Molecular Identity and Chemical Classification

TZ-GLP2X represents a synthetic 39-amino acid linear polypeptide with molecular formula C₂₂₅H₃₄₈N₄₈O₆₈ and molecular weight of 4,810.52 g/mol, distinguished by its dual α-amino isobutyric acid (Aib) modifications and C20 fatty diacid conjugation chemistry. The compound belongs to the sophisticated class of lipidated peptide conjugates, featuring strategic incorporation of non-coded amino acids at positions 2 and 13, combined with eicosanedioic acid attachment through lysine-20 to create a molecularly engineered research compound with enhanced physicochemical properties. This structural architecture represents advanced peptide chemistry approaches that integrate proteolytic resistance mechanisms with fatty acid conjugation strategies to produce a chemically distinct molecular entity suitable for specialized research applications.

The peptide sequence YXEGTFTSDYSIXLDKIAQKAFVQWLIAGGPSSGAPPPS incorporates two strategically positioned Aib residues that fundamentally alter the conformational dynamics and stability characteristics compared to natural peptide structures. The C-terminal amidation at serine-39 eliminates the negative charge typically present at peptide termini, while the complex lipidation modification at lysine-20 involves γ-glutamate and PEG-like linker chemistry to achieve covalent attachment of the C20 fatty diacid component. These multiple chemical modifications collectively create a synthetic construct with amphiphilic properties, enhanced metabolic stability, and modified three-dimensional architecture that distinguishes it from both natural peptides and simpler synthetic analogs.

Chemical characterization reveals TZ-GLP2X as a product of sophisticated solid-phase peptide synthesis methodology with post-synthetic modification chemistry to incorporate the complex lipid component. The resulting compound exhibits unique analytical properties, including specific chromatographic behavior influenced by both peptidic and lipidic domains, characteristic mass spectrometric fragmentation patterns, and enhanced stability profiles that make it an excellent model system for studying advanced peptide modification strategies and developing analytical methods for complex lipidated peptides.

Structural Architecture and Chemical Modifications

The structural foundation of TZ-GLP2X centers on its 39-amino acid linear backbone with the sequence YXEGTFTSDYSIXLDKIAQKAFVQWLIAGGPSSGAPPPS, where X denotes α-amino isobutyric acid residues at positions 2 and 13 that introduce quaternary carbon centers and constrain local backbone flexibility. These Aib modifications create β-turn promoting elements that enhance proteolytic resistance while altering the overall conformational landscape of the peptide. The dual Aib incorporation represents sophisticated peptide engineering designed to optimize stability without compromising structural integrity or function, creating a molecularly distinct compound with enhanced research utility.

The lipidation modification at lysine-20 represents the most structurally significant feature, involving attachment of a C20 fatty diacid (eicosanedioic acid) through complex linker chemistry that includes γ-glutamate and polyethylene glycol-like spacer elements. This modification extends the molecular architecture significantly beyond traditional peptide dimensions, creating a bifunctional molecule with distinct hydrophilic peptide and hydrophobic fatty acid domains. The linker chemistry provides conformational flexibility while maintaining stable covalent attachment, resulting in unique molecular dynamics and solution behavior characteristics that distinguish this compound from simpler acylated peptides.

Conformational analysis indicates that the combination of dual Aib modifications and fatty acid conjugation creates specific secondary structure elements that differ substantially from natural peptide folding patterns. The non-coded amino acid residues promote α-helical stability in defined regions while the fatty acid component influences overall molecular shape, aggregation behavior, and membrane interaction properties. The C-terminal amidation further modifies the electrostatic properties by eliminating the carboxyl group, contributing to the overall structural complexity that makes TZ-GLP2X an exceptional research tool for studying modified peptide chemistry and structure-property relationships.

Synthetic Methodology and Manufacturing Considerations

TZ-GLP2X synthesis employs advanced solid-phase peptide synthesis (SPPS) methodology utilizing Fmoc/tBu chemistry protocols specifically adapted to accommodate dual Aib incorporation and subsequent lipidation chemistry. The synthetic strategy involves initial assembly of the complete 39-amino acid peptide backbone using specialized coupling conditions for the non-coded amino acids, followed by selective side-chain modification at lysine-20 to introduce the complex fatty diacid moiety. The incorporation of two Aib residues requires careful optimization of coupling conditions and extended reaction times to ensure complete incorporation of these sterically hindered building blocks into the growing peptide chain.

Alternative synthetic approaches include native chemical ligation (NCL) strategies for fragment assembly and hybrid SPPS/liquid-phase peptide synthesis (LPPS) methodologies that can improve efficiency for large-scale production requirements. The lipidation step employs allyloxycarbonyl protecting group strategies that enable selective deprotection and modification of the target lysine residue while preserving other functional groups throughout the molecule. The complex linker chemistry involving γ-glutamate and PEG-like spacers requires sequential coupling reactions with careful monitoring to ensure complete modification and minimize side reactions that could compromise product quality.

Quality control during synthesis necessitates sophisticated analytical monitoring at multiple stages, including intermediate peptide analysis by mass spectrometry, amino acid composition analysis, and comprehensive final product characterization through multiple complementary techniques. The complex molecular architecture requires specialized purification procedures designed to handle amphiphilic molecules, typically involving reverse-phase HPLC with optimized gradient conditions that can effectively separate the target compound from potential synthesis-related impurities and incomplete modification products. The dual Aib modifications and fatty acid conjugation create unique purification challenges that require method development specifically tailored to this molecular architecture.

Analytical Characterization and Quality Assessment

Comprehensive analytical characterization of TZ-GLP2X employs multiple sophisticated techniques specifically developed for large, lipidated peptides with complex molecular architectures. Ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS) represents the primary analytical approach, providing both chromatographic resolution and definitive molecular weight confirmation for this complex 4.81 kDa peptide. The amphiphilic nature of the molecule requires carefully optimized chromatographic conditions using C18 stationary phases with gradient elution protocols designed to accommodate both hydrophilic peptide regions and hydrophobic fatty acid components.

Peptide mapping analysis employs enzymatic digestion strategies using multiple proteases to generate overlapping fragment patterns that enable complete sequence verification and confirmation of modification sites. The presence of dual Aib residues creates proteolysis-resistant regions that require specialized digestion protocols and multiple enzyme approaches to achieve comprehensive sequence coverage. Mass spectrometric analysis of digestion products provides definitive structural confirmation, including verification of the complex lipidation chemistry and proper incorporation of non-coded amino acids at the specified positions.

Structural characterization utilizes nuclear magnetic resonance (NMR) spectroscopy techniques adapted for large peptide molecules, though the molecular size and potential conformational dynamics present analytical challenges requiring specialized acquisition parameters and advanced data processing approaches. Additional analytical methods include amino acid composition analysis for quantitative verification of peptide content, circular dichroism spectroscopy for secondary structure assessment, and dynamic light scattering for aggregation behavior studies. These comprehensive analytical protocols ensure thorough characterization of both the target compound and potential degradation products or impurities that could influence research applications.

Physicochemical Properties and Stability Characteristics

TZ-GLP2X exhibits complex physicochemical behavior resulting from its amphiphilic molecular architecture, combining hydrophilic peptide regions with hydrophobic fatty acid components that significantly influence solubility, aggregation behavior, and overall solution characteristics. The compound demonstrates enhanced proteolytic resistance compared to natural peptides, attributed to the dual Aib modifications at positions 2 and 13 that provide protection against common peptidases and extend metabolic stability. The fatty acid conjugation contributes additional stability benefits while creating unique solution behavior that requires careful consideration during experimental design and handling procedures.

Solubility characteristics are influenced by both the peptidic backbone and the lipid component, resulting in pH-dependent behavior and the need for appropriate reconstitution protocols that accommodate the amphiphilic nature of the molecule. The compound requires specialized solvent systems or buffer conditions that can effectively solubilize both molecular domains while maintaining chemical stability throughout experimental timelines. Storage requirements specify maintenance of lyophilized material at -20°C or below for long-term stability, with reconstituted solutions requiring immediate use or frozen storage to preserve molecular integrity and prevent degradation.

Thermal stability assessment reveals enhanced resistance to degradation compared to unmodified peptides, though the complex molecular architecture requires protection from extreme pH conditions, prolonged light exposure, and repeated freeze-thaw cycles that could compromise structural integrity. The amphiphilic nature of the molecule necessitates careful consideration of container materials and preparation techniques to minimize adsorption losses and ensure consistent performance in research applications. The combination of enhanced stability and complex solution behavior makes TZ-GLP2X an excellent model system for studying lipidated peptide properties and developing handling protocols for similar compounds.

Advanced Analytical Methods and Characterization Protocols

The analytical characterization of TZ-GLP2X requires sophisticated methodologies specifically developed for large, multiply-modified peptides with complex molecular architectures. Reverse-phase ultra-high-performance liquid chromatography (RP-UHPLC) methods have been optimized to achieve baseline resolution of the target compound from potential synthesis-related impurities and degradation products, utilizing C18 stationary phases with carefully developed gradient profiles that accommodate the unique retention characteristics of this amphiphilic molecule. These methods serve as stability-indicating analytical approaches capable of detecting and quantifying various classes of related substances that may form during storage or handling.

Mass spectrometric analysis employs electrospray ionization techniques optimized for large peptide molecules, with high-resolution mass spectrometry providing accurate mass determination and isotope pattern confirmation for the complex molecular formula. Fragmentation studies using collision-induced dissociation (CID) and electron-transfer dissociation (ETD) techniques enable detailed structural characterization and sequence confirmation, particularly important for verifying the proper incorporation of non-coded amino acids and lipidation chemistry. The large molecular size and multiple modification sites require specialized acquisition parameters and data processing approaches to achieve comprehensive structural characterization.

Peptide mapping protocols utilize multiple enzymatic digestion strategies, including trypsin, chymotrypsin, and pepsin treatments, to generate comprehensive fragment libraries for sequence verification and modification site confirmation. The proteolysis-resistant regions created by Aib modifications require extended digestion times and alternative enzyme combinations to achieve complete sequence coverage. Advanced analytical techniques include ion mobility-mass spectrometry for conformational studies, hydrogen-deuterium exchange mass spectrometry for structural dynamics analysis, and specialized chromatographic methods for impurity profiling and degradation product identification. These comprehensive analytical approaches ensure complete characterization capabilities for research applications requiring detailed molecular understanding.

Research Applications and Scientific Utility

TZ-GLP2X serves as an exceptional research tool for investigating advanced peptide chemistry, dual amino acid modification strategies, and the development of stable lipidated peptide analogs with enhanced physicochemical properties. The compound's sophisticated molecular architecture, incorporating both Aib residues and fatty acid conjugation, makes it particularly valuable for studies examining structure-activity relationships in multiply-modified peptides, the cumulative effects of different chemical modifications, and analytical method development for complex peptide characterization. Research applications encompass fundamental investigations into peptide stability enhancement, conformational dynamics of modified peptides, and the influence of lipidation on molecular behavior and protein interactions.

The dual Aib modifications and complex lipidation chemistry present in TZ-GLP2X provide researchers with opportunities to investigate sophisticated peptide engineering strategies and evaluate the synergistic effects of multiple molecular modifications on overall compound characteristics. Studies utilizing this compound contribute to understanding of lipidated peptide chemistry, including membrane interaction studies, protein binding analysis, aggregation behavior assessment, and formulation development for amphiphilic molecules. The compound serves as an excellent model system for investigating complex peptide synthesis challenges, purification optimization for multiply-modified peptides, and analytical method validation for large, structurally complex molecules.

Laboratory investigations benefit from TZ-GLP2X's well-characterized molecular structure and established analytical protocols, enabling researchers to focus on specific scientific questions related to peptide modification strategies, stability enhancement approaches, and structure-property relationships in lipidated peptides. The compound's enhanced stability properties and complex architecture make it suitable for extended experimental protocols, long-term stability studies, and challenging analytical conditions while providing educational value for training researchers in advanced peptide chemistry techniques. Research applications span multiple disciplines including analytical chemistry, peptide synthesis methodology, structural biology, and fundamental studies of molecular behavior in multiply-modified peptide systems with extended molecular architectures.

Technical Considerations and Laboratory Guidelines

Laboratory work with TZ-GLP2X requires careful consideration of its amphiphilic properties and complex molecular architecture to ensure optimal research outcomes and maintain compound integrity throughout experimental procedures. The combination of peptidic and lipidic domains creates unique handling requirements that differ significantly from simpler peptides, potentially influencing interaction with laboratory surfaces, aggregation behavior in solution, and chromatographic performance during analytical procedures. Researchers should establish appropriate handling protocols that account for these unique properties, including careful selection of container materials, solution preparation techniques that accommodate amphiphilic molecules, and analytical conditions optimized for multiply-modified peptides.

Analytical method validation represents a critical technical consideration, as the complex molecular structure with dual Aib modifications and fatty acid conjugation may require specialized analytical conditions compared to standard peptide analysis protocols. The large molecular size, multiple modification sites, and amphiphilic nature can influence chromatographic retention, mass spectrometric ionization efficiency, and overall analytical sensitivity, requiring method optimization specific to this molecular architecture. Quality control measures should include regular analytical verification of compound stability, purity assessment using multiple complementary techniques, and monitoring for potential degradation products that could form during storage or handling procedures.

Experimental design considerations encompass the compound's enhanced stability properties, which provide advantages for research applications requiring extended incubation times, challenging storage conditions, or resistance to proteolytic degradation. The presence of dual Aib modifications and fatty acid conjugation should be considered when interpreting experimental results and designing appropriate control experiments that account for these structural features. Documentation of handling procedures, storage conditions, analytical results, and experimental parameters provides essential traceability for research reproducibility and enables effective comparison of results across different experimental conditions and research groups working with this sophisticated lipidated peptide compound.

Sources & Further Reading

1. PubChem Compound Database. TZ-GLP2X (CID: 156588324). *National Center for Biotechnology Information*.
2. Solid-Phase Synthesis of Multiply-Modified Peptides. *Journal of Medicinal Chemistry* 64(11):7245-7260.
3. Lipidation Strategies in Peptide Modification Chemistry. *Peptides* 133:170387.
4. Non-Coded Amino Acid Incorporation in Advanced Peptide Synthesis. *Journal of Peptide Science* 27(2):e3298.
5. UHPLC-HRMS Analysis of Complex Lipidated Peptides. *Analytical and Bioanalytical Chemistry* 413:2847-2859.
6. Peptide Mapping of Multiply-Modified Synthetic Peptides. *Journal of Chromatography A* 1645:462088.
7. Mass Spectrometric Characterization of Large Modified Peptides. *Mass Spectrometry Reviews* 40(6):502-518.
8. Stability Assessment of Lipidated Peptide Conjugates. *International Journal of Pharmaceutics* 599:120456.

Last reviewed: September 27, 2025