Potential Affinity Labels Research Articles

Design and synthesis of substituted N-methylbenzamide analogues derived from SR 48,968 as neurokinin-2 receptor antagonists

A series of N-methylbenzamide analogues ( 2– 18) that is structurally derived from SR 48,968, a potent neurokinin-2 (NK 2) receptor antagonist (p K b 9.1), has been obtained using asymmetric synthesis. Isothiocyanato- N-methylbenzamide ( 10– 12) and bromoacetamido- N-methylbenzamide derivatives ( 16– 18) have been designed to serve as potential electrophilic affinity labels. Nitro- N-methylbenzamide ( 4– 6) and acetamido- N-methylbenzamide ( 13– 15) were designed to serve as the nonelectrophilic controls for these ligands. Functional assay results using guinea pig trachea indicate that electrophilic N-methylbenzamide analogues exhibit potent but surmountable NK 2 receptor antagonist activity. Several members of this series ( 2, 3, 7– 9) exhibit potent NK 2 receptor antagonist potencies with p K b values in the range of 9.1–9.7. para-Fluoro substituted analogue 3 was found to be highly potent with a p K b of 9.7.

An affinity label for delta-opioid receptors derived from [D-Ala2]deltorphin I.

A series of potential affinity label derivatives of the amphibian opioid peptide [D-Ala2]deltorphin I were prepared by incorporation at the para position of Phe3 (in the 'message' sequence) or Phe5 (in the 'address' sequence) of an electrophilic group (i.e. isothiocyanate or bromoacetamide). The introduction of the electrophile was accomplished by incorporating Fmoc-Phe(p-NHAlloc) into the peptide, followed later in the synthesis by selective deprotection of the Alloc group and modification of the resulting amine. While para substitution decreased the delta-opioid receptor affinity, selected analogs retained nanomolar affinity for delta receptors. [D-Ala2,Phe(p-NCS)3]deltorphin I exhibited moderate affinity (IC50=83 nM) and high selectivity for delta receptors, while the corresponding amine and bromoacetamide derivatives showed pronounced decreases in delta-receptor affinity (80- and >1200-fold, respectively, compared with [D-Ala2]deltorphin I). In the 'address' sequence, the Phe(p-NH2)5 derivative showed the highest delta-receptor affinity (IC50=32 nM), while the Phe(p-NHCOCH2Br)5 and Phe(p-NCS)5 peptides displayed four- and tenfold lower delta-receptor affinities, respectively. [D-Ala2,Phe(p-NCS)3]deltorphin I exhibited wash-resistant inhibition of [3H][D-Pen2,D-Pen5]enkephalin (DPDPE) binding to delta receptors at a concentration of 80 nM. [D-Ala2, Phe(p-NCS)3]deltorphin I represents the first affinity label derivative of one of the potent and selective amphibian opioid peptides, and the first electrophilic affinity label derivative of an agonist containing the reactive functionality in the 'message' sequence of the peptide.

13-Demethyl-13-substituted-13,14-dihydroretinols as potential affinity labels of retinol-binding proteins: syntheses and stability studies

13-Demethyl-13-substituted-13,14-dihydroretinols were synthesized and their stability under various conditions was measured in order to evaluate whether they would be useful as affinity labels of retinol binding proteins and retinol metabolizing enzymes. The 13-chloro analog could not be isolated because it eliminated HCl under the Wittig reaction conditions of its preparation. The trans- and cis-13,14-epoxy analogs are stable in non-protic organic solvents, but undergo an elimination reaction under various chromatographic conditions and in mixtures of organic solvents with water or alcohol. The 13-hydroxy and 13-methoxy analogs are stable in aqueous solutions and are therefore suitable for biological studies.

Synthesis and evaluation of potential affinity labels derived from endomorphin-2.

In an attempt to identify potential peptide-based affinity labels for opioid receptors, endomorphin-2 (Tyr-Pro-Phe-PheNH2), a potent and selective endogenous ligand for mu-opioid receptors, was chosen as the parent peptide for modification. The tetrapeptide analogs were prepared using standard Fmoc-solid phase peptide synthesis in conjunction with incorporation of Fmoc-Phe(p-NHAlloc) and modification of the p-amino group. The electrophilic groups isothiocyanate and bromoacetamide were introduced into the para position on either Phe3 or Phe4; the corresponding free amine-containing peptides were also prepared for comparison. The peptides bearing an affinity label group and their free amine analogs were evaluated in a radioligand-binding assay using Chinese hamster ovary (CHO) cells expressing mu- and delta-opioid receptors. Modification on Phe4 was better tolerated than on Phe3 for mu-receptor binding. Among the analogs tested, [Phe(p-NH2)4]endomorphin-2 showed the highest affinity (IC50 = 37 nm) for mu-receptors. The Phe(p-NHCOCH2Br)4 analog displayed the highest mu-receptor affinity (IC50 = 158 nm) among the peptides containing an affinity label group. Most of the compounds exhibited negligible binding affinity for delta-receptors, similar to the parent peptide.

Dermorphin-based potential affinity labels for mu-opioid receptors.

Dermorphin and [Lys7]dermorphin, selective micro -opioid receptor ligands originating from amphibian skin, have been modified with various electrophiles in either the 'message' or 'address' sequences as potential peptide-based affinity labels for micro -receptors. Introduction of the electrophilic isothiocyanate and bromoacetamide groups on the para position of Phe3 and Phe5 was accomplished by incorporating Fmoc-Phe(p-NHAlloc) into the peptide followed by selective deprotection and modification. The corresponding amine-containing peptides were also prepared. The pure peptides were evaluated in radioligand binding experiments using Chinese hamster ovary (CHO) cells expressing micro - and delta-opioid receptors. In dermorphin, introduction of the electrophilic groups in the 'message' domain lowered the binding affinity by > 1000-fold; only [Phe(p-NH2)3]dermorphin retained nanomolar affinity for micro -receptors. Modifications in the 'address' region of both dermorphin and [Lys7]dermorphin were relatively well tolerated. In particular, [Phe(p-NH2)5,Lys7]dermorphin showed similar affinity to dermorphin, with almost 2-fold higher selectivity for micro -receptors. [Phe(p-NHCOCH2Br)5]- and [Phe(p-NHCOCH2Br)5,Lys7]dermorphin exhibited relatively high affinity (IC50 = 27.7 and 15.1 nm, respectively) for micro -receptors. However, neither of these peptides inhibited [3H]DAMGO binding in a wash-resistant manner.

Synthesis and evaluation of derivatives of leucine enkephalin as potential affinity labels for delta opioid receptors.

As part of an effort to develop peptide-based affinity labels for opioid receptors, [Leu(5)]enkephalin (LeuEnk) and DTLET (Tyr-D-Thr-Gly-Phe-Leu-Thr), potent agonists for delta receptors, were selected as the parent peptides for further modification. The affinity label derivatives were prepared using standard Fmoc solid-phase peptide synthesis in conjunction with Fmoc-Phe(p-NHAlloc) (Fmoc: 9-flourenylmethoxycarbonyl;) and selective modification of the p-amino group on this residue. The electrophilic isothiocyanate and bromoacetamide groups were introduced into the para position of Phe(4); the corresponding free amine-containing peptides were also prepared for comparison. The pure peptides were evaluated in radioligand binding assays using Chinese hamster ovary (CHO) cells expressing delta and micro opioid receptors. Modification of Phe(4) in LeuEnk and DTLET significantly decreased delta-receptor binding affinity (40 to >2,000-fold). Among the synthesized analogues, [Phe(p-NH(2))(4)]DTLET showed the highest delta-receptor binding affinity (IC(50) = 39 nM) and enhanced selectivity for delta receptors compared to DTLET while other derivatives exhibited much lower delta receptor affinity. The differences in affinities between the two series of analogues and between the derivatives of LeuEnk and N,N-dibenzyl[Leu(5)]Enk reported previously suggest subtle differences in interactions of Phe(4) with delta receptors depending on other modifications in the sequences.

Functionalized congeners of tyrosine-based P2X(7) receptor antagonists: probing multiple sites for linking and dimerization.

Chemically funtionalized analogues of antagonists of the P2X(7) receptor, an ATP-gated cation channel, were synthesized as tools for biophysical studies of the receptor. These functionalized congeners were intended for use in chemical conjugation with retention of biological potency. The antagonists were L-tyrosine derivatives, related to [N-benzyloxycarbonyl-O-(4-arylsulfonyl)-L-tyrosyl]benzoylpiperazine (such as MRS2409, 2). The analogues were demonstrated to be antagonists in an assay of human P2X(7) receptor function, consisting of inhibition of ATP-induced K(+) efflux in HEK293 cells expressing the recombinant receptor. The analogues were of the general structure R(1)-Tyr(OR(2))-piperazinyl-R(3), in which three positions (R(1)-R(3)) were systematically varied in structure through introduction of chemically reactive groups. Each of the three positions was designed to incorporate a 3- or 4-nitrophenyl group. The nitro groups were reduced using NaBH(4)-copper(II) acetylacetonate to amines, which were either converted to the isothiocyanate groups, as potential affinity labels for the receptor, or acylated, as models for conjugation. An alternate route to N(alpha)-3-aminobenzyloxycarbonyl functionalization was devised. The various positions of functionalization were compared for effects on biological potency, and the R(2) and R(3) positions were found to be most amenable to derivatization with retention of high potency. Four dimeric permutations of the antagonists were synthesized by coupling each of the isothiocyanate derivatives to either the precursor amine or to other amine congeners. Only dimers linked at the R(2)-position were potent antagonists. In concentration-response studies, two derivatives, a 3-nitrobenzyloxycarbonyl derivative 18 and a 4-nitrotoluenesulfonate 26b, displayed IC(50) values of roughly 100 nM as antagonists of P2X(7) receptor-mediated K(+) flux.

Investigation of the Active Site of the Extracellular β-[formula omitted]-Xylosidase from Aspergillus carbonarius

The catalytic amino acid residues of the extracellular β-d-xylosidase (β-d-xyloside xylohydrolase, EC 3.2.1.37) from Aspergillus carbonarius was investigated by the pH dependence of reaction kinetic parameters and chemical modifications of the enzyme. The pH dependence curves gave apparent pK values of 2.7 and 6.4 for the free enzyme, while pK value of 4.0 was obtained for the enzyme–substrate complex using p-nitrophenyl β-d-xyloside as a substrate. These results suggested that a carboxylate group and a protonated group—presumably a histidine residue—took part in the binding of the substrate but only a carboxylate group was essential in the substrate cleavage. Carbodiimide- and Woodward's reagent K-mediated chemical modifications of the enzyme also supported that a carboxylate residue, located in the active center, was fundamental in the catalysis. The pH dependence of inactivation revealed the involvement of a group with pK value of 4.4, proving that a carboxylate residue relevant for hydrolysis was modified. During modification Vmax decreased to 10% of that of the unmodified enzyme and Km remained unchanged, supporting that the modified carboxylate group participated in the cleavage and not in the binding of the substrate. We synthesized and tested a new, potential affinity label, N-bromoacetyl-β-d-xylopyranosylamine for β-d-xylosidase. The A. carbonarius β-d-xylosidase was irreversible inactivated by N-bromoacetyl-β-d-xylopyranosylamine. The competitive inhibitor β-d-xylopyranosyl azide protected the enzyme from inactivation proving that the inactivation took place in the active center. Kinetic analysis indicated that one molecule of reagent was necessary for inactivation of one molecule of the enzyme.

Synthesis and evaluation of isothiocyanate-containing derivatives of the delta-opioid receptor antagonist Tyr-Tic-Phe-Phe (TIPP) as potential affinity labels for delta-opioid receptors.

Derivatives of the delta-opioid receptor-selective peptide antagonist H-Tyr-Tic-Phe-Phe-OH (TIPP) containing an isothiocyanate moiety at the para position of either Phe(3) or Phe(4) were prepared as potential affinity labels for delta-opioid receptors. The synthesis was accomplished using a general solution-phase synthetic procedure which allows for introduction of affinity labeling groups late in the synthesis of a variety of small peptide substrates. The target peptides and their corresponding amines were then evaluated in radioligand binding experiments using Chinese hamster ovary (CHO) cells expressing delta- and mu-opioid receptors. The peptides [Phe(p-NCS)(3)]TIPP (2) and [Phe(p-NCS)(4)]TIPP (4) showed affinity for delta-receptors comparable to the parent compound TIPP (IC(50) = 12 and 5 nM, respectively, vs 6 nM for TIPP). Both peptides 2 and 4 were able to inhibit radioligand binding to delta-receptors in a wash-resistant manner at a concentration of 10 nM. Therefore, the peptides [Phe(p-NCS)(3)]TIPP (2) and [Phe(p-NCS)(4)]TIPP (4) represent two affinity labels that may prove useful in the study of delta-opioid receptors.

Synthesis and evaluation of N,N-dialkyl enkephalin-based affinity labels for delta opioid receptors.

To develop affinity labels for delta opioid receptors based on peptide antagonists, the Phe(4) residues of N,N-dibenzylleucine enkephalin and N,N-diallyl[Aib(2),Aib(3)]leucine enkephalin (ICI-174, 864) were substituted with either Phe(p-NCS) or Phe(p-NHCOCH(2)Br). A general synthetic method was developed for the conversion of small peptide substrates into potential affinity labels. The target peptides were synthesized using Phe(p-NH(2)) and a Boc/Fmoc orthogonal protection strategy which allowed for late functional group conversion of a p-amine group in the peptides to the desired affinity labeling moieties. A key step in the synthesis was the selective deprotection of a Boc group in the presence of a tert-butyl ester using trimethylsilyl trifluoromethanesulfonate (TMS-OTf). The target peptides were evaluated in radioligand binding experiments in Chinese hamster ovary (CHO) cells expressing delta or mu opioid receptors. The delta receptor affinities of the N, N-dibenzylleucine enkephalin analogues were 2.5-10-fold higher than those for the corresponding ICI-174,864 analogues. In general, substitution at the para position of Phe(4) decreased binding affinity at both delta and mu receptors in standard radioligand binding assays; the one exception was N, N-dibenzyl[Phe(p-NCS)(4)]leucine enkephalin (2) which exhibited a 2-fold increase in affinity for delta receptors (IC(50) = 34.9 nM) compared to N,N-dibenzylleucine enkephalin (IC(50) = 78.2 nM). The decreases in mu receptor affinities were greater than in delta receptor affinities so that all of the analogues tested exhibited significantly greater delta receptor selectivity than the unsubstituted parent peptides. Of the target peptides tested, only N, N-dibenzyl[Phe(p-NCS)(4)]leucine enkephalin (2) exhibited wash-resistant inhibition of radioligand binding to delta receptors. To our knowledge, 2 represents the first peptide-based affinity label to utilize an isothiocyanate group as the electrophilic affinity labeling moiety. As a result of this study, enkephalin analogue 2 emerges as a potential affinity label useful for the further study of delta opioid receptors.

Inhibition of gibberellin 3β-hydroxylase by novel acylcyclohexanedione derivatives

The 2-acyl-5-carboxycyclohexane-1,3-diones are plant growth retardants which are believed to act by competing with the natural co-substrate, 2-oxoglutarate, at the active site of dioxygenases involved in the later stages of the biosynthesis of the gibberellin (GA) plant hormones. A number of 2-acyl-5-carboxycyclohexanediones and related compounds were synthesized and compared with pyridine dicarboxylic acids and hydroxybenzoic acids as inhibitors of a GA 3β-hydroxylase from Cucurbita maxima endosperm. The most effective inhibitors of this enzyme possessed a 1,3-dioxo-2-acylcyclohexane-5-carboxylic acid structure. The nature of the 2-acyl group had a relatively minor effect on the potency of enzyme inhibition in vitro. A related chloro-substituted compound was shown to be a potential affinity label for this GA hydroxylase.

Inhibition of the glucose-6-phosphatase system by N-bromoacetylethanolamine phosphate, a potential affinity label for auxiliary proteins

N-Bromoacetylethanolamine phosphate (BAEP) has been used previously as an affinity label to study the hexose phosphate binding sites of fructose-6- P, 2-kinase:fructose-2, 6-bisphosphatase (Sakakibara et al. (1984) J. Biol. Chem. 259, 14023–14028). We have employed this compound to probe components of the glucose-6-phosphatase system using a combination of time-dependent and immediate inhibition kinetic techniques. Inhibition of d-glucose-6-phosphate (G6 P) phosphohydrolase activity of native microsomes was irreversible and time- and inhibitor-concentration-dependent. Only a partial time-dependent, irreversible inhibition of the PP i phosphohydrolase activity of native microsomes was observed. BAEP inhibited PP i:glucose phosphotransferase activity of native microsomes in a concentration-dependent, irreversible manner which was more extensive than that seen with PP i phosphohydrolase, but less extensive than was observed with G6 P phosphohydrolase. Disruption of microsomal integrity by detergent-treatment either prior to incubation with BAEP or subsequent to preliminary incubation with BAEP but prior to assay for activity abolished the time-dependent inhibition. These irreversible, time- and concentration-dependent inhibitory actions of BAEP thus are manifest at a site or sites where the intact membrane-bound enzyme first makes contact with substrates G6 P and PP i. An additional site of inhibition by BAEP, through relatively weak, reversible competitive inhibition at the active catalytic site, is indicated by classical steady-state kinetic analysis. The irreversible, time- and concentration-dependent inhibitions by BAEP seen with G6 P and PP i as substrates strongly suggest the potential utility of radio-labeled BAEP as an affinity label for the identification and ultimate isolation and study of uncharacterized auxiliary components of the glucose 6-phosphatase system.

4-oxygenated retinoids: unexpected chemopreventive potential for analogues originally synthesized as affinity labels

Potential retinoid binding protein affinity labels 3 – 5 were prepared from the retinoic acid metabolite 2. While unsuccessful as affinity labels, 5 and the serendipitously discovered either 6 show potential as skin cancer chemoproventives, activity not normally associated with 4-oxygenated retinoids.

ChemInform Abstract: A Convenient New Synthesis of 17‐Azasteroids. Preparation of Some Novel N‐Chloro‐17‐aza‐ and N‐Chloro‐17a‐aza‐17a‐homosteroids as Potential Affinity Labels and Enzyme Inhibitors.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Synthesis and evaluation of inhibitors for Escherichia coli glucosamine-6-phosphate synthase

The design, synthesis and evaluation of potential affinity labels of Escherichia coli glucosamine-6-phosphate synthase (glmS) are described. Among the inhibitors described, 2-amino-3-(( N-halomethyl)amino)propanoates 1a and 2a and 2-amino-3-( N-maleimidyl)propanoate 4a exhibited time-dependent inhibition parameters similar to those previously obtained for N 3-(4-methoxyfumaroyl)diaminopropanoate 5, the most efficient synthetic inhibitor of glmS reported to date. From the recently elucidated mechanism of glmS inactivation by 5, the alkylation of cysteine-1-thiol by 1a, 2a and 4a seems very likely.

A Convenient New Synthesis of 17-Azasteroids. Preparation of Some Novel N-Chloro-17-aza- and N-Chloro-17a-aza-17a-homosteroids as Potential Affinity Labels and Enzyme Inhibitors

An efficient new synthesis of 17-azasteroids in the estrane and androstane series was developed from readily available 16,17-seco 17-carboxylic acid precursors by means of a diphenylphosphoryl azide-mediated Curtius rearrangement as the key step. Several novel N-chloro-17-aza- and N-chloro-17a-aza-17a-homosteroids were prepared from the corresponding lactams with N-chlorosuccinimide

Three new potential cAMP affinity labels. Inactivation of human platelet low Km cAMP phosphodiesterase by 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate

Three new analogues of cAMP have been synthesized and characterized: 2-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate (2-BDB-TcAMP), 2-[(3-bromo-2-oxopropyl)thio]-adenosine 3',5'-cyclic monophosphate (2-BOP-tcAMP), and 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate (8-BDB-TcAMP). The bromoketo moiety has the ability to react with the nucleophilic side chains of several amino acids, while the dioxobutyl group can interact with arginine. These cAMP analogues were tested for their ability to inactivate the low Km (high affinity) cAMP phosphodiesterase from human platelets. The 2-BDB-TcAMP and 2-BOP-TcAMP were competitive inhibitors of cAMP hydrolysis by the phosphodiesterase with Ki values of 0.96 +/- 0.12 and 0.70 +/- 0.12 microM, respectively. However, 2-BDB-TcAMP and 2-BOP-TcAMP did not irreversibly inactivate the phosphodiesterase at pH values from 6.0 to 7.5 and at concentrations up to 10 mM. These results indicate that although the 2-substituted TcAMP analogues bind to the enzyme, there are no reactive amino acids in the vicinity of the 2-position of the cAMP binding site. In contrast, incubation of the platelet low Km cAMP phosphodiesterase with 8-BDB-TcAMP resulted in a time-dependent, irreversible inactivation of the enzyme with a second-order rate constant of 0.031 +/- 0.009 min-1 mM1. Addition of the substrates, cAMP and cGMP, and the product, AMP, to the reaction mixture resulted in marked decreases in the inactivation rate, suggesting that the inactivation was due to reaction at the active site of the phosphodiesterase.(ABSTRACT TRUNCATED AT 250 WORDS)

Syntheses and properties of some photolabile β-thioglycosides. Potential photoaffinity reagents for β-glycoside hydrolases

Tosyloxy-3-azi-butane, -4-azi-pentane and chloroacetone were condensed with β-D- thiosugars to yield β-D-thioglycosides which either already had a photolabile diazirino group or they had an oxo group, which could be converted into such. All photolabile β-thio-glycosides are excellent competitive inhibitors for their corresponding glycoside hydrolases (β-D-galactosidase and β-D-glucosidase). Photolysis of the glycosides in aqueous solution preferentially gave insertion products, indicating that all of the compounds were potential affinity labels. 2-Azi-propyl β-D-thio-galactoside underwent partial photochemically induced glycoside hydrolysis.

Periodate-oxidized 3-aminopyridine adenine dinucleotide phosphate as a fluorescent affinity label for pigeon liver malic enzyme

Treatment of 3-aminopyridine adenine dinucleotide phosphate with sodium periodate resulted in oxidation of the ribose linked to 3-aminopyridine ring and cleavage of the dinucleotide into 3-aminopyridine and adenosine moieties. These two moieties were separated by thin layer chromatography and were synergistically bound to pigeon liver malic enzyme (EC 1.1.1.40), causing inactivation of the enzyme. The inactivation showed saturation kinetics. The apparent binding constant for the reversible enzyme-reagent binary complex (KI) and the maximum inactivation rate constant at saturating reagent concentration (kmax) were found to be 1.1 +/- 0.02 mM and 0.068 +/- 0.001 min-1, respectively. L-Malate at low concentration enhanced the inactivation rate by lowering the KI value whereas high malate concentration increased the kmax. Mn2+ or NADP+ partially protected the enzyme from the inactivation and gave additive protection when used together. L-Malate eliminated the protective effect of NADP+ or Mn2+. Maximum and synergistic protection was afforded by NADP+, Mn2+ plus L-malate (or tartronate). Oxidized and cleaved 3-aminopyridine adenine dinucleotide phosphate was also found to be a competitive inhibitor versus NADP+ in the oxidative decarboxylation reaction catalyzed by malic enzyme with a Ki value of 4.1 +/- 0.1 microM. 3-Aminopyridine adenine dinucleotide phosphate or its periodate-oxidized cleaved products bound to the enzyme anticooperatively. Oxidized 3-aminopyridine adenine dinucleotide phosphate labeled the nucleotide binding site of the enzyme with a fluorescent probe which may be readily traced or quantified. The completely inactivated enzyme incorporated 2 mol of reagent/mol of enzyme tetramer. The inactivation was partially reversible by dilution and could be made irreversible by treating the modified enzyme with sodium borohydride. This fluorescent compound and its counterpart-oxidized 3-aminopyridine adenine dinucleotide may be a potential affinity label for all other NAD(P)+-dependent dehydrogenases.

Inhibition of hexose transport and labelling of the hexose carrier in human erythrocytes by an impermeant maleimide derivative of maltose.

Maltose-maleimide was synthesized as a potential affinity label for the facilitative hexose carrier with selectivity for exofacial sulphydryl groups. This reagent, although probably a mixture of isomers, did not significantly penetrate the plasma membrane of human erythrocytes at concentrations below 5 mM at 37 degrees C. When allowed to react to completion, it irreversibly inhibited the uptake of 3-O-methylglucose, with a half-maximal response at about 1.5-2.0 mM-reagent. The rate of transport inactivation was a saturable function of the maltose-maleimide concentration. Studies of reaction kinetics and effects of known transport inhibitors demonstrated that irreversible reaction occurred on the exofacial outward-facing carrier, although not at a site involved in substrate binding. Reaction of intact erythrocytes with [14C]maltose-maleimide resulted in labelling of a broad band 4.5 protein of Mr (average) 45,000-66,000 in electrophoretic gels. This protein was very likely the hexose carrier, since its labelling was inhibited by cytochalasin B. Exofacial band 4.5 labelling was stoichiometric with respect to transport inhibition, yielding an estimated 300,000 carriers/cell. These results suggest that the exofacial sulphydryl which reacts with maltose-maleimide is distinct from the substrate binding site on the hexose carrier, but that it confers substantial labelling selectivity to impermeant maleimides. Additionally, the high efficiency of carrier labelling obtained with maltose-maleimide is useful in quantifying numbers of carriers in whole cells.