Large-scale production of peptides using the solid-phase continuous flow method. Preparative synthesis of the novel tachykinin antagonist MEN 10627

Abstract

The large-scale solid-phase continuous flow synthesis of the bicyclic peptide MEN 10627, a new potent Neurokinin A receptor antagonist, is described using the Fmoc-polyamide method on both macrosorb 125 and Macrosorb 250 resin. A new synthesizer designed in-house was realized by assembling Whitey valves and Waters pump in order to allow small-scale (0.0001 mol; 1 x 10 cm Omnifit columns) synthetic studies which were strongly predictive of the conditions required for large-scale (0.01-0.10 mol; 3.6 or 5.9 x 46 cm Büchi columns) production, performed on the same apparatus.

Large-scale production of peptides using the solid phase continuous flow method. Part 2: preparative synthesis of a 26-mer peptide thrombin inhibitor

“…In fact, with this resin loading, a theoretical weight of pepide/weight of starting resin ratio (tP w /R w ) of 0.295 could be obtained at the end of the synthesis of I against a value of 0.146 calculated for MEN 10627 (M.W. =760) synthesized with the described 0.192 mmoles/g of first amino acid functionalization [3]. This 0.295 (tP w /R w ) corresponds to 36.5% of resin weight increase during the synthesis, calculating also for the protective groups.…”

“…In the case of a large peptide like I (M.W. =2957 as medium average mass) an esterification of the first amino acid on the resin of 0.1 mmoles/g does not represent a poor volumetric characteristic of the solid support such as in the case of small peptides like MEN 10627 [3]. In fact, with this resin loading, a theoretical weight of pepide/weight of starting resin ratio (tP w /R w ) of 0.295 could be obtained at the end of the synthesis of I against a value of 0.146 calculated for MEN 10627 (M.W.…”

“…A new resin package, incorporating the volume expansion, is successively restored by normal-flow. In longer resin package of about 40 cm, which we previously employed [1,3], the breakage of the packed resin bed was more difficult. For this reason we chose 23 cm long Bü chi columns packed, at the start of the synthesis, with a resin bed of 15 -16 cm.…”

“…The dead volume of these packed columns was exactly regulated with suitable pistons that were adjusted after each coupling cycle during the synthesis. These packed columns were applied at our already described synthesiser [3] and used as usual.…”

“…Small peptides like the 163-171 fragment of Interleukin-1i [1], the Leu 5 -enkephalin (pentapeptide), the human angiotensin I (decapeptide) [2], and the double-cyclized MEN 10627 (hexapeptide) [3] have been successfully produced in preparative amounts by solid phase continuous flow methodology.…”