On-line monitoring of deprotection reaction in peptide automated synthesizer comprising UV detector
A method for on-line monitoring of deprotection reaction in a peptide automated synthesizer comprising UV detector is disclosed. A UV source, detector, electronics, and housing are integrated with a line or tube through which liquid reactants flow for periodic measurements in the peptide synthesizer. The method includes determining the progression of the reaction, completion point of the reaction, and the modification of the reaction times and repetitions in real time.
1. A method for real-time monitoring the deprotection reaction of a Fmoc protecting group in a peptide automated synthesizer to obtain feedback on reaction completion during said deprotection reaction, comprising the steps of:
(a) within said automated synthesizer, which includes an ultraviolet (UV) source and UV sensor, periodically pushing a deprotection solution comprising dibenzofulvene and related adducts and byproducts from said reaction to the UV sensor in an area that is adapted to transmit UV light and irradiating it with UV light at a wavelength of about 301 nm;
(b) detecting the absorption of said UV light and determining the rate at which the reaction is progressing by calculating a floating average of a specified number of previous readings; and
(c) determining the reaction completion when the floating average does not change more than a preselected delta.
2. The method of claim 1 , wherein said deprotection reaction time is extended based on absorption data recorded during said detecting in step (b), with said data used to determine the extent of completion of the deprotection reaction.
3. The method of claim 1 , wherein said deprotection reaction is repeated based on absorption data recorded during said detecting in step (b), with said data used to determine the extent of completion of the deprotection reaction.
4. The method of claim 1 , wherein the time of a subsequent amino acid or monomer coupling reaction is extended based on the time of said deprotection reaction, itself dependent on the absorption data recorded during said detecting in step (b).
5. The method of claim 1 , wherein said area is a UV transmissive tube.
6. The method of claim 1 , wherein steps (a) and (b) are performed at specified time intervals during said reaction.
7. The method of claim 1 , further comprising the step of providing a visual output of absorption data acquired during the Fmoc deprotection steps of the synthesis.
8. The method of claim 7 , wherein said visual output of absorption data comprises a graphical synthesis summary or a graphical representation of an individual deprotection reaction.
9. A method for real time monitoring the deprotection reaction of a Fmoc protecting group in a peptide automated synthesizer to obtain feedback on reaction completion during said deprotection reaction, the synthesizer having a reaction vessel, an ultraviolet (UV) source and UV sensor within said synthesizer, and a UV-transmissive tube disposed in fluid connection with and proximally to said reaction vessel, comprising the steps of:
(a) periodically drawing a liquid containing dibenzofulvene and realated adducts and byproducts from said reaction vessel into said UV-transmissive tube,
(b) irradiating said liquid with UV light at a wavelength of about 301 nm,
(c) detecting through said UV sensor the absorption of said UV light and determining the rate at which the reaction is progressing by calculating a floating average of a specified number of previous readings; and
(d) returning said liquid to the reaction vessel unless reaction completion is indicated when the floating average does not change more than a preselected delta.
10. The method of claim 9 , wherein the step of drawing a liquid from the reaction vessel occurs during said reaction.
11. The method of claim 9 , wherein steps (a) thru (d) are performed at specified time intervals during said reaction.
12. The method of claim 9 , wherein the time of the deprotection reaction is extended based on the absorption data recorded during said detecting in step (c), with said data used to determine the extent of completion of the deprotection reaction.
13. The method of claim 9 , wherein the deprotection reaction is repeated based on the absorption data recorded during said detecting in step (c), with said data used to determine the extent of completion of the deprotection reaction.
14. The method of claim 9 , wherein the time of a subsequent amino acid or monomer coupling reaction is extended based on the time of said deprotection reaction, itself dependent on the absorption data recorded during said detecting in step (c).
15. The method of claim 9 , further comprising the step of providing a visual output of the absorption data during the synthesis.
16. The method of claim 15 , wherein said visual output of absorption data comprises a graphical synthesis summary or a graphical representation of an individual deprotection reaction.