O

CONH2

Figure 37

to the growing chain, fashioned after a membrane-bound oligomer. It was anticipated that these combined factors would stabilize backbone hydrogen bonding interactions and lead to relatively small molecules with stable secondary structures.

Caproamide was immobilized on Rink resin, and Fmoc-protected ^-methoxy neuraminic acids were condensed by using BOP and DIEA in V-methylpyrrolidinone (NMP). Flaherty et al. had earlier reported that solution phase coupling of neuraminic acids was sluggish, and only poor yields were realized (48 h, 25-30%) [40]. In contrast, the solid phase syntheses were completed in a few hours, as determined by the Kaiser test, and combined yields ranged from 44 to 55% indicating that each coupling step was far more efficient (Fig. 38).

^-Methoxy NeuAc was used as a sugar amino acid in the construction of oligomers for three primary reasons. First, it was readily available from a naturally occurring S-amino acid. Second, it was hypothesized that the trihydroxy side chain would increase water solubility in higher order oligomers. Finally, O-glycoside oligomers of NeuAc were known to have stable secondary structure in aqueous solution, and it seemed possible that amide-linked analogs would exhibit similar properties

The second hypothesis was proved by the chemical syntheses of the oligomers, which were all shown to be highly water soluble. The oligomer secondary structures were probed using a combination of NH/ND exchange studies and CD, patterned after the studies of Gellman and Seebach. The exchange studies were originally performed in dimethyl sulfoxide (DMSO) because they were too rapid to be observed in water. The half-lives of NH/ND exchange were determined for the series of oligomers ranging from dimer to octamer. The dimer exchanged rapidly (half-life ~ 30 min). Two different exchange rates were observable for the trimer; the amino terminus amide exchanged fastest, on the same order as the dimer. The half-life of the internal amide was approximately 6 h. This was also true of the tetramer; the reducing end amide exchanged relatively quickly, but the internal amides took several hours. The rapid exchange of the reducing end amide was attributed to fraying. For the most part, as the oligomer length increased, the exchange rates of the internal amides slowed (the octamer was the slowest).

Figure 38

CD spectra were recorded in water at neutral pH, and they correlated with the NH/ND studies (performed in DMSO) surprisingly well. The dimer, lacking an internal amide, did not show a signature CD. The other oligomers displayed an ab-sorbance maximum at ~200 nm with a zero crossover at ~212 nm and a minimum at 200 nm, returning to zero at ~240 nm. The intensity of the maximum at 200 nm consistently increased with increasing length. These data completely correlated increasing secondary structural stability with increasing oligomer length. Shortly after this report, Fleet and coworkers reported that furanosyl-derived amido-linked sugar amino acids also adopt stable secondary structures in solution (Fig. 39) [68].

Gregar and Gervay-Hague recently prepared a series of a-methoxy NeuAc amido-linked oligomers and compared them to the ^-methoxy series [69]. The first noticeable difference was the NH/ND exchange rates, which were slow enough in water (pH = 3.0 phosphate buffer) to be measured. The exchange rates were fast for dimer and trimer, and essentially disappeared before the NMR spectra were acquired.

Figure 39

The rates slowed for the tetramer, which exchanged similarly to the pentamer through octamer. CD spectra were also recorded in pH = 3.0 phosphate buffer solution. The CD signatures of these compounds showed a reverse trend from the 0-methoxy series, with an absorbance minimum at ~195 nm and a maximum at ~230 nm, returning to zero at ~260 nm (Fig. 40). Interestingly, there was a dramatic increase in the intensity of the absorbencies in going from trimer to tetramer, but the spectra of the longer oligomers (5-mer through 8-mer) were similar to the tetramer, which correlated exactly with the NH/ND studies. The CD and NMR studies indicated that the a-methoxy series formed stable secondary structures with as few as four residues.

10 —,—,—i—,—,—,—i—,—,—,—i—,—,—,—i—■—,—.—

200 220 240 260 280

wavelength

Figure 40 CD spectra of a-methoxy NeuAc amido-linked oligomers recorded in phosphate buffer solution (pH 3).

10 —,—,—i—,—,—,—i—,—,—,—i—,—,—,—i—■—,—.—

200 220 240 260 280

wavelength

Figure 40 CD spectra of a-methoxy NeuAc amido-linked oligomers recorded in phosphate buffer solution (pH 3).

10 ■ ■ i i ' ■ ■ i i ■ i | . i ■ | ■ ■ ■ } i i ■ i i i ■ |

10 ■ ■ i i ' ■ ■ i i ■ i | . i ■ | ■ ■ ■ } i i ■ i i i ■ |

Figure 41 CD spectra of ¡-methoxy NeuAc amido-linked oligomers recorded in phosphate buffer solution (pH 3).

r. . . i . . . j . . . j . . . i . . . i . . . i . . . i . . . j

192 200 208 216 224 232 240 248 wavelength

Figure 41 CD spectra of ¡-methoxy NeuAc amido-linked oligomers recorded in phosphate buffer solution (pH 3).

For comparison purposes, CD spectra of the ¡-methoxy series were recorded in pH 3.0 phosphate buffer solution. In contrast to the a-methoxy series, the octamer was clearly distinguishable from shorter oligomers, suggesting that it is significantly more stable (Fig. 41). NH/ND exchange studies in pH 3.0 buffer showed that all oligomers exchanged rapidly with the exception of the octamer. These combined studies clearly showed that both a- and ¡-methoxy neuraminic acid-derived oligomers adopt stable secondary conformations in aqueous solution. However, the a series required only four residues for stability, whereas the ¡-series required eight.

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