Practical Protein Chemistry - A. Darbre 1989

The latest methods of solid-phase and liquid-phase amino acid sequence determination
Solid-Phase Analysis. The Latest Approaches
Supports

Solid-phase determinations are carried out almost exclusively on Peptides covalently linked to modified matrices of one of three types:

1) polystyrene (1% cross-linked with divinylbenzene);

2) porous Glass;

3) polyacrylamide derivatives.

The preparation Methods and properties of these supports have been described [70].

An ideal matrix intended for peptide immobilization should possess the following properties:

1) low flow resistance in the Column, ensured by both high mechanical strength of the particles and minimal volume change of the support throughout the Cleavage cycle;

2) chemical stability;

3) a high specific surface area readily accessible to the surrounding liquid;

4) sufficient surface chemical capacity of the sorbent particles for efficient peptide attachment;

5) kinetic conditions of the peptidyl-support reactions should closely approach The kinetics of Reactions in Solution.

None of the currently known supports satisfies all the aforementioned criteria simultaneously. Low-cross-linked polystyrene and polyacrylamide must be thoroughly pre-swelled in a solvent so that the Functional groups of the support or the peptide attached to it are accessible to Reagents. Therefore, the choice of Solvents is crucial when working with such sorbents. Solvent changes often cause “shrinkage” or Swelling of these gel-like polymers, leading to channeling or column “clogging”. To prevent these phenomena, the peptidyl-polymer particles are thoroughly mixed with much larger volumes of small glass beads. However, increasing the total volume of the reaction mass requires higher consumption of reagents and washing solvents, and leads to the sorption of reagents and reaction by-products. Consequently, the Background level in the analyzed samples increases, which can become a serious obstacle for Microscale analysis.

In contrast, porous glasses have a rigid Structure and do not present these problems. Therefore, a wide range of solvents can be used with them without the risk of generating high pressure in the column (provided that mechanical damage to the fragile particles is avoided during peptide attachment, since grinding large particles into smaller ones results in high backpressures during subsequent column operation).

Unfortunately, glass is soluble to some extent in alkaline media, so solutions with pH>8.5:9.0 should be avoided.

Despite the drawbacks inherent in Glass-Based Supports, their use enables successful Sequence Determination.

Low-cross-linked polystyrene is an unsuitable matrix for attaching Proteins and very large peptides. The frequent incompatibility between solvents and peptides (or the matrix) leads to structural Changes in the peptidyl-support that are unfavorable for reagent access to N-terminal amino groups. To solve this problem, a matrix based on polyacrylic acid amidated with sulfonated p-phenylenediamine was used [20]. The isothiocyanate groups obtained during further polymer modification are used for protein attachment, while adjacent sulfonate ions create the local hydrophilic environment necessary for solvent compatibility.

All commonly used supports contain structural elements that contribute to the chemical background; therefore, chemical Modification of the support is required to lower the background level. Since stepwise yields rarely exceed 95%, these structures obviously make a significant contribution to non-ideal reaction kinetics.

Further progress in developing supports depends on a deeper understanding of The Nature of Chemical Reactions in multiphase systems. We must abandon the notion that a solid support is simply an inert “wall” to which a peptide is anchored. Research in solid-phase Peptide Synthesis currently relies on METABOLISM/2.html">THE CONCEPT OF a dynamic support-peptide-solvent system governed by various kinetic and thermodynamic rules [35]. Non-ideal reaction kinetics depend not so much on slowed diffusion or mass transport into the bead as on poor solvation of the peptide and the support matrix [39]. Solvation is likely significantly affected by local phase Separation. Within a specific microenvironment, the peptide and the matrix may interact so strongly that this interaction becomes equivalent to precipitation, removing the substance from the reaction.

In other cases, liquid-liquid reagent partitioning occurs. For this reason, the reagent concentration in the microenvironment of the N-terminal part of the peptide is particularly low. This problem is easily avoided by using a single-solvent system [80]. Local redistributions occurring via any of the aforementioned mechanisms arise during sequential cleavage and cause a temporary or permanent decrease in the reactivity of individual chains. This drawback is exacerbated by the presence of inherent inhomogeneities in the matrix structure (uneven cross-link distribution, etc.). Chemical modification of porous glass results in a surface covered with a thin film of cross-linked siloxane polymer rather than a chain attached to the sorbent at a single point. Therefore, glass-based supports must exhibit the same local reaction “barriers” as polymeric gel structures.

There are several reports on the preparation, properties, and use in solid-phase peptide synthesis of acrylic copolymers consisting mainly of poly-N,N-dimethylacrylamide [23, 95, 96] or poly-N-acryylpyrrolidone [93, 97]. These polymers are compatible with a much larger number of polar and moderately polar solvents (and apparently also with attached peptides) than polyacrylamide or polystyrene supports. Therefore, it is tempting to use these polymers as supports for peptide TF analysis. When choosing a support, we focus primarily on the rigid matrix design, which avoids major complications associated with sorbent swelling and column blocking. To allow the PITC coupling reaction to be carried out in a strongly alkaline medium, we chose macroporous polystyrene instead of glass supports. An additional advantage of polystyrene is that multiple chemical modification reactions can be performed on it stably and reproducibly. It turned out that this rigid, highly cross-linked matrix nevertheless exhibits some flexibility at THE MOLECULAR LEVEL and provides a wide range of microenvironments (including unfavorable ones) within the Hydrophobic surface structures.

Our first goal was to introduce “spacers” (“legs”) protruding from the surface with functional amino groups necessary for subsequent peptide attachment. If the spacers are polymers of the required size and composition, they can also act as co-solvents and facilitate The formation of a matrix-separated phase in which the peptide and the solvents used are compatible. In principle, conditions can be found to ensure both the necessary degree of solvation of all peptide chains and the required degree of interaction between the latter and reagents. Polyethylene glycol was chosen as the spatially modifying polymer, having been found empirically as a co-solvent in TF analysis [87, 88]. Peptide synthesis has been successfully carried out with sequential elongation of the peptide chain attached to the ends of polyethylene glycol chains [79]. In this case, the coupling kinetics are similar to those observed in free solution [9].

Macroporous polystyrene derivatives were synthesized According to the Procedure detailed in [51]. Preliminary sequence analysis results have been reported [1].

These supports were found to be well permeable to solvents and to exhibit a low level of impurity background. However, during subsequent comparative Edman Degradation of peptides attached to short spacers and the same peptides “anchored” to small polyethylene glycol chains (containing up to 13 oxyethylene units), no significant differences were observed in either initial or stepwise yields of amino acid ATZ derivatives. We concluded that in this case, to create a co-solvation effect or prevent strong polystyrene-peptide interactions, either an increase in the sample weight or an increase in the Molecular Weight of the modified polymer is necessary.

In Conclusion, it should be noted that a probable ideal support would be a graft copolymer in which one component serves as a rigid framework supporting The structure of a second copolymer to which the peptide is covalently attached. Due to complexities caused by its surface nature, a polymer with a rigid framework would by itself be an unreliable “anchor” for peptide Condensation. The second component should be chosen taking into account its compatibility with the various solvents and reagents used both for peptide attachment and for determining the STRUCTURE OF THE latter. Due to its solvent compatibility and accessible internal structure, this solvated polymer by itself would be mechanically unsuitable for column use. To summarize the above briefly, the aforementioned macroporous system with a rigid framework is a pellicular construct whose successful application depends on the proper choice of the grafted solvated polymer.