Deletions, production

Limitations in product size were investigated by preparing several peptides of various lengths. Peptides containing 7, 9,13, and 19 amino acid residues were prepared on a synthesis robot employing Fmoc-protected amino acids and carbodiimide-hydroxybenzotriazol activation (0.25 M, 1 h) without detection of deletion products. To illustrate the remarkable economy of the ULTRA resin, 3.4 mg of the starting resin 15 sufficed for the synthesis of 42 mg resin with fully protected tridecapeptide. The raw product was obtained by ethereal trituration in excellent purity and yield (90% purity in the raw product, 78% yield, 13.1 mg after preparative HPLC). 

In order to facilitate the generation of peptide thioesters, several groupst have developed generalized versions of the thioester linker pioneered by Hojo and Aimoto.f A 3-sulfanylpropanoic acid residue is generated on an acid-labile linker such as Boc-Leu-PAM-resin or Boc-Leu-MBHA-resin. It is important to have a one-residue spacer between the MBHA linker and the thiol for optimal acid stability of the amide bond.f All twenty Boc-protected amino acids can be coupled to this thiol on the solid support to generate the thioester.Despite the potential reactivity of the thioester to the amino terminus, the formation of a dioxopiperazine is not generally observed when using in situ neutralization protocols. However, when the sequence was Leu-Tyr-Arg-Ala-Pro, 20% of a dipeptide deletion product, Leu-Tyr-Arg, was observed. It is likely that sequences such as C-terminal Pro-Gly would also be subject to this side reaction.t ... 

Fmoc removal from PNA monomers is more rapid than from amino acids, presumably because of reduced hindrance in the vicinity of the amine. The PNA synthesis protocol on the Expedite 8909 (Table 2) employs a deprotection time of 2 min to ensure complete iV-Fmoc removal of PNA monomers and allow efficient incorporation of amino acids into the sequence. Deblocking the synthesis handles Fmoc-PAL and Fmoc-XAL is also slow compared with PNA monomers. Longer initial deprotection of universal supports is incorporated into the synthesis protocols of the Expedite 8909 to avoid C-terminal deletion products. 

The 20-mer peptide prepared also contained an Asp-Gly sequence, this potentially leading to aspartamide formation, another well documented potential side reaction when performing peptide synthesis. This sequence-dependent side reaction occurs during the Fmoc deprotection step of Asp-containing peptides where the adjacent residue is Gly, Asn, Ser, or Ala. Each subsequent deprotection after the inclusion of Asp will result in an iterative decrease in the peptide purity. In the initial synthesis of the test peptide, deprotection with 20% piperidine at 80 °C led to considerable amounts of aspartimide formation. Addition of 0.1 M HOBt to the deprotection mixture together with piperidine reduced the aspartimide formation and resulted in a 10% increase in crude purity. The use of piperazine in place of piperidine resulted in an even further decrease in the aspartimide formation, and no deletion products were observed despite the decreased pKa of the deprotection reagent. 

FSCE can also show impurities present in solid-phase synthesis products that are not clearly evident by conventional purification methods. RP-HPLC with ultra violet (UV) detection is traditionally used to analyze solid-phase synthesis products such as peptoids and peptides as well as to separate them preparatively away from any deletion products. Since each synthetic monomer addition cycle is less than 100% efficientj products that are one or two monomer units shorter than the desired product must be purified out to ensure monodispersity of the final polypeptide or polypeptoid. The separating power of FSCE was compared with analytical RP-HPLC traces after RP-HPLC purification (Figure 12.7). The one peak in the RP-HPLC trace actually separates into one large peak... 

Clearly, the complex Horn Km, Kn) can be viewed as a deleted product of m copies of n — l)-dimensional simplices. In this context, the special case m = 2, which is dealt with in Proposition 19.8, is well known. 

Fig. 2. HPLC and MALDI-MS analysis of a crude synthesis product. (A) Analytical HPLC trace of crude Im-Py-Py-Py-y-Py-Py-Py-Py-p-Dp after cleavage from resin and ether precipitation. Immediately prior to preparative HPLC, 25 iL of the sample was subjected to analytical HPLC with a linear gradient of 20-50% Buffer B over 30 min (detection at 254 nm). Peaks labeled A-E correspond to the masses observed in the MALDI spectrum using CHCA as matrix (B). A, unknown impurity of -1-12 amu as well as oxidation products B, double Py deletion product C, single Py deletion D, full length polyamide and its sodium adduct E, tert-huty adducts of B, C, and D. Note that the t-butyl adducts elute in a very broad band on the analytical HPLC, and are often overlooked as insignificant impurities.

A number of different types of effervescent granules were once official in the B.P.C. but although most of these have been deleted, products of this type are still very popular. The ingredients of principal therapeutic value are mixed with sodium bicarbonate and both tartaric and citric acids sometimes sucrose is added. 

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