Methods of manufacture of pteroyl-amino acid-fluorescent dyes

1. A method for synthesizing a compound of the formula:

or a racemic mixture thereof, wherein:

X is selected from the group consisting of:

or a racemic mixture thereof; and

Y is represented by the formula:

wherein:

R 1 is independently selected from the group consisting of O, S, NH, CH 2 and CH 2 CH 2 ; and

R 2 is independently selected from the group consisting of CH 2 and CH 2 CH 2 ;

comprising the steps of:

(a) reacting a compound of the formula 1:

 with a compound of the formula:

 or a racemic mixture thereof,

 in the presence of a polar solvent and trifluoroacetic acid to provide a compound of the formula:

 or a racemic mixture thereof;

(b) reacting the compound of the formula:

 or a racemic mixture thereof;

 with sodium hydroxide and a dye compound of the formula:

 wherein:

 R 1 is independently selected from the group consisting of O, S, NH, CH 2 and CH 2 CH 2 ; and

 R 2 is independently selected from the group consisting of CH 2 and CH 2 CH 2 ; and

(c) isolating the compound of the formula:

 or a racemic mixture thereof,

 wherein:

 R 1 is independently selected from the group consisting of O, S, NH, CH 2 and CH 2 CH 2 ; and

 R 2 is independently selected from the group consisting of CH 2 and CH 2 CH 2 .

2. The method of claim 1 , wherein the dye compound in step (b) is selected from the group consisting of:

3. The method of claim 1 , wherein the polar solvent is selected from the group consisting of dimethylformamide, dimethylsulfoxide and water.

4. A method for synthesizing a compound of the formula:

 wherein:

 W, X, Y and Z are independently selected from the group consisting of H + , Na + , K + or NH 4 + ;

 comprising the steps of:

(a) reacting a compound of the formula:

 with a compound of the formula:

 in the presence of dimethylformamide and trifluoroacetic acid to provide a compound of the formula:

 wherein:

 W is independently selected from the group consisting of H + , Na + , K + or NH 4 + ;

(b) reacting the compound of the formula:

 with sodium hydroxide and a dye compound of the formula:

 wherein:

 X, Y and Z are independently selected from the group consisting of H + , Na + , K + or NH 4 + ; and

(c) isolating the compound of the formula:

 wherein:

W, X, Y and Z are independently selected from the group consisting of H + , Na + , K + or NH 4 + .

5. A method for synthesizing an isotopic form of a compound of the formula:

 wherein said isotopic form comprises one or more carbon and/or hydrogen isotopes selected from the group consisting of 2 H, 3 H, 13 C and 14 C, and further wherein:

 W, X, Y and Z are independently selected from the group consisting of H + , Na + , K + or NH 4 + ;

 comprising the steps of:

(a) reacting a compound of the formula:

 wherein:

 W is selected from the group consisting of H + , Na + , K + or NH 4 + ;

 with an isotopic form of a compound of the formula:

 wherein said isotopic form comprises one or more carbon and/or hydrogen isotopes selected from the group consisting of 2 H, 3 H, 13 C and 14 C, and further wherein:

 W is selected from the group consisting of H + , Na + , K + or NH 4 + ;

 in the presence of dimethylformamide to provide an isotopic form of a compound of the formula:

 wherein said isotopic form comprises one or more carbon and/or hydrogen isotopes selected from the group consisting of 2 H, 3 H, 13 C and 14 C, and further wherein:

 W is selected from the group consisting of H + , Na + , K + or NH 4 + ;

(b) reacting the isotopic form of the compound of the formula:

 wherein said isotopic form comprises one or more carbon and/or hydrogen isotopes selected from the group consisting of 2 H, 3 H, 13 C and 14 C, and further wherein:

 W is selected from the group consisting of H + , Na + , K + or NH 4 + ;

 with sodium hydroxide and a dye compound of the formula:

 wherein

 X, Y and Z are independently selected from the group consisting of H + , Na + , K + or NH 4 + ; and

(c) isolating the isotopic form of the compound of the formula:

 wherein said isotopic form comprises one or more carbon and/or hydrogen isotopes selected from the group consisting of 2 H, 3 H, 13 C and 14 C, and further wherein:

 W, X, Y and Z are independently selected from the group consisting of H + , Na + , K + or NH 4 + .

6. A method for the solid phase synthesis of a compound of the formula:

wherein:

W, X, Y and Z are independently selected from the group consisting of H + , Na + , K + or NH 4 + ;

comprising in a solid phase peptide synthesizer, the steps of:

(a) reacting a compound of the formula:

 wherein:

 W is selected from the group consisting of H + , Na + , K + or NH 4 + ;

 with a compound of the formula:

 wherein:

is a resin bead;

 in the presence of dimethylformamide to provide a compound of the formula:

 wherein:

is a resin bead;

(b) reacting the compound of the formula:

 in the presence of trifluoroacetic acid:H 2 O:triisopropylsilyl alcohol to provide a compound of the formula:

 wherein:

 W is selected from the group consisting of H + , Na + , K + or NH 4 + ;

(c) reacting the compound of the formula:

 with sodium hydroxide and a dye compound of the formula:

 wherein:

 X, Y and Z are independently selected from the group consisting of H + , Na + , K + or NH 4 + ; and

(d) isolating the compound of the formula:

 wherein:

 W, X, Y and Z are independently selected from the group consisting of H + , Na + , K + or NH 4 + .