Semaglutide Molecular Weight Da, Sequence, and Chemical Specifications

Direct Answer: Semaglutide Molecular Weight Da and Chemical Formula

The standard semaglutide molecular weight da is 4113.58 Da (grams per mole), corresponding to the empirical chemical formula C187H291N45O59. This monoisotopic mass accounts for the 31-amino-acid backbone derivative derived from human glucagon-like peptide-1 (GLP-1), modified by an alpha-aminobutyric acid substitution and a specialized C18 diacid fatty acylation chain.

In high-resolution liquid chromatography-mass spectrometry (LC-MS) assays, research laboratories typically register the monoisotopic peak at 4111.12 Da, with multicharged species ([M+3H]3+, [M+4H]4+, and [M+5H]5+) resolved based on the ionization matrix and mobile phase composition. Accurate mass verification ensures that synthetic lot-to-lot consistency is maintained across in vitro receptor binding assays and cell culture platforms.

Primary Amino Acid Sequence and Structural Modifications

Semaglutide is a modified linear peptide analog modeled after native human GLP-1 (7-37). Its primary amino acid sequence consists of 31 residues featuring key structural modifications engineered to increase enzymatic stability against dipeptidyl peptidase-4 (DPP-4) cleavage and extend plasma albumin binding affinity in non-human biological models.

The single-letter amino acid sequence for semaglutide is expressed as follows: H-X-E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A-K(*)-E-F-I-A-W-L-V-R-G-R-G-OH, where X represents 2-aminoisobutyric acid (Aib) at position 8, replacing native L-alanine. The asterisk (*) indicates position Lys26, which is covalently conjugated via a hydrophilic linker to a lipophilic side chain.

The complete chemical name of the Lys26 modification is N-epsilon-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]lysine. This modification comprises two 8-amino-3,6-dioxaoctanoic acid (AEEAc) spacer groups, a gamma-glutamic acid linker, and a terminal C18 fatty diacid (17-carboxyheptadecanoyl) moiety. In addition, native Lys34 is substituted with Arg34 to prevent non-specific acylation during chemical synthesis. A complete breakdown of these modifications is available in our semaglutide structural analysis documentation.

Physicochemical Specifications and Mass Spectrometry Analysis

Establishing baseline analytical parameters for semaglutide 5mg requires evaluating specific physical and chemical properties under standardized laboratory conditions. Because semaglutide features a hydrophobic C18 fatty acid moiety balanced by hydrophilic PEG-like spacers and polar amino acid residues, its chromatographic behavior exhibits unique retention characteristics compared to unmodified peptides.

Key physicochemical properties include an isoelectric point (pI) calculated at approximately 4.5 to 4.8, reflecting the abundance of acidic glutamic and aspartic acid residues alongside the terminal carboxylate of the fatty acid chain. Reversed-phase high-performance liquid chromatography (RP-HPLC) typically utilizes a C18 column with a gradient of water/acetonitrile containing 0.1% trifluoroacetic acid (TFA) or formic acid to resolve semaglutide from truncation sequences.

Electrospray ionization mass spectrometry (ESI-MS) spectra for semaglutide consistently yield prominent multi-charged ion clusters. Researchers quantifying raw material purity evaluate the spectral purity by verifying the absence of unacylated backbone fragments (MW ~3297 Da) or truncated acetylated species in the mass domain. Comprehensive reference data across our catalog can be explored via the PX1 research library.

Molecular Weight Comparison: Semaglutide vs. Related Incretin Peptides

When designing comparative in vitro binding assays, researchers must account for differences in molecular mass, primary sequence length, and acylation strategies across the incretin peptide class. Semaglutide differs structurally from dual and triple receptor agonists, as well as earlier single-agonist analogs.

For example, tirzepatide possesses a molecular weight of 4813.45 Da and contains 39 amino acid residues featuring a C20 fatty acid side-chain conjugated to a Lys residue at position 20, acting as a dual GLP-1 and GIP receptor agonist. Similarly, retatrutide has a calculated molecular weight of 4731.33 Da across 39 residues, incorporating triple agonist activity at the GLP-1, GIP, and glucagon receptors. In contrast, liraglutide displays a lower molecular weight of 3751.20 Da due to its shorter C16 fatty acid chain and single gamma-glutamic acid spacer without AEEAc units.

Understanding these mass differences is critical when preparing equimolar concentrations for comparative receptor transactivation, cAMP accumulation, or competitive binding studies in cell culture lines.

PX1 Research Quality Verification and Sourcing Standards

Assay reproducibility depends directly on the structural purity and lot-to-lot consistency of synthetic compounds. PX1 Research implements rigorous quality control protocols for all solid-phase peptide synthesis (SPPS) lots manufactured for laboratory evaluation.

Every production lot undergoes independent, third-party analytical testing to confirm compound identity and purity before release. PX1 Research standards require:

• RP-HPLC Purity: Greater than or equal to 99.0% main peak area purity. • Mass Spectrometry (ESI-MS): Exact mass confirmation matching the calculated 4113.58 Da weight. • Endotoxin Quantification: Tested via Chromogenic LAL assay to guarantee < 0.01 EU/mg, minimizing cell culture cytotoxicity. • Moisture & Trifluoroacetate Content: Quantified to ensure correct molar mass calculation during reconstitution.

Laboratories sourcing compounds for analytical protocols can review batch-specific Certificates of Analysis (COAs) directly from our all peptides hub or establish custom supply channels through our wholesale lab portal.

Reconstitution and Laboratory Handling Protocols

Proper reconstitution technique is crucial to maintain structural integrity and prevent aggregation of acylated peptides. Semaglutide's lipophilic fatty diacid chain increases its tendency to form self-associated micellar structures if dissolved under unfavorable pH or ionic strength conditions.

For optimal solubility, lyophilizates should be brought to room temperature prior to reconstitution. Reconstitute using sterile reconstitution solution 30ml (bacteriostatic water) or phosphate-buffered saline (PBS) adjusted to pH 7.4. Avoid intense vortexing or high-shear mechanical agitation, which can induce physical shearing or surface-induced denaturation; gentle inversion or swirl is recommended.

Reconstituted aliquots intended for short-term in vitro assays should be stored at 2°C to 8°C and protected from light. For long-term stock storage, freeze aliquots at -80°C to prevent freeze-thaw degradation cycles. Always calculate final molar concentrations based on net peptide content rather than gross lyophilized mass to ensure precise assay dosing.

Preclinical Applications and Receptor Binding Kinetics

In preclinical settings, semaglutide serves as a reference standard for evaluating GLP-1 receptor (GLP-1R) orthosteric interactions. In vitro radioligand binding assays using recombinant human or rodent GLP-1R membrane preparations demonstrate nanomolar binding affinity (Ki ~0.38 nM to 1.2 nM depending on albumin presence).

The incorporation of the Aib residue at position 8 provides sterical hindrance against cleavage by DPP-4 endopeptidases, increasing enzymatic half-life in non-human plasma models from minutes to several hours or days. Simultaneously, the C18 fatty diacid chain promotes reversible non-covalent binding to serum albumin, shielding the peptide from renal clearance.

Researchers investigating intracellular signaling cascades utilize semaglutide to measure downstream cyclic adenosine monophosphate (cAMP) accumulation, beta-arrestin recruitment, and receptor internalization kinetics across diverse cell models. Additional mechanistic studies are detailed in our section on GLP-1 receptor agonists overview.