words: 2,3-sialyl-T antigen, glycopeptides, MUC1, regio-
selective sialylation, solid-phase synthesis
For the 3-b-galactosylation of Fmoc-Thr(aGalNAc)-O-t-
Bu 1, protection of the 4- and 6-OH groups as benzylidene
acetal was necessary to give 2 (yield: 83%).8 The subse-
quent glycosylation using 6-O-benzyl protected galacto-
In contrast to normal cells, tumor cells are significantly al-
tered in their glycoprotein profile on the outer cell sur-
face.1 This difference constitutes the basis for a selective
immunological attack at certain cancer cells.
9
syl bromide 5 activated with Hg(CN)2 in a mixture of
The epithelial mucin MUC12 is a heavily O-glycosylated
glycoprotein present at the interface between many epi-
thelia and their extracellular environments.3 The extracel-
lular domain consists of tandem repeats comprising 20
amino acids of the sequence HGVTSAPDTRPAPG-
STAPPA, including five potential O-glycosylation sites.
In epithelial tumor cells, the expression of MUC1 is dras-
tically increased which is combined with an incomplete
formation of the glycan side-chains due to a premature
sialylation. The aberrant glycosylation results in the expo-
sure of additional peptide epitopes which hence become
accessible to the immune system. The 2,3-sialyl-T antigen
(aNeuNAc-2,3-bGal-1,3-aGalNAc-O-Ser/Thr) consti-
tutes an important tumor-associated saccharide which was
found on breast cancer cells.4 Glycopeptides, containing
partial structures of cancer-associated cell-surface glyco-
proteins, are considered promising candidates for the con-
struction of tumor-selective immunostimulating antigens.
In particular, glycopeptides from MUC1 have been shown
to be processed by antigen-presenting dendritic cells and
presented via MHC molecules – leaving the glycan side-
chain intact5 – thus bearing the potential of inducing both
humoral as well as cellular immune responses. For the de-
velopment of antitumor vaccines, the generation of syn-
thetic glycopeptides, combining both saccharide as well
as peptide epitopes, is therefore a feasible strategy.
nitromethane/dichloromethane (3:2) furnished the disac-
charide conjugate 6 in a yield of 85%. Galactosyl bromide
5 was prepared from 1,2:3,4-diisopropylidene galactose 3,
which was subsequently benzylated at the 6-OH function
(87%). Following acidolysis of the acid-labile isopropyl-
idene protecting groups, acetylation (4, 89% over two
steps) and careful treatment with HBr in glacial acid af-
forded 55% of the galactosyl donor 5 after silica gel filtra-
tion.10
Due to steric hindrance, O-deacetylation of the conjugate
6 proved difficult. Only careful, prolonged treatment with
NaOMe/MeOH at pH = 8.5 furnished the acceptor 7 in a
yield of 62%. The key step of the synthetic strategy in-
volved the stereo- and regioselective sialylation of the T
antigen disaccharide 7 at the 3¢-hydroxy group. For this
purpose, xanthate 8 of the N-acetylneuraminic acid benzyl
ester11 was activated with phenylsulfenyl triflate, generat-
ed in situ from phenylsulfenyl chloride and silver tri-
flate.12 Di-tert-butylpyridine (DTBP) served as proton
scavenger.12 Low temperature and the use of acetonitrile/
dichloromethane (2:1) as solvent favored the stereoselec-
tive formation of the a-2,3-sialyl-T threonine conjugate 9
in a yield of 50% (a/b, 97:3).13 Cleavage of the ben-
zylidene acetal with acetic acid (10, 82%) and O-acetyla-
tion with pyridine/acetic anhydride (2:1) catalyzed by
N,N-dimethylamino pyridine (DMAP)14 afforded the pro-
tected compound 11 in a yield of 87%. Subsequent careful
acidolysis of the tert-butyl ester quantitatively furnished
the Fmoc-2,3-ST threonine building block 12.15
Synlett 2003, No. 11, Print: 02 09 2003. Web: 11 08 2003.
Art Id.1437-2096,E;2003,0,11,1623,1626,ftx,en;G13903ST.pdf.
DOI: 10.1055/s-2003-41009
© Georg Thieme Verlag Stuttgart · New York

1624
S. Dziadek, H. Kunz
LETTER
OH
O
OBn
O
AcO
AcO
O
O
O
b
3
O
OAc
AcO
4
c
OBn
O
OAc
OAc
AcO
AcO
OR
RO
BnO2C
S
AcO
O
O
OBn
RO
RO
O
O
AcHN
S
OEt
HO
AcO
O
O
5
8
1 R = H
AcO
Br
AcHN
O
O
a
OR
AcHN
d
2 R, R = CH-Ph
f
O
OtBu
FmocHN
6 R = Ac
7 R = H
OtBu
e
O
FmocHN
O
OAc
OAc
OAc
OR2
O
OBn
OAc
O
BnO2C R1O
R2O
O
BnO C
AcO
2
OBn
AcO
AcO
AcO
OAc
O
O
O
O
AcHN
AcHN
O
O
O
OR1
AcHN
AcO
AcO
OAc
AcHN
O
O
h
9 R1 = H; R2,R2 = CH-Ph
10 R1 = R2 = H
11 R = tBu
12 R = H
OtBu
OR
O
i
FmocHN
FmocHN
g
O
Scheme 1 Reagents and conditions: a) a,a-dimethoxytoluene, p-TsOH, CH3CN, 3 h, 83%. b) i) NaH, BnBr, DMF, 24 h, 87%; ii) 1. AcOH
(60%), 85 °C, 2. pyridine/Ac2O (2:1), DMAP, 6 h, 89%. c) HBr in AcOH (33%), CH2Cl2, 0 °C, 30 min, 55%. d) Hg(CN)2, CH3NO2/CH2Cl2
(3:2), MS 3 Å, 25 h, 85%. e) NaOMe/MeOH, pH = 8.5, 12 h, 62%. f) CH3CN/CH2Cl2 (2:1), MS 3Å, PhSCl, AgOTf, DTBP, –68 °C, 4 h, 50%
(a/b, 97:3). g) AcOH (80%), 80 °C, 1 h, 82%. h) pyridine/Ac2O (2.3:1), DMAP, 6h, 87%. i) TFA, anisole, quant. DTBP = di-tert-butylpyridine.
The solid-phase synthesis of a glycododecapeptide from 2.5 equivalents TBAF·3H2O in dichloromethane (45 min
the tandem repeat region of MUC1 carrying the 2,3-sialyl- each) to furnish the protected glycopeptide 14 in a yield of
T antigen was performed in a peptide synthesizer16 ac- 39% (based on the loaded resin 13) after purification by
cording to the Fmoc-strategy. NovaSyn Tenta Gel resin semi-preparative RP-HPLC. Subsequently, the acid-labile
equipped with the novel fluoride-labile PTMSEL linker17 side-chain protecting groups were removed by acidolysis
was employed and loaded with the starting amino acid with a mixture of trifluoroacetic acid, triisopropylsilane
Fmoc-proline. This anchor can be cleaved under almost and water (15:0.9:0.9). The partially deprotected glyco-
neutral conditions by applying tetrabutylammonium fluo- conjugate 15 was precipitated into diethyl ether and thor-
ride trihydrate (TBAF·3 H2O) in dichloromethane. Under oughly washed. Cleavage of the sialic acid benzyl ester as
these conditions aspartate structures do not rearrange via well as the galactose benzyl ether was accomplished by
aspartimides, and most of the common protecting groups hydrogenolysis (23 h) in methanol using palladium on ac-
in Fmoc-peptide chemistry are not affected. The first eight tivated charcoal (10%) as catalyst. Complete final
amino acids of the glycododecapeptide were coupled ac- deacetylation was only achieved by treatment with aque-
cording to the standard protocol. After removal of the ous NaOH (5 mM). At first, a derivative occurred as the
Fmoc-protecting group of 13 with piperidine in N-meth- sole product still carrying one acetyl group, presumably at
ylpyrrolidone (NMP), the extension of the peptide chain the hindered 4¢-OH group of the galactose portion. Ex-
was performed by iterative coupling using a 10-fold ex- tended reaction time slowly furnished the desired product.
cess of each amino acid activated by HBTU18/HOBt and The reaction was carefully monitored by analytical
diisopropylethylamine (DIPEA) in NMP (Scheme 2). Un- HPLC. After 59 h the reaction was terminated with a con-
reacted amino groups were capped after each step with version of 89%. Neither b-elimination of the glycan por-
Ac2O/DIPEA/HOBt (4:1:0.12) in NMP. The 2,3-sialyl-T tion nor epimerization were detectable. The pure,
threonine building block 12 (1.4 equiv) was coupled man- completely deprotected glycododecapeptide 1620 was ob-
ually over a period of 4 h using HATU/HOAt19 and N-me- tained after purification by semipreparative RP-HPLC in
thylmorpholine (NMM) for activation. The final two a yield of 56% over three steps.
amino acids were attached according to the standard pro-
tocol. After completed synthesis the N-terminal Fmoc
group was exchanged for an acetyl group. The glycocon-
The immunological evaluation of this synthetic 2,3-sialyl-
T glycopeptide is currently in progress.
jugate was detached from the resin by treatment with 2 ×
Synlett 2003, No. 11, 1623–1626 © Thieme Stuttgart · New York

LETTER
Synthesis of a 2,3-Sialyl-T Glycododecapeptide from MUC1
1625
PTMSEL
O
O
O
O
N
H
O
N
O
Si(CH3)3
a SPGS
13
2,3-sialyl-T
PTMSEL
Ac-Gly-Val-Thr-Ser(tBu)-Ala-Pro-Asp(OtBu)-Thr(tBu)-Arg(Pmc)-Pro-Ala-Pro
b
OAc
OAc
OBn
O
OAc
O
BnO2C AcO
AcO
O
AcO
O
AcHN
O
AcO
R1O
O
OAc
AcHN
O
O
O
O
O
H
N
H
H
H
N
N
OH
N
N
N
N
AcHN
N
N
N
H
O
N
H
H
H
O
O
O
O
O
O
O
R1O
OR1
NH
14 R1 = tBu, R2 = Pmc
15 R1 = H, R2 = H
HN
NHR2
c
OH
OH
OH
d,e
HO2C
O
HO
O
HO
O
HO
OH
HO
O
O
AcHN
OH
AcHN
O
O
HO
O
O
O
O
H
N
H
N
H
H
N
OH
N
N
N
N
AcHN
N
N
N
H
O
N
H
H
H
O
O
O
O
O
O
O
HO
16
OH
NH
HN
NH2
Scheme 2 Reagents and conditions: a) solid-phase glycopeptide synthesis (SPGS): Fmoc-removal [piperidine/NMP (20%)], coupling (1.-8.,
10.-11.: Fmoc-AA-OH (10 equiv), HBTU/HOBt/DIPEA, DMF, 9. 12 (1.4 equiv), HATU/HOAt/NMM, DMF, 4 h), capping: Ac2O, DIPEA,
HOBt (4:1:0.12). b) TBAF·3H2O (2 × 2.5 equiv), CH2Cl2, 45 min (each time), 39% (with respect to 13). c) TFA/TIS/H2O (15:0.9:0.9), 2 h. d)
H2, Pd/C (10%), MeOH, 23 h. e) NaOHaq (5 mM), 59 h (56% over 3 steps). HATU = O-(7-aza-benzotriazole-1-yl)-N,N,N¢,N¢-tetramethyluro-
nium hexafluorophosphate, HBTU = O-(1H-benzotriazol-1-yl)-N,N,N¢,N¢-tetramethyluronium hexafluorophosphate, HOAt = N-hydroxy-7-
azabenzotriazole, HOBt = N-hydroxybenzotriazole; NMM = N-methylmorpholine; TBAF = tetrabutylammonium fluoride; TIS = triisopropyl-
silane; Pmc = 2,2,5,7,8-pentamethylchroman-6-sulfonyl-; PTMSEL = (2-phenyl-2-trimethylsilyl)ethyl-linker.
Acknowledgment
References
This work was supported by the Deutsche Forschungsgemeinschaft
and by the Stiftung Rheinland-Pfalz für Innovation. S. D. is grateful
for a doctoral fellowship provided by the Fonds der Chemischen In-
dustrie and the Bundesministerium für Bildung und Forschung
(BMBF).
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Graham, R.; Correa, I.; Miles, D.; Smith, M. J. Mammary
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Synlett 2003, No. 11, 1623–1626 © Thieme Stuttgart · New York

1626
S. Dziadek, H. Kunz
LETTER
169.76, 167.77 (C=O), 156.83 (C=O-urethane), 143.71
Müller, S. Glycobiology 2000, 10, 439. (c) von Mensdorff-
Pouilly, S.; Snijdewint, F. G. M.; Verstraeten, A. A.;
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Miles, D. W.; Dalziel, M. Biochim. Biophys. Acta 1999, 301.
(C1a-, C8a-Fmoc), 141.29, 141.15 (C4a-, C5a-Fmoc),
138.07, 137.51 (Cq-Bn), 134.21 (Cq-Bn), 128.97, 128.84,
128.78, 128.43, 127.51 (Car-Bn), 128.78, 128.04, 126.44
(Car-Bzn), 127.79 (C3-, C6-Fmoc), 127.09, 126.97 (C2-, C7-
Fmoc), 125.17, 124.94 (C1-, C8-Fmoc), 120.03 (C4-, C5-
Fmoc), 106.55 (C1¢), 100.96 (CH-Bzn), 100.45 (C1), 97.47
(C2¢¢), 83.13 (Cq-t-Bu), 78.15 (C3), 75.94, 75.15 (Tb, C4,
C3¢), 73.60 (CH2-Bn), 73.43, 72.92 (C6¢¢,C5¢), 69.72 (C8¢¢,
C6¢), 69.07 (C6), 68.61 (C4¢¢), 68.22, 68.11 (CH2-Bn,
C2¢,C4¢,C7¢¢), 67.25 (CH2-Fmoc), 63.64 (C9¢¢), 63.52 (C5),
59.24 (Ta), 49.08 (C5¢¢), 47.79 (C2), 47.09 (C9-Fmoc), 37.42
(C3¢¢), 28.06 (CH3-t-Bu), 23.16, 23.07 (CH3-NHAc), 21.33,
21.21, 21.01, 20.78, 20.64 (CH3-OAc), 19.47 (Tg), HR-ESI-
TOF-MS (positive ion mode): calcd. for C77H91N3O27Na:
1512.5737, found: 1512.5729 [M + Na]+.
(3) (a) Gum, J. R. Biochem. Soc. Trans. 1995, 23, 795.
(b) Carlstedt, I.; Davies, J. R. Biochem. Soc. Trans. 1997, 25,
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(4) Brockhausen, I.; Yang, J.-M.; Burchell, J.; Whitehouse, C.;
Taylor-Papadimitriou, J. Eur. J. Biochem. 1995, 233, 607.
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Jr.; Hanisch, F.-G.; Finn, O. J. J. Exp. Med. 2002, 11, 1435.
(6) (a) Nakahara, Y.; Nakahara, Y.; Ito, Y.; Ogawa, T.
Carbohydr. Res. 1998, 309, 287. (b) Komba, S.; Meldal,
M.; Werdelin, O.; Jensen, T.; Bock, K. J. Chem. Soc., Perkin
Trans. 1 1999, 415. (c) Schwarz, J. B.; Kuduk, S. D.; Chen,
X.-T.; Sames, D.; Glunz, P. W.; Danishefsky, S. J. J. Am.
Chem. Soc. 1999, 121, 2662.
(7) (a) Liebe, B.; Kunz, H. Angew. Chem. Int. Ed. Engl. 1997,
36, 618. (b) Liebe, B.; Kunz, H. Helv. Chim. Acta 1997, 80,
1473. (c) Dziadek, S.; Espínola, C. G.; Kunz, H. Aust. J.
Chem. 2003, 56, 519.
(8) George, S. K.; Holm, B.; Reis, C. A.; Schwientek, T.;
Clausen, H.; Kihlberg, J. J. Chem. Soc., Perkin Trans. 1
2001, 880.
(14) Neises, B.; Steglich, W. Angew. Chem., Int. Ed. Engl. 1978,
17, 522.
(15) 12: [a]D27 = 24.75 (c = 1, CHCl3), HR-ESI-TOF-MS
(positive ion mode): calcd. for C74H86N3O31Na2: 1558.5040,
found: 1558.5024 [M – H + 2 × Na]+.
(16) Perkin Elmer ABI 433A (Applied Biosystems).
(17) (a) Wagner, M.; Kunz, H. Angew. Chem. Int. Ed. 2002, 41,
317. (b) Wagner, M.; Kunz, H. Z. Naturforsch. 2002, 57b,
928.
(9) Helferich, B.; Wedemeyer, F. Liebigs Ann. Chem. 1949,
563, 139.
(18) Knorr, R.; Trzeciak, A.; Bannwarth, W.; Gillessen, D.
Tetrahedron Lett. 1989, 30, 1927.
(10) Lergenmüller, M.; Ito, Y.; Ogawa, T. Tetrahedron 1988, 54,
1381.
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(b) Keil, S.; Claus, C.; Dippold, W.; Kunz, H. Angew. Chem.
Int. Ed. 2001, 40, 366.
(19) (a) Carpino, L. A. J. Am. Chem. Soc. 1993, 115, 4397.
(b) Carpino, L. A.; El-Faham, A. S.; Minor, C.; Albericio, F.
J. Chem. Soc., Chem. Commun. 1994, 210.
(20) 16: [a]D26 = –40.68 (c = 0.86, H2O), Rt = 14.52 min
[Phenomenex Jupiter C18, grad.: CH3CN/H2O + 0.1% TFA
(5:95) → (30:70), 40min]; 1H NMR (400 MHz, D2O, COSY,
TOCSY, HMQC): d (ppm) = 4.92 (d, 1 H, H1, JH1,H2 = 3.72
Hz), 4.69 (t, 1 H, Da, JDa,Db = 6.36 Hz), 4.65–4.58 (m, 2 H,
(12) Martichonok, V.; Whitesides, G. M. J. Org. Chem. 1996, 61,
1702.
(13) 9: [a]D27 = 32.01 (c = 1, CHCl3); Rt = 30.07 min
[Phenomenex Luna C18 (2), grad.: CH3CN/H2O (60:40) →
(90:10), 40min]; 1H NMR (400 MHz, CDCl3, COSY,
a
TSTa {4.63}, Ra {4.62}), 4.57–4.49 (m, 1 H, A1 ), 4.49–4.41
(m, 3 H, H1¢ {4.47}, Sa {4.45}, A2a {4.44}), 4.40–4.34 (m,
a
HMQC): d (ppm) = 7.74 (d, 2 H, H4-, H5-Fmoc, JH4,H3
=
=
3 H, 3 × Pa), 4.33–4.26 (m, 3 H, TSTb {4.31}, Va {4.29}, T1
b
J
H5,H6 = 7.84 Hz), 7.58 (dd, 2 H, H1-, H8-Fmoc, JH1,H2
{4.28}), 4.22–4.14 (m, 3 H, H4 {4.21}, H2 {4.19}, T1
JH8,H7 = 7.80 Hz), 7.49 (d, 2 H, Har-Bzn, JHa,Hb = 5.84), 7.42–
7.35 (m, 2 H, H3-, H6-Fmoc), 7.35–7.24 (m, 15 H, H2-, H7-
Fmoc, Har-Bzn (3 H), Har-Bn (10 H)), 6.59 (d, 1 H, NH-
GalNAc, JNH,H2 = 9.40 Hz), 6.12 (d, 1 H, NH-T, JNH,Ta = 9.76
Hz), 5.45 (s, 1 H, CH-Bzn), 5.40 (t, 1 H, H8¢¢, JH8¢¢,H9¢¢ = 6.64
Hz), 5.28–5.13 (m, 2 H, H7¢¢, NH-NeuNAc), 5.16 (s, 2 H,
CH2-Bn), 4.98 (d, 1 H, H1, JH1,H2 = 3.52 Hz), 4.92–4.81 (m,
1 H, H4¢¢), 4.74–4.63 (m, 1 H, H2), 4.55 (s, 2 H, CH2-Bn),
4.52–4.03 (m, 10 H, H9a¢¢{4.46}, CH2-Fmoc {4.39, 4.34},
Tb {4.33}, H4 {4.26}, Ta {4.25}, H9-Fmoc {4.19}, H1¢
{4.15}, H6a {4.13}, H5¢¢ {4.06}), 3.99–3.84 (m, 4H, H6¢¢
{3.93}, H9b¢¢ {3.92}, H3¢ {3.90}, H6b {3.87}), 3.72–3.55
(m, 4 H, H3 {3.67}, H2¢ {3.61}, H6a¢ {3.59}, H5 {3.57}),
3.54–3.39 (m, 2 H, H6b¢{3.87}, H5¢{3.41}), 3.24 (s, 1 H,
{4.18}), 4.08–3.95 (m, 3 H, H3¢ {4.04}, H7¢¢ {4.03}, H3
{4.00}), 3.94–3.87 (m, 3 H, Ga {3.91}, H4¢{3.90}), 3.86–
3.52 (m, 20 H, H5¢, H8¢¢ {3.83}, H6a {3.80}, Sb {3.76}, 3 ×
Pad {3.73}, H5¢¢ {3.71}, H9a¢¢, H9b¢¢, H4¢¢ {3.67}, 3 × Paa
{3.63}, H6b {3.62}, H6¢¢, H5, H6a¢, H6b¢), 3.48 (dd, 1 H, H2¢,
J
H1¢,H2¢ = 7.92 Hz), 3.21–3.14 (m, 2 H, Rd), 2.99–2.83 (m, 2
H, Db), 2.71 (dd, 1 H, H3eq¢¢, JH3eq¢¢,H3ax¢¢ = 12.20 Hz,
J
H3eq¢¢,H4¢¢ = 4.40 Hz), 2.35–2.19 (m, 3 H, 3 × Pab), 2.10–1.94
(m, 8 H, Vb {2.03}, 3 × Pg {2.01}, Rab), 2.02, 1.99, 1.97 (3 ×
s, 9 H, 3 × CH3-NHAc), 1.93–1.77 (m, 4 H, 3 × Pbb {1.88},
H3ax¢¢ {1.80}), 1.77–1.59 (m, 3 H, Rb {1.70}, Rg {1.63}),
b
1.38–1.31 (m, 6 H, 2 × Ab), 1.24 (d, 3 H, TSTg, JTg,Tb = 6.12
Hz), 1.15 (d, 3 H, T1 , JTg,Tb = 6.36 Hz), 0.94 (d, 6 H, Vg);
g
MALDI-TOF-MS (dhb, positive ion mode): calcd. for
C76H124N17O37: 1867.9, found: 1868.8 [M + H]+, 1890.9 ([M
+ Na]+, calcd.: 1889.9), 1907.1 ([M + K]+, calcd.: 1905.9),
1912.8 ([M – H + 2 × Na]+, calcd.: 1911.9); ESI-MS
(positive ion mode): calcd. for [C76H121N17O37Na4]2+:
977.88, found: 977.87 [M – 2 H + 4 Na]2+.
H4¢), 2,78 (sb, 1 H, OH), 2.71 (dd, 1 H, H3eq¢¢, JH3eq¢¢,H3ax¢¢
=
12.72 Hz, JH3eq¢¢,H4¢¢ = 4.28 Hz), 2.36 (s, 1 H, OH), 2.03–2.00
(m, 1 H, H3ax¢¢), 2.09, 2.07, 2.02, 2.00, 1.92, 1.82 (5*s, 18H,
6* CH3-Ac), 1.42 (s, 9 H, CH3-t-Bu), 1.26 (d, 3 H, Tg,
J
Tg,Tb = 5.92 Hz); 13C NMR (100.6 MHz, CDCl3, BB,
HMQC): d = 171.53, 170.91, 170.82, 170.24, 170.09,
Synlett 2003, No. 11, 1623–1626 © Thieme Stuttgart · New York