Chloramphenicol Analogues Research Articles

Development and characterisation of an ultrasensitive monoclonal antibody for chloramphenicol

In this study, a highly sensitive monoclonal antibody (McAb) against chloramphenicol (CAP) was successfully raised and characterised by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). Three antigens coupled with different proteins were synthesised and determined by the UV-vis method. After five rounds of immunisation, antisera were collected from six Balb/C mice and screened by ic-ELISA. The standard inhibition curve of one CAP McAb (labelled 6F11) showed the best sensitivity to CAP after optimisation of the principal working conditions. The half-maximal inhibition concentration (IC50) was calculated to be 0.01 ng/mL and the linear working range was from 0.0028 ng/mL to 0.040 ng/mL with a correlation coefficient of 0.996. This McAb showed high specificity, with negligible cross-reactivities detected towards CAP analogues (florfinicol, florfinicol amine, thiamphenicol). The recovery rate was 98.9–106.8% while the coefficient of variation was 4.45–6.05%. The IC50 value and other parameters obtained for this CAP McAb suggest that it is likely to have a promising future in further applications in combination with different analytical techniques.

Two Novel Alkylating Structural Analogues of Chloramphenicol Suitable for Topical Treatment of Dermal Neoplasms

Aims: To evaluate the efficacy of two alkylating structural analogues of chloramphenicol that have potential for application for treatment of dermal sited neoplasms. Study Design: Two compounds have been shown to alkylate guanosine-5’-diphosphate (GDP) at physiological conditions. These same compounds are evaluated for dermal penetration based on Kp and compared to other alkylating agents applied for treatment of skin cancer. Place and Duration of Study: University of Nebraska, Omaha Nebraska from December 2013 to May 2014 and March to August of 2002. Methodology: Two analogues of chloramphenicol were synthesized and shown to alkylate GDP at pH 7.4 and 37oC. Various pharmacological properties of these two analogues, such as Log P, molecular weight, polar surface area, etc, were determined and compared. The dermal permeability coefficient Kp was determined for two analogues based on their molecular weight and partition coefficient Log P. The numerical values of Kp were used to prediction of the distance each analogue is expected to travel in penetration of a dermal barrier. Result was plotted and compared to the anticancer agent’s carmustine, mustargen, and 5-fluorouracil. Evaluation of the analogues included findings of previous studies. Results: Two analogues of chloramphenicol alkylate sites on GDP. The properties of Original Research Article Journal of Advances in Medical and Pharmaceutical Sciences, 1(1): 11-23, 2014 12 compound 1 and compound 2 were determined and when compared to the parent structure chloramphenicol were found to have favorable drug likeness. Values of Log P and permeability coefficient Kp for compounds 1 and 2 are; 3.343, 3.312, 0.00244 cm/hour, 0.000768 cm/hour, respectively. Values of Kp for both compound 1 and 2 were greater than that of chloramphenicol at 0.000131 cm/hour. Plots of skin penetration showed compounds 1 and 2 to be superior to 5-fluorouracil. Conclusion: Analogues 1 and 2 were shown to have alkylation activity and properties suitable for drug likeness. Both compounds have high penetration rates of dermal layers.

Synthesis, Characterization, and Antimicrobial Studies of Novel Benzodipyran Analog of Chloramphenicol

A synthesis, characterization, and antimicrobial study of a novel benzodipyran analog of chloramphenicol was carried out. Structure–antimicrobial activity relationship study indicates that benzodipyran analog of chloramphenicol was most active against Gram‐positive bacteria, Staphylococcus aureus, and the fungal strains Rhizoctonia bataticola and Penicillium even at minimum inhibitory concentration 10 µg/mL and showed moderate activity in other bacterial and fungal organisms. All the compounds synthesized during the present investigation were characterized by IR, 1H NMR, 13C NMR, ESI‐MS, and elemental analysis.

Versatile synthesis of probes for high-throughput enzyme activity screening

Mass spectrometry based technologies are promising as generalizable high-throughput assays for enzymatic activity. In one such technology, a specialized enzyme substrate probe is presented to a biological mixture potentially exhibiting enzymatic activity, followed by an in situ enrichment step using fluorous interactions and nanostructure-initiator mass spectrometry. This technology, known as Nimzyme, shows great potential but is limited by the need to synthesize custom substrate analogs. We describe a synthetic route that simplifies the production of these probes by fashioning their perfluorinated invariant portion as an alkylating agent. This way, a wide variety of compounds can be effectively transformed into enzyme activity probes. As a proof of principle, a chloramphenicol analog synthesized according to this methodology was used to detect chloramphenicol acetyltransferase activity in cell lysate. This verifies the validity of the synthetic strategy employed and constitutes the first reported application of Nimzyme to a non-carbohydrate-active enzyme. The simplified synthetic approach presented here may help advance the application of mass spectrometry to high-throughput enzyme activity determination. FigureThe Nimzyme high-throughput enzyme activity assay allows for the detection of enzyme activity in cell lysate. Fluorous interactions between a specialized substrate probe and a nanostructure-initiator mass spectrometry surface allow for in situ cleanup and the subsequent collection of unambiguous mass spectra. One of the main hurdles that prevents the widespread adoption of this technology is the need to chemically synthesize the required probes. Here, we present a simplified route to derive Nimzyme probes from a wide variety of biologically interesting substrates. Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-013-6888-z) contains supplementary material, which is available to authorized users.

Quantitative trace analysis of eight chloramphenicol isomers in urine by chiral liquid chromatography coupled to tandem mass spectrometry

Chloramphenicol is a broad-spectrum antibiotic with, apart from its human medicinal use, veterinary abuse in all major food-producing animals. Chloramphenicol occurs in four stereoisomers (all para-nitro substituted) and furthermore four meta-nitro analogs of chloramphenicol exist. In this paper these are referred to as eight chloramphenicol isomers. According to EU regulations an analytical method should be able to discriminate the analyte from interfering substances that might be present in the sample, including isomers. For the first time a quantitative method for the analysis of trace levels of eight chloramphenicol isomers in urine by chiral liquid chromatography in combination with tandem mass spectrometric detection is reported. The separation of the isomers on the analytical column, the clean-up of urine and the selectivity of the monitored product ions turned out to be critical parameters. To obtain reproducible retention isocratic elution on a chiral AGP column was applied. For urine samples matrix compounds present in the final extract caused decreased retention of the isomers on the chiral stationary phase and a lack of chromatographic resolution. Therefore an extended clean-up procedure that combines solid phase extraction and liquid–liquid extraction had to be developed. The final method was fully validated and showed satisfactory performance for all isomers with decision limits (CCα) ranging from 0.005 to 0.03 μg L −1 and within-laboratory reproducibility of all isomers below 20% at the minimum required performance limit level of 0.3 μg L −1.

Inhibition of Helicobacter pylori aminoacyl-tRNA amidotransferase by chloramphenicol analogs

Genomic studies revealed the absence of glutaminyl-tRNA synthetase and/or asparaginyl-tRNA synthetase in many bacteria and all known archaea. In these microorganisms, glutaminyl-tRNA Gln (Gln-tRNA Gln) and/or asparaginyl-tRNA Asn (Asn-tRNA Asn) are synthesized via an indirect pathway involving side chain amidation of misacylated glutamyl-tRNA Gln (Glu-tRNA Gln) and/or aspartyl-tRNA Asn (Asp-tRNA Asn) by an amidotransferase. A series of chloramphenicol analogs have been synthesized and evaluated as inhibitors of Helicobacter pylori GatCAB amidotransferase. Compound 7a was identified as the most active competitive inhibitor of the transamidase activity with respect to Asp-tRNA Asn ( K m = 2 μM), with a K i value of 27 μM.

Chromatographic evaluation of polymers imprinted with analogs of chloramphenicol and application to selective solid-phase extraction

To obtain a highly selective material for the antibiotic chloramphenicol, which has several harmful side effects in humans, different molecularly imprinted polymers (MIPs) were prepared. In order to avoid a major traditional drawback associated with MIPs of residual template bleeding, molecules that are structurally related to chloramphenicol were used as templates for polymer synthesis. Chromatographic evaluation indicated that the employed template imparted a significant influence on the recognition properties of the corresponding polymer. A strong retention of chloramphenicol under nonpolar elution conditions (k = 68.03, IF = 17.72) and under aqueous elution conditions (k = 92.44, IF = 1.35) was achieved. After chromatographic evaluation, the MIP was utilized as the recognition sorbent in a solid-phase extraction to determine chloramphenicol using either an organic or aqueous washing solvent. Recoveries of nearly 100% from the chloramphenicol standard solution and nearly 90% from honey samples spiked with chloramphenicol were attained. Furthermore, the applicability of the MIP for sample cleanup was demonstrated.

A Versatile Photocleavable Bifunctional Linker for Facile Synthesis of Substrate−DNA Conjugates for the Selection of Nucleic Acid Catalysts

Covalent photocleavable attachment of small molecules or peptides to oligonucleotides is an integral strategic element in the selection of novel nucleic acid enzymes. Here, we report the synthesis of a multipurpose, photocleavable bifunctional linker (PCBL) suitable for nucleic acid selections and other biotechnology applications. PCBL contains a photocleavable O-nitrobenzyl group flanked on one side by an N-hydroxysuccinimidyl ester (reactive toward primary amines) and on the other side by a sulfhydryl. To demonstrate the utility of PCBL, the linker was used to couple an analog of the antibiotic chloramphenicol (Cam) to the 5' end of an amino-modified 8-mer DNA oligo. Coupling was confirmed by MALDI-TOF spectrophotometry. Decoupling was performed by irradiating the coupled species with near-UV light (approximately 360 nm), regenerating the original amino-modified oligo. Ligation of the Cam-PCBL-DNA conjugate to random-sequence RNA generated a diversity library appropriate for the selection of new ribozymes that catalyze reactions involving the tethered substrate. Coupling and decoupling of the Cam analog from the library was monitored on a trilayered organomercurial polyacrylamide gel. The coupling/decoupling strategy described here is readily generalized to many combinations of macromolecules and small molecules. For example, analogs of this small molecule-DNA conjugate can be generated as synthons for ligation to nucleic acid diversity libraries during each round of novel ribozyme selections, or they can be immobilized onto chips for addresssably reversible microarray analysis.

Continuous synthesis of aminophenols from nitroaromatic compounds by combination of metal and biocatalyst

The combined action of immobilized hydroxylaminobenzene mutase and zinc in a flow-through system catalyzes the conversion of nitroaromatic compounds to the corresponding ortho-aminophenols, including a novel analog of chloramphenicol.

Pharmacokinetics of thiamphenicol in pigs.

Pharmacokinetics of thiamphenicol in pigs.

Serum and lung levels of thiamphenicol after administration of its glycinate N-acetylcysteinate ester in experimentally infected guinea pigs

Thiamphenicol is an analogue of chloramphenicol and is characterised by a broad spectrum of action. In this study, serum and lung levels of thiamphenicol (TAP) were studied in infected guinea pigs after the administration of thiamphenicol glycinate N-acetylcysteinate (TGA). Animals received a single dose of TGA (15 mg/kg, subcutaneously) immediately after intra-tracheal infection with Haemophilus influenzae (about 10 7 CFU/animal). Serum and lung concentrations of TAP were determined at 0, 1, 3, 6, 12 and 24 h after drug administration by means of HPLC. TAP serum levels were elevated at 1 h and remained detectable for 24 h after drug administration. Tissue lung levels were comparable to peak serum concentrations but remained higher and decreased more slowly than serum concentrations.

Aminoacyl and peptidyl analogs of chloramphenicol as slow-binding inhibitors of ribosomal peptidyltransferase: a new approach for evaluating their potency.

In a model system derived from Escherichia coli, acetylphenylalanyl-puromycin is produced in a pseudo-first-order reaction between the preformed acetylphenylalanyl/tRNA/poly(U)/ribosome complex (complex C) and excess puromycin. Two aminoacyl analogs [3, Gly-chloramphenicol (CAM): 4, L-Phe-CAM] and two peptidyl analogs (2, L-Phe-Gly-CAM: 5, Gly-Phe-CAM) of CAM (1) were tested as inhibitors in this reaction. Detailed kinetic analysis suggests that these analogs (I) react competitively with complex C and form the complex C*l, which is inactive toward puromycin. C*l is formed via a two-step mechanism in which C*l is the product of a slow conformational change of the initial encounter complex Cl according to the equation C + l reversible Cl reversible C*l. Furthermore, we provide evidence that analog 5 may react further with C*l forming the species C*l2. The values of the apparent association rate constant (K(assoc)) are 1.42 x microM-1 min-1 for 2, 0.55 x microM-1 min-1 for 3, and 0.18 x microM-1 min-1 for 4 and 0.038 x microM-1 min-1 for 5 [corrected]. In the case of analog 5, K(assoc) is a linear function of the inhibitor concentration; when [I] approaches zero, the K(assoc) value is equal to 3.8 x 10(2) M-1 sec-1. Such values allow the classification of CAM analogs as slow-binding inhibitors. According to K(assoc) values, we could surmise that analog 2 is 2.5-fold more potent than 3 and 8-fold more potent than 4. The relative potency of analog 5 is the lowest among the analogs and is dependent on its concentration. The results are compared with previous data and discussed on the basis of a possible retro-inverso relationship between CAM analogs and puromycin.

Structural determinants of cytochrome P450 2B1 specificity: evidence for five substrate recognition sites.

Twelve site-directed mutants of rat cytochrome P450 2B1 distributed over seven positions and four putative substrate recognition sites (SRS) were constructed and expressed in COS cells. Function was examined using androstenedione and testosterone as substrates. Substitutions at positions 303, 360, and 473 did not markedly affect the regio- or stereoselectivity of androgen metabolism, whereas mutants in positions 206 (SRS-2), 302 (SRS-4), and 363 and 367 (SRS-5) exhibited markedly different steroid metabolite profiles compared with parental P450 2B1. In particular, the Phe-206-->Leu substitution conferred androgen 6 alpha- and testosterone 7 alpha-hydroxylase activities, and the Thr-302-->Ser substitution suppressed androgen 16 beta-hydroxylation in favor of androstenedione 16 alpha- and testosterone 15 alpha-hydroxylation. Replacement of Val-363 or Val-367 with Ala conferred androgen 15 alpha-hydroxylase and 6 beta-hydroxylase activities, respectively, and suppressed susceptibility to mechanism-based inactivation by the P450 2B1-selective chloramphenicol analog N-(2-p-nitrophenethyl)chlorofluoroacetamide. The Val-367-->Ala mutant was also resistant to chloramphenicol itself. The Leu mutant at position 363 exhibited increased specificity for androstenedione and testosterone 16 beta-hydroxylation, whereas the Leu mutant at position 367 exhibited decreased stereospecificity. Most interestingly, the size of key residues identified plays a critical role in governing steroid hydroxylation from the alpha-face or beta-face and hydroxylation on the D-ring or the B-ring.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminoacyl analogs of chloramphenicol: examination of the kinetics of inhibition of peptide bond formation.

Two aminoacyl analogs and one peptidyl analog of chloramphenicol (1, Cl2CHCO-CA) were prepared. These are 2 (L-Phe-CA), 3 (Gly-CA), and 4 (L-Phe-Gly-CA). The kinetics of inhibition of peptide bond formation by these analogs were examined in a cell-free system which was derived from E. coli and used previously for the study of 1 (Drainas; et al. Eur. J. Biochem. 1987, 164, 53-58). In the absence of inhibitor, the reaction follows first-order kinetics for the entire course of the reaction. In the presence of the analog the reaction gives biphasic log-time plots. The kinetic information pertaining to the initial slope of the plot is analyzed (initial-slope analysis). This information differentiates the analogs from the parent compound 1. The parent compound 1 gives complex inhibition kinetics; increasing the concentration of 1 changes the inhibition from competitive to mixed noncompetitive (Drainas; et al. Eur. J. Biochem. 1987, 164, 53-58). In contrast, the analogs give competitive kinetics even at high concentrations of the inhibitor. The following Ki values have been determined: 18.0 microM for 2, 5.5 microM for 3, 1.5 microM for 4. If we were to assume that compounds 2, 3 and 4 behave as classical competitive inhibitors, we could say that 4 is 12 times more potent than 3 and 4 times more potent than 2. On this assumption we could also compare 1 with 4 and see that 4 is 2 times weaker than 1. It is suggested that as compared with 1, the two aminoacyl analogs and the dipeptidyl analog have increased structural similarity to the 3'-terminus of aminoacyl-tRNA or of peptidyl-tRNA and that this similarity results in a more pronounced competitive inhibition.

Role of residue 478 as a determinant of the substrate specificity of cytochrome P450 2B1.

Two allelic variants and eight site-directed mutants of cytochrome P450 2B1 differing at residue 478 have been expressed in COS cells and assayed for androstenedione hydroxylase activities. The 478Gly and 478Ala variants and five mutants (Ser, Thr, Val, Ile, and Leu) exhibited 16 beta-OH:16 alpha-OH ratios ranging from 0.7 to 9.3, whereas the Pro, Glu, and Arg mutants were expressed but inactive. The seven samples active toward androstenedione also exhibited testosterone 16 beta-OH:16 alpha-OH ratios ranging from 0.4 to 2.3. With both steroids, the Gly variant had the highest 16 beta-hydroxylase activity, and the 16 beta-OH:16 alpha-OH ratio increased with the size of aliphatic size chains (Ala, Val, and Ile/Leu). The highest ratio of androgen 15 alpha:16-hydroxylation was observed with the Ser mutant. On the basis of previous work indicating decreased susceptibility of the 478Ala variant in liver microsomal and reconstituted systems to inactivation by chloramphenicol analogs, methodology was refined for monitoring enzyme inactivation in COS cell microsomes. The Gly and Ala variants were inactivated by chloramphenicol with similar rate constants, whereas the Ser and Val mutants were inactivated more slowly, and the Leu mutant was refractory. Only the Gly variant was inactivated by the chloramphenicol analog N-(2-p-nitrophenethyl)chlorofluoroacetamide. Thus, the side chain of residue 478 appears to be a major determinant of enzyme inactivation as well as of androgen hydroxylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Intrinsic fluorescence of chloramphenicol acetyltransferase: responses to ligand binding and assignment of the contributions of tryptophan residues by site-directed mutagenesis

Replacement by tyrosine or phenylalanine was used to assign the additive contributions of each of the three tryptophan residues of chloramphenicol acetyltransferase (CAT) to its intrinsic fluorescence on excitation at 295 nm. During the assessment of the fluorescence responses of the wild-type enzyme to the binding of ligands, it was found that the overlapping absorption spectra of chloramphenicol and tryptophan, with an attendant inner filter effect, required the use of a displacement technique involving an alternative substrate (the p-cyano analogue of chloramphenicol) without significant absorption at 295 nm. By the use of two-Trp, one-Trp, and Trp-less variants, in combination with this displacement technique, it was possible to demonstrate that Trp-86 and Trp-152 are involved in the fluorescence quenching associated with the binding of chloramphenicol, most likely via nonradiative energy transfer from these residues to the bound substrate. Trp-152 is mainly responsible for the fluorescence enhancement accompanying the binding of acetyl-CoA (and CoA) through proximity effects and solvent exclusion on substrate association.

A nonradioactive assay for transfected chloramphenicol acetyltransferase activity using fluorescent substrates

Studies of the transcriptional activity of gene promoters have been greatly assisted by the widespread use of the chloramphenicol acetyltransferase (CAT) gene as a reporter gene. Previous techniques for assaying CAT enzymatic activity have utilized radioactive substrates or cofactors with the resulting complications of handling radioactive materials. We report here the development of fluorescent substrates for the CAT enzyme which form the basis of a CAT enzyme assay of enzyme kinetic parameters ( K m and V max) and sensitivity similar to those based on radioactive substrates. Fluorescent substrates were designed as analogs of chloramphenicol and were based on the structure-function requirements of the enzyme. Several fluorophores were used to derivatize chloramphenicol base; one of the most effective was the borondipyrromethene difluoride (BODIPY) fluorophore. One BODIPY-chloramphenicol analog was found to have a K m for the purified CAT enzyme of 2 μ m (compared to 12 μ m for 14C-labeled chloramphenicol) and a V max of 120 pmole/min (compared to 180 pmol/min for the radioactive substrate). To verify its usefulness, a BODIPY-chloramphenicolbased CAT assay was used to measure transient transfection of primary cultures of ovarian granulosa cells in serum-free medium. This experimental system requires a highly sensitive assay for detecting transfected CAT gene activity. Robust expression of CAT activity was easily detected in crude cellular extracts using FluoReporter FAST CAT, a kit containing the BODIPY-chloramphenicol analog. The expression was precisely quantified by methanol extraction of the substrate and products from TLC plates and subsequent measurement of fluorescence using excitation-emission spectroscopy. These data demonstrate that the new fluorescent BODIPY-chloramphenicol analogs are highly effective as substrates in CAT enzyme assays and offer the advantages of providing results quickly, eliminating the handling and disposal of radioactivity, and furnishing an assay of a sensitivity comparable to those based on radioactive substrates.

Alternative binding modes for chloramphenicol and 1-substituted chloramphenicol analogs revealed by site-directed mutagenesis and x-ray crystallography of chloramphenicol acetyltransferase

Leucine-160 of chloramphenicol acetyltransferase (CAT) has been replaced by site-directed mutagenesis to investigate enzyme-ligand interactions at the 1-hydroxyl substituent of the substrate chloramphenicol. The consequences of the substitution of Leu-160 by glutamine and by phenylalanine were deduced from the steady-state kinetic parameters for acetyl transfer from acetyl-CoA to the 3-hydroxyl of chloramphenicol and its analogues 1-deoxychloramphenicol and 1-acetylchloramphenicol. The acetyl group of the latter, which is a substrate both in vivo and in vitro, could potentially bind in a similar position to the 1-hydroxyl of chloramphenicol, in close proximity to the side chain of Leu-160. In the case of Gln-160 CAT, large increases in Km for the three acetyl acceptors were accompanied by small decreases in kcat and in apparent affinity for acetyl-CoA. Such results are consistent with the introduction of the relatively hydrophilic amide in place of the delta-methyl groups of Leu-160. The kinetic properties of Phe-160 CAT were unexpected in that Km for each of the three acetyl acceptors was unchanged or reduced, compared to the equivalent parameters for the wild-type enzyme, whereas kcat fell significantly (44-83-fold) in each case. The ratios of specificity constants (kcat/Km) for the acetylation of chloramphenicol compared with the alternative acyl acceptors were similar for wild-type and mutant enzymes. As the residue substitutions for Leu-160 do not result in enhanced discrimination against the binding and acetylation of 1-acetylchloramphenicol, it appears unlikely that the 1-acetyl group binds to the CAT active site in the same position as that occupied by the 1-hydroxyl of chloramphenicol.(ABSTRACT TRUNCATED AT 250 WORDS)

Porphyrinogenic effects in chick embryo liver cell culture of chloramphenicol analogues that are mechanism-based inactivators of cytochrome P-450

Structural analogues of chloramphenicol (CAP) cause mechanism-based inactivation of rat liver cytochrome P-450 (P450) either via protein acylation or destruction of the heme prosthetic group. The goal of the present work was to determine whether CAP analogues that cause loss of the P450 heme moiety also cause porphyrin accumulation in chick embryo liver cell culture. The porphyrin profiles produced by exposure of cells to CAP analogues (160 microM) were determined by high-performance liquid chromatography with fluorescence detection. Of three CAP analogues that do not cause loss of the heme moiety of rat liver P450IIB1, two dichloroacetamides were not porphyrinogenic. The third compound, a chlorofluoroacetamide, caused porphyrin accumulation. This result may be due to the presence of P450 isozymes in chick embryo hepatocytes, distinct from rat liver P450IIB1, that are susceptible to destruction by this analogue. Of four CAP analogues that inactivate rat liver P450IIB1 with concomitant heme loss, a dichloroacetamide and two chlorofluoroacetamides caused porphyrin accumulation. The remaining compound, a monochloroacetamide, was not porphyrinogenic, perhaps because the P450 apoprotein cannot be reconstituted with fresh heme drawn from the regulatory "free heme pool" following inactivation by this analogue. Alternatively, there may be no P450 isozyme in chick embryo liver cell culture that is susceptible to inactivation by this compound.

Adamantylmethyl analogues of chloramphenicol

The synthesis and biological activity of novel adamantylmethyl analogues of chloramphenicol are described. Substituting the polar para-nitro group on the phenyl ring with a non-polar lipophilic adamantylmethyl moiety resulted in a diminished antimicrobial activity. At a daily dose of 3 mg/kg the threo-4-[2′-tricyclo[3.3.1.1 3,7]decylidene)-methyl-1-[1″, 3″-dihydroxy-2″-(α,α-dichloroacetamido)propyl]benzene caused a significant loss of humoral immuno-competence in the T-dependent Kennedy plaque assay in mice.