Multiple column solid phase glycopeptide synthesis

Article abstract of DOI:10.1016/S0040-4039(00)93429-4

The simultaneous synthesis of 40 different α-linked O-glycopeptides from human intestinal mucin and porcine submaxillary gland mucin in a manual multiple column peptide synthesizer using the new glycosylamino acid building blocks N(α)-Fmoc-Ser(Ac₃

Full text of DOI:10.1016/S0040-4039(00)93429-4

Tetrahedron Letters, Vol.32, No.38, pp 5067-5070, 1991
Printed in Great Britain
0040-z1039/91 $3.00 + .GO
Pergamon Press plc
MULTIPLE COLUMN SOLID PHASE GLYCOPEPTIDE SYNTHESIS
Stefan Petersa, Tim Bielfeldta, Morten Meldalb, Klaus Bockb and Hans Pauisena*
a) Institut f'tir Organische Chemic der Universit~t Hamburg, Martin-Luther-King-Platz 6, D-2000 Hamburg 13, Germany
b) Department of Chemistry, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, Denmark
Abstract: The simultaneous synthesis of 40 different s-linked O-glycopeptides from human intestinal mucin and porcine
submaxillary gland mucin in a manual multiple column peptide synthesizer using the new glycosylamino acid building blocks Na-
Fmoc-Ser(Ac3-~-D-GalpNAc)-OPfp (3) and Na-Fmoc-Thr(Ac3-~-D-GalpNAc)-OPfp (4) is described.
The important role in biological systems played by the surface carbohydrates on glycoproteins and cell membranes
in recognition and transport processes has increased the interest and demand for a variety of glycopeptides as
model compounds 1. For the study of the biosynthesis2 of mucin type glycoproteins synthetic O-glycopeptides,
which carry an a-linked GalNAc-sugar on serine or threonine, are required. Recently, Brockhausen3 has shown
that the enzymatic synthesis of O-glycan core structures is controlled by the peptide moiety. The activity of the
133-Galactosyltransferase, which glycosylates the 3-OH position in the GalNAc unit, is dependent of the peptide
sequence, the chain length, the attachment site of the GalNAc and the number of GalNAc-residues in the substrate.
N-terminally acetylated glycopeptide amides were much better substrates than the corresponding molecules with
free amino and carboxy termini especially in the case of partially purified enzymes. For a more detailed enzymatic
investigation a large number of synthetic glycopeptides 4 is needed. The concept to synthesize O-glycopeptides by
using glycosylated hydroxy amino acids as building blocks for solid phase synthesis has been used successfully
several times5.
In this paper the application of the two new building blocks 3 and 4 is reported. These key compounds were
obtained by esterification of the acids 1 and 26 using pentafluorophenol (Pfp-OH) and dicyclohexylcarbodiimide
(DCCI) in dry ethyl acetate at 0°C. After chromatographic purification on silica gel under dry conditions5d and
precipitation from diethyl ether the activated ester 3 and 4 were both obtained in 75% yield as stable solid
materials (- 24% of the valuable starting material could be recovered from each separation). The active esters 3
and 4 were characterized by 1D-and 2D-1H-NMR spectroscopy and FAB-MS7.
AcO ~..OAc
AeO f ~ O A c
AcO
Pfp-OH
AcO
AcNHI
DCC I
AcNHI
O.
o ~ R
R
F
FmoC\N H~/O H
Fmo
c
-
~
.
N
~
O
~
F
F
I
I
0
0
F
~
"
F
~
I
R
-
H
$
R - H
II
R
-
CHa
4
R - CHs
5067

5068
The protecting group pattem of these building blocks is very suitable for solid phase glycopeptide synthesis.
Pentafluorophenyl ester show very fast coupling rates in the presence of 3,4-dihydro-3-hydroxy-4-oxo-l,2,3-
benzotriazine (Dhbt-OH) and the progress of the acylation reaction could be followed visually by the displacement
of the yellow ion pair formed between Dhbt-OH and resin bound amino groups5d'e;8. Application of the fluoren-9-
yl-methoxycarbonyl (Fmoc) group allows an a-amino deprotection under mild conditions with morpholine9. The
O-acetyl groups of the carbohydrate part can easily be removed at the end of the synthesis with catalytic amounts
of sodium methoxide in methanol10. The two octapeptides PTTTPIST (5) and GSSSGSPG (43) which represent
parts of the repeating units from human intestinal mucin11 and porcine submaxillary gland mucin12 were selected
as target molecules. Based on these two sequences a series of N-terminally acetylated O-glycopeptide amides were
designed and synthesized as shown in figure 1.
5:
[ - P T T T P I S T - I
18: Ae-GT~TGIST-NH 2
2 7 : A e - P ~ T P I S T - N H 2
28: Ac-P~T~PIST-NH 2
29: A c - P ~ P I S T - N H z
30: I c - P T T S P I S T - N H 2
37: Ae-P3TTPIST-NH z
5: A c - P T T ~ P I S T - N H z
17: Ac-CTTTGIST-NH z
38: A c - P S T S P I S T - N H s
'
,
7: A e - P T T P I S T - N H2
IS: Ae-PTTGPIST-NH z
39: Z c - P T T S P I S T - N X z
8:
A e - P ~ T T P I B T - N H z
1O: Ac-P~TGPIST-NH 2
40: Ac-PTSTPIST-NH z
e
o
e
O: A e - P T T GIST-NH z
20: Ac-PTGTPIST-NH 2
81: I c - P T S T P I S T - N H ~
41: I c - P T S S P I S T - N H ~
L0:
Ae-PT~TGIST-NH 2
tl: A~-P~rrclsr-Na z
12: Ac-GTr~PIST-Na,
13: Ac-CT~YPIST-Na z
21: Ae-P~GTPIST-NH z
22: Ao-Pcr~PIsr-~H z
28: Ac-Pe~TPIST-NH 2
24: Ac-PrP~PIST-NH 2
25: Ac-PTPTPIST-NH z
32: Ac-P~TTPIST-NHz
88: , ~-Prs~P18r-Nn~
34: Ae-PSr~PISr-NH z
35: Ac-PSS~PlSr-NHz
35: Ac-PTTSPIST-NH~
42: Ac-PTT~SIST-NH~
48:
[ - c s s s e s P c - ]
44: Ze-GSSSeSPC-NH,
45: Ac-GSSSCSPC-Naz
46: I c - C S S S C S P C - N Hz
1 4 :
Ac-GTTTPIST-NH z
o
ee
15: Ac-GTTTGIST-NH z
28: Ac-PTTTPIST-NH z
HO ~OH
HO
F i g u r e 1.
The multiple column peptide synthesizer13 used for simukaneous assembly of 40 glycopeptides consist of a steel
frame equipped with two parallel 96 channel dispensers, one connected to a dispensing bottle with DMF and one
to a dispensing bottle with deprotection reagent. The synthesis is carried out in a block of teflon with 96 wells
in a ELISA type of arrangement all connected via a filter to a pressure/vacuum regulated chamber beneath. Resin
columns (25 mg each) are packed to the wells and addition of DMF or deprotection reagent from dispensing
bottles can be carried out by mounting the block on elevator tables under the washers. The acylating reagents are
dissolved in a ELISA plate of teflon and added to the wells by a 8 channel multi pipette. The resin was washed
4 times after acylations and 5 times after deprotectinns.
The 5-(4-aminomethyl-3,5-dimethoxyphenoxy)-valeric acid (PAL)TM, which has successfully been applied in
glycopeptide synthesis of morphiceptin analogues5f and glycosyl enkephalinamides5g, was used as acid labile
amide linker.
The Fmoc protected PAL group was coupled to a kieselguhr supported polydimethyl acrylamide resin carrying
norleucine as reference amino acid by activation with O-(benzotriazol-l-yl)-N,N,N',N'-tetramethyluronium
tetrafluoroborate (TBTU)15 and 4-ethylmorpholine. After removing the Fmoc group with 20% piperidine in DMF

5069
the first amino acid was coupled as Dhbt-ester to the linker in a quantitative yield as shown by amino acid
analysis. All the following Fmoc deprotections were effected under mild conditions by treatment of the resin with
50% morpholine in DMF. The Fmoc amino acids were introduced into the peptide chain as Dhbt-esters. The
hydroxy groups of non glycosylated serine and threonine were protected as tBu ether. In case of the glycosylated
amino acids the pentafluorophenyl ester building blocks 3 and 4 were used with the addition of Dhbt-OH as
auxiliary nucleophile. In order to assure complete acylation reactions the activated esters were allowed to react
ovemight. The progress of the peptide bond formation was indicated by the decrease in coloration of the solid
support. After attachment of the last amino acid and Fmoc removal the terminal amino groups were acetylated with
acetic anhydride in DMF (1:7). The resins were simukaneously transferred into small glass vials. All 40
glycopetides were cleaved off the resin by treatment with 95% aqueous TFA with concurrent removal of the O-tBu
groups. Finally, the deacetylation of the carbohydrate unit was performed with sodium methoxide in methanol at
pH 8.5 for 3 hours. Racemization or ~-elimination of the glycosyl moiety was not observed.
The crude products were purified by preparative reversed phase HPLC. The pure N~t-acetyl-O-glycopeptide amides
were obtained in yields of 4 to 6 mg after lyophilization. The characterization of all compounds was performed
by 1D- and 2D-1H-NMR spectroscopy and all proton signals were assigned as exemplified for compound 1016.
The amino acid analysis were all correct within 8%. The full experimental data will be published elsewhere.
This paper describes an efficient strategy for the synthesis of many different O-glycopeptides of biological interest
in a short period of time by multiple column peptide synthesis.
Acknowledgements: We would like to thank the Studienstiftung des Deutschen Volkes and the Stiftung Stipendien-
Fonds des Verbandes der Chemischen Industrie for their interest and financial support.
References and Notes
1.
2.
3.
4.
5.
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5070
k) J. Knolle, W. K6nig and G. Breipohl, in Peptides 1990, Proc. Eur. Pept. Symp., 21st 414-415 (E. Giralt,
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J. Thurin, Tetrahedron Lett. 31, 5889 (1990) •
6.
7.
H. Paulsen and K. Adermann, Liebigs Ann. Chem., 751 (1989).
Compound 3: m.p. 96 C (not corr.); [(x] ]~ = + 43.8 (c = 1.0, CHC13);
FAB-MS found MH+: 823.0; calc. for C38H35F5N2013:822.2
Compound 4: m.p. I10C (not corr.); [¢x]~ = + 34.0 (c = 1.0, CHC13);
FAB-MS found MH+: 837.3; calc. for C39H37FsN2013: 836.7.
8.
9.
a) E. Atherton, J.L. Holder, M. Meldal, R.C. Sheppard and R.M. Valerio, J. Chem. Soc. Perkin Trans I,
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de Gruyter, Berlin) (1989) .
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16. Compound 10 (Ac-P-T-T(o~-D-GalNAc)-T-G-I-S-T-NH2). 400 MHz 1H-NMR; 6, ppm (in D20, ref DOH=
4.80 ppm at 300 K) J, Hz: ot-D-GalNAc 4.98 (d, 3.8 Hz, H-l); 4.17 (dd, 11.0 Hz, H-2); 4.08 (H-5); 4.03
(dd, 0.8 Hz, H-4); 3.95 (dd, 3.2 Hz, H-3); 3.83-3.77 (H-6, H-6'); 2.18a (Ac); Thr.s 4.73 (d, 2.0 Hz, H4xl);
4.57 (d, 5.0 Hz, H-~2);4.43(d, 4.4 Hz, H-o.3); 4.43 (H-I]I); 4.39 (H-co4); 4.37 (H-[~4); 4.31 (H-~2); 4.26 (H-
133); 1.34 (d, 6.6 Hz, H-y1); 1.31 (d, 6.6 Hz, H-y2); 1.29 (d, 6.8 Hz, H-y3); 1.27 (d, 6.8 Hz, H-~/4); Ser 4.62
(t, H-c~); 3.97 (dd, 6.0 and 11.4 Hz, H-I]); 3.92 (dd, 6.0 Hz, H-~'); Ile 4.30 (d, 7.6 Hz, H-c0; 1.99-1.89 (H-
I3); 1.59-1.46 (H-y); 1.33-1.18 (H-'/'); 0.98 (d, 6.8 Hz, H-7"); 0.93 (t, 7.4 Hz, H-fi); Gly 4.10 (d, 16.8 Hz,
H-(x); 4.01 (d, H-~'); Pro 4.54 (dd, 4.0 and 8.0 Hz, H-cx); 3.83-3.61 (2 HS); 2.43-2.32 (H-15); 2.12-1.99 (H-
I]', 2 H-y); 2.08a (Ac); a: The assignment can be interchanged.
(Received in Germany 1 July 1991)