4-(9-Fluorenylmethyloxycarbonyl)phenylhydrazine (FmPH):  A New Chromophoric Reagent for Quantitative Monitoring of Solid-Phase Aldehydes^1-3

Abstract

A direct method for quantifying solid-phase aldehydes has been developed, using a new reagent, 4-(9-fluorenylmethyloxycarbonyl)phenylhydrazine (FmPH). The FmPH reagent was synthesized in three steps (24% overall yield) from commercially available p-hydrazinobenzoic acid. Resin-bound aldehydes reacted quantitatively with FmPH, in the presence of trimethylorthoformate (TMOF) as a dehydrating agent, to form a highly conjugated, immobilized FmPH-hydrazone. Next, mild treatment of the hydrazone with an excess of piperidine-N,N-dimethylformamide (1:1) released the piperidine-dibenzofulvene adduct chromophore (epsilon(301nm) = 7800 M(-1) cm(-1)) from the support. FmPH quantitation of aldehydes proved to be a straightforward, sensitive, and reproducible technique for monitoring resin-bound aldehydes [albeit insufficiently reactive to allow reliable quantification of ketones]. The FmPH aldehyde assay is applicable to a range of solid supports, as demonstrated specifically for poly(ethylene glycol)-polystyrene (PEG-PS), aminomethylpolystyrene (AMP), and cross-linked ethoxylate acrylate resin (CLEAR).

Synthesis and Immobilization of erythro‐C14‐ω‐Aminosphingosine‐1‐phosphate as a Potential Tool for Affinity Chromatography

“…Thus, reaction of 9 (2 equiv) with Affi-Gel 10 in NMP in the presence of excess DIPEA produced immobilized product 12 (Scheme 2). Following a procedure reported by Shannon et al, [17] treatment with excess piperidine/DMF then led to cleavage of the Fm group to give free phosphate 13 and quantitative formation of the piperidine-dibenzofulvene adduct 14 which could be identified by LC/MS. To determine the percentage of sphingosine phosphate bound to the gel, the concentration of 14 was determined by UV spectroscopy measuring absorbance at 301 nm and using the known extinction coefficient for the dibenzofulvene chromophore.…”

“…To determine the percentage of sphingosine phosphate bound to the gel, the concentration of 14 was determined by UV spectroscopy measuring absorbance at 301 nm and using the known extinction coefficient for the dibenzofulvene chromophore. [17] According to this calculation, the effective loading was 66 %, corresponding to 10 mmol mL À1 gel. Under the chosen deprotection conditions, unreacted NHS esters on the resin were concomitantly blocked during repeated washing with piperidine.…”

Collagen immunoassay as a method to optimise surface functionalisation

“…For example, colourimetric methods that rely on the reducing ability of aldehydes are usually performed immediately. [42][43][44][45][46][47][48][49][50][51][52][53][54] But such derivativisation techniques, being performed immediately and without a period stressing the conjugation bonds, give limited information. In addition, optimum protein conjugation may require different parameters compared to optimum marker molecule conjugation.…”

“…The imine bond was reduced to a stable amine bond (-CH 2 -NH-) by the addition of sodium cyanoborohydride reagent, [45,46] Fehling's solution, [47] , Benedict's solution [48] and hydrazines in Wolff-Kishner reductions. [49][50][51][52][53][54] For proteins, all such methods are limited because conjugation is not simply a matter of labelling one aspect of surface reactivity, such as reducing ability; protein conjugation depends on several surface properties, including wettability, surface charge and pK a . [1] It was hypothesised that in situations where protein bonding is the aim of surface functionalisation, optimising functionalisation by optimising the resistance of conjugated protein to degradation in 8 M urea may serve a twofold purpose: (1) it may be sufficient surface analysis and (2) it is essential for the interpretation of later biological responses.…”

Polystyrolsulfonylchlorid – ein hochorthogonales Linkerharz für die Synthese von Stickstoffheterocyclen

“…Der Erfolg der Immobilisierung wurde qualitativ durch FT-IR-Spektroskopie (CarbonylStreckschwingung bei 1730 cm À1 ) und quantitativ mithilfe der FmPH-Methode [21] ermittelt. Auf diese Weise wurde das Piperidonharz 10 mit einer Beladung von 1.…”