330
1. The peptide might be difficult to synthesize, especially when
the target peptide consists of more than 30 amino acids. The
interactions of the side chains of the amino acids cause problems during the synthesis, as the new amino acids to be
attached require higher reaction times, and the final peptide is
usually impure. An effective way to surpass this hurdle is by
shortening the sequence and/or substituting the hydrophobic
residues. The aforementioned problem is usually encountered
during the manual solid-phase peptide synthesis; in some cases
it can be addressed by utilizing microwave-assisted synthesis.
2. The synthesized peptide might possess low aqueous solubility,
which renders its purification extremely hard. Insolubility
issues can be surpassed by shortening the sequence through
elimination of hydrophobic residues or by lengthening the
sequence through the addition of polar amino acids. Another
effective way to face the low solubility is by altering the C-/Nterminus and/or substituting specific hydrophobic residues.
3. During the conjugation between the peptide and the drug or
the linker, some problems may arise, derived from the utilized
coupling reagents. For instance, HATU is known to react with
the N-terminus of the peptide and recently, its participation in
the formation of side products on the side chains of specific
peptide residues has been described [9].
In this chapter, we provide the procedure to assemble a peptidedrug conjugate consisting of D-Lys
6
-GnRH (peptide) and gemcitabine (anticancer agent), tethered via various bonds (ester,
amide, carbamate and oxime) derived from the utilization of different linkers (succinyl, carbamate, and PEG-aminooxy). The utilized
peptide (D-Lys
6
-GnRH-II) is gonadotropin-releasing hormone
that binds selectively on type II GnRH-receptor (GnRH-R), which
is overexpressed in various cancer types including prostate, lung,
and breast [10]. D-Lys
6
-GnRH possesses a lysine that can be utilized for orthogonal coupling in liquid phase with the linker and
consequently the drug. Gemcitabine belongs to the antimetabolite
family of anticancer agents and is active against various solid malignant
tumors including ovarian, prostate, lung, breast, and pancreatic.
Gemcitabine possesses three possible conjugation sites: (1) a
primary –OH that is the site of intracellular phosphorylation leading to the active metabolites difluorodeoxycytidine diphosphate
(dFdCDP) and difluorodeoxycytidine triphosphate (dFdCTP), (2)
a secondary –OH, and (3) a primary –NH 2 which mediates its metabolic inactivation toward 2′,2′-difluorodeoxyuridine (dFdU) via
the cytidine deaminase [11]. Herein, it will be described the synthetic procedure concerning the primary or the secondary –OH,
where different linkers can be incorporated.
Eirinaios I. Vrettos and Andreas G. Tzakos
1. The peptide might be difficult to synthesize, especially when
the target peptide consists of more than 30 amino acids. The
interactions of the side chains of the amino acids cause problems during the synthesis, as the new amino acids to be
attached require higher reaction times, and the final peptide is
usually impure. An effective way to surpass this hurdle is by
shortening the sequence and/or substituting the hydrophobic
residues. The aforementioned problem is usually encountered
during the manual solid-phase peptide synthesis; in some cases
it can be addressed by utilizing microwave-assisted synthesis.
2. The synthesized peptide might possess low aqueous solubility,
which renders its purification extremely hard. Insolubility
issues can be surpassed by shortening the sequence through
elimination of hydrophobic residues or by lengthening the
sequence through the addition of polar amino acids. Another
effective way to face the low solubility is by altering the C-/Nterminus and/or substituting specific hydrophobic residues.
3. During the conjugation between the peptide and the drug or
the linker, some problems may arise, derived from the utilized
coupling reagents. For instance, HATU is known to react with
the N-terminus of the peptide and recently, its participation in
the formation of side products on the side chains of specific
peptide residues has been described [9].
In this chapter, we provide the procedure to assemble a peptidedrug conjugate consisting of D-Lys
6
-GnRH (peptide) and gemcitabine (anticancer agent), tethered via various bonds (ester,
amide, carbamate and oxime) derived from the utilization of different linkers (succinyl, carbamate, and PEG-aminooxy). The utilized
peptide (D-Lys
6
-GnRH-II) is gonadotropin-releasing hormone
that binds selectively on type II GnRH-receptor (GnRH-R), which
is overexpressed in various cancer types including prostate, lung,
and breast [10]. D-Lys
6
-GnRH possesses a lysine that can be utilized for orthogonal coupling in liquid phase with the linker and
consequently the drug. Gemcitabine belongs to the antimetabolite
family of anticancer agents and is active against various solid malignant
tumors including ovarian, prostate, lung, breast, and pancreatic.
Gemcitabine possesses three possible conjugation sites: (1) a
primary –OH that is the site of intracellular phosphorylation leading to the active metabolites difluorodeoxycytidine diphosphate
(dFdCDP) and difluorodeoxycytidine triphosphate (dFdCTP), (2)
a secondary –OH, and (3) a primary –NH 2 which mediates its metabolic inactivation toward 2′,2′-difluorodeoxyuridine (dFdU) via
the cytidine deaminase [11]. Herein, it will be described the synthetic procedure concerning the primary or the secondary –OH,
where different linkers can be incorporated.
Eirinaios I. Vrettos and Andreas G. Tzakos