Aligned peptoid-based macrodiscs for structural studies of membrane proteins by oriented-sample NMR.

Development of a robust, uniform, and magnetically orientable lipid mimetic will undoubtedly advance solid-state NMR of macroscopically aligned membrane proteins. Here, we report on a novel lipid membrane mimetic based on peptoid belts. The peptoids, composed of 15 residues, were synthesized by alternating N-(2-phenethyl)glycine with N-(2-carboxyethyl)glycine residues at a 2:1 molar ratio. The chemically synthesized peptoids possess a much lower degree of polydispersity versus styrene-maleic acid polymers, thus yielding uniform discs. Moreover, the peptoid oligomers are more flexible and do not require a specific folding, unlike lipoproteins, in order to wrap around the hydrophobic membrane core. The NMR spectra measured for the membrane-bound form of Pf1 coat protein incorporated in this new lipid mimetics demonstrate a higher order parameter and uniform linewidths compared with the conventional bicelles and peptide-based macrodiscs. Importantly, unlike bicelles, the peptoid-based macrodiscs are detergent free.

On-resin Cα-functionalization of N-arylglycinyl peptides with boronic acids.

A late-stage α-C-H functionalization reaction of resin-bound, electron-rich N-aryl peptides with boronic acid nucleophiles under mild conditions is reported. We explore the impact of the N-arylglycinyl peptide structure on reactivity, and present a scope of the optimized reaction where both the peptide sequence and nature of boronic acid derivatives are varied.

Peptoid nanosheets exhibit a new secondary-structure motif.

A promising route to the synthesis of protein-mimetic materials that are capable of complex functions, such as molecular recognition and catalysis, is provided by sequence-defined peptoid polymers--structural relatives of biologically occurring polypeptides. Peptoids, which are relatively non-toxic and resistant to degradation, can fold into defined structures through a combination of sequence-dependent interactions. However, the range of possible structures that are accessible to peptoids and other biological mimetics is unknown, and our ability to design protein-like architectures from these polymer classes is limited. Here we use molecular-dynamics simulations, together with scattering and microscopy data, to determine the atomic-resolution structure of the recently discovered peptoid nanosheet, an ordered supramolecular assembly that extends macroscopically in only two dimensions. Our simulations show that nanosheets are structurally and dynamically heterogeneous, can be formed only from peptoids of certain lengths, and are potentially porous to water and ions. Moreover, their formation is enabled by the peptoids' adoption of a secondary structure that is not seen in the natural world. This structure, a zigzag pattern that we call a Σ('sigma')-strand, results from the ability of adjacent backbone monomers to adopt opposed rotational states, thereby allowing the backbone to remain linear and untwisted. Linear backbones tiled in a brick-like way form an extended two-dimensional nanostructure, the Σ-sheet. The binary rotational-state motif of the Σ-strand is not seen in regular protein structures, which are usually built from one type of rotational state. We also show that the concept of building regular structures from multiple rotational states can be generalized beyond the peptoid nanosheet system.

N-Arylation of Amino Acid Esters to Expand Side Chain Diversity in Ketoxime Peptide Ligations.

Palladium-catalyzed N-arylations of amino acid tert-butyl esters using 4-bromo-N,N-dimethylaniline as a coupling partner are reported. The resulting N-aryl amino acid esters are suitable building blocks for the synthesis of electron-rich N-aryl peptides, which undergo oxidative couplings to aminooxy groups to afford ketoxime peptides under mild conditions. N-aryl amino acid tert-butyl esters possessing unnatural side chains were also accessed via glycine Schiff base alkylation, further increasing the scope of Cα-substitution in ketoxime peptides.

Adiponectin has a pivotal role in the cardioprotective effect of CP-3(iv), a selective CD36 azapeptide ligand, after transient coronary artery occlusion in mice.

CD36 is a multiligand receptor involved in lipid metabolism. We investigated the mechanisms underlying the cardioprotective effect of CP-3(iv), an azapeptide belonging to a new class of selective CD36 ligands. The role of CP-3(iv) in mediating cardioprotection was investigated because CD36 signaling leads to activation of peroxisome proliferator-activated receptor-γ, a transcriptional regulator of adiponectin. CP-3(iv) pretreatment reduced infarct size by 54% and preserved hemodynamics in C57BL/6 mice subjected to 30 min coronary ligation and reperfusion but had no effect in CD36-deficient mice. The effects of CP-3(iv) were associated with an increase in circulating adiponectin levels, epididymal fat adiponectin gene expression, and adiponectin transcriptional regulators ( Pparg, Cebpb, Sirt1) after 6 h of reperfusion. Reduced myocardial oxidative stress and apoptosis were observed along with an increase in expression of myocardial adiponectin target proteins, including cyclooxygenase-2, phospho-AMPK, and phospho-Akt. Moreover, CP-3(iv) increased myocardial performance in isolated hearts, whereas blockade of adiponectin with an anti-adiponectin antibody abrogated it. CP-3(iv) exerts cardioprotection against myocardial ischemia and reperfusion (MI/R) injury and dysfunction, at least in part, by increasing circulating and myocardial adiponectin levels. Hence, both paracrine and endocrine effects of adiponectin may contribute to reduced reactive oxygen species generation and apoptosis after MI/R, in a CD36-dependent manner.-Huynh, D. N., Bessi, V. L., Ménard, L., Piquereau, J., Proulx, C., Febbraio, M., Lubell, W. D., Carpentier, A. C., Burelle, Y., Ong, H., Marleau, S. Adiponectin has a pivotal role in the cardioprotective effect of CP-3(iv), a selective CD36 azapeptide ligand, after transient coronary artery occlusion in mice.

Aza-Amino Acids Disrupt ß-Sheet Secondary Structures.

Cα to N substitution in aza-amino acids imposes local conformational constraints, changes in hydrogen bonding properties, and leads to adaptive chirality at the nitrogen atom. These properties can be exploited in mimicry and stabilization of peptide secondary structures and self-assembly. Here, the effect of a single aza-amino acid incorporation located in the upper ß-strand at a hydrogen-bonded (HB) site of a ß-hairpin model peptide (H-Arg-Tyr-Val-Glu-Val-d-Pro-Gly-Orn-Lys-Ile-Leu-Gln-NH2) is reported. Specifically, analogs in which valine3 was substituted for aza-valine3 or aza-glycine3 were synthesized, and their ß-hairpin stabilities were examined using Nuclear Magnetic Resonance (NMR) spectroscopy. The azapeptide analogs were found to destabilize ß-hairpin formation compared to the parent peptide. The aza-valine3 residue was more disruptive of ß-hairpin geometry than its aza-glycine3 counterpart.

Azapeptide Synthesis Methods for Expanding Side-Chain Diversity for Biomedical Applications.

Mimicry of bioactive conformations is critical for peptide-based medicinal chemistry because such peptidomimetics may augment stability, enhance affinity, and increase specificity. Azapeptides are peptidomimetics in which the α-carbon(s) of one or more amino acid residues are substituted by nitrogen. The resulting semicarbazide analogues have been shown to reinforce ß-turn conformation through the combination of lone pair-lone pair repulsion of the adjacent hydrazine nitrogen and urea planarity. Substitution of a semicarbazide for an amino amide residue in a peptide may retain biological activity and add benefits such as improved metabolic stability. The applications of azapeptides include receptor ligands, enzyme inhibitors, prodrugs, probes, and imaging agents. Moreover, azapeptides have proven therapeutic utility. For example, the aza-glycinamide analogue of the luteinizing hormone-releasing hormone analogue Zoladex is a potent long-acting agonist currently used in the clinic for the treatment of prostate and breast cancer. However, the use of azapeptides was hampered by tedious solution-phase synthetic routes for selective hydrazine functionalization. A remarkable stride to overcome this bottleneck was made in 2009 through the introduction of the submonomer procedure for azapeptide synthesis, which enabled addition of diverse side chains onto a common semicarbazone intermediate, providing a means to construct azapeptide libraries by solution- and solid-phase chemistry. In brief, aza residues are introduced into the peptide chain using the submonomer strategy by semicarbazone incorporation, deprotonation, N-alkylation, and orthogonal deprotection. Amino acylation of the resulting semicarbazide and elongation gives the desired azapeptide. Since the initial report, a number of chemical transformations have taken advantage of the orthogonal chemistry of semicarbazone residues (e.g., Michael additions and N-arylations). In addition, libraries have been synthesized from libraries by diversification of aza-propargylglycine (e.g., A3 coupling reactions, [1,3]-dipolar cycloadditions, and 5-exo-dig cyclizations) and aza-chloroalkylglycine residues. In addition, oxidation of aza-glycine residues has afforded azopeptides that react in pericyclic reactions (e.g., Diels-Alder and Alder-ene chemistry). The bulk of these transformations of aza-glycine residues have been developed by the Lubell laboratory, which has applied such chemistry in the synthesis of ligands with promising biological activity for treating diseases such as cancer and age-related macular degeneration. Azapeptide analogues of growth hormone-releasing peptide-6 (His-d-Trp-Ala-Trp-d-Phe-Lys-NH2, GHRP-6) have for example been pursued as ligands of the cluster of differentiation 36 receptor (CD36) and show promising activity for the development of treatments for angiogenesis-related diseases, such as age-related macular degeneration, as well as for atherosclerosis. Azapeptides have also been employed to make a series of conformationally constrained second mitochondria-derived activator of caspase (Smac) mimetics that exhibit promising apoptosis-inducing activity in cancer cells. The synthesis of cyclic azapeptide derivatives was used to make an aza scan to study the conformation-activity relationships of the anticancer agent cilengitide, cyclo(RGDf-N(Me)V), and its parent counterpart cyclo(RGDfV), which exhibit potency against human tumor metastasis and tumor-induced angiogenesis. Innovations in the synthesis and application of azapeptides will be presented in this Account, focusing on the creation and use of side-chain diversity in medicinal chemistry.

Design, Synthesis, Assembly, and Engineering of Peptoid Nanosheets.

Two-dimensional (2D) atomically defined organic nanomaterials are an important material class with broad applications. However, few general synthetic methods exist to produce such materials in high yields and to precisely functionalize them. One strategy to form ordered 2D organic nanomaterials is through the supramolecular assembly of sequence-defined synthetic polymers. Peptoids, one such class of polymer, are designable bioinspired heteropolymers whose main-chain length and monomer sequence can be precisely controlled. We have recently discovered that individual peptoid polymers with a simple sequence of alternating hydrophobic and ionic monomers can self-assemble into highly ordered, free-floating nanosheets. A detailed understanding of their molecular structure and supramolecular assembly dynamics provides a robust platform for the discovery of new classes of nanosheets with tunable properties and novel applications. In this Account, we discuss the discovery, characterization, assembly, molecular modeling, and functionalization of peptoid nanosheets. The fundamental properties of peptoid nanosheets, their mechanism of formation, and their application as robust scaffolds for molecular recognition and as templates for the growth of inorganic minerals have been probed by an arsenal of experimental characterization techniques (e.g., scanning probe, electron, and optical microscopy, X-ray diffraction, surface-selective vibrational spectroscopy, and surface tensiometry) and computational techniques (coarse-grained and atomistic modeling). Peptoid nanosheets are supramolecular assemblies of 16-42-mer chains that form molecular bilayers. They span tens of microns in lateral dimensions and freely float in water. Their component chains are highly ordered, with chains nearly fully extended and packed parallel to one another as a result of hydrophobic and electrostatic interactions. Nanosheets form via a novel interface-catalyzed monolayer collapse mechanism. Peptoid chains first assemble into a monolayer at either an air-water or oil-water interface, on which peptoid chains extend, order, and pack into a brick-like pattern. Upon mechanical compression of the interface, the monolayer buckles into stable bilayer structures. Recent work has focused on the design of nanosheets with tunable properties and functionality. They are readily engineerable, as functional monomers can be readily incorporated onto the nanosheet surface or into the interior. For example, functional hydrophilic "loops" have been displayed on the surfaces of nanosheets. These loops can interact with specific protein targets, serving as a potentially general platform for molecular recognition. Nanosheets can also bind metal ions and serve as 2D templates for mineral growth. Through our understanding of the formation mechanism, along with predicted features ascertained from molecular modeling, we aim to further design and synthesize nanosheets as robust protein mimetics with the potential for unprecedented functionality and stability.

Assembly and molecular order of two-dimensional peptoid nanosheets through the oil-water interface.

Peptoid nanosheets are a recently discovered class of 2D nanomaterial that form from the self-assembly of a sequence-specific peptoid polymer at an air-water interface. Nanosheet formation occurs first through the assembly of a peptoid monolayer and subsequent compression into a bilayer structure. These bilayer materials span hundreds of micrometers in lateral dimensions and have the potential to be used in a variety of applications, such as in molecular sensors, artificial membranes, and as catalysts. This paper reports that the oil-water interface provides another opportunity for growth of these unique and highly ordered peptoid sheets. The monolayers formed at this interface are found through surface spectroscopic measurements to be highly ordered and electrostatic interactions between the charged moieties, namely carboxylate and ammonium residues, of the peptoid are essential in the ability of these peptoids to form ordered nanosheets at the oil-water interface. Expanding the mechanism of peptoid nanosheet formation to the oil-water interface and understanding the crucial role of electrostatic interactions between peptoid residues in nanosheet formation is essential for increasing the complexity and functionality of these nanomaterials.

On-resin N-terminal peptoid degradation: Toward mild sequencing conditions.

A novel approach to sequentially degrade peptoid N-terminal N-(substituted)glycine residues on the solid-phase using very mild conditions is reported. This method relies on the treatment of resin-bound, bromoacetylated peptoids with silver perchlorate in THF, leading to an intramolecular cyclization reaction to liberate the terminal residue as a N-substituted morpholine-2,5-dione, resulting in a truncated peptoid upon hydrolysis and a silver bromide byproduct. Side-chain functional group tolerance is explored and reaction kinetics are determined. In a series of pentapeptoids possessing variable, non-nucleophilic side-chains at the second position (R(2) ), we demonstrate that sequential N-terminal degradation of the first two residues proceeds in 87% and 74% conversions on average, respectively. We further demonstrate that the degradation reaction is selective for peptoids, and represents substantial progress toward a mild, iterative sequencing method for peptoid oligomers. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 726-736, 2016.

Molecular Engineering of the Peptoid Nanosheet Hydrophobic Core.

The relationship between the structure of sequence-defined peptoid polymers and their ability to assemble into well-defined nanostructures is important to the creation of new bioinspired platforms with sophisticated functionality. Here, the hydrophobic N-(2-phenylethyl)glycine (Npe) monomers of the standard nanosheet-forming peptoid sequence were modified in an effort to (1) produce nanosheets from relatively short peptoids, (2) inhibit the aggregation of peptoids in bulk solution, (3) increase nanosheet stability by promoting packing interactions within the hydrophobic core, and (4) produce nanosheets with a nonaromatic hydrophobic core. Fluorescence and optical microscopy of individual nanosheets reveal that certain modifications to the hydrophobic core were well tolerated, whereas others resulted in instability or aggregation or prevented assembly. Importantly, we demonstrate that substitution at the meta and para positions of the Npe aromatic ring are well tolerated, enabling significant opportunities to tune the functional properties of peptoid nanosheets. We also found that N-aryl glycine monomers inhibit nanosheet formation, whereas branched aliphatic monomers have the ability to form nanosheets. An analysis of the crystal structures of several N,N'-disubstituted diketopiperazines (DKPs), a simple model system, revealed that the preferred solid-state packing arrangement of the hydrophobic groups can directly inform the assembly of stable peptoid nanosheets.

Accelerated Submonomer Solid-Phase Synthesis of Peptoids Incorporating Multiple Substituted N-Aryl Glycine Monomers.

N-Aryl glycines are a chemically diverse class of peptoid monomers that have strong structure-inducing propensities. Yet their use has been limited due to the sluggish reactivity of the weakly nucleophilic aniline submonomers. Here, we report up to a 76-fold rate acceleration of the displacement reaction using aniline submonomers in solid-phase peptoid synthesis. This is achieved by adding halophilic silver salts to the displacement reaction, facilitating bromide abstraction and AgBr precipitation. Mechanistic insight derived from analysis of a series of 15 substituted anilines reveals that the silver-mediated reaction proceeds through a transition state that has considerably less positive charge buildup on the incoming nucleophile and an enhanced leaving group. This straightforward enhancement to the submonomer method enables the rapid room temperature synthesis of a wide variety of N-aryl glycine-rich peptoid oligomers, possessing both electron-withdrawing and -donating substituents, in good yields.

Analysis of N-amino-imidazolin-2-one peptide turn mimic 4-position substituent effects on conformation by X-ray crystallography.

N-Amino-imidazolin-2-ones, a new class of turn mimics onto which side chains of different amino acids may be added conveniently, have been analyzed by X-ray crystallography. 4-Methyl and 4-benzyl N-amino-imidazolin-2-one tetrapeptide models 2 and 3 were examined to study the influence of the 4-position substituent on turn conformation and the chi dihedral angle of the neighbouring C-terminal residue side-chain. The nature of the 4-position substituent caused substantial effects on the ψ(i + 2) main chain and C-terminal Phe χ(1) side-chain dihedral angle values, giving a preference for ß- and γ-turn conformers for the methyl- and benzyl-substituted imidazolin-2-ones, respectively. Conformational analysis in the solid state has provided insight to guide application of N-amino-imidazolin-2-ones in peptide mimicry.

Peptoid-based macrodiscs of variable lipid composition for structural studies of membrane proteins by oriented-sample solid-state NMR.

Solid-state Nuclear Magnetic Resonance (NMR) in combination with magnetically aligned discoidal lipid mimics allows for studying the conformations of membrane proteins in planar, lipid-rich bilayer environments and at the physiological temperature. We have recently demonstrated the general applicability of macrodiscs composed of DMPC lipids and peptoid belts, which yield magnetic alignment and NMR spectroscopic resolution comparable or superior to detergent-containing bicelles. Here we report on a considerable improvement in the magnetic alignment and NMR resolution of peptoid-based macrodiscs consisting of a mixture of the zwitterionic and negatively charged lipids (DMPC/DMPG at the 85% to 15% molar ratio). The resulting linewidths are about 30% sharper due to the higher orientational order parameter likely arising from the stabilizing electrostatic repulsion between the discs. Moreover, highly aligned, detergent-free macrodiscs can be formed with a longer-chain lipid, DPPC. Interestingly, the spectra of Pf1 in the two lipid mimetics are almost indistinguishable, which would mean that the overall transmembrane helix tilt might be governed not only by the hydrophobic matching but also possibly by the interactions of the flanking lysine and arginine residues at the membrane interface.

Late-Stage Chloride Displacements Enable Access to Peptoids with cis-Inducing Alkylammonium Side Chains.

The synthesis of peptoids possessing multiple cis-inducing monomers with alkylammonium side chains is reported, where chloropropyl side chains are diversified on a solid support by late-stage SN2 displacements with amines. The conditions were optimized for a wide variety of primary, secondary, and tertiary alkyl amine nucleophiles. We also demonstrated that multiple chloride displacements could be achieved on sequences possessing trans-inducing N-aryl- and N-imino glycine monomers.

Late-Stage N-Alkylation of Azapeptides.

Azapeptides undergo on-resin, late-stage N-alkylations to install side chains with high chemoselectivity for the hydrazide nitrogen atoms. The major product is the N1-alkylated "azapeptoid", with only small amounts (<10%) of alkylation occurring at the other aza-amino acid nitrogen (N2). Dialkylations are also possible and afford highly functionalized, disubstituted azapeptides with side chains installed on both aza-amino acid nitrogen atoms. The site-selectivity was determined using Edman degradation, MS/MS sequencing, and comparative LCMS and NMR analyses.

Reliability of Electric Pulp Test, Cold Pulp Test or Tooth Transillumination to Assess Pulpal Health in Permanent Dog Teeth.

The aim of this study was to evaluate the reliability of electric pulp test (EPT), cold pulp test (CPT) and tooth transillumination (TTI) in the assessment of pulpal health in dog teeth. Forty-five client-owned dogs requiring tooth extraction or pulpectomy were included. For each patient, one affected and two control healthy teeth were evaluated with EPT, followed by CPT and TTI. Direct pulp inspection was used as a gold standard. The real pulpal health (vital or necrotic) was determined by the presence or absence of bleeding after creating access to the pulp chamber. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy of EPT, CPT and TTI were obtained for each pulp test using the binomial Clopper-Pearson exact method to establish confidence intervals. Forty-five affected teeth were tested. Forty-three were tested with EPT, CPT, and TTI, and two were tested solely with EPT and CPT. All dogs tested with EPT and TTI were included in the study whereas 21 out of 45 (47%) dogs tested with CPT were excluded. The sensitivity, specificity, PPV, NPV and accuracy were respectively 0.96, 1.00, 1.00, 0.96 and 0.98 for EPT; 1.00, 0.89, 0.92, 1.00 and 0.95 for CPT; and 0.59, 0.95, 0.94, 0.67 and 0.76 for TTI. This study concluded that EPT is a highly reliable diagnostic test to evaluate pulpal health in dogs. The high accuracy of CPT is conditional on the patient's responsiveness to stimulation applied to its control healthy teeth. TTI was the least reliable test in the study.

Structure-activity relationships of GHRP-6 azapeptide ligands of the CD36 scavenger receptor by solid-phase submonomer azapeptide synthesis.

The cluster of differentiation 36 (CD36) class B scavenger receptor binds a variety of biologically endogenous ligands in addition to synthetic peptides (i.e., growth hormone-releasing peptides, GHRPs), which modulate biological function related to anti-angiogenic and anti-atherosclerotic activities. Affinity labeling had previously shown that GHRP-6 analogues such as hexarelin, [2-Me-W(2)]GHRP-6 (1), bind to the lysine-rich domain of the CD36 receptor. Moreover, the azapeptide analogue [aza-F(4)]GHRP-6, 2, exhibited a characteristic ß-turn conformation as described by CD and NMR spectroscopy and a slightly higher CD36 binding affinity relative to hexarelin (1.34 and 2.37 µM, respectively), suggesting receptor binding was mediated by the conformation and the aromatic residues of these peptide sequences. Ligand-receptor binding interactions were thus explored using azapeptides to examine influences of side-chain diversity and backbone conformation. In particular, considering that aromatic cation interactions may contribute to binding affinity, we have explored the potential of introducing salt bridges to furnish GHRP-6 azapeptide ligands of the CD36 receptor. Fifteen aza-glutamic acid analogues related to 2 were prepared by submonomer solid-phase synthesis. The azapeptide side chains were installed by novel approaches featuring alkylation of resin-bound semicarbazone with Michael acceptors and activated allylic acetates in the presence of phosphazene base (BTPP). Moreover, certain Michael adducts underwent intramolecular cyclization during semicarbazone deprotection, leading to novel pyrrazoline and aza-pyroglutamate N-terminal residues. Structural studies indicated that contingent on sequence the [aza-Glu]GHRP-6 analogues exhibited CD spectra characteristic of random coil, polyproline type II and ß-turn secondary structures in aqueous media. In covalent competition binding studies with the GHRP-6 prototype hexarelin bearing a radiotracer, certain [aza-Glu]GHRP-6 azapeptides retained relatively high (2-27 µM) affinity for the CD36 scavenger receptor.

Modified peptide monolayer binding His-tagged biomolecules for small ligand screening with SPR biosensors.

A peptide self-assembled monolayer (SAM) was designed to bind His-tagged biomolecules for surface plasmon resonance (SPR) bioanalysis, which was applied for the determination of K(d) for small ligand screening against CD36. Nonspecific adsorption could be minimized using penta- and hexa-peptide monolayers. In particular, monolayers consisting of 3-mercaptopropionyl-leucinyl-histidinyl-aspartyl-leucinyl-histidinyl-aspartic acid (3-Mpa-LHDLHD) exhibited little (12 ng cm(-2)) nonspecific adsorption in crude serum. Modification of this peptide monolayer with Nα,Nα-bis(carboxymethyl)-L-lysine gave a surface competent for binding His-tagged proteins, as demonstrated using enzyme (human dihydrofolate reductase), protein/antibody and receptor (CD36) examples. Immobilization featured chelation of copper and the His-tagged protein by the peptide monolayer, which could be recycled by removing the copper using imidazole washes prior to reuse.

Solid phase submonomer azapeptide synthesis.

Azapeptides contain at least one aza-amino acid, where the α-carbon has been replaced by a nitrogen atom, and have found broad applicability in fields ranging from medicinal chemistry to biomaterials. In this chapter, we provide a step-by-step protocol for the solid phase submonomer synthesis of azapeptides, which includes three steps: (1) hydrazone activation and coupling onto a resin-bound peptide, (2) chemoselective semicarbazone functionalization for installation of the aza-amino acid side chain, and (3) orthogonal deprotection of the semicarbazone to complete the monomer addition cycle. We focus on semicarbazone functionalization by N-alkylation with primary alkyl halides, and describe conditions for coupling onto aza-amino acids. Such divergent methods accelerate the synthesis of peptidomimetics and allow the rapid introduction of a wide variety of natural and unnatural side chains directly on solid support using easily accessible submonomers.