Synthesis of Gadolinium-DOTA-Peptide substrates-JS Linear sequences

2.1.2.1. Synthesis of Gadolinium-DOTA-Peptide substrates-JS Linear sequences

The rationale for the synthesis of the gadolinium-DOTA-peptide substrates was to image atherosclerotic plaques by specifically identifying the biomarkers (legumain) over expression in these plaques. Hence two series of compounds were synthesized and this section explains in detail the chemical synthesis of the JS-linear series. The JS-linear series of JS compounds had a design: Paramagnetic gadolinium complex-AA4-AA3-AA2-AA1-Spacer-AQ, where AA represents amino

- 105 - acids and the complex consisted of Gd-DOTA chelate. The linear sequences were synthesized following the procedure for solution peptide synthesis. The JS-linear library of compounds consisted of a hydrophobic group comprising of an aminoanthraquinone moiety, which was conjugated to the C-terminus of a tetra peptide sequence in order to, potentially, increase the retention time of the contrast agent (DOTA-Gd) within the atherosclerotic plaque environment. This increase in retention time of the contrast agent could perhaps enhance the quality/resolution of the image from a MRI scanner. The presence of a short diamino chain between the carboxyl end of the peptide sequence and anthraquinone moiety was incorporated to produce an amphiphilic compound in order to improve the solubility of the compound. Furthermore, the intense reddish-pink colour of the aminoanthraquinone chromophore helped to identify the required compound on a silica gel chromatographic column; thus making the process of identifying the required fraction easier. The chelating agent-DOTA was either covalently attached to the side chain of a lysine residue (JS-lysine series) or to the terminal proline (JS-linear series). The rationale for such a design was based on the concept that the aminoanthraquinone-peptide-contrast agent conjugate can accumulate and bind to the extracellular protein moieties in the ECM and when cleaved by the target enzyme, the intact contrast agent may be released locally. The released contrast agent (DOTA-Gd), along with the hydrophobic site (proline, β-alanine, and AQ-spacer-lysine) attached to them, could increase the retention time of the CA. This may enhance the contrast between the normal and abnormal regions of investigation. As discussed in previous sections, DOTA is a favourable bifunctional chelating agent (BFCA) when compared to DTPA mainly because of its thermodynamic stability and kinetic inertness preventing in vivo dissociation of free metal ions into the blood stream. Furthermore, most of the clinically relevant MRI contrast agents are based on DOTA-gadolinium complexes such as Gadovist^®, ProHance^®, Dotarem^® and Gadomer-17^®. Several derivatives of DOTA-based BFCA’s are available commercially such as active DOTA esters, DOTA with a coupling unit present either at the α- site of one carboxylate arm (McMurry et al. 1992; James et al. 2012) or into the macrocycle (Kruper et al.

1993) and protected DOTA species (Jamous et al. 2013). However, the protected DOTA derivative (DOTA-tris-^tBu ester) has been used in this research project as it can be readily coupled to the peptide sequence following a standard coupling procedure during both solution and solid phase peptide synthesis.

- 106 - A. Synthesis of Gd(III)DOTA-Pro-Ala-Asn-Leu-Spacer-AQ (Compound 49) I. Synthesis of intermediate compound DOTA(^t Bu)-Pro-Ala-Asn(Trt)-Leu-Spacer-AQ (Compound 47)

The tert-butyl protected compound (47) was synthesized by reacting

DOTA-tris-tBu-ester with compound 43 in DMF along with the in situ coupling agents:

TBTU/HOBT and DIPEA. The crude sample was purified by column chromatography and the yield of the product (47) post column chromatography was 43%. After purification the compound 47 was characterized by high resolution ESI (+) mass spectrometry, which gave a signal at m/z 1568.8402 (M+Na)⁺ corresponding to the molecular mass of 1545 and also a signal at 784 [(M+Na)/2]²⁺.

II. Synthesis of intermediate compound DOTA-Pro-Ala-Asn-Leu-Spacer-AQ (Compound 48)

The γ-trityl and tert-butyl protected aminoanthraquinone-tetra peptide sequence (47) was deprotected using trifluoroacetic acid. The reaction conditions and reagents were the same as detailed in section (2.1.1.3). Reaction progress was monitored by TLC analysis, which showed the presence of a single new component close to the base line confirming the polar nature of the deprotected sample. The residual TFA was evaporated under reduced pressure and any remaining solvent was re-evaporated with ethanol. The condensate was triturated with diethyl ether

- 107 - followed by cooling on ice to facilitate further precipitation. The yield of the crude sample 48 was found to be 87%. The product (48) was characterized by high resolution ESI (+) mass spectrometry, which gave a doubly-charged signal at m/z 568.7844 [(M+2H)/2]²⁺ corresponding to a molecular mass of 1135 and also a signal at m/z 587 corresponding to the potassium adduct [(M+H+K)/2]²⁺.

III. Synthesis Gd(III)DOTA-Pro-Ala-Asn-Leu-Spacer-AQ (Compound 49)

Synthesis of the target gadolinium complex of DOTA-tetrapeptide-amino anthraquinone spacer conjugate (49) was performed in accordance to the reaction conditions proposed by Digilio and co-workers (2010). The Gd complex was prepared in a neutral aqueous solution by reacting GdCl3(6H2O) with compound 48.

Figure 51: Expansion of the ESI (+) mass spectrum of the Gd-DOTA-peptide sequence (49) that shows the isotopic pattern of the molecular ion (doubly charged at half mass).

[(M+2H)/2]²⁺

- 108 - The reaction pH was adjusted to 7 by adding a solution of ammonia 0.35% drop wise. After two hours the reaction progress was checked by TLC analysis using chloroform: MeOH: pyridine:35%ammonia (20:55:15:10), which showed the presence of two components with one corresponding to the starting material. After 13 hours the reaction mixture was reduced to low volume by evaporation at room temperature and then purified by column chromatography using the same solvent system as for the TLC analysis. Analytical HPLC for the target compound 49 was performed on a Shimadzu Prominence system equipped with UV-Vis detector using an eclipse XDB-C18 RP column and the purity of the sample was found to be 72%.

The product (49) was characterized by high resolution ESI (+) mass spectrometry, which gave a signal at m/z 1291 (M+H)⁺ corresponding to a molecular mass of 1290 and also a signal at m/z 1313 corresponding to the sodium adduct (M+Na)⁺. A doubly-charged signal at m/z 646.2351 [(M+2H)/2]²⁺ was also observed (figure 51).

B. Synthesis of Gd(III)DOTA-Pro-Gly-Asn-Leu-Spacer-AQ (Compound 53)

HN

DOTA-Gd³⁺ Pro Gly Asn Leu Spacer AQ

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