Rational Design of Fluorophores for In Vivo Applications - Progress in Molecular Biology and Translational Science - page 66

analogous derivatives of Cy7 (cybate), 34 have been prepared and characterized
by Achilefu and coworkers. The carboxylate groups in cypate have been
subsequently used for attachment of a variety of functional moieties, such
as polycarboxylic acids, 35,41 glucosoamine, 35,42 peptides, 37,39,42,43 and poly
(ethylene oxide) dendrimers. 44 Cyanine derivatives containing carboxylate
groups at the benzo ring of each terminal indole moiety have been prepared
by Licha 45 and Tung (compound NIR-820, Chart 3.1 ) 31 and used for

attaching glucosoamid 45 and transferrin, respectively. 31
The polyvalent symmetrical derivatives described above are equipped
with two identical carboxylic groups. These groups can be selectively
functionalized, so that only one is activated and derivatized 31,42,45,46 ;
however, having two identical carboxylic groups complicates the
derivatization and purification procedure because of the formation of the
bis-derivatized ester as a side product. Therefore, monovalent derivatives
(i.e., derivatives with one bioconjugatable group) have been developed.
One strategy for the preparation of the monovalent cyanine derivatives
entails the preparation of nonsymmetrical derivatives, where the functional
derivatizable group is located only on the one indole moiety (either as a
substituent on indole nitrogen or on the benzo ring, Fig. 3.1 ). A series of
nonsymmetrical carboxylate derivatives of Cy5.5, 47 Cy7, 48-50 and benzo-
heptamethine cyanines 51,52 have been prepared and conjugated with
proteins, 49 peptides, 50,53,54 poly(ethylene) glycols, 55 sugars, 56,57 fluorescence
quenchers, 58 and other targeting agents. 59 Tung and coworkers prepared the
nonsymmetrical monovalent carboxylate-substituted hybrid cyanine with
benzoindole moiety on one side and carboxylate-substituted indole on the
other side of the heptamethine chain. 60 This derivative has been
subsequently conjugated to the PEGylated graft polymer (to make enzyme-
activatable fluorescent probe) 60 or to the folate residue (to prepare folate
receptor expressing cancer cells). 61
Preparation of nonsymmetrical cyanine derivatives causes purification
problems because of the inevitable formationof the corresponding symmetrical
side product. Therefore, recently a new strategy for the preparation of mono-
functional cyanine fluorophores has been pursued ( Fig. 3.2 ). This strategy relies
on the synthesis and derivatization of cycloheptamethine cyanines (

Cl-cyclo-

) with chlorine atom on the cyclohexene ring. 62-64 In these new cyanine
derivatives, in which part of the polymethine chain is embedded in the
cyclohexane ring, the chlorine substituent on the cyclohexane ring can be
further derivatized by nucleophilic substitution, using phenolates (to form
C

Cy

O bond), thiols (to form C S bond), or amines (to form C N