Review on N-methylation

Veteran peptide chemist Horst Kessler (TU Munich) has a good review on the effects of N-methylation of peptides and proteins in a recent issue of Angewandte Chemie. N-methylation has been an interesting and frequently productive strategy for a long time, but the main problem was that the chemistry needed to implement it wasn't there yet. But thanks to new developments chemists have caught up and selective N-methylation of amides no longer needs to be the rate-limiting step that it was.

N-methylation has a variety of interesting and potentially very useful effects on small molecule and peptide conformation and function. For one thing, N-methylated amide bonds have a different distribution of cis and trans forms which is somewhat more evenly distributed than that in non N-methylated amide bonds which dominantly prefer the trans conformation. This can significantly tweak the distribution of conformations in solution.

From a biological standpoint things get even more interesting. N-methylation makes the molecule more lipophilic and therefore more membrane-permeable, improving cell penetration. It gets rid of one hydrogen bonding N-H bond. This can sometimes have an unfavorable effect on permeability if that N-H forms an intramolecular hydrogen bond, but often it can help. Intramolecular hydrogen bonds are another valuable tactic for hiding polar surface area and improving permeability. The ideal situation is a combination of both N-methylation and intramolecular hydrogen bonding, exemplified by the archetypal "large", complex, biologically active drug, cyclosporine. Recent studies by the Jacobson (UCSF) and Lokey (groups) have described strategies for both specific N-methylation chemistry and for predicting permeability using computational calculations.

Finally, N-methylation can prevent recognition and cleavage by peptidases which recognize "normal" amide bonds, especially when the N-methylated amides are part of a cyclic peptide. All these factors can significantly improve bioavailability. Kessler talks about all of them and illustrates these principles with a few striking examples, including somatostatin, amanitin and melanocortin. Many of these sport similar motifs, leading to ideas for possible design of standardized building blocks for improving permeability and bioavailability. The piece is worth a look if you are into developing peptides and peptidomimetics as drugs or even more generally if you are interested in peptide and small molecule conformations.

Conformations of the stevastelins: A reassessment

Shameless self-promotion: my paper on the conformational analysis of cyclic antiviral peptides called stevastelins is now online on the Biopolymers site. Here's a brief overview.

The stevastelins are cyclic peptides that show promising antiviral activity against the vaccinia viral VHR phosphatase. These peptides are phosphorylated in vivo before they can inhibit their target protein. A group in Germany previously did a meticulous analysis of four diastereometric analogs of these peptides which included their synthesis, biological characterization and conformational analysis. However, the conformational analysis was done using force field conformational searches from a single force field, constrained by variables from the NMR data (coupling constant derived dihedral angles and NOESY derived distances). Using such a protocol, the group concluded that each of the four diastereomers exists as a single conformational family in solution.

The problem with constrained conformational searches (or constrained molecular dynamics for that matter) is that they constitute a rather self-fulfilling exercise, with the assumption that there is in fact a single conformation of the molecule under question. However, as I have often discussed on this blog, any molecule with a couple of rotatable bonds is going to exist as multiple conformers in solution, so an assumption of a single conformation would be fuzzy unless supported by more data. NMR by itself is of scant value in determining these conformations for thermodynamic and kinetic reasons. Plus, analyzing conformations using a single force field can be fraught with ambiguity, since every force field comes with its own set of parameters and convergence criteria. Especially trusting energies from force fields can be dangerous. In this case, the stevastelin peptides have 9 rotatable bonds each, so I thought it worthwhile to apply our previously developed and applied NAMFIS (NMR Analysis of Molecular Flexibility In Solution) methodology combining NMR variables with structures from extensive conformational searches to the enumeration of the conformational behavior of these interesting molecules.

The paper essentially describes the conformational variability obtained for each of the diastereomers. Many of the conformations are very similar to the previously postulated families, but some are quite different. There are also some striking observations that are corroborated; for instance, the use of a d-serine truly seems to 'lock' the peptides in a single conformation. Such a lock could be effected to counter the entropic penalty that a multiconformational ensemble of molecules might have to pay. The instructive general observation is that subtle changes in sterechemistry at one or two chiral centers can dramatically affect conformational behavior, a fact that continues to surprise and confound medicinal chemists. I also note that if the NMR data for the phosphorylated peptides were available, an interesting comparison of the conformational pool for the phosphorylated and unphosphorylated counterparts could be attempted. This would shed light on whether phosphorylation leads to less conformational variability or simply increases the proportion of a chosen subset of conformations of the peptides.

Comments, criticism and general feelings of chagrin are welcomed.

Jogalekar, A. (2010). Conformations of stevastelin C3 analogs: Computational deconvolution of NMR data reveals conformational heterogeneity and novel motifs Biopolymers DOI: 10.1002/bip.21504

Remote control of peptide screw sense

As is well-known, peptides helices can be right or left handed. Many details of structure, amino acid identity and orientation can control this screw sense, and sometimes the controlling factors can be quite subtle. In a JACS communication, Jonathan Clayden (yes, the co-author of the amazing organic chemistry textbook) and his group uncover a surprising factor that controls the helical screw sense and also incorporate a neat "reporter group" to monitor the screw sense.

But this reporter group is nothing fancy and is simply a gycine installed in the middle of a long sequence of amino acids which consists of alpha-aminoisobutyric acid or Aib. Aib is simply alanine with an extra methyl at the alpha carbon. It is well known to impart helical propensities to peptides and has been used several times as a helical 'lock'.

In this case the Gly is in the center of a 20 amino acid peptide where all other residues are Aib. The peptide is clearly helical, but what's the screw sense? That's where the power of NMR spectroscopy comes in. The two protons in Gly are diastereotopic which means that in principle they could have different chemical shifts and signals in the NMR spectrum. In practice though, rapid interconversion between the left and right handed helices leads to an average and gives a single signal in the spectrum.

However if interconversion between the two screw senses could be 'biased' by making the equilibrium constant favor one of them, then one could presumably observe two separate signals for the two Gly protons even if the transition is fast on the NMR time scale. To accomplish this, Clayden et al. do something peculiar; they incorporate a L-Phe residue at the N-terminal of the helix. This group, even if far away from the central Gly, somehow seems to remotely interact differently with each of the two Gly protons. The incorporation of this terminal group leads to a considerable splitting in the signals of the two protons (up to 100 ppm), easily distinguishing them apart. Also for some reason, N-terminal groups seem to work better than C-terminal groups.

The reasons for the transmission of this effect over no less than 27 bonds are not clear, but they probably have something to do with the subtle change in conformational behavior that dictate helix folding. The authors even observe small differences for amide vs ester bonds as capping groups. Finally, they obtain an x-ray structure of this helix which turns out to be a 3/10 helix and confirm their observations.

These days there is a drive to 'tether' certain parts of oligopeptides to lock the resulting conformation in a helical form. Sometimes, even constraining end groups covalently (by metathesis for instance) seems to ensure a critical 'nucleation' structure that then zips up the rest of the helix. The exact percentage of the helix in solution could be a matter for discussion, but this study seems to indicate similiar end-group influenced conformational organization. I thought it was neat and points to further challenges and questions in our understanding of the deceptively simple question, "Why are helices stable in solution"?

Solà, J., Helliwell, M., & Clayden, J. (2010). N- versus C-Terminal Control over the Screw-Sense Preference of the Configurationally Achiral, Conformationally Helical Peptide Motif Aib-Gly-AibJournal of the American Chemical Society DOI: 10.1021/ja100662d