The Mechanism of the Alkaline Hydrolysis of p-Nitrophenyl N-Methylcarbamate<sup>1</sup>

Bender, Myron L.; Homer, Roger B.

doi:10.1021/jo01022a528

Cited by 61 publications

(24 citation statements)

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“…Second, the locus of the nitrogen mustard in prodrug 1 was selected to define the position of the linker arm in order to minimize prodrug alkylation of the abzymes. Third, we chose to promote the disfavored BAC2 mechanisms of carbamate hydrolysis rather than the spontaneous E1cB process to obtain the maximum catalytic rate enhancement for a weakly acidic phenol leaving group (11). Accordingly, a phosphonamidate tetrahedral center (12) the BAC2 cleavage process in anticipation that its N-H group might also promote selection in favor of a hydrogen bond donor-acceptor system in the abzyme for enhanced transitionstate stabilization.…”

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confidence: 99%

Toward antibody-directed "abzyme" prodrug therapy, ADAPT: carbamate prodrug activation by a catalytic antibody and its in vitro application to human tumor cell killing.

Wentworth¹,

Datta²,

Blakey³

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

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Antibody-directed enzyme prodrug therapy, ADEPT, is a recent approach to targeted cancer chemotherapy intended to diminish the nonspecific toxicity associated with many commonly used Antibody-directed enzyme prodrug therapy, ADEPT, has been developed to overcome the unwanted nonspecific toxicity associated with anticancer agents (1-5). There are two components to such therapy: an antibody-enzyme conjugate and an anticancer prodrug of low toxicity. The conjugate is administered first and accumulates predominantly at the tumor site through antibody binding to tumor-associated antigenic determinants. Once the conjugate has been cleared from the plasma, the prodrug is administered to the patient. Cleavage of the prodrug to generate the active cytotoxic agent by the enzyme component of the conjugate occurs selectively at the tumor site and so leads both to enhanced efficacy of the anticancer agent and to reduced peripheral cytotoxicity (Fig. 1).To diminish the extent of peripheral hydrolysis of the prodrug, the enzyme component selected has commonly been of bacterial origin in order to achieve the necessary degree of specificity of activation (3): a human enzyme would be less specific. Unfortunately, such a choice imposes a dose-limiting immunogenicity on the ADEPT conjugate which could reduce the clinical potential of this therapy (6).Antibodies generated against appropriate transition-state analogues (TSAs) can catalyze a variety of chemical transfor-The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. mations (7,8). Furthermore, murine antibodies can be "humanized" by existing technologies to reduce their inherent in vivo mammalian immunogenicity (9). These concepts have been juxtaposed by Bagshawe (6), who has proposed that humanized catalytic antibodies ("abzymes") could replace the enzyme component of ADEPT in an improved targeted therapy. We here demonstrate the feasibility of that proposition, which we have named antibody-directed abzyme prodrug therapy, ADAPT. We have identified an antibody that can efficiently hydrolyze a carbamate prodrug and so effect cytotoxicity in vitro. This provides a major extension to the potential for application of catalytic antibodies to clinical therapy.Our TSA design takes account of three requirements. First, we needed antibodies that could operate on carbamates derived from L-glutamic acid (Fig. 2) to permit direct comparisons of antibody performance with those of the bacterial CPG2 exopeptidase enzyme previously reported to convert this prodrug and give antitumor activity (10). Second, the locus of the nitrogen mustard in prodrug 1 was selected to define the position of the linker arm in order to minimize prodrug alkylation of the abzymes. Third, we chose to promote the disfavored BAC2 mechanisms of carbamate hydrolysis rather than the spontaneous E1cB process to obtain the maximum catalytic rate enhancement f...

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confidence: 99%

Toward antibody-directed "abzyme" prodrug therapy, ADAPT: carbamate prodrug activation by a catalytic antibody and its in vitro application to human tumor cell killing.

Wentworth¹,

Datta²,

Blakey³

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

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“…[15][16][17][18] To clarify this point, ZSarNHPr (Sar = sarcosine or N-methylglycine) was used as substrate. This amino acid derivative contains a N,N-disubstituted carbamate and therefore cannot participate in an E1cB mechanism due to the lack of a deprotonable NH unit.…”

Section: Resultsmentioning

confidence: 99%

Mechanistic Insight into the Lability of the Benzyloxycarbonyl (Z) Group in N‐Protected Peptides under Mild Basic Conditions

2014

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The unexpected lability of Z protecting group under mild basic conditions at room temperature is explained by a mechanism based on anchimeric assistance. It is found that the vicinal amide group stabilizes the tetrahedral intermediate formed after the nucleophilic addition of hydroxide to the carbonyl of the Z group. This effect operates in N-protected tripeptides and tetrapeptides but Zprotected dipeptides are stable under the same conditions due to the blockage of the vicinal amide NH by intramolecular Hbonding with terminal carboxylate moiety.____________ [a] Departament de Química Inorgànica i Orgànica, Universitat Jaume I, Avda. Sos Baynat s/n, Castelló, 12071 Spain Fax: +34964728214 E-mail: miravet@uji.es; escuder@uji.es Homepage: http://supra.uji.es Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/ejoc.xxxxxxxxx. IntroductionAnchimeric assistance or neighbouring group participation constitutes a classic topic in organic chemistry and in particular in organic reaction mechanisms. Widespread examples of unusual reactivity associated to anchimeric assistance have been reported in the last decades and the relevance of this effect is still often highlighted nowadays in different areas of organic chemistry such as saccharides, 1 synthesis of natural products, 2 reactive intermediates, 3 biochemical reactions, 4 nucleotide chemistry 5 or the reactivity of mustard reagents. 6 Here we report on the lability of benzyloxycarbonyl (Z thereafter) N-protecting group in diluted aqueous NaOH at room temperature and on a mechanistic rationale for this behaviour based on anchimeric assistance of a vicinal amide group. The lability of Z group in cold, diluted sodium hydroxide solutions is unexpected if one considers that the literature on this protecting group in peptide and organic chemistry does not report such property. It is well known that the Z group can be cleaved by catalytic hydrogenolysis, acidolysis or, less commonly, by dissolving metal reduction. 7,8 However, Z group is described to tolerate basic conditions 9 and only a few reports are available of its removal under rather harsh conditions such as, refluxing with Ba(OH) 2 in glyme/H 2 O for 40 h or using 40% NaOH in MeOH/H 2 O. 10,11 Results and DiscussionFollowing our previous work on supramolecular hydrogels, 12,13 three isomeric ionizable Z-protected tetrapeptides derived from aspartic acid and phenylalanine were prepared to be used as hydrogelators (Scheme 1). Unexpectedly, we noticed that upon dissolving the peptides in basic water (0.1 M NaOH) the Z groups were rapidly depleted from these molecules at room temperature. 14 1 H NMR spectra showed that the signal corresponding to the benzyl protons of the Z group progressively disappeared giving place to a new signal at at 4.57 ppm which was assigned unambiguously to the benzylic protons of benzyl alcohol (Figure 1; notice that bottom spectrum is poorly resolved due to peptide aggregation. Well resolved spectra were obtained for smaller compounds shown in Tab...

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“…Also acetylcholine esterase, which is carbamate hydrolysis [30] implicates the pulling up of the inhibited by many carbamates [44], did not lose its activity on hydrogen atom beared by the nitrogen atom by a nucleoacetylcholine after incubation in the presence of carbamate 9. philic site of the enzyme. The anionic intermediate leads…”

Section: Active-site Titrationmentioning

confidence: 99%

“…The aim of this study was to test a series of (more or less) stable carbamates which allowed us to design the best inhibitory structure specific for bile-salt-dependent lipase active-site titration. We were also able to determine whether the reactive species of carbamate compounds is an isocyanate intermediate, as suggested by Bender and Homer [30].…”

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confidence: 90%

N‐Butyl‐N‐methyl‐4‐nitrophenyl carbamate as a specific active site titrator of bile‐salt‐dependent lipases

Fourneron

Abouakil²,

Chaillan³

et al. 1991

European Journal of Biochemistry

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carbamates were not even recognized by the enzyme. The N-alkyl chain length is essential for the enzyme inhibition and Nbutyl-(4-nitrophenyl) or N-pentyl-O-phenyl carbamates are more potent inhibitors than N-methyl-O-phenyl or N-isopropyl carbamates. The inhibition by reactive carbamates fits the criteria for mechanism-based inhibition: the inhibition is first-order with time, shows saturation kinetics with increasing carbamate concentration and leads to an inactive stoichiometric enzyme-inhibitor complex ; the enzyme activity can be protected by a competitive inhibitor. Evidence is shown that the enzymatic nucleophilic attack of carbamates is directed at the carbonyl carbon atom and not the nitrogen atom. The inhibition of bile-salt-dependent lipase does not occur consecutive to the formation of a reactive isocyanate derivative of carbamate but via a tetrahedral intermediate involving essential residues implicated in the enzyme catalytic site. This intermediate evolves by liberation of alcohol (or phenol) and formation of an inactive carbamyl enzyme. Among the carbamates tested, N-butyl-N-methyl-(4-nitrophenyl) carbamate specifically inhibits the bile-salt-dependent lipase; the release of 4-nitrophenol from this carbamate is directly proportional to the enzyme inhibition and it may be defined as a specific active-site titrator for bile-salt-dependent lipases.Efficient digestion of fat is achieved through the concerted action of three lipolytic enzymes secreted along the digestive tract. These are the bile-salt-dependent lipase (cholesterol esterase) [I], the pancreatic colipase-dependent lipase [2] and the preduodenal acid lipase [3]. Bile-salt-dependent lipase originating from milk or pancreatic juice [4] presents a wide substrate specificity, since it has been shown to be involved in dietary hydrolysis of triacylglycerol esters, cholesterol esters and lipid-soluble vitamin esters [l, 51. This enzyme, therefore, has been referred to as a non-specific lipase [6], cholesterol ester hydrolase [7], vitamin esterase [8], pancreatic carboxylester hydrolase [9, 101 and bile-salt-dependent (activated or stimulated) lipase [ l l -151. The wide specificity of this enzyme indicates that in the presence of primary bile salts, it is able to hydrolyze each class of lipid esters. As a consequence, a correlation was observed between the enzymatic activity in vivo and adsorption of cholesterol [14, 151, but also with the growth rate of a kitten [16]. Nevertheless, the physiological role of this enzyme is still uncertain and the design of specific inhibitors would assist its elucidation.

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The Mechanism of the Alkaline Hydrolysis of p-Nitrophenyl N-Methylcarbamate¹

Cited by 61 publications

References 0 publications

Toward antibody-directed "abzyme" prodrug therapy, ADAPT: carbamate prodrug activation by a catalytic antibody and its in vitro application to human tumor cell killing.

Toward antibody-directed "abzyme" prodrug therapy, ADAPT: carbamate prodrug activation by a catalytic antibody and its in vitro application to human tumor cell killing.

Mechanistic Insight into the Lability of the Benzyloxycarbonyl (Z) Group in N‐Protected Peptides under Mild Basic Conditions

N‐Butyl‐N‐methyl‐4‐nitrophenyl carbamate as a specific active site titrator of bile‐salt‐dependent lipases

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The Mechanism of the Alkaline Hydrolysis of p-Nitrophenyl N-Methylcarbamate1

Cited by 61 publications

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Toward antibody-directed "abzyme" prodrug therapy, ADAPT: carbamate prodrug activation by a catalytic antibody and its in vitro application to human tumor cell killing.

Toward antibody-directed "abzyme" prodrug therapy, ADAPT: carbamate prodrug activation by a catalytic antibody and its in vitro application to human tumor cell killing.

Mechanistic Insight into the Lability of the Benzyloxycarbonyl (Z) Group in N‐Protected Peptides under Mild Basic Conditions

N‐Butyl‐N‐methyl‐4‐nitrophenyl carbamate as a specific active site titrator of bile‐salt‐dependent lipases

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The Mechanism of the Alkaline Hydrolysis of p-Nitrophenyl N-Methylcarbamate¹