Pentagastrin CAS 5534-95-2

Target sequence: Boc-β-Ala-Trp-Met-Asp-Phe-NH₂. A C-to-N terminal sequential condensation strategy is adopted, with L-phenylalaninamide serving as the C-terminal starting material.

Selective side-chain protection is implemented for individual amino acids: the β-carboxyl group of aspartic acid is protected via benzyl esterification, the indole moiety of tryptophan undergoes mild capping, and Boc is used for transient amino-group shielding.

Using classic DCC/HOBt as condensation reagents, coupling proceeds at low temperature in DMF, with sequential incorporation of Asp, Met and Trp, followed by final conjugation with Boc-β-alanine.

Post-condensation, low-temperature hydrogenolysis removes benzyl-based side-chain protecting groups, and residual trace Boc groups are cleaved under mild TFA treatment.

The concentrated crude product is recrystallized from an acetonitrile-water mixed solvent system. Featuring low capital investment on production equipment, this process was widely adopted for early bulk drug manufacturing with an overall yield ranging from 32% to 38%. Its main drawbacks include cumbersome intermediate isolation and excessive waste discharge, leading to gradual replacement by solid-phase synthesis technology.

2-chlorotrityl chloride resin (CTC resin) is selected as the solid support. Fmoc-Phe-NH₂ is first anchored onto the resin matrix, and unoccupied reactive resin sites are capped with methanol.

With DMF as reaction medium, Fmoc deprotection is completed using piperidine, and carboxyl activation is realized with HBTU/HOBt. Fmoc-Asp(OBzl), Fmoc-Met and Fmoc-Trp are sequentially coupled from the C-terminus toward the N-terminus, and terminal Boc-β-Ala (Fmoc-free) is introduced in the final step.

Cleavage from resin and global side-chain deprotection are performed at ambient temperature with TFA cleavage cocktail; the resin is removed by filtration, and the concentrated filtrate is subjected to preparative reversed-phase HPLC purification on a C18 column with gradient elution of acetonitrile-water, followed by lyophilization to afford high-purity bulk pharmaceutical raw material with purity ≥98.5% and total yield between 52% and 60%.

This method features minimal side reactions and straightforward purification, complying with manufacturing specifications for pharmacopoeia-grade active pharmaceutical ingredients. In recent years, optimized eco-friendly procedures adopting fluorine-free cleavage reagents have drastically cut waste-water pollution and become the preferred production technology for pharmaceutical manufacturers worldwide.

In 1905, British physiologist Edkins put forward the hypothesis that the antral ventricular mucosa secretes gastrin, a humoral factor that modulates stomach acid output via blood circulation.

Restricted by insufficient purification technologies, this hypothesis remained controversial and unproven for nearly half a century. In 1926, Chinese physiologist Lin Kesheng validated the existence of gastrointestinal endocrine substances via animal experiments and supplemented the physiological framework of the gastrin hypothesis, yet native monomeric gastrin was not isolated.

It was not until 1959 that Gregory and Tracy from the University of Liverpool, UK, purified natural heptadecapeptide gastrin G17 from porcine antral mucosa using countercurrent distribution and Sephadex gel chromatography. Their verification of its acid-stimulating activity laid solid chemical and physiological foundations for gastrin research.

In 1964, the same research team finished full amino acid sequencing of G17. Segmental synthesis and screening confirmed that the C-terminal tetrapeptide Trp-Met-Asp-Phe-NH₂ constitutes the pharmacophoric core, fully retaining native gastrin's receptor-binding capacity and stomach acid-stimulating activity, whereas the long N-terminal amino acid chain only regulates in-vivo metabolism with no essential pharmacological function.

Against the backdrop of urgent clinical demand for standardized diagnostic reagents for stomach acid function and the high cost plus limited supply of extracted natural heptadecapeptide, ICI Pharmaceuticals collaborated with physiological researchers to launch a development project.

By conjugating Boc-protected β-alanine to the amino terminus of the active tetrapeptide, a more stable pentapeptide derivative was constructed to resist rapid enzymatic hydrolysis in vivo.

Full chemical synthesis of pentagastrin was accomplished by Konturek's research group in 1967, with in-vitro and animal assays confirming its acid-secretagogue potency equivalent to native gastrin. The agent obtained marketing approval in Europe the same year and has since become the gold-standard reagent for stomach acid output testing.

Subsequent approvals were granted in the United States, Japan and China for gastrinoma screening and parietal cell function assessment. Starting from the 1990s, its application expanded into fundamental research as a pharmacological tool compound targeting the CCK-B receptor, and it remains a classic reference compound in digestive pharmacology to date.