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A novel class of isotope-coded affinity tag is proposed possessing a fluorescent feature, referred to as fluorescent isotope-coded affinity tag (FCAT), to provide a new tool for quantitative proteomics. The label is designed to bind cysteine containing proteins or peptides. The FCAT reagent comprises four functional elements: a specific chemical reactivity group toward sulfhydryl groups; a linker that can incorporate the stable isotopes; a hydroxymethylbenzoic residue (base labile group) to cleave off a large part of the label before MS analysis; and a fluorescent tag for absolute quantification. The fluorescent part of the tag is also planned to be utilized to isolate the FCAT-labeled peptides via antibody based pull-down method. In this paper, we report on the solid phase organic synthesis of the light isotope containing FCAT molecule. The new labeling reagent showed good reactivity with model cysteine containing peptides. The fluorophore group was also effectively cleaved off from the labeled products to accommodate easier MS based analysis.

In the design of polyethyleneimine derivatives for use as catalysts and binding media, the placement of reactive and hydrophobic groups previously has been limited by the specificity of the addition reaction. In this paper is described a protocol to limit the sites of addition of nucleophiles and long-chain alkanes to tertiary amines and the less reactive of the secondary amines. Three blocking groups for the primary and secondary amines were tested, but only trifluoroacteylating reagents left the polymer reactive to substitution on the tertiary amines with halogenated alkanes. The secondary amines that resisted trifluoroacetylation were blocked with either tert-butyloxy carbonate or trimethylsilyl carbonate. The tertiary amines were quaternized with iodododecane or dodecyl benzyl chloride. After removal of the trifluoroacetyl groups, the polymer amines were inactivated by methylation, which proceeded to 93% completion. The 7% of the amines that were not quaternized were largely tertiary, since propylene sulfide, which reacts only with secondary and primary amines, was substituted onto the polymer only to the extent of 0.2% total amine, as quantified by the indirect method of P. H. Butterworth, F. Baum, and J. W. Porter (1967, Arch. Biochem. Biophys., 118, 716–723). The sulfhydryl group did not oxidize over at least 14 days. This is the first stable sulfhydryl-containing synthetic polymer that has been reported.

This paper reports: (i) the facile synthesis of a cysteine synthon incorporating both a fluorescent group and a triphenylphosphonium derivative (TBTP) via the formation of a disulphide bond, which can subsequently undergo facile intracellular scission, (ii) the direct conjugation of this synthon to a non-permeable drug, (a cyclic PNA (peptide nucleic acid)-based compound has been chosen as a model), and (iii) that this conjugation enables the efficient homogenous delivery of the otherwise non-permeable cyclic PNA into the cytoplasm of cells, as demonstrated by fluorescence microscopy. Our results indicate that this fluorescent-labelled cysteine-TBTP synthon can provide a very useful tool for exploring the cellular uptake of a large range of molecules of biological interest, containing only a single reactive function. The preparation of an activated TBTP derivative is also described and this procedure could be widely used to introduce a TBTP cation to any thio-containing molecule.

The galactofuranose moiety found in many surface constituents of microorganisms is derived from UDP-D-galactopyranose (UDP-Galp) via a unique ring contraction reaction catalyzed by a FAD-dependent UDP-Galp mutase. When the enzyme is reduced by sodium dithionite, its catalytic efficiency increases significantly. Since the overall transformation exhibits no net change in the redox state of the parties involved, how the enzyme-bound FAD plays an active role in the reaction mechanism is puzzling. In this paper, we report our study of the catalytic properties of UDP-Galp mutase reconstituted with deaza-FADs. It was found that the mutase reconstituted with FAD or 1-deazaFAD has comparable activity, while that reconstituted with 5-deazaFAD is catalytically inactive. Because 5-deazaFAD is restricted to net two-electron process, yet FAD and 1-deazaFAD can undergo concerted two-electron as well as stepwise one-electron redox reactions, the above results support a radical mechanism for the mutase catalyzed reaction. In addition, the activity of the mutase reconstituted with FAD was found to increase considerably at high pHs. These observations have allowed us to propose a new mechanism involving one-electron transfer from the reduced FAD to an oxocarbenium intermediate generated by C-1 elimination of UDP to give a hexose radical and a flavin semiquinone. Subsequent radical recombination leads to a coenzyme-substrate adduct which may play a central role to facilitate the opening and recyclization of the galactose ring. A deprotonation step, accompanied or followed the electron transfer step, to increase the nucleophilicity of the flavin radical anion may account for the activity enhancement at pH > 8.

Acyl phosph(on)ates represent a new class of inhibitors of β-lactam—recognizing enzymes. Previously described members of this class were aroyl phosph(on)ates. These compounds have been shown to acylate and/or phosphylate the active site serine residue, leading to either transient or essentially irreversible inhibition [Li, N., and Pratt, R. F. (1998) J. Am. Chem. Soc. 120 , 4264–4268]. The present paper describes the synthesis and evaluation as inhibitors of an inverse pair of acyl phosph(on)ates that incorporate the amido side chain that represents a major substrate specificity determinant of these enzymes. Thus, N -(phenylacetyl)glycyl phenyl phosphate and benzoyl N -(benzyloxycarbonyl)aminomethyl phosphonate were prepared. The former of these compounds was found to be a substrate of typical class A and C β-lactamases and of the DD-peptidase of Streptomyces R61; it thus acylates the active site serine. In contrast, the latter compound was an irreversible inhibitor of the above enzymes, probably by phosphonylation of the active site serine. With each of these enzymes therefore, the amido side chain rather than the acyl group dictates the orientation of the bound phosph(on)ate and thus the mode of reaction.

This contribution describes the biogenetic-type syntheses of some isoquinoline alkaloids and related compounds which, without duplicating our previous review ( 1 ), is based on papers published after 1970.

The fluorescent 1,N6-ethenoadenosine derivatives of adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, 3′:5′-cyclic adenosine monophosphate, adenosine and nicotinamide adenine dinucleotide have been prepared. Paper and thin layer chromatographic purification methods have been developed. Nuclear magnetic resonance and mass spectrum data indicate that only the purine ring has been modified. The 1,N6-ethenoadenosine triphosphate had about 70% of the activity of adenosine triphosphate as a substrate for total adenosine triphosphatase activity of hypophysectomized rat liver membranes. The 1,N6-ethenoadenosine diphosphate had about 86% of the activity of adenosine diphosphate as a substrate for adenosine diphosphatase of hypophysectomized rat liver membranes. The 1,N6-etheno derivative of nicotinamide adenine dinucleotide had about 8% of the activity of nicotinamide adenine dinucleotide as a substrate for nicotinamide adenine dinucleotide glycohydrolase and about 54% of the activity of nicotinamide adenine dinucleotide as a substrate for nicotinamide adenine dinucleotide pyrophosphatase of hypophysectomized rat liver membranes. Km's for the ATPase, ADPase and yeast alcohol dehydrogenase using e-ATP and e-ADP and e-NAD as substrates are presented.

The enzymes phosphotransacetylase (EC 2.3.1.8) or carnitine acetyltransferase (EC 2.3.1.7) and their respective substrates (acetyl phosphate and l -acetylcarnitine) provide the basis for efficient recycling systems for acetyl-CoA. This paper also surveys syntheses and substrate activities of analogs of these two substances.

This paper describes the first examples of asymmetric induction in the oxidative coupling of phenols using chiral oxidants. When chiral cupric-amine complexes were used as oxidants, low asymmetric induction was achieved in the coupling of naphthols. The formation of optically active d -dehydrogriseofulvin and l -Licarin A using the cupric- l-a -phenylethylamine complex perhaps mimics the action of copper-containing enzymes known to catalyze phenol coupling.

The catalytic mechanism of the enzyme carbonic anhydrase has recently been the subject of renewed debate. The generally accepted picture of the mechanism of the enzyme's action has been thrown into doubt by new evidence derived from spectroscopic techniques. In this paper some of the points of conflict are examined and the degree is assessed to which new evidence is incompatible with the previously held picture of CA action. Reinterpretations are put forward of the mechanisms of proton transfer between enzyme and solution and of the detailed mechanism of ligand displacement at the zinc ion. These show that the generally accepted picture of the mechanism of CA action is by no means disproved and remains plausible.

The C-terminal cyanogen bromide fragment of VIP, Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH 2 (all l ), was synthesized to provide evidence for the correctness of the sequence proposed by Mutt and Said ( 1 ). The synthesis of this hendecapeptide (VIP 18–28 ) was carried out by coupling Z-Ala-Val-(Z)Lys to (Z)Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH 2 . After removal of the protecting groups and purification, the synthetic material was indistinguishable from the natural fragment on paper chromatograms and electropherograms. Their identity was further confirmed by comparison of the products formed on enzymatic hydrolysis.

This paper describes the synthesis of 3- S -benzylmercapto-3-methyl- d 8 -butanoic-2,2,4,4,4- d 5 acid ( S -benzyl-deaminopenicillamine- d s ) and [1-deaminopenicillamine- d s ]-oxytocin. Within experimental error the deuterated oxytocin analog possessed the same anti-oxytocic activity as the protio derivative [1-deaminopenicillamine]-oxytocin.