Employment of on-line FT-IR spectroscopy to monitor the deprotection of a 9-fluorenylmethyl protected carboxylic acid peptide conjugate of doxorubicin.
A method for accurately determining the end-point, >98% conversion, of the deprotection reaction of a highly toxic 9-fluorenylmethyl (Fm) ester 1b to its corresponding carboxylate 1d in real time by FT-IR spectroscopy is reported. Advantages of this method over analysis by conventional chromatographic means include real time determination of the end-point of a reaction that is time sensitive to by-product formation, and elimination of sampling a highly toxic reaction mixture. The FT-IR method is based on monitoring, in real time, the disappearance of the Fm ester carbonyl band for 1b at 1737 cm(-1), during deprotection by piperidine, and calibration models were established by Partial Least Squares (PLS) regression analysis with high performance liquid chromatography (HPLC) as reference. The best calibration model was built with 5 PLS factors in the spectral range of 1780-1730 and 1551-1441 cm(-1) and resulted in a standard error of cross validation (SECV) of 0.63 mM 1b and a standard error of prediction (SEP) of 0.51 mM 1b in the range of 0-25 mM. This error of prediction is approximately 0.8% of the initial concentration of 1b and is well within our specifications of <2% initial concentration.
Cameron
Mark
M
Department of Process Research, Merck Research Laboratories, Merck & Co. Inc., PO Box 2000, Rahway, NJ 07065, USA. mark_cameron@merck.com
Zhou
George X
GX
Hicks
Micheal B
MB
Antonucci
Vincent
V
Ge
Zhihong
Z
Lieberman
David R
DR
Lynch
Joesph E
JE
Shi
Yao-Jun
YJ
D000970:Antineoplastic Agents / Q000032:analysis / Q000737:chemistry; D002138:Calibration; D002264:Carboxylic Acids / Q000032:analysis* / Q000737:chemistry; D004317:Doxorubicin / Q000032:analysis* / Q000737:chemistry; D005449:Fluorenes / Q000032:analysis* / Q000737:chemistry; D009842:Oligopeptides / Q000032:analysis* / Q000737:chemistry; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods
D000970:Antineoplastic Agents; D002264:Carboxylic Acids; D005449:Fluorenes; D009842:Oligopeptides; D004317:Doxorubicin