We have shown that cyclic peptides can be selectively N-methylated on the solid phase under conditions that for linear peptides are known to induce complete N-methylation19, 20
. The fact that the observed selectivity was highly dependent on scaffold geometry points toward conformation as the key selectivity determinant. The H/D exchange results suggest that backbone N-methylation rigidifies the conformation of the peptide, since the free amide N-H groups exchanged much more slowly in 3
than in their nonmethylated precursors. This observation is consistent with the prediction that certain patterns of N-methylation stabilize hydrogen-bonded conformations by decreasing flexibility in the peptide backbone.
The H/D exchange results for the non-methylated peptides 1
suggested that they are in conformational flux among multiply hydrogen-bonded states on a time scale of seconds to minutes. However, the high degree of selectivity observed in the on-resin N-methylation for these compounds would seem to require restricted conformational freedom on this time scale. While this discrepancy may be due to solvent differences between the H/D exchange (chloroform) and N-methylation reactions (THF), we postulate that the amide deprotonation events are sequential and cooperative, such that deprotonation of the most exposed N-H limits the flexibility of the molecule and renders one or more of the remaining exposed amides more acidic. In other words, initial deprotonation of the most exposed N-H could shift the conformational equilibrium in the precursor toward the hydrogen-bonded conformation found in the product. This hypothesis is supported by a report showing that, while hydrogen bonding protects the involved amide N-H from H/D exchange, it increases
the exchange rate of the amide whose carbonyl is participating as the H-bond acceptor30
The permethylated versions of both 1 and 2 were significantly less permeable than their partially N-methylated counterparts, 3 and 4, in the PAMPA permeability measurements. For compound 1, the same trend was observed in the RRCK permeability assay, in which 3 was more permeable than either 1 or 5. This was likely due to the greater solvent exposure of hydrophilic carbonyl groups in the permethylated derivatives, which were sequestered in hydrogen bonds in the partially methylated compounds. Since none of the amide carbonyls in permethylated cyclic peptides can participate in intramolecular hydrogen bonding, we predict that these compounds will be less permeable, in general, than those partially methylated derivatives that can achieve optimal internal hydrogen bonding. Because the synthetic strategy outlined here afforded preferential N-methylation of exposed NH groups over those involved in internal hydrogen bonds, we postulate that these conditions can lead to methylated cyclic peptides with optimal or nearly optimal passive membrane permeability. In many cases, the most optimal conformation in terms of permeability may not be the predominant conformation selected by the N-methylation chemistry. However, given that hydrogen bonding is favorable to both permeability and selectivity, it is likely that the products of the on-resin N-methylation reaction will be among the most permeable of the many possible N-methyl variants.
For the compound 2 series, the cell-based permeability results are in partial conflict with the PAMPA data. In cells, 6 was more permeable than either 4 or the parent compound 2, while in PAMPA the 2 series followed the same trend as 1, with the partially methylated species showing the greater permeability. The discrepancy between the relative PAMPA and cell-based permeability values between 6 and 4 may lie in the differences between these two assays. The former is comprised of a relatively thick alkane layer impregnated with phospholipids, while the latter is a bona fide cell membrane. Where PAMPA and RRCK permeabilities disagree, the cell-based assay should be considered the more biologically relevant result with respect to passive membrane diffusion and is likely to be the better predictor of oral bioavailability.
We found that replacing a single Leu residue with Ser caused a significant loss in permeability. The magnitude of that loss, however, was dependent on the position of the substitution, indicating that the effect of polar side chains on permeability is dependent not only on the intrinsic polarity of the particular functional group, but also on its location along the backbone. This effect could result from a position-dependent interaction between the side chain hydroxyl and the backbone in a way that perturbs the backbone conformation, although preliminary calculations suggest that the beta structure of 3 is robust to side chain substitutions (data not shown).
While the cell-based permeabilities of the Leu-to-Ser substitutions were significantly lower than those of the all-Leu analogs, their PAMPA permeabilities were so low that they were undetectable. The threshold of detection for this assay put an upper limit on these values at 0.08% T, which, while consistent with the RRCK results in relative terms, predicted a more profound loss in permeability for these compounds than the cell-based results indicate. This difference in magnitude between the two results may also reflect the differences in the physical properties between the relatively thick hydrophobic spacer in the PAMPA vs. the true bilayer of a cell.
It is worth noting that the scaffold and N-methylation pattern found in 3
is similar to that identified by Biron, et al.4
in their N-methyl scan of the Veber-Hirschmann somatostatin analog cyclo[Pro–Phe–D-Trp–Lys–Thr Phe]31
. In their study, an extensive N-methyl scan led to 30 analogs, of which a subset cyclized efficiently to yield the desired N-methyl cyclic peptide. Of the 7 N-methyl variants whose affinity toward the somatostatin receptor was similar to that of the wild type sequence, only the parent sequence and a single trimethyl variant, cyclo[Pro–Phe–DTrp(NMe)–Lys(NMe)–Thr–Phe(NMe)], showed significant in vivo
uptake. Although the relative placement of the conformation-determining D-Trp and Pro residues in this trimethylated version of the Veber-Hirschmann peptide differ from that of 3
, both peptides adopt similar backbone structures in which the D-residue and the Pro form two opposing β-turns with the nonmethylated amides participating in transannular hydrogen bonds. This conformation, in which the positioning of the two D-residues templates the formation of two opposing β-turns, is likely the key determinant of both the N-methylation regiochemistry in compound 1
and the membrane permeability of its trimethyl adduct. The reported oral bioavailability of this peptide is 9%6
, a value comparable to that of 28% for 3
While the strategy reported here may help identify novel membrane permeable scaffolds, its ability to confer bioavailability to peptide sequences with known bioactivity is limited. In many cases, N-methylation is likely to diminish biological activity, either by directly blocking interactions of the backbone with its receptor or by driving the peptide into an inactive conformation. However, varying both stereochemistry and N-methyl placement for a given sequence may yield novel scaffolds that retain biological activity while showing improved permeability.
The observation that 3 is significantly more permeable than 5 may provide insight into the relative scarcity of permethylated cyclic peptide natural products. A survey of the online Dictionary of Natural Products revealed that, among ~353 cyclic peptide and depsipeptide natural products whose chemical structures have been deposited, 146 have partially N-methylated backbones whereas only two are completely N-methylated. While the biosynthesis of N-methyl cyclic peptides by non-ribosomal peptide synthetases (NRPS) may disfavor permethylated species simply due to steric strain in the cyclization step, it is tempting to speculate that the pattern of N-methylation observed in cyclic peptides of natural origin may be driven, at least in part, by selective pressure to maximize membrane permeability. Such selective pressure may exist for peptide natural products created by bacteria to interact with eukaryotic host organisms.
Large cyclic peptide natural products, and macrocycles in general, may occupy “islands of bioactivity”*
in chemical space that become increasingly sparse with increasing molecular mass. We postulate that many of these islands are populated with bioactive natural products whose properties have been optimized by the action of natural selection, although it is likely that many other islands remain undiscovered. A combination of the synthetic and computational approaches described here may open these regions of chemical space up to the discovery of new macrocyclic scaffolds with drug-like oral bioavailability similar to 3
, a 755-molecular weight compound that nonetheless achieves acceptable oral bioavailability.