**2.2 Solid-phase peptide synthesis**

Total chemical protein synthesis is an attractive alternative to biological protein production. Chemical peptide synthesis can be divided in: (I) liquid-phase peptide synthesis and (II) solid-phase peptide synthesis. Liquid-phase peptide synthesis is a classical approach to peptide synthesis and since the beginning of the 20th century this technique has developed considerably. Although liquid-phase peptide synthesis has some limitations due to its time consuming nature, solubility issues and the need for lengthy purification procedures, it is still useful for large-scale peptide production and for specialized laboratory applications [10].

Solid-phase peptide synthesis (SPPS) is currently the preferential technique to establish access to synthetic peptides. The general process for SPPS is based on sequential addition of α-amino and reactive side chain protected amino acids to a solid support (resin). The *C*-terminal residue is coupled to the resin and after removal of the *N<sup>α</sup>* -protecting group, the next *C*-terminally activated *N<sup>α</sup>* -protected amino acid is coupled and so on, until full chain assembly is reached. The most commonly used *N<sup>α</sup>* -protecting groups are the acid-labile Boc (*tert*-butyloxycarbonyl) group and the base-labile Fmoc (9-fluorenylmethoxycarbonyl) group. Side chain protecting groups are ideally orthogonal to the *N<sup>α</sup>* -protecting group, which means that they are removable under completely different reaction conditions.

The use of synthetic chemistry allows infinite variation of the polypeptide chain by for example incorporation of unnatural amino acids such as β-amino acids, *N*-methyl amino acids, peptoids, stable isotope-labeled amino acids, and D-amino acids. Furthermore, the use of orthogonal amino acid side chain protecting groups during the sequential elongation of the peptide chain in SPPS, allows conjugation of fluorescent tags, chelators, biotin, etc., at single specific sites. Current optimized SPPS chemistry protocols enable effective peptide synthesis of 30–50 amino acids. For the synthesis of peptides and proteins bigger than 30–50 amino acids, various chemical ligation techniques were developed that enable the formation of a peptide bond between two unprotected peptides resulting in larger synthetic proteins with a fully native peptide backbone [11, 12].

#### **2.3 Site-specific incorporation of chelators and/or fluorescent tags**

Functionalization of peptides and proteins still heavily relies on amine or thiol functionalities, present in proteins as lysine and cysteine side chains, respectively. New ligation techniques are emerging that are moving away from amines or use of protected thiols. The functionalization of lysine side chains can be achieved by reacting them with activated esters such as NHS-DTPA or –DOTA that are commercially available (**Figure 2**). The most appropriate derivatives of these chelators for conjugation to a peptide or protein are those which are *t*Bu (*tert*-butyl)-protected at all functional acid groups, except one. This acid group can either be activated *in situ* using a proper coupling agent or be obtained as a preactivated NHS ester in DOTA-tris(*t*Bu)ester NHS ester, NOTA-bis(*t*Bu) ester NHS ester, or DTPA-tetrakis(*t*Bu) ester NHS ester.

The main advantage of these activated ester chelators is their ease of use, while the main disadvantage of this technique is their unspecific labeling. A protein generally contains more than one lysine residue and thus more than one position for chelator conjugation. It is difficult to predict the site of coupling, which will

#### **Figure 2.**

*Conjugation at N<sup>ε</sup> -amine group of a lysine residue with an amine reactive NHS ester (N-hydroxysuccinimide ester) resulting in an amide bond.*

often lead to heterogenous labeling of the compound of interest. With the use of Boc SPPS this problem can be circumvented by using orthogonally ε-amino Fmocprotected lysine residues. Deprotection of the Fmoc group can be performed on resin and directly be followed by functionalization of the desired lysine with an NHS-activated label of choice. In case of Fmoc SPPS, orthogonally ε-amino allyloxycarbonyl (Alloc) protected lysine residues can be used.

Conjugation at cysteine residues can be realized by reactions with maleimide containing compounds or 2-azidoacrylate-derivatives [13]. Similar to the amine reactive NHS esters, commercial compounds with maleimides are widespread. Maleimide-DOTA or –DTPA are coupled to free cysteine-containing proteins (**Figure 3**). Although the reaction is specific and easy to use, maleimides have their disadvantages. The first being the availability of a free cysteine in a protein of interest; the major part of cysteines present in proteins are paired with a second cysteine to form a disulfide bridge. Moreover, these cysteines are often buried within the core of the protein making them inaccessible for maleimides. To overcome this problem an additional cysteine can be incorporated into the protein specifically for labeling. This will, however, lead to problems with oxidative folding of the protein and can lead to improperly folded proteins with loss of activity.

However, this does not mean that thiol reactive compounds cannot be useful in protein labeling. The introduction of an encrypted cysteine that can be deprotected after correct folding of the protein can offer a solution. Recently *N<sup>ε</sup>* -(thiazolidine carboxyl)-lysine was applied for this purpose [14], the thiazolidine carboxylic acid (Thz) that was originally designed to facilitate sequential one-pot native chemical ligation (NCL) [15–17], was used as a handle for late stage site-specific modification of proteins under mild conditions [14]. The encrypted cysteine was introduced on a lysine side chain which after opening under mild conditions with MeONH2 or NH2OH resulted in a free cysteine enabling reactions with maleimide groups (**Figure 4**).

Furthermore, it was shown that this technique is fully compatible with established techniques of peptide synthesis and NCL. The chemokine CCL5 was synthesized from an *N*- and *C*-terminal part that were joined by native chemical ligation. The *C*-terminus contained a lysine with an orthogonal Fmoc protective group. After completion of the synthesis of the *C*-terminus, the Fmoc group was removed and the thiazolidine residue was site-specifically introduced. After cleavage from resin and

*Strategies for Site-Specific Radiolabeling of Peptides and Proteins DOI: http://dx.doi.org/10.5772/intechopen.99422*

#### **Figure 4.**

*Deprotection of thiazolidinecarboxyl coupled to N<sup>ε</sup> -amine group of a lysine residue using methoxylamine.*

#### **Figure 5.**

*Schematic representation of the synthesis of chemokine CCL5. In the first step two unprotected peptide fragments are ligated using native chemical ligation. Subsequently, the peptide is folded into an active protein. In the last step the thiazolidine ring is opened while simultaneously the newly formed thiol moiety is modified with a maleimide.*

#### **Figure 6.**

*Reaction of an aminooxy with a ketone which results in an oxime bond.*

subsequent purification, the two parts were ligated to obtain the full length protein and subsequently the protein was folded under oxidative conditions. After obtaining the folded protein, the thiazolidine ring was opened by treatment with MeONH2.

Upon treatment with MeONH2 to convert the thiazolidine functionality into a free cysteine and subsequent modification with a maleimide label, unwanted disulfide shuffling can occur in proteins containing disulfide bonds [18]. To circumvent this problem a synchronized protocol for thiazolidine deprotection and maleimide coupling can be best used (**Figure 5**).

A chemoselective conjugation approach that does not use any of the naturally occurring functional groups in proteins is oxime ligation. The reaction is comprised of a ketone or aldehyde reacting with an aminooxy group to yield an oxime bond (**Figure 6**). The reaction can be performed in aqueous media at neutral pH but is faster at slightly acidic pH [19]. The reaction can be catalyzed with aniline or derivatives thereof, to facilitate fast reactions [20, 21].

Although the oxime reaction itself can be performed relatively easy, the more challenging part is the incorporation of a ketone or aminooxy in the peptide/ protein of interest. Since a ketone is virtually inert to most chemical reactions, the ketone is mostly chosen over the aminooxy component to be incorporated in the protein of choice, while the aminooxy component is used to modify the label. The increased attention for the oxime bond in the last decade has led to the development of several methods to incorporate ketones or aldehydes in proteins. An overview of the available techniques was previously reviewed, here we will briefly highlight methods useful in chemical synthesis [22]. Historically, oxidation of peptides/proteins containing an *N*-terminal 2-amino alcohol residue (serine/ threonine) is among the most used to obtain an aldehyde in the form of a glyoxyloyl moiety [23]. Site-specific incorporation using chemical synthesis, however, can be achieved using suitably protected unnatural amino acids such as p-acetylphenylalanine or keto-proline [24, 25]. A large amount of flexibility in both site of introduction and distance from active regions can be achieved with modification of amine moieties with keto-acids [26, 27]. Care has to be taken, however, in the choice of keto-acid as some are less efficient in subsequent oxime formation [28].

In summary, several techniques are available for the modification of proteins to include chelators used for PET and SPECT. Amine and thiol reactive compounds are easy to use through orthogonally protected lysine side chains or through thiazolidine deprotection in pre-folded proteins. Oxime conjugation can be used for chelator incorporation but is also used for covalent radiolabeling approaches, such as introducing 18F-containing prosthetic groups (see for an overview [29]).

#### **3. Radiolabeling of peptides**

A variety of labeling techniques can be applied to peptides and proteins, but according to George De Hevesy's definition of a tracer the radiolabeling procedure should not affect the biological properties, the affinity to the target, or the physicochemical properties (e.g. charge, hydrophilicity, size). In the following part a list of radiosynthesis techniques for commonly used isotopes is described, omitting isotopes that are used less frequently. For example, 11C is a widespread isotope for labeling small organic compounds, but it is used less frequently in peptides or larger structures and so will therefore not be further discussed here.
