We wish to report a serious side reaction which occurs during the preparation of 9-fluorenylmethyloxycarbonyl (Fmoc) amino acid derivatives 1. Because of their base lability, Fmoc-amino acids are potentially amongst the most versatile intermediates for peptide synthesis, especially when used in conjunction with acid-labile sidechain protecting groups.lS2 1 However, when these compounds are prepared by standard pro~edures,~.3 we have found that the majority of Fmoc-amino acids are contaminated by variable but often substantial amounts of Fmoc-oligopeptides. We have established this by HPLC, amino acid analysis, nmr, and the synthesis of a number of standards. The Fmoc-oligopeptides are likely formed via the intermediacy of relatively stable mixed anhydrides formed during Fmoc-chloride-mediated synthesis of the Fmoc-amino acid derivatives.'T3The oligopeptide contaminants were first noted during routine TLC analyses to assess the homogeneity of commercial samples of Fmoc-amino acids prior to their use. While the literature TLC systems' (e.g. ch1oroform:methanol:acetic acid, 9:l:0.1) did not necessarily separate the contaminants in all cases, we were able to demonstrate reproducible separations using the TLC system to1uene:acetic acid, 10:l. The impurities were invariably slower-moving in this TLC system, were strongly uv-absorbing, chlorine-tolidine4 positive, and also difficult or impossible to remove completely by recrystallization techniques. This suggested that the impurities were both Fmoc-blocked and amino-acid containing. One of the most severely contaminated amino acid derivatives, Fmoc-glycine, was chosen for careful, in-depth study.When Fmoc-glycine was synthesized by the standard literature procedure,'13 using Fmoc-chloride in dioxane/aqueous sodium carbonate, the product was found by TLC to be contaminated by a major (10-20%), slower-moving contaminant. Fractional crystallization using ethyl acetate/hexane provided a sample that was
We describe a direct colorimetric assay for alpha-amylase, with 2-chloro-4-nitrophenyl-alpha-maltotrioside as substrate. Both human pancreatic and salivary amylase split this substrate without the use of helper enzymes, yielding free 2-chloro-4-nitrophenol, which is monitored at 405 nm. The performance of this reagent compares well with that of Pantrak Amylase (Behring Diagnostics) for both serum and urine samples. The reagent is very stable in dry powder form and is stable for one month at 2 to 8 degrees C after reconstitution. Because of the rapid color development and linear kinetics (less than 30 s), the assay is easily automated. Results can be obtained in less than 5 min.
According to a personal communication of R. D. G. Cooper the chemical shifts of the methyl groups of compound 4 in CDCI3 and of compound 6 in C6D5 should be inverted in Table II of this publication. After correction of » and ß for these groups, one obtains As -4 for 2/3-Me -0.07 and for 2ct-Me +0.21 and As -4 for 2/3-Me +0.29 and for 2a-Me -0.04. The values for the methyl groups in Table 111 also should be corrected. (4) G.
Apolipoprotein C-I, a protein constituent of the very low density lipoproteins of human plasma, consists of a single chain of 57 amino acids. The total synthesis of a protein corresponding to apolipoprotein C-I in physical properties and composition was accomplished by solid phase techniques employing a modified polystyrene incorporating spacer groups between the point of attachment of the first residue and the polymer matrix. The synthetic apoprotein was shown to activate lecithin:cholesterol acyltransferase to the same extent as the native protein. Comparative lipidbinding studies with dimyristoyl phosphatidylcholine gave complexes for native and synthetic apoprotein which floated at the same density after ultracentrifugation in KBr gradients and had virtually the same lipid:protein ratios. The very low density lipoproteins (VLDL) of human plasma are primarily responsible for the transport of endogenously synthesized triglycerides in the blood stream. Delipidation of VLDL gives a protein fraction which can be separated by gel-filtration chromatography into apolipoprotein B (apoB), which is also the major constituent of low density lipoprotein (LDL) (1), and a mixture of apoproteins, collectively referred to as apolipoprotein C (apoC) (2). DEAE-cellulose chromatography of the C-proteins yields four major components and several minor ones (2, 3). The first apoprotein to be eluted from the DEAE-cellulose column, apoC-I or aposerine, consists of a single chain of 57 amino acids, the sequence of which has been determined by Schulman et al. (4,5) and confirmed by Jackson and coworkers (6) (Fig. 1).The binding of apoC-I to phosphatidylcholine has been studied (7). Several potential phospholipid-binding regions in the sequence have been identified by model building experiments (7,8).Recent work by Soutar et al. has shown that apoC-I, as well as apolipoprotein A-I (apoA-I) (Gln-I) of high density lipoproteins (HDL), can activate the serum enzyme, lecithin: cholesterol acyltransferase (LCAT) in vitro when defined phosphatidylcholine:cholesterol vesicles are used as substrates (9). The extent of activation for the two apoproteins differs depending on the nature of the acyl donor.In order to investigate the sequence determinants for both binding of phosphatidylcholine and activation of LCAT by apoC-I, a program was initiated to synthesize fragments from the carboxyl-terminus of varying lengths up to and including the total sequence. We report here the synthesis of a protein corresponding in physical properties and chemical composition to apoC-I, its complex formation with dimyristoyl phosphatidylcholine, and its activation of LCAT. MATERIALS AND METHODSSynthesis Procedures. Chloromethylated polystyrene resin, 1% cross-linked with divinyl benzene (0.75 meq of Cl per g) (Bio-Rad) was converted to the modified resin of Sparrow (10), p-bromomethylphenylacetamido-1 1-hendecanamido-1 1-hendecanamido-methyl resin. N-tert-butyloxycarbonyl-O-benzyl-L-serine was esterified to the resin as its cesium salt according to the me...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.