SYNTHESIS OF MULTI-EPITOPIC GLYCOPEPTIDE-BASED CANCER VACCINES - Biologically-Responsive Hybrid Biomaterials - page 146

without protein carrier or immunoadjuvant [44]. Later, the incorporation of
T-cell epitope peptide into a three-component vaccine candidate was suggested
by Boons and co-workers to enhance to production of IgG antibodies [45-46]. In
order to improve the synthetic access to such lipoglycopeptide conjugate, Boons
developed for the first time a methodology based on a sequential Native
Chemical Ligation (NCL) process [47]. NCL results generally from the
condensation between two peptides, one bearing N -terminal cysteine residue and
the second a C -terminal thioester [48]. In a first reversible step, a trans-
thioesterification occurs between the thioester and the thiol group to provide a
thioester-linked intermediate. This intermediate undergoes an irreversible
rearrangement to form a native amide bond at the ligation site. In Boons strategy,
a Tn containing MUC1 was first prepared as B-cell epitope, using a glycosylated
Fmoc-protected amino acid building block to be incorporated to the MUC1
peptide sequence containing a free cysteine at N -terminal end 19 (Figure 9). A
helper T-epitope (YAF sequence) 20 bearing Acm-protected cystein and benzyl
thioester on both sides was simultaneously synthesized on safety-catch linker and
conjugated in sodium phosphate buffer containing guanidinium hydrochloride
and thiophenol as catalyst. A first B- and T-cell bi-conjugate was thus obtained
after 18h in a modest 48% yield. After Acm removal, attempts to couple B/T
conjugate with Pam 3 CysSK 4 α-thioester 21 using a similar procedure failed due
to the low solubility of the lipopeptide. To circumvent this problem, the authors

O

O

H 2 N

Y-A-F-K-Y-A-R-H-A-N-V-G-R-N-A-F-E-L-F-L-G

SR

20 (YAF sequence)

AcmS

OH

O O

HO

O

S-K-K-K-K-G

SBn

O S

HO

O

O HN

AcHN

O H 2 N

14

T-S-A-P-D-T-R-P-A-P
19 (MUC1)

21 (Pam 3 CysSK 4 )

O

O 14 1 4

HS

OH

HO

O

HO

O

AcHN

O S

S-K-K-K-K-G-C-Y-A-F-K-Y-A-R-H-A-N-V-G-R-N-A-F-E-L-F-L-G-C-T-S-A-P-D-T-R-P-A-P

O HN

O

SH SH

1 4

22

O

O

1 4 14

Lipopeptide adjuvant Helper T-epitope B-epitope

Fig. 9. Synthesis of three-component vaccine by sequential NCL.