Fmoc Solid-phase Method Research Articles

Direct preparation of peptide thioesters using an Fmoc solid-phase method

Aiming at the direct preparation of peptide thioesters by an Fmoc solid-phase method, we searched a new deblocking reagent, which efficiently removed Fmoc groups while keeping the thioester intact. The deblocking reagent, which contains 1-methylpyrrolidine, hexamethyleneimine and HOBt in a one to one mixture of NMP and DMSO, realized the preparation of peptide thioesters by an Fmoc solid-phase method in a yield equivalent to that obtained by a Boc solid-phase method.

Chemical synthesis and characterization of the sweet protein mabinlin II.

The sweet protein mabinlin II isolated from the seeds of Capparis masaikai consists of the A chain with 33 amino acid residues and the B chain composed of 72 residues. The B chain contains two intramolecular disulfide bonds and is connected to the A chain through two intermolecular disulfide bridges. The A chain was synthesized by the stepwise fluoren-9-ylmethoxycarbonyl (Fmoc) solid-phase method in a yield of 5.9%, while the B chain was synthesized by a combination of the stepwise Fmoc solid-phase method and fragment condensation in a yield of 6.0%. Disulfide formation and combination of the A and B chains followed by purification by ion-exchange high-performance liquid chromatography (HPLC) gave mabinlin II in a yield of 47.4%. The characterization of the synthetic mabinlin II by HPLC, electrospray ionization mass spectrometry, amino acid analysis, and disulfide bond determination fully supported the expected structure. A 0.1% solution of the synthetic mabinlin II had an astringent-sweet taste.

固相合成による甘味タンパク質モネリンの構造活性相関

Monellin, an intensely sweet protein, was isolated from the fruit of the West African plant, Dioscoreophyllum cumminsii Monellin consists of two noncovalently associated polypeptide chains, the A chain with 44 amino acid residues and the B chain with 50 residues. The tertiary structure has been determined, and the native conformation is essential for the sweet taste. Recently, the primary structure of monellin was unambiguously determined, and monellin was synthesized by the Fmoc solid-phase method. In an attempt to delineate the active site(s) of monellin, amino acid residues within potential active sites were replaced. Replacement of CysB41 by Ser led to a small change in sweetness. Replacement of IleB6, AspB7 or IleB8 by different amino acids resulted in a complete or marked loss of sweetness. Replacement of AspA16 or TyrA13 by other amino acids did not reduce the substantial sweetness. Replacement of each Lys residue by L-2-aminohexanoic acid (L-norleucine) significantly diminished the sweetness potency, but did not completely remove the sweetness. Analysis of the synthetic analogs by circular dichroism (CD) showed that the major structural features of monellin were not significantly altered. These findings suggest that IleB6, AspB7 and IleB8 are responsible for eliciting a sweet taste, and that one or more basic residues may also be important for eliciting a sweet taste. In the course of the investigation, the D-enantiomer of monellin was synthesized by the Fmoc solid-phase method, and crystallized by the hanging drop vapor diffusion method. The D-isomer was shown by CD to be the mirror image of the synthetic L-monellin, and was devoid of any sweetness (essentially tasteless). DL-Monellin was crystallized from a 1 : 1 mixture of the synthetic L- and D-monellin, and its CD spectrum did not show any absorption. DL-Monellin was 2000 times sweeter than sucrose on a weight basis (cf., monellin is 4000 times sweeter than sucrose) These results indicate that the crystals consist of the racemates of DL-monellin.

FMOC solid phase synthesis of an endothelin converting enzyme substrate. Use of allyl ester as the third orthogonal protecting group.

The synthesis of a substrate for the endothelin converting enzyme using the continuous flow FMOC solid phase method has been accomplished. The allyl group, employed to protect Glu side chain, was cleanly removed during the synthesis in the presence of tert.-butyl based protecting groups to enable the introduction of the Edans moiety. The substrate was obtained in 31% yield after reverse phase hplc purification.

Solid-phase synthesis of crystalline monellin, a sweet protein.

The sweet protein, monellin, consists of two noncovalently associated polypeptide chains, the A chain of 44 amino acid residues and the B chain of 50 residues. The B chain was synthesized by the stepwise Fmoc solid-phase method in an overall yield of 6.2%. The synthetic B chain was combined with the synthetic A chain, which was left over from a previous work, to give monellin in a yield of 25.7%. The synthetic monellin was approximately 4000 times as sweet as sucrose, while the previously synthesized [Asn49, Glu50] B-chain monellin was also approximately 4000 times as sweet as sucrose. Exchanging Glu49 and Asn50 in the B chain did not affect the sweetness potency. Crystallization was performed by a vapor diffusion method.

Solid-Phase Synthesis of Crystalline (Ser41) B-Chain Monellin, an Analogue of the Sweet Protein Monellin.

The sweet protein monellin consists of two noncovalently associated polypeptide chains, the A chain of 44 amino acid residues and the B chain of 50 residues. Since blocking the free SH group of Cys41 in the B chain or treating the adjacent Met42 with CNBr removed its sweetness, this part of the molecule has been suggested to be essential for the sweetness. The [Ser41] B chain, an analogue of the B chain, was synthesized by the stepwise Fmoc solid-phase method in an overall yield of 1.9%. The synthetic B chain analogue was combined with the A chain, which was left over from a previous work, to give [Ser41] B-chain monellin in a yield of 31.0%. This synthetic monellin analogue was 2000 times as sweet as sucrose. Changing the Cys41 residue to the Ser residue significantly decreased the sweetness potency and stability of the molecule in solution. Crystallization was carried out by a vapor diffusion method.