Tumour-associated glycopeptide antigens and their modification in anticancer vaccines - Carbohydrate Chemistry: Chemical and Biological Approaches - page 513

building blocks were carried out as will briefly be outlined for the next
example (Scheme 6). After completion of the glycopeptide assembly in a
peptide synthesizer and final N-acetylation, the MUC4-tandem repeat
glycopeptide 32 was detached from the resin by treatment with TFA/
triisopropylsilane (TIS) and water (17 : 1 : 1). 28
The trityl linker 34 48 was used, for example, in the synthesis of a MUC1
tandem repeat glycopeptide carrying the sialyl-Tn-antigen at serine of the
GSTA sequence (Scheme 6). 49 Starting from resin-linked Fmoc-alanine 34
the automated solid-phase synthesis included three typical steps ac-
cording to a well-proven protocol: 28 removal of the Fmoc group using
piperidine (20%) in N-methylpyrrolidone (NMP); after washing, coupling
of Fmoc amino acids (10 eq.) activated with O-(benzotriazol-1-yl)-
N,N,N 0 ,N 0 -tetramethyluronium hexafluorophosphate (HBTU) 50 and 1-
hydroxy-benzotriazol (HOBt); after washing, capping of unreacted amino
functions using Ac 2 O, catalytic HOBt and diisopropylethylamine (DIPEA)
in NMP. The Fmoc sialyl-Tn building block 35 (2 eq.) was coupled
in a prolonged reaction time (4 h) using the more reactive
O-(7-azobenzotriazol-1-yl)-N,N,N 0 ,N 0 -tetramethyluronium hexafluorophos-
phate (HATU) 51 and 7-aza-1-hydroxybenzotriazol (HOAt). A triethylene
glycol-derived spacer amino acid 27 was coupled as the N-terminal com-
ponent. After removal of the Fmoc group and acidolytic release from
resin, the MUC1(22) glycopeptide 36 still protected in the carbohydrate
portion was isolated and purified (overall yield 29%). 49
The carbohydrate protecting groups were finally removed by treatment
with aq. NaOH solution at pH 11. Under these conditions, base-catalysed
b-elimination of the carbohydrate does not occur. Purification by semi-
preparative HPLC gave the pure glycopeptide antigen 37 in a 20mg scale. 49
Applying the same methodology, a series of MUC1 tandem repeat
glycopeptide antigens were synthesised on solid phase. For example,
using the Fmoc-protected T-antigen-threonine building block 15, the
T-antigen-threonine containing MUC1 glycopeptide 38 was obtained
(Scheme 7). 33,52
There was no change in the reaction conditions required in the solid-
phase synthesis, release reaction and removal of the protecting groups in
order to accomplish the synthesis of the analogous 6,6 0 -difluoro-T-anti-
gen MUC1 glycopeptide 39 in likewise good yield. 33,53
C-Glycosidically linked glycosyl amino acids of type 30 (Scheme 4) are
less sensitive to basic conditions. Logically, mimics of natural MUC1
glycopeptide antigens, as for example C-glycopeptide 40 43 containing the
C-glucosyl-tyrosine (Scheme 8) was accessible through solid-phase
syntheses as outlined for antigen 37.
In addition to these glycopeptide antigens, we also synthesised the non-
glycosylated MUC1(22)-peptide 41 according to the same procedure in
order to compare the immunological results and demonstrate the influence
of the carbohydrates on these MUC1 antigen vaccines (Scheme 9). 54
The peptide and glycopeptide antigens displayed in Schemes 6-9 con-
tain an N-terminal spacer amino acid which is derived from triethylene
glycol. Such hydrophilic, flexible structural elements are not immuno-
genic, but useful for the coupling to other components which enhance the