Ultrasonic-Assisted Solid-Phase Peptide Synthesis of DOTA-TATE and DOTA-linker-TATE Derivatives as a Simple and Low-Cost Method for the Facile Synthesis of Chelator–Peptide Conjugates

Abstract

Peptides have been widely adopted as biological targeting vectors for applications in molecular imaging and peptide-receptor radionuclide therapy (PRRT). Somatostatin (SST) analogues such as octreotate (TATE) are exogenous ligands for somatostatin receptors (SSTRs), which are highly expressed on neuroendocrine tumors (NETs). Recently, both [68Ga]­Ga-DOTA-TATE (NETSPOT) and [177Lu]­Lu-DOTA-TATE (LUTATHERA) received U.S. Food and Drug Administration approval for positron emission tomography (PET) imaging and PRRT of NETs, respectively. However, to the best of our knowledge a well-described synthesis of DOTA-TATE has not been reported in the literature. Herein, we report a fully reoptimized DOTA-TATE synthesis, including the application of a simple ultrasonic bath to greatly improve yields, reduce coupling times, and decrease the amount of reagents required for each coupling step by a half. The most prevalently used cyclizing agents such as iodine, thallium­(III) trifluoroacetate, hydrogen peroxide, and dimethyl sulfoxide were compared. On-resin cyclizations using mechanical agitation showed higher yields (23% and 25% using I2 and Tl­(III), respectively) than off-resin (1.3% and 11% using DMSO and H2O2, respectively), and the total synthesis time of DOTA-TATE was ∼540 min excluding the cyclization step, with a total synthesis yield of ∼23%. The same manual SPPS methods/reagents were reoptimized with ultrasonic (US) agitation, resulting in an immense reduction in the total synthesis time by ∼8-fold to ∼70 min for DOTA-TATE with a higher yield (∼29% yield), and ∼13-fold to 105 min for DOTA-PEG4-TATE (∼29% yield). Also, the use of US agitation reduces the need for excess molar equivalents of the reagents to a half, which is particularly important when coupling expensive or custom-synthesized groups such as bifunctional chelators and linkers. Finally, the synthesized DOTA-TATE was successfully radiolabeled with [68Ga]­Ga3+ (t 1/2 = 68 min) with high radiochemical yields (30 min, 95 °C). We believe this work opens the door to the facile and low-cost synthesis of many new chelator-linker-peptide conjugates that were previously cumbersome or cost-prohibitive to produce with manual SPPS.

Ultrasonication Improves Solid Phase Synthesis of Peptides Specific for Fibroblast Growth Factor Receptor and for the Protein-Protein Interface RANK-TRAF6

“…Additionally, the same authors reported considerable time reduction in amide bond formation. Similar relevant results on the application of ultrasonication in peptide synthesis have been reported by other authors [ 20 , 21 ]. Remarkably, sonication does not cause the racemization of sensible residues as clearly demonstrated by Merlino et al and Wołczański et al and can be applied to assemble peptides bearing amino acid residues prone to racemization [ 19 , 20 ].…”

Toward Personalized Medicine: One Chelator for Imaging and Therapy with Lutetium-177 and Actinium-225

“…The Fmoc-TATE peptide was synthesized by an automated solid-phase peptide synthesizer using the standard HATU/DIPEA protocols . The peptide was cyclized in the synthesizer with iodine-mediated disulfide formation by addition of 5 equiv of iodine in DMF (0.25 M) for 1 h. , After washing with DMF twice, the Fmoc group was removed by 20% piperidine in DMF for 10 min (2 cycles). The Fmoc-protected mini PEG linker was added to the deprotected TATE-Wang resin via standard coupling procedures .…”

Toward the Development of GE11-Based Radioligands for Imaging of Epidermal Growth Factor Receptor-Positive Tumors

“…For the coupling of the activated building blocks, two different approaches were followed for some of the compounds: the standard mechanical agitation-aided conjugation and the ultrasound-assisted conjugation using a conventional ultrasonic bath. In contrast to the frequently used microwave-assisted peptide synthesis, the application of ultrasound for more efficient coupling performance is still a rather new field. − Although all peptide syntheses were successful using both approaches, the ultrasound-assisted conjugation reactions required lower excesses of the activated building blocks (only 2 equiv instead of 4) and shorter reaction times to complete reactions (15 instead of 60 min for standard building blocks to be coupled), usually giving the products in considerably higher yields (1.5 to 2-fold).…”