Design, synthesis, and structural elucidation of spiroligomers for targeted molecular recognition

Spiroligomers are rigid, shape-programmable, sequence-defined molecular scaffolds assembled from stereochemically pure bis-amino acid building blocks. By controlling backbone stereochemistry, spiroligomers enable precise and predictable three-dimensional presentation of side chains, offering a level of conformational control that extends beyond conventional peptides. This dissertation integrates scalable monomer synthesis, automated solid-phase spiroligomer synthesis, functional scaffold design, and X-ray crystallography to establish how spiroligomer stereochemistry governs molecular space, side-chain orientation, and target engagement across host-guest systems and next-generation functional systems. Scalable synthetic routes were developed to access stereochemically pure, functionalized bis-amino acid building blocks. Compatible with automated solid-phase synthesis, enabling rapid generation of diverse spiroligomer architectures. Building on our group’s earlier development of solution-phase spiroligomer anion receptors, this work advances structural validation by obtaining crystall structures for all stereoisomeric receptors and a high-affinity phosphate-bound complex. Together with previously acquired NMR titration data, these structures reveal a strong stereochemical preference for optimal anion binding and demonstrate how specific backbone configurations pre-organize hydrogen-bonding geometries to match the guest, providing a direct structural basis for observed affinity trends. To expand their function toward catalysis and therapeutic targeting, larger and more structurally complex constructs of spiroligomers were designed and synthesized and efforts were taken to determine their structure via X-ray crystallography. Because these spiroligomers commonly fall within ~1-3 kDa range and can resist structure solution by direct methods, crystallographic strategies were extended to include molecular replacement (MR) using computationally generated search models. Although MR is underutilized and often challenging for small-molecule systems, applying CANDO-generated models establishes an initial framework for solving spiroligomer structures that are otherwise inaccessible, enabling experimental validation of predicted conformations and side-chain projections. Collectively, this dissertation establishes synthetic strategies, structural insights, and crystallographic methodologies that advance spiroligomers as programmable platforms for molecular recognition, metal coordination, catalysis, and therapeutic design while providing a foundation for next generation spiroligomer development guided by combined experimental structure determination and computational modelling. Chapter 2 establishes reproducible routs to functionalized bis amino acid building blocks and provides comprehensive structural validation of the synthetic pathway. Single crystal X-ray diffraction was used to determine every major intermediate from proline-4-ketone starting materials through the functionalized Fmoc-bis amino acid monomers, providing unambiguous confirmation of structure and stereochemistry at each stage. A persistent impurity observed during hydantoin purification was identified as trichloroacetamide, clarifying a major source of material heterogeneity. In parallel, large-scale synthesis of unfunctionalized Fmoc-bis amino acids supported continuous avalibility of stereochemically diverse monomers for library production. Crystallization based on purification and X-ray validation enabled efficient, reproducable access to enantiopure building blocks that form the synthetic and structural foundation for spiroligomer assembly. Chapter 3 defines optimized conditions for automated spiroligomer synthesis on Chorus peptide synthesizers and evaluates how coupling reagent choice impacts reproducibility across diverse bis-amino acid building blocks. Through systematic reagent comparisons, PyOxim was identified as the most effective reagent for the majority of building blocks, delivering consistent coupling performance, while other building blocks are better suited for using PyAOP. The observed dimerization of monomers is consistent with premature PyOxim activation for sufficiently basic monomers, enabling side reactions prior to productive couplings to the resin. An aged coupling solution experiment provided strong evidence for why the MultiPep2 synthesizer robot failed to produce spiroligomers in reliable yields. Together, these results establish a practical reagent selection for high throughput spiroligomer synthesis and support instrument choices that match the sensitivity of coupling reagents. Chapter 4 advances structural validation of spiroligomer anion receptors by obtaining X-ray crystal structures for the full stereoisomeric series. Prior NMR titration experiments conducted by Yanfeng Fan showed that the RRRR configuration is optimal for presenting urea side chains for anion binding. The unbound structure of the optimal anion receptor was previously determined by Owen O’Sullivan. In this work, crystal structures of the remaning stereoisomers were obtained, along with a phosphate-bound complex of the best binder. Together, these structures provide a structural basis for the stereochemical preference observed in solution and support the conclusion that the RRRR configuration best pre-organizes the urea donors for cooperative anion engagement. Chapter 5 describes efforts to crystalize head-to-tail spiroligomers and establish a workflow for structure determination. Because these spiroligomers occupy an intermediate regime between conventional small molecules and macromolecules, macromolecular-style crystallization screening and macromoleular phasing techniques were evaluated. Diffraction data was collected for enantiomeric spiroligomers 5-1 and 5-1a at various synchrotron beamlines.When resolution and/or data quality were insufficient for solution by direct methods, alternative approaches were explored, including MAD/SAD phasing and molecular replacement using CANDO-generated conformational models as search templates. Although these approaches did not produce a solved structure for these spiroligomers using the datasets obtained, the results define practical constraints and establish an experimental framework for future structure determination efforts, including improving crystal quality and strengthening anomalous signal.

Grantor dc:publisher

Temple University. Libraries

Year dc:date.issued

2026

Author dc:creator

• Brunner, Kyle

Advisor dc:contributor.advisor

• Schafmeister, Christian

Committee members dc:contributor.committeemember

• Kim, Daniel K.
• Zdilla, Michael
• Yatsunyk, Liliya

• Chemistry
• Organic chemistry

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• IN COPYRIGHT- This Rights Statement can be used for an Item that is in copyright. Using this statement implies that the organization making this Item available has determined that the Item is in copyright and either is the rights-holder, has obtained permission from the rights-holder(s) to make their Work(s) available, or makes the Item available under an exception or limitation to copyright (including Fair Use) that entitles it to make the Item available.

Licence dc:rights.uri

• http://rightsstatements.org/vocab/InC/1.0/

Language dc:language.iso

eng

Repository record dc:identifier.uri

https://scholarshare.temple.edu/handle/20.500.12613/12112

OAI identifier oai:identifier

oai:scholarshare.temple.edu:20.500.12613/12112