Practical Protein Chemistry - A. Darbre 1989

Latest methods for solid-phase and liquid-phase amino acid sequence determination
Solid-Phase Analysis: The Latest Approaches
Attachment of peptides to supports and "blocking" of the remaining reactive sites of the support

The SP approach generates its own challenges stemming from the need to anchor the peptide to a solid support (typically via covalent bonds). Although Peptides can also be bound through adsorption forces, such immobilization is unreliable because liquid-phase elution, occurring at least once in every amino acid Cleavage cycle, can lead to peptide leaching and loss. Reliable adsorption requires a careful Selection of well-matched peptide samples and adsorbing surfaces. Multiple and extremely strong adsorption bonds hinder solvation and reduce peptide mobility, thereby impairing reaction kinetics (see Section 16.2.2 for a Structure/133.html">Discussion of Supports). Taking these potential drawbacks into account, a support consisting of a Polybrene-coated Glass-fiber disc was developed and successfully used in a miniaturized Sequencer [42]. The test sample is adsorbed onto the disc (Chapter 17). This type of support has been reported as successfully used in structural studies of A wide variety of Peptides and Proteins (determining sequences comprising >30 residues).

Peptide loss is minimized due to their sorption onto a large support surface area and The Use of the most polar Reagents (trimethylamine and TFA) in the gas phase.

Covalent attachment presents numerous complications. Furthermore, there is no satisfactory general method for attaching peptides exclusively via their C-terminal residues.

In special cases, several well-established variants of attachment via the α-carboxyl group, which are widely used in practice, can be employed. These specific attachment techniques, as well as several general Condensation Methods not involving terminal Amino Acids, have been discussed in [64, 106] and in this book (Chapter 12).

Direct attachment of peptides to the support proceeds via a bimolecular mechanism. When condensing very small quantities of a peptide, a high reaction yield is only possible at a very high effective concentration of the support's nucleophilic groups (typically amino groups). Since the latter are present in a large molecular excess, a pseudo-first-order reaction is kinetically feasible. However, the concentration of the peptide's amino groups is low, and therefore their protection during attachment is unnecessary (except in special cases [64]). Amino groups should be blocked when the peptide is pre-activated prior to The addition of the support. Single-step attachments rarely require protection of N-terminal groups. If such protection is indeed necessary, a 2-tert-butyloxycarbonyloxyimino-2-phenylacetonitrile reagent [1] is used to introduce the 2-tert-butyloxycarbonyl (Boc) group [1]. Similar amino group protection is also required prior to carboxyl Group activation with a carbodiimide during C-terminal carboxyl condensation [85], since subsequent incubation under alkaline conditions converts the carboxyl Functions of amino acid side chains into stable N-acylureas, while a fraction of the activated C-terminal amino acids is converted into reactive oxazolinones.

Under standard conditions, carbodiimide Activation of a peptide leads to its condensation with the support through the peptide's side-chain carboxyl groups. During subsequent Sequence Determination, gaps may appear in the sequence at positions where carboxyl-containing amino acid residues are quantitatively attached to the support via their side-chain carboxyl groups. If these residues are not the terminal ones in the chain and are followed by other attachment points to the support, Peptide Structure determination can still proceed. Mild condensation conditions preserve the free side-chain carboxyl groups of certain internal Asp or Glu residues. There are numerous Examples of peptide structure analyses where it was possible to identify Asp (Glu) residues located within the peptide molecule that possessed free side-chain carboxyl groups. For instance, the Amino Acid Sequence of A large number of such short peptides was determined by the SP method following their attachment to aminopolystyrene at pH 5.0 using a Water-soluble carbodiimide [67]. This technique is based on the work of [109] with some modifications. Thus, The problem of side-chain attachment can generally be solved thermodynamically and/or statistically by carefully controlling the yield at the peptide condensation stage.

An improved variant of protein attachment to porous glass beads activated with DITC has been described [22].

The same paper proposes the immobilization of large peptides via His and Tyr residues on diazotized arylamino supports or via Cys residues on iodoacetamidated glass beads. Methods have also been developed for attaching peptides or intermediates bearing an amino group to a support via an ester bond [14].

Carbodiimide reagents are sometimes used in combination with activating additives that promote ester bond formation (e.g., N-hydroxysuccinimide or 1-hydroxybenzotriazole) [1, 28, 64]. Rapid formation of active esters prevents the appearance of inactive N-acylureas. To ensure the required reaction rate, carbodiimides and activating additives must be present in large excess, but in doing so, they may react with each other to yield undesirable by-products, as observed in the joint use of dicyclohexylcarbodiimide (DCC) [38, 69] and N-hydroxysuccinimide [55].

Recently, a new activator—N-hydroxy-5-norbornene-2,3-dicarboximide—has come into use in Peptide Synthesis, proving to be exceptionally stable in the presence of dicyclohexylcarbodiimide (DCC) [37]. This activator can be successfully employed in SP attachments; additionally, it is soluble in both water and organic Solvents.

A simple and convenient method for activating the peptide carboxyl group prior to immobilization has recently been proposed. Dissolving and keeping the peptide in a mixture of TFA and TFA anhydride apparently generates a mixed anhydride. The volatile compounds are then removed. The activated peptide is dissolved in DMF, and The amino acid is added. Pre-dissolving the sample in TFA facilitates subsequent dissolution in DMF [70]. The yields for the attachment step of small- to medium-sized peptides range from 55% to 98%; high yields indicate that the trifluoroacetyl group is a good leaving group [66]. Treatment of Gln-containing peptides may result in partial deamidation of the latter, while for Asp-containing peptides, cyclic imide formation is possible. These problems can be mitigated by finding a way to activate only a fraction of the carboxyl groups. It may subsequently prove possible to achieve selective activation of C-terminal residues with The formation of intermediate oxazolinones.

To improve the DITC attachment method for lysyl-containing tryptic peptides, a heterobifunctional reagent, p-isothiocyanatobenzoyl-D,L-homoserine lactone, has been proposed [40, 101]. First, the amino groups of the peptide react with the N=C=S groups of the reagent. Treatment of the resulting compound with TFA leads to lactone formation and yields an amino-acid-truncated peptide with a free N-terminal amino group. The peptide is then condensed with an amino-group-bearing support, during which the lactone remaining attached to the Lys side chain reacts with the support's amino groups. Activating the peptide as a lactone-fragment-containing compound has proven highly effective in increasing the yield at the peptide attachment stage [45]. The large excess of reagent typically required to prevent peptide cross-linking is unnecessary, as the two Functional groups of the reagent differ markedly in reactivity.

Peptides can be attached to the support in two steps: first, by "anchoring" it to the support via a temporary bond using a highly efficient, well-established method, followed by a rigid attachment via the desired bond through an intramolecular reaction. For example, Lys-free peptides were first attached to the support via their α-amino group using DITC, and then via their carboxyl group using the generally less efficient carbodiimide condensation [90]. Preliminary temporary attachment sharply increased the effective concentration of the peptide on the support surface, thereby raising the yield of the second attachment. Subsequent TFA treatment causes Cleavage of the N-terminal amino acid; the resulting shortened peptide is ready for sequence determination. A truly intramolecular attachment mechanism was utilized in [105], employing the previously proposed four-component condensation method [104]. Both of these approaches represent some of the most sophisticated and promising conceptual developments.

The nucleophilic groups of the support can also participate in intramolecular "assistance" of the attachment reaction. This may explain the enhanced reactivity of 1,2-diamines compared to monoamines [45]. For instance, matrices bearing ethylenediamine or triethylenetetramine (TETA) are more reactive toward homoserine lactone than ω-alkylamines. Therefore, when choosing a peptide condensation method, The Nature of the support's nucleophilic groups must be taken into account, selecting them so that attachment proceeds efficiently with the chosen carboxyl group activation method.

An excess of unreacted support amino groups can cause complications. If these groups remain unmodified after peptide attachment, they will be carbamoylated at the very first step of the Edman Degradation. According to our data, this leads to poorer cleavage results and a high Background level [64]. Using MITC instead of FITC in the first cleavage cycle significantly increases the yield of cleaved Amino Acids and lowers the background level. For successful micro-scale sequence determination, finding an even more efficient reagent for "capping" residual support amino groups is critical. At the same time, the reagent must not irreversibly block the N-terminal amino acid's amino group or alter the Structure of Amino acid side chains. If the peptide's N-terminal amino group is pre-protected with Boc or another easily removable group, an effective "capping" method is the Acetylation of excess reactive support amino groups using mild reagents, such as acetic acid esters. Following the attachment of Boc-peptides to macroporous polystyrene resins, we treat the support with methyl acetimidate (G. C. DuBois, V. Alvarez, E. Apella, unpublished data). The positively charged amidine groups appearing on the resin after capping presumably enhance support hydrophilicity and solvation. Overall, the capping Procedure helps improve the surface STRUCTURE OF THE support.

However, when planning and carrying out such surface treatment, one must anticipate and rule out the possibility of subsequent Side Reactions between the functional groups of the modifying agent and the attached peptide, which lead to decreased sequencing yields and an elevated background level.