355
Cyclic Peptides as Scaffolds for
Multivalent Presentation of Carbohydrates:
A Combinatorial Approach
Sonja Seeberger and Valentin Wittmann
Institut für Organische Chemie, Johann Wolfgang Goethe-Universität, Marie-Curie-Straße 11, 60439 Frankfurt/Main, Germany
Introduction
Carbohydrate–lectin interactions mediate a large variety of intercellular recognition events. Since the interactions of individual saccharide groups are usually of low affinity and broad specificity, subsite and subunit multivalency is often employed by nature in order to enhance the attractive binding forces [1]. Accordingly, the synthesis of multivalent carbohydrate ligands has recently attracted interest [2]. Binding studies with these multivalent ligands revealed the importance of the spacer between the monomeric epitopes. Attachment of the saccharides to rigid scaffolds can result in particularly pronounced affinity enhancements provided that the sugars are presented in the “correct” spatial arrangement. However, in contrast to the utilization of flexible linkers, a larger number of ligands has to be screened in order to find a ligand with the appropriate orientation of the carbohydrates.
Here we present a synthetic approach that enables the preparation of libraries of conformationally restricted cyclic peptides which can serve as scaffolds for the multivalent presentation of carbohydrate ligands [3]. Such libraries are useful to (1) identify high affinity lectin ligands based on multivalent interactions and (2) to determine the required orientation of the carbohydrates via conformational analysis of the cyclopeptide scaffold.
Strategy:
A library of cyclic peptides with regioselectively addressable side chain amino groups is synthesized by combinatorial solid phase synthesis (split and combine method [4]).
Following peptide assembly, several copies of a carbohydrate ligand are attached to the side chain amino groups via a new Alloc-based urethane-type linker. In contrast to glycosylation reactions employing resin bound peptides, the formation of an urethane bond proceeds in virtually quantitative yields. The members of the obtained library differ in the conformation of the peptide scaffold and the number and location of carbohydrates attached to it.
The neoglycopeptide library can then be applied in on-bead lectin binding assays.
Identification of high affinity ligands can be accomplished by standard peptide analytical methods (Edman degradation) after Pd(0) catalyzed cleavage of the
First publ. in: Innovation and Perspectives in Solid Phase Synthesis & Combinatorial Libraries 2000 / ed. Roger Epton. Kingswinford: Mayflower 2001, pp. 355-358
Konstanzer Online-Publikations-System (KOPS) URL: http://www.ub.uni-konstanz.de/kops/volltexte/2008/5509/
URN: http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-55090
356
carbohydrate-peptide linker. Attachment of the saccharides after the cyclopeptide synthesis has the conceptual advantage that once a library is prepared, it can be used to study several carbohydrate binding proteins by simply connecting different sugars to it.
Results and Discussion
Figure 1 outlines the synthetic strategy using a cyclic model neoglycopeptide. Solid phase peptide synthesis was carried out on TentaGel functionalized with the Sieber linker and followed the Fmoc strategy. Only in the last step, an Nα-Boc-protected amino acid was applied. Cyclization of the resin bound nonapeptide was carried out between the side-chain carboxyl group of glutamic acid (Allyl protection) and the ε- NH2 group of lysine (Alloc protection) after simultaneous deprotection with catalytic amounts of tetrakistriphenylphosphine palladium(0) and morpholine in DMF/DMSO (1:1). Addition of HBTU/HOBt/DIEA resulted in clean cyclization product as judged by HPLC analysis of the crude cleavage product obtained from a small resin sample by treatment with dichloromethane/TFA/tri-iso-propyl silane (98:1:1) followed by ESIMS.
Boc-Lys(Alloc)-Orn(Ddv)-Gly-Ala- -Lys(Ddv)-Orn(Ddv)- -Val-Glu(OAl)-Bal-NHD D Sieber Coupling cycle:
Last cycle:
1. 20% piperidine, DMF
2. Fmoc-Xaa-OH, HBTU, HOBt, DIEA 1. 20% piperidine, DMF
2. Boc-Xaa-OH, HBTU, HOBt, DIEA
Ddv = O
O
RPHPLC profiles of crude peptides.
Column: Nucleosil 100-5 C18 PPN, 250 x 4 mm; 20–80% B in 30 min, A: 0.1%
TFA , B: 0.1% TFAin CH CN; 1mL/min.aq 3
Boc-Lys-Orn(Ddv)-Gly-Ala- -Lys(Ddv)-Orn(Ddv)- -Val-Glu-Bal-NHD D Sieber
Boc-Lys-Orn( )-Gly-Ala- -Lys( )-Orn( )- -Val-Glu-Bal-NHR D R RD Sieber
O O
NHAc OAc AcOAcO
= O
O R
1% TFA NH
O O Fmoc-HN
O Fmoc-Bal-NH Sieber
TentaGel
1. Pd(PPh ) (0.1 eq), morpholine, DMF/DMSO 2. HBTU (4 eq.), HOBt, DIEA3 4
1. 4% H N-NH • H O2 2 2
2. O
NHAcO OAc
AcOAcO O
O O
NO2
1
10 t(min) 20 30
A(220nm)
0 1% TFA
10 t(min) 20 30
A(220nm)
0
(M+H ) : 1630.0 (M+H ) : 1630.7
+ +calcd
found
(M+H ) : 2341.1 (M+H ) : 2342.1
+ +calcd
found
Figure 1. Solid phase synthesis and HPLC analysis of a cyclic model neoglycopeptide.
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Combinatorial solid phase peptide synthesis (Fmoc strategy, split and combine method)
1. Pd(PPh ) (0.2 eq.), morpholine, DMF/DMSO 2. HBTU (4 eq.), HOBt, DIEA3 4
1. 20 % piperidine, DMF
2. Boc-Lys(Alloc)-OH, HATU, HOAt, DIEA Val Lys(
ProIle
Fmoc Ala
D-Lys(Ddv)Leu
Ddv) Gly
D-Lys(Ddv)
Pro Leu Glu(O l) Bal NHA Sieber Fmoc-Bal-NH Sieber
Val Lys(
ProIle
AlaLeu
D-Lys(Ddv)
Ddv) Gly
D-Lys(Ddv)
Pro Leu Glu(O l) Bal NHA Sieber Boc-Lys(Alloc)
HN CO
Val Lys(
ProIle
AlaLeu
D-Lys(Ddv)
Ddv) Gly
D-Lys(Ddv)
Pro Leu GluBal NHSieber Boc-Lys
60
0 10 20 30 40 50
40-80 % B in 60 min
1 2
1718 1516
14 3
4 5
7 13 8
6 9 101112
A(220nm)
t(min)
RPHPLC profiles of crude peptides.
Column: Nucleosil 100-5 C18 PPN, 250 x 4 mm; 1 mL/min. A: 0.1%
TFA , B: 0.1% TFA in CH CN.aq 3 40-80 % B in 60 min
5 1213 11
60
0 10 20 30 40 50
1 1718
1516 23 4 789 14
6 10
t(min)
A(220nm)
60
0 10 20 30 40 50
30-70 % B in 60 min
12 3 4
5
8 18
17 1615 1314 1211 10 69 7
t(min)
A(220nm)
*
*= non peptidic
Figure 2. Synthesis and HPLC analysis of an 18-membered cyclic-peptide library. Peaks of corresponding peptides are labeled with the same number.
Subsequently, the 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)isovaleryl (Ddv) groups [5] which were used to protect the attachment points of the carbohydrate residues were removed with 4% hydrazine hydrate in DMF. Treatment with the p- nitrophenyl active carbonate 1 finally gave the target neoglycopeptide which was cleaved from the solid support. HPLC analysis of the crude peptide confirmed the high efficiency of the overall process.
We next turned our attention to the question weather the reaction conditions, especially those of the critical cyclization step, would be successful with other peptide sequences. Therefore, we prepared an 18-membered cyclopeptide library (split and combine method) and followed the course of the synthesis by HPLC in combination with ESIMS (Figure 2).
It turned out that all 18 peptides underwent cyclization without forming major side products. Particularly, we were not able to detect any linear or cyclic peptide dimers.
The assignment of each peak of the HPLC profile of the cyclopeptide library is given in Table 1.
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Table 1. Calculated and experimentally determined masses of the cyclic-peptide library.
[M + H]+ Peak Compound
calcd found 1 cyclo[Boc-Lys-Pro-Lys(Ddv)-Ala-Pro-Gly-Leu-Glu]-Bal-NH2 1197.7 1198.3 2 cyclo[Boc-Lys-Val-Lys(Ddv)-Ala-Pro-Gly-Leu-Glu]-Bal-NH2 1199.7 1200.1 3 cyclo[Boc-Lys-Ile-Lys(Ddv)-Ala-Pro-Gly-Leu-Glu]-Bal-NH2 1213.8 1214.1 4 cyclo[Boc-Lys-Pro-Lys(Ddv)-Leu-Pro-Gly-Leu-Glu]-Bal-NH2 1239.8 1240.4 5 cyclo[Boc-Lys-Val-Lys(Ddv)-Leu-Pro-Gly-Leu-Glu]-Bal-NH2 1241.8 1242.2 8 cyclo[Boc-Lys-Pro-Lys(Ddv)-D-Lys(Ddv)-Pro-Gly-Leu-Glu]-Bal-NH2 1460.9 1461.5 7 cyclo[Boc-Lys-Ile-Lys(Ddv)-Leu-Pro-Gly-Leu-Glu]-Bal-NH2 1255.8 1256.2 6 cyclo[Boc-Lys-Pro-Lys(Ddv)-Ala-Pro-D-Lys(Ddv)-Leu-Glu]-Bal-NH2 1474.9 1475.4 9 cyclo[Boc-Lys-Val-Lys(Ddv)-Ala-Pro-D-Lys(Ddv)-Leu-Glu]-Bal-NH2 or
cyclo[Boc-Lys-Ile-Lys(Ddv)-D-Lys(Ddv)-Pro-Gly-Leu-Glu]-Bal-NH2
1476.9 1477.5 10 cyclo[Boc-Lys-Val-Lys(Ddv)-D-Lys(Ddv)-Pro-Gly-Leu-Glu]-Bal-NH2 1462.9 1463.5 12 cyclo[Boc-Lys-Ile-Lys(Ddv)-D-Lys(Ddv)-Pro-Gly-Leu-Glu]-Bal-NH2 or
cyclo[Boc-Lys-Val-Lys(Ddv)-Ala-Pro-D-Lys(Ddv)-Leu-Glu]-Bal-NH2
1476.9 1477.4 11 cyclo[Boc-Lys-Ile-Lys(Ddv)-Ala-Pro-D-Lys(Ddv)-Leu-Glu]-Bal-NH2 1491.0 1491.6 13 cyclo[Boc-Lys-Pro-Lys(Ddv)-Leu-Pro-D-Lys(Ddv)-Leu-Glu]-Bal-NH2 1517.0 1517.5 14 cyclo[Boc-Lys-Val-Lys(Ddv)-Leu-Pro-D-Lys(Ddv)-Leu-Glu]-Bal-NH2 1519.0 1519.6 16 cyclo[Boc-Lys-Pro-Lys(Ddv)-D-Lys(Ddv)-Pro-D-Lys(Ddv)-Leu-Glu]-Bal-NH2 1738.1 1739.2 15 cyclo[Boc-Lys-Ile-Lys(Ddv)-Leu-Pro-D-Lys(Ddv)-Leu-Glu]-Bal-NH2 1533.0 1533.5 17 cyclo[Boc-Lys-Val-Lys(Ddv)-D-Lys(Ddv)-Pro-D-Lys(Ddv)-Leu-Glu]-Bal-NH2 1740.1 1740.8 18 cyclo[Boc-Lys-Ile-Lys(Ddv)-D-Lys(Ddv)-Pro-D-Lys(Ddv)-Leu-Glu]-Bal-NH2 1754.1 1755.0
Conclusion
We have demonstrated an efficient process for the generation of libraries of cyclic- peptide scaffolds suitable for a conformationally defined multivalent presentation of carbohydrate ligands. Currently, we are using this strategy to screen neoglycopeptide libraries for lectin binding properties.
References
1. a) Rini, J.M., Annu. Rev. Biophys. Biomol. Struct. 24 (1995), 551-577; b) Dwek, R.A., Chem. Rev. 96 (1996) 683-720; c) Mammen, M., Choi, S.-K. and Whitesides, G.M., Angew.
Chem. 110 (1998) 2908-2953.
2. a) Neoglycoconjugates: Preparation and Applications, Lee, Y.C. and Lee, R.T. Eds., Academic Press, San Diego, 1994; b) Kiessling, L.L. and Pohl, N.L., Chem. Biol. 3 (1996) 71-77; c) Roy, R., Curr. Opin. Struct. Biol. 6 (1996) 692-702.
3. For other examples of cyclic glycopeptides see: a) Franzyk, H., Christensen, M.K., Jørgensen, R.M., Meldal, M., Cordes, H., Mouritsen, S. and Bock, K., Bioorg. Med. Chem.
5 (1997) 21-40; b) Sprengard, U., Schudock, M., Schmidt, W., Kretzschmar, G. and Kunz, H., Angew. Chem. 108 (1996) 359-362. For use of cyclic peptides as scaffolds see: c) Tuchscherer, G. and Mutter, M., Pure Appl. Chem. 68 (1996) 2153-2162.
4. Lam, K.S., Lebl, M. and Krchnák, V., Chem. Rev. 97 (1997) 411-448.
5. Chhabra, S.R., Hothi, B., Evans, D.J., White, P.D., Bycroft, B.W. and Chan, W.C., Tetrahedron Lett. 39 (1998) 1603-1606.
