CJC-1295: Structure & Chemistry
Molecular Formula and Basic Properties
CJC-1295 with DAC is a modified peptide with a molecular weight of approximately 3,647 Daltons (exact value depends on the specific formulation and DAC attachment chemistry). This makes it a medium-sized peptide—larger than many hormone peptides but smaller than proteins like antibodies. The molecule consists of approximately 29 amino acids in the peptide backbone plus the Drug Affinity Complex (DAC) modification attached to a specific lysine residue.
As a peptide, CJC-1295 is composed primarily of amino acids linked by peptide bonds (amide bonds between the carboxyl group of one amino acid and the amino group of the next). The peptide backbone provides the basic structure and biological activity, while the amino acid side chains give the molecule its specific properties. The DAC modification adds a hydrophobic chemical group that enables albumin binding, fundamentally altering the peptide's pharmacokinetic properties.
CJC-1295 without DAC (Modified GRF 1-29) has a molecular weight of approximately 3,367 Daltons, reflecting the absence of the DAC group. This 280 Da difference represents the mass of the maleimidoproprionic acid derivative that comprises the DAC modification. Despite this relatively small mass difference, the DAC modification has profound effects on the peptide's behavior in the body.
Amino Acid Sequence
CJC-1295's amino acid sequence is based on the first 29 amino acids of natural growth hormone releasing hormone (GHRH), with strategic modifications to enhance stability and enable DAC attachment. The exact sequence is proprietary to ConjuChem Biotechnologies, but the general structure and key features are known from published research and patents.
N-Terminal Region (Positions 1-10)
The N-terminus is critical for GHRH receptor binding and activation. This region is highly conserved relative to natural GHRH, as modifications here could abolish biological activity. Position 1 is typically tyrosine, matching natural GHRH. Position 2 contains an amino acid substitution (often alanine or another small amino acid) that provides resistance to dipeptidyl peptidase-4 (DPP-4), the enzyme that rapidly degrades natural GHRH.
Positions 3-10 closely resemble natural GHRH, maintaining the structural features required for high-affinity receptor binding. These amino acids form part of an alpha-helical structure that fits into the GHRH receptor binding pocket. Any modifications in this region must preserve the helix and the spatial arrangement of key side chains that interact with the receptor.
Mid-Region (Positions 11-20)
This region contains amino acids important for receptor activation and includes the lysine residue (typically position 15 or 20) to which the DAC modification is attached. The sequence in this region may differ from natural GHRH to optimize properties for DAC attachment and to enhance overall stability. The lysine selected for DAC attachment must be positioned such that the attached DAC group doesn't interfere with receptor binding while still being accessible for albumin binding.
The mid-region also contributes to the peptide's alpha-helical structure, which extends from the N-terminus through much of the peptide length. This extended helix is important for maintaining the correct three-dimensional structure for receptor interaction. Amino acid substitutions in this region are carefully selected to preserve helical structure while improving pharmaceutical properties.
C-Terminal Region (Positions 21-29)
The C-terminal region is less critical for receptor binding than the N-terminus, allowing more flexibility for modifications. This region may contain amino acid substitutions designed to enhance stability, improve solubility, or optimize other pharmaceutical properties. The C-terminus contributes to overall peptide structure and may influence receptor binding kinetics or signaling, though its role is less well-defined than the N-terminal region.
Natural GHRH extends to position 44, but research showed that amino acids 30-44 are not essential for biological activity. CJC-1295 uses only positions 1-29, reducing the peptide's size while maintaining full potency. This truncation also simplifies synthesis and may improve stability by removing potentially problematic sequences.
The DAC Modification: Chemical Structure and Function
The Drug Affinity Complex (DAC) modification is the defining feature of CJC-1295, distinguishing it from other GHRH analogs and enabling its extended half-life. Understanding the DAC's chemistry is essential for appreciating how CJC-1295 works.
Chemical Composition
The DAC consists of a maleimidoproprionic acid (MPA) derivative attached to the epsilon-amino group of a lysine side chain. The MPA contains a maleimide functional group—a five-membered ring containing two carbonyl groups and a nitrogen. This maleimide is highly reactive toward thiols (sulfur-containing groups), particularly the cysteine residues found on serum albumin.
The attachment chemistry involves forming an amide bond between the MPA's carboxylic acid and the lysine's amino group. This creates a stable covalent linkage between the peptide and the MPA. The maleimide group on the MPA then remains available to react with albumin's cysteine-34 residue, forming a thioether linkage that binds the peptide to albumin.
Albumin Binding Mechanism
When CJC-1295 is injected, the maleimide group reacts with cysteine-34 on serum albumin, the most abundant protein in blood plasma. This reaction forms a reversible covalent bond—the thioether linkage can slowly break and reform, creating an equilibrium between albumin-bound and free CJC-1295. The equilibrium strongly favors the bound state, with the majority of CJC-1295 molecules bound to albumin at any given time.
Albumin has a half-life of approximately 19 days, much longer than small peptides which are rapidly cleared by the kidneys. By binding to albumin, CJC-1295 effectively "hitchhikes" on albumin's long circulation time. The albumin-CJC-1295 complex is too large for renal filtration, preventing kidney clearance. Additionally, albumin binding protects CJC-1295 from enzymatic degradation, as the bound peptide is less accessible to proteases.
The reversible nature of the albumin binding is important—CJC-1295 must dissociate from albumin to exert its biological effects by binding to GHRH receptors. The equilibrium ensures a steady supply of free CJC-1295 available for receptor activation while maintaining a large reservoir of bound peptide. This creates sustained, relatively constant levels of active peptide over days.
Spatial Considerations
The DAC modification must be positioned such that it doesn't interfere with GHRH receptor binding. The lysine selected for DAC attachment is typically in the mid-region of the peptide, away from the N-terminal receptor-binding domain. When CJC-1295 binds to the GHRH receptor, the DAC group extends away from the receptor-peptide interface, minimizing steric interference.
Similarly, when CJC-1295 is bound to albumin, the peptide portion must be accessible for dissociation and receptor binding. The DAC acts as a tether, keeping CJC-1295 associated with albumin while allowing the peptide to extend into solution where it can interact with receptors. This spatial arrangement is critical for the DAC strategy to work—the modification must enable albumin binding without preventing receptor activation.
Three-Dimensional Structure
CJC-1295's biological activity depends not just on its amino acid sequence but on its three-dimensional structure. Like other peptide hormones, CJC-1295 adopts specific conformations that enable receptor binding and activation.
Secondary Structure
Structural studies of GHRH and its analogs suggest that CJC-1295 contains significant alpha-helical structure, particularly in the N-terminal and mid-regions. Alpha-helices are common in peptide hormones and are often important for receptor binding. The helical structure presents amino acid side chains in a specific spatial arrangement that fits into the receptor binding pocket.
The extent and stability of helical structure depend on the amino acid sequence and the environment (solution vs receptor-bound). Certain amino acids (alanine, leucine, glutamic acid) promote helix formation, while others (proline, glycine) disrupt it. CJC-1295's sequence is designed to maintain appropriate helical structure for receptor binding while incorporating modifications for stability and DAC attachment.
Receptor-Bound Conformation
When CJC-1295 binds to the GHRH receptor, it likely adopts a specific conformation that enables optimal receptor activation. The N-terminal region inserts into the receptor's binding pocket, with key amino acid side chains forming specific interactions (hydrogen bonds, electrostatic interactions, hydrophobic contacts) with receptor residues. These interactions trigger the conformational changes in the receptor that initiate intracellular signaling.
The receptor-bound conformation may differ from the solution conformation, with the peptide undergoing conformational changes upon binding. This induced-fit mechanism is common for peptide-receptor interactions. The peptide must be flexible enough to adopt the receptor-bound conformation while maintaining sufficient structural stability to avoid degradation.
Solution Conformation
In solution (including in the bloodstream and in formulations), CJC-1295 likely exists as an ensemble of conformations in dynamic equilibrium. The peptide may have some helical structure but is probably not fully helical in solution. The DAC modification may influence solution conformation by affecting the peptide's hydrophobicity and aggregation tendency.
When bound to albumin, CJC-1295's conformation is constrained by the albumin interaction. The peptide portion may adopt different conformations than when free in solution. Understanding these conformational dynamics is important for optimizing formulation and predicting biological activity, though detailed structural studies of CJC-1295 have not been published.
Chemical Properties
Solubility
CJC-1295's solubility is influenced by its amino acid composition and the DAC modification. The peptide backbone contains both hydrophilic (charged and polar amino acids) and hydrophobic (nonpolar amino acids) residues, giving it amphipathic character. The DAC modification adds significant hydrophobic character, potentially reducing aqueous solubility compared to unmodified peptide.
In practice, CJC-1295 is soluble in aqueous buffers at appropriate pH and concentration. The lyophilized powder readily dissolves in bacteriostatic water or saline for injection. However, at high concentrations or inappropriate pH, the peptide may aggregate or precipitate. Formulation development must balance solubility with stability and other pharmaceutical properties.
Stability
CJC-1295's chemical stability is affected by multiple factors. The peptide bonds are susceptible to hydrolysis, particularly at elevated temperatures or extreme pH. Certain amino acids are prone to specific degradation pathways: asparagine and glutamine can undergo deamidation, methionine can be oxidized, and cysteine (if present) can form disulfide bonds or undergo oxidation.
The DAC modification may affect stability in complex ways. The maleimide group is reactive and could potentially undergo side reactions. The albumin binding may protect the peptide from degradation, but the modification site itself could be a point of vulnerability. Proper storage (refrigeration or freezing, protection from light) is essential for maintaining CJC-1295 stability.
Charge and Isoelectric Point
CJC-1295's net charge depends on pH due to ionizable amino acid side chains (aspartic acid, glutamic acid, lysine, arginine, histidine). At physiological pH (7.4), the peptide likely carries a net positive charge due to basic amino acids, though the exact charge depends on the specific sequence. The isoelectric point (pI)—the pH at which net charge is zero—is likely in the range of pH 8-10, though this depends on the amino acid composition.
Understanding charge properties is important for purification (ion exchange chromatography), formulation (pH selection), and predicting interactions with other molecules. The DAC modification may affect overall charge distribution, particularly if it's attached near charged residues.
Hydrophobicity
The DAC modification significantly increases CJC-1295's hydrophobicity compared to unmodified GHRH analogs. This increased hydrophobicity is essential for albumin binding but also affects other properties like aggregation tendency, membrane interactions, and chromatographic behavior. The hydrophobicity can be quantified by parameters like logP (partition coefficient) or retention time in reversed-phase HPLC.
The increased hydrophobicity may contribute to CJC-1295's tendency to aggregate at high concentrations or during storage. Aggregation can reduce biological activity and potentially increase immunogenicity. Formulation strategies (pH adjustment, excipients, concentration limits) help minimize aggregation.
Receptor Binding Chemistry
CJC-1295 binds to GHRH receptors through multiple molecular interactions between amino acid side chains and the receptor binding pocket. These interactions include hydrogen bonds (between polar groups), electrostatic interactions (between charged groups), hydrophobic contacts (between nonpolar groups), and van der Waals forces (weak attractions between atoms in close proximity).
The N-terminal region is particularly important for receptor binding, with specific amino acids making critical contacts. Tyrosine at position 1 is essential—modifications at this position typically abolish activity. Other key residues in the N-terminal region contribute to binding affinity and receptor activation. The mid-region and C-terminus play supporting roles, contributing to overall binding and potentially influencing signaling kinetics.
The binding affinity of CJC-1295 for GHRH receptors is similar to natural GHRH, typically in the nanomolar range. This high affinity ensures that even low concentrations of free CJC-1295 (dissociated from albumin) can effectively activate receptors. The binding is reversible, with CJC-1295 associating and dissociating from receptors in dynamic equilibrium.
Comparison to Related Molecules
CJC-1295 vs Natural GHRH
CJC-1295 shares approximately 70-80% sequence identity with natural GHRH(1-29). The key differences are amino acid substitutions for DPP-4 resistance and stability, the DAC modification (absent in natural GHRH), and truncation at position 29 (natural GHRH extends to position 44). These changes transform a hormone with a 6-8 minute half-life into a therapeutic agent with a 6-8 day half-life—a 1,000-fold increase.
CJC-1295 vs Modified GRF 1-29
Modified GRF 1-29 has the same or very similar amino acid sequence as CJC-1295 but lacks the DAC modification. This 280 Da difference in molecular weight translates into a dramatic difference in half-life (30 minutes vs 6-8 days). The two peptides have similar receptor binding and activation properties but fundamentally different pharmacokinetics and use patterns.
CJC-1295 vs Sermorelin
Sermorelin is GHRH(1-29) with minimal modifications—essentially the first 29 amino acids of natural GHRH. It lacks both the DPP-4 resistance modifications and the DAC modification of CJC-1295. Sermorelin has a half-life of approximately 10-20 minutes (longer than natural GHRH due to some DPP-4 resistance but much shorter than CJC-1295). CJC-1295 represents a more advanced generation of GHRH analog with superior pharmaceutical properties.
Analytical Characterization
Comprehensive chemical characterization of CJC-1295 requires multiple analytical techniques, each providing different information about structure and properties.
Mass Spectrometry
High-resolution mass spectrometry determines CJC-1295's exact molecular weight (approximately 3,647 Da for the DAC version) and confirms the amino acid sequence and DAC modification. Tandem mass spectrometry (MS/MS) can sequence the peptide by fragmenting it and analyzing the fragments. This verifies that the correct amino acids are present in the correct order and that the DAC is attached at the correct position.
Nuclear Magnetic Resonance (NMR)
NMR spectroscopy provides detailed structural information including secondary structure, conformational dynamics, and interactions between different parts of the molecule. Two-dimensional NMR techniques can determine which amino acids are close in space (even if distant in sequence), revealing the three-dimensional structure. However, NMR studies of CJC-1295 have not been published, limiting detailed structural knowledge.
Circular Dichroism (CD) Spectroscopy
CD spectroscopy measures the secondary structure content (alpha-helix, beta-sheet, random coil). This technique is useful for comparing different batches of CJC-1295 to ensure consistent structure, for studying conformational changes with pH or temperature, and for detecting aggregation or misfolding. CD can quickly assess whether a peptide sample has the expected structural characteristics.
Chromatographic Techniques
Reversed-phase HPLC separates CJC-1295 from impurities based on hydrophobicity. The DAC-modified peptide elutes later than unmodified peptide due to increased hydrophobicity. Ion exchange chromatography separates based on charge. Size exclusion chromatography separates based on size, detecting aggregates and fragments. These techniques are essential for purity assessment and quality control.