In-situ neutralization

Electron-Induced Reactions—HREELS Measurements. Novel LEE-induced chemistry has also been observed in HREEL measurements of molecular solids and molecules physisorbed on the surface of RGS. For example, Lepage et ah, building on the initial observations of Jay-Gerin et al. [141], have employed HREELS to measure in situ, neutral dissociation products arising from the impact of low-energy electrons on thin multilayer films of methanol [37] and acetone [38]. The technique is similar to that developed earlier by Martel et al. [258] for chemisorbed systems, in that the same electron beam is used for both the production and the detection of the neutral fragments. However, in the work of Lepage... 

Approximately 100 mg of resin was distributed to each of the reaetion bloek wells (of an ACT block or a Bohdan block) by pipetting a slurry of the resin in DMF/DCM (3 1) or as dry resin into each IRORI kan. The peptides were then assembled by the combinatorial chemistry apparatus suited for parallel or split-and pool-synthesis (34) using in situ neutralization/HBTU activation protocols for BOC chemistry. The resin was initially washed with DCM and the BOC protecting group removed by washing twice with a 40% solution of TFA in DCM. 

Almost all of these conclusions are applicable in the case of halophosphonium reagents, including the one-pot reaction, in situ neutralization and the influence of the amount of base on the reaction yield.P However, a case of BroP-mediated TFA activation has been reported (see footnote 65 in refy ). 

The following experimental procedures describe the solid-phase synthesis of peptides by means of Fmoc chemistry and TBTU activation (Scheme 8), as well as by HBTU-mediated coupling of Boc amino acid derivatives and in situ neutralization (Scheme 9). Segment condensations on solid phase with C-ternoinal glycine or proline residues can be easily achieved, even on a multigram scale (Scheme 10). Segment condensations involving the activation of other amino acids require the careful optimization of reaction conditions in order to suppress extensive epimerization (see Scheme 11 and Section 3.8.1.2). 

Scheme 9 SPPS of ACP (65-74) by the HBTU In Situ Neutralization Protocolb l...

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 ... 

The use of phenacyl handles 15 is only compatible with the Boc/Bzl strategy since the peptide-resin anchor is not stable to the nucleophilic conditions used in the Fmoc/tBu strategy. Besides the formation of piperazine-2,5-diones, another side reaction can take place on incorporation of the second residue. Attack by the free amino group of the first amino acid on the carbonyl group of the phenacyl resin leads to a cyclic Schiff base.P Both types of side reaction can be minimized using the in situ neutralization protocol. Photolysis is carried out as for nitrobenzyl resins. 

Where convenient, the reaction can be avoided in Fmoc chemistry by using a suitable orthogonally stable ester and employing a Boc amino acid at the second residue. Coupling the third amino acid as the Fmoc derivative with in situ neutralization of the salt formed during Boc removal will avoid the reaction completely. Clearly this type of approach is not suitable if the second amino acid requires TFA-labile side-chain protection for the duration of the synthesis. 

The in situ neutralization of gastric juice certainly prevents intragastric proteolysis and permits studying the macromolecular components of gastric secretion without demonstrable peptic degradation, as they are secreted into the gastric lumen. However, the basic shortcomings of this method are as follows ... 

From the information just cited, it appears that serum proteins are normal constituents of gastric juice and can be demonstrated even in acid secretion if proteolysis is prevented by in situ neutralization of gastric juice. 

Gullberg used the intragastric in situ neutralization of gastric juice technique to study B12 binders of the gastric juice from normals and patients with pernicious anemia by paper electrophoresis (G34). Phos-... 

This results in more basic pH and thus more negative mobility. Usually the pH is measured before but not during or after the electrokinetic measurements. The electrolysis products can be in situ neutralized using buffer solutions rather than strong 1-1 electrolytes. However, such solutions often contain strongly adsorbing ions that shift the lEP. 

P. Alewood, D. Alewood, L. Miranda, et al. Rapid in situ neutralization protocols for t-Boc and Fmoc solid-phase chemistries. Methods in Enzy-mology 289, 14 (1997). 

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