Peptide solvent systems

It is worthwhile to present some examples of the types of results available from Monte Carlo simulations of peptide solvent systems. In Figure 6 we present the convergence of the energy of hydration of the lysozyme crystal over a million configurations. 

Fluorinated alcohols are not only interesting as model systems for weak hydrogen bonds with implications in the life sciences [254] and as chemical sensor materials [255], but also provide excellent reaction media [256, 257] and peptide solvents [258 260] with conformation-modulating properties. In both cases, molecular aggregates are thought to play an important role. One of the most widely used fluorinated alcohols is 2,2,2-trifluoroethanol, which will be in the focus of the following section. 

FIGURE 5.4 Schematic representation of a continous-flow system for the solid-phase synthesis of peptides. Solvent is forced through the system by a pump. The support is in the form of a column that is stationary. A reaction is monitored by measuring the change in absorbance of the solvent stream. 

H Kuroda, Y-N Chen, T Kimura, S Sakakibara. Powerful solvent systems useful for synthesis of sparingly-soluble peptides in solution. Ini J Pept Pmt Res 40, 294, 1992. 

Amino acids and peptides can be separated and their purity estimated rapidly and economically using TLC. In fact, until not long ago, TLC was to the peptide chemist what the hammer was to the carpenter, and extensive literature exists that describes all aspects of TLC including supports and solvent systems as well as the theory behind the separations. 4"14 TLC supports and solvent systems were developed since the beginning of this century to assess the purity of protected and unprotected amino acids and peptides reproducibly and quantitatively. 

Since phosphate and borate buffers are well described in the literature, the power of CZE in peptide chemistry will be illustrated with two examples that emphasize less conventional conditions than those offered by the manufacturers, such as using TEAP solvent systems or standard buffers in the presence of MeCN. These were utilized for some unusual separations. 

The use of nonpolar chemically bonded stationary phases with a polar mobile phase is referred to as reverse-phase HPLC. This technique separates sample components according to hydrophobicity. It is widely used for the separation of all types of biomolecules, including peptides, nucleotides, carbohydrates, and derivatives of amino acids. Typical solvent systems are water-methanol, water-acetonitrile, and water-tetrahydrofiiran mixtures. Figure 3.15 shows the results of protein separation on a silica-based reverse-phase column. 

Period 2 Part B—Work up the dansyl hydrolysate and spot on the TLC plate with standard dansyl amino acids. Part A. 1—Work up peptide hydrolysate and prepare FMOC derivatives of amino acids for analysis by HPLC or CE. Part A.2-If applicable, develop paper chromatogram in solvent system. 

Fig. 6 Reversed-phase chromatography of peptide fractions from curd slurries on a C, 8 (50 X 5 mm) column. Neutrase added (a) followed by starter peptidase (b). Elution was with the following solvent systems solvent A, 0.1% v/v TFA in H20 solvent B, 0.1% v/v TFA in methanol.

The reaction of diazomethane with a mixed anhydride is generally complete within 15 minutes. If the peptide contains Tyr the reaction should be worked up immediately to avoid methylation of the Tyr side-chain hydroxy group. In the assessment of purity of peptidyl diazomethyl ketones using HPLC, it should be noted that the peptidyl diazomethyl ketone, R1CHN2, is converted into RlCH COCFj if TFA is used in the HPLC solvent system. The peak seen in HPLC is the trifluoroacetate derivative, sometimes two peaks appear if the reaction is slowJ71... 

Although some investigators consider free 1-aminoalkylphosphonic adds as poor substrates for the synthesis of peptides with a C-terminal 1-aminoalkylphosphonic acid moiety, successful procedures are reported in the literature with yields higher than 80% J6 7 The competitive formation of a mixed anhydride between the 1-aminoalkylphosphonic acid and the acylating agent (e.g., N-protected active ester of an amino acid) followed by its fast hydrolysis could be minimized in some solvent systems. The free 1-aminoalkylphosphonic acids could also be N-protected and esterified to provide various esters suitable for the peptide synthesis. 

It may be determined whether known or new peptide alkaloids are present and whether or not the laborious isolation of pure constituents is warranted. The mass spectrum is particularly useful for purposes of identification because the physical properties are sometimes indefinite and often similar and nearly identical Iif numbers in several solvent systems are becoming very common. On the other hand, mass spectra are specific and only rarely require further chemical studies (adouetine-Y and frangufoline). 

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