In the next step, the trimers 95 and 96 were deprotected at the C-terminus via hydrogenolysis (Scheme 24). The reaction proceeded smoothly and gave the desired products 97 and 98 in quantitative yields after purification via column chromatography on silica. Subsequent coupling with C-protected leucine 72
gave the all-L- and L-(alt)-D-ester-(alt)-urea-tetramers 92 and 99 in high yields and purities.
Scheme 24: Extension to protected L-(alt)-D- and all-L-ester-(alt)-urea-tetramer.
The tetramers 92 and 99 include the repeat unit of the 50% amide containing structures, protected with two orthogonal protecting groups, rendering the elongation of the tetramers via a divergent/convergent synthesis possible (Scheme 25).
Scheme 25: Divergent/convergent synthesis to L-(alt)-D- and all-L -ester-(alt)-urea-octamers102 and 103.
Both deprotections proceeded smoothly and gave the resulting L-(alt)-D- and all-L-ester-(alt)-urea-tetramers 93, 94, 100, and 101 in quantitative yields and high purity. The subsequent coupling of the fragments with EDC and HOBT gave the desired octamers 102 and 103 in high yields and purity after precipitation and column chromatography. The octamers were now subjected to another split/pool-synthesis cycle to yield the hexadecamers (Scheme 26).
Scheme 26: Divergent/convergent synthesis to L-(alt)-D- and all-L -ester-(alt)-urea-hexadecamers108 and 109.
The cleavage of the benzyl ester proceeded smoothly to the octamers 106 and 107. The Boc cleavage in methylene chloride also gave the desired product. In order to couple both fragments, excess triethylamine had to be added to neutralize the solution as some residual TFA remained. By this route both hexadecamers could be isolated in good yields.
4.2.5 Synthesis Of The Peptide
To complete the series of pseudopeptides and peptides with varying stereochemistry and degree of isostere incorporation, D-(alt)-L- and all-L-leucine oligomers were synthesized. Encouraged by the experiences in the synthesis of
D-(alt)-L-lysine octapeptide, the divergent/convergent synthesis in solution was chosen for the synthesis of leucine oligopeptides.
Scheme 27: Synthesis of Boc-L-Leu-L-Leu-Bn (110) and Boc-D-Leu-L-Leu-Bn (111).
In the first reaction of the synthesis, L-Leu-Bn 72 was coupled with Boc-L-Leu or Boc-D-Leu to give the dipeptides Boc-L-Leu-L-Leu-Bn (110) and Boc-D-Leu-L -Leu-Bn (111) in high yields and purity after column chromatography. The protected dipeptides were then selectively deprotected and coupled to the tetrapeptides (Scheme 28).
Scheme 28: Divergent/convergent synthesis to Boc-L- and Boc-D-(alt)-L-Leu-Bn tetrapeptides 116 and 117.
The deprotection reactions gave the desired dipeptides in quantitative yields.
Subsequent coupling to the tetrapeptides proceeded smoothly and gave the resulting peptides in good to quantitative yields and high purities. Further growth of the peptide chain was first explored with the L-isomer 116 (Scheme 29).
Scheme 29: Divergent/convergent synthesis attempt of Boc-L-Leu-Bn octapeptide.
Both deprotections yielded the desired peptides (L-Leu)4-Bn (118) and Boc-(L -Leu)4 (119) in quantitative yields. Since both fragments were insoluble in methylene chloride, the solvent for the coupling reaction had to be changed to DMF. TLC monitoring of the reaction was not possible due to the use of DMF.
Aqueous work-up was tedious, due to extensive emulsion formation. The resulting crude product was fractioned by column chromatography. Since the product fractions were hardly soluble, their handling was unpleasant. No pure fraction could be isolated and in no fraction, the product mass could be detected by ESI-MS. For these reasons, no further growth of the peptide chain was
attempted. Final products of the synthesis are the peptides Boc-(L-Leu-L-Leu)2 -Bn and Boc-(D-Leu-L-Leu)2-Bn.
4.2.6 Aggregation Studies
All compounds synthesized are small, leucine based oligomers, differing in the stereochemistry of the neighboring units and in the connectivity of the building blocks. Compounds 116 and 117 are leucine tetrapeptides with all-L- and D -(alt)-L-stereochemistry. In compounds 92 and 99, 50% of the amide bonds are replaced by an ester-(alt)-urea moiety, resulting in a unique backbone structure that has not been reported so far. In these compounds, the stereochemistry varies from all-L- to D-(alt)-L. In compounds 90 and 85, every amide bond was replaced by an ester-(alt)-urea moiety, resulting in a unique backbone structure that has not been reported so far either. The stereochemistry in the backbone was varied as in the other compounds. The replacement of amide bonds by ester-(alt)-urea moieties was expected to significantly change the hydrogen bonding pattern in the compound. Since the oligomers are expected to be too short for intramolecular secondary structure formation, the aim of these studies was the investigation of their aggregation. The eminent differences of these compounds, regarding stereochemistry and connectivity were expected to display a significant change in aggregation behavior.
4.2.6.1 NMR Studies
Proton NMR spectroscopy is a versatile tool for aggregation studies of small oligomers, since the protons involved in hydrogen bonding interactions are expected to display a detectable downfield shift in the spectrum. Aggregation as such is a concentration dependent process.[16-19] Once, every relevant proton in the spectrum has correctly been attributed to the corresponding signal, aggregation can be monitored as function of the concentration dependent downfield shift of the amide or urea protons.
The choice of the solvent for these studies was crucial for several reasons. The protons involved in aggregation processes were all exchanging protons. This fact excluded all protic solvents such as water or alcohols, since they would have undergone fast proton exchange, vanishing all relevant signals. The solvent had to dissolve all six compounds. This further eliminated acetonitrile
and all apolar solvents, such as hexane or toluene. On the other hand, the solvation of the compounds in the solvent should not disfavor the aggregation process. This excluded strong solvating solvents, such as DMF or DMSO. All these restrictions limited the number of possible solvents to only a few. One possible solvent is CDCl3, however, due to its potenital decomposition, liberating HCl, CDCl3 is not suitable as the aggregation process is also pH-dependent. For this reason, CD2Cl2 was chosen as the solvent for the aggregation studies. Major drawbacks of CD2Cl2 are its low boiling point, excluding experiments at temperatures higher than 25 °C.
All compounds were measured at concentrations of 7.5, 15, 30, 60 and 120 mmol/L. The assignment of all signals was achieved via COSY and NOESY measurements at a concentration of 7.5 mmol/L, where no significant aggregation was expected to occur.
The dilution series of the all-L-tetrapeptide 116 is shown in Figure 7.