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The bifunctional reagent ethyl 4-azidobenzoylaminoacetimidate was used to crosslink specifically ribosomal protein S1 to 16-S RNA within 30-S subunits. The reagent was attached to isolated protein S1. The modified protein was reassociated with protein-S1-depleted 30-S subunits and then crosslinked to the RNA molecule. The covalently bound 16-S RNA-protein S1 complex was isolated and the RNA fragment C-U-A-A-C-G-C-G-U-U-A-A-G-U-C-G-A-C-C-G-C-C-U-G-G-G-G-A-G (positions 861-889) was characterized to be crosslinked to protein S1.

α-Acetylenic esters are able to react under mild experimental conditions with the base moiety of adenosine and cytidine, while guanosine is unreactive. A double reaction of the triple bond and the ester group of the reagent with the NH 2 group and the adjacent ring nitrogen of the base yields derivatives in which an additional pyrimidone ring is fused to the original base. These derivatives can exist in two isomeric forms. In alkaline solution, or by prolonged heating in water, the medium pyrimidine ring of adenosine derivatives opens by loss of carbon 5

Alkoxycarbonylation of propargyl chloride: methyl 4-chloro-2-butynoate product: methyl 4-chloro-2-butynoate product: 4-Chloro-2-butynoic (chlorotetrolic) acid product: ethyl 4-chloro-2-butynoate product: tert-butyl 4-chloro-2-butynoate Keywords: acylation; grignard and related reactions; grignard and related reactions; metalation reactions, lithium; diethyl ether; lithium chloromethylacetylide; benzene; methyl 4-chloro-2-butynoate, irritant; methyl chloroformate, irritant; methyllithium, ethereal solution of; propargyl chloride, irritant; bath, cooling, refrigerated

Two isomeric tricyclic adenine derivatives can form by reaction of methyl 4—chloro-2—butynoate with single-stranded nucleic acids, depending on the degree of base stacking. An evaluation of the conformation of the polynucleotide can thus be easily performed.

The NH2 group and the adjacent ring nitrogen of adenosine and cytidine react with α-acetylenic esters by addition across the triple bond and formation of a lactam with the ester group.

A new compound, dithiobis[9-(2-ethylenecarbamoylethylamino)-2,3-dimethoxy-6-azido-acridine], was synthesized and used in some preliminary experiments to form cleavable complexes between nucleic acids and proteins. In a first step the azidoacridine moiety of the reagent intercalates between the bases of nucleic acids and is then bound by reaction of the azido group. The disulfide group of the reagent is simultaneously converted under reducing conditions into a thiol which, in a second step, can be bound by oxidation to -SH groups of a vicinal protein (additional -SH groups can be inserted in the protein using 2-iminothiolane). The nucleic acid-protein complexes thus formed can be redissociated by reduction. The potential applications of this new cleavable crosslinking reagent could be extended to topographical investigations of any biological structure composed of nucleic acids and proteins.

The properties of the chloro- o,o′ -dinitrophenyl group, more suitable as a building block for the design of chromogenic bifunctional protein reagents than the corresponding known o , p derivatives, are investigated by model reactions with small molecules and with proteins. This study shows that (1) the chlorine substituent can be easily substituted by protein nucleophiles, (2) arylation of SH and NH 2 groups leads to chromophores in which the differences between the λ max (330 and 430 nm, respectively) are larger than in the case of o,p -dinitro compounds, (3) afler substitution by an SH group, transarylation to a vicinal NH 2 , whenever possible, occurs faster than in the case of o , p derivatives (4) substitution by imidazole or phenol can be reversed by a thiol yielding an alkylthioaryl derivative; however, when mercaptoethanol is used in this reaction, o,o′ -dinitrophenol is obtained, probably resulting from a S to O transfer in the initially formed hydroxyethylthiophenol, followed by hydrolysis of the labile alkoxyphenyl derivative.

The reaction of yeast tRNAPhe with methyl chlorotetrolate, ClCH2-C≡C-COOCH3, was studied. This reagent converts adenine and cytosine rings into derivatives in which an additional heterocycle bearing the alkylating chloromethyl group is fused to the original base; these derivatives can exist in two isomeric forms. Modified nucleosides of this type can be easily identified by reverse-phase HPLC. It was found that under native conditions, the modification of tRNA involves the anticodon loop and the 3′-end. The isomers of adenine derivatives formed in the anticodon loop were different from those formed in the 3′-end. It is suggested that the isomeric structure of the derivatives is related to the fine conformational differences between these two regions of tRNAPhe. Methyl chlorotetrolate could thus be used as a conformational probe of single-stranded nucleic acids. Preliminary assays showed that modified tRNAPhe binds irreversibly to yeast phenylalanyl-tRNA synthetase.

The possibility of using the acetylenic function for chemical modification of proteins was tested on chlorotetrolic acid and methyl chlorotetrolate. These compounds react under mild conditions with different functional groups analogous to those present in proteins. The ease with which the same nucleophile adds to the triple bond and substitutes the chlorine was compared, and the stability of the addition products was checked. The modification of amino groups by addition across the triple bond can be reversed in acidic medium. In reactions with small model molecules and with proteins, the tested compounds behave like bifunctional protein reagents.

Ethyl bromoacetimidate was designed as a potential reagent for cross-linking protein NH2 groups with a vicinal nucleophile. The chemical properties of this compound were studied by model reactions with small molecules. Ethyl bromoacetimidate amidinates lysine residues in ribonuclease at pH 9. In a second step, at lower pH values, one of the bromoacetamidino groups bound to the enzyme alkylates a proximal histidine residue. This substitution is pH-dependent with a sharp optimum at 5.6, the same as was earlier observed for alkylation of histidine-119: histidine-12 by halogenoacetates and halogenoacetamides. A common mechanism is suggested for all these types of alkylation. Ethyl bromoacetimidate thus appears as a short-distance crosslinker which can be used, for example, to explore chemically the microenvironment of an essential lysine residue of an enzyme within the active site.

tRNAPhe in which the adenine and cytosine rings in the aminoacyl arm and in the anticodon loop were converted to alkylating derivatives by mild treatment with methyl chlorotetrolate was used to study the tRNAPhe-yeast phenylalanyl-tRNAPhe synthetase interaction. At neutral pH, modified tRNA inhibited the enzyme competitively. At pH 9 this binding is accompanied by irreversible inactivation of the enzyme due to alkylation of the α subunit of the synthetase. Such a derivatization of tRNA could probably be used to investigate the interaction of other tRNAs with their cognate synthetases.

The title compounds were investigated as examples of potential hetero-bifunctional protein reagents having an electrophilic moiety in addition to a maleimide or isomaleimide ring. The synthesis of such products by cyclization of the corresponding maleamic acids gives lower yields in the maleimide than in the isomaleimide series, especially when the second function is labile, owing to the more drastic reaction conditions necessary to form the symmetric isomer. On the other hand, a comparison of the isomeric imides showed that despite some differences in reactivity, isomaleimides can be used instead of maleimides for chemical modification of proteins. Therefore, if the synthesis of new maleimide reagents containing a second fragile functional group is difficult, they can be designed in the isoimide form.

A new reagent, ethyl 4-azidobenzoylaminoacetimidate, was prepared in a four-step synthesis starting from 4-aminobenzoic acid. This compound was used to cross-link RNA with proteins within the Escherichia coli 30-S ribosomal subunits. Following the reaction of the imidoester function with protein NH2 groups, photoactivation of the azide binds the other end of the reagent to RNA. The cross-linked proteins were labelled with 125I and identified by bidimensional gel electrophoresis. Proteins S3, S4, S5, S7, S9, S17, S18, and in a lower and more variable yield, S12, S13, S14 and S16 were bound to 16-S RNA. These results were confirmed by isolating cross-linked protein-oligonucleotide complexes from 30-S subunits containing 32P-labelled RNA.

α-Acetylenic esters react with the NH2 group and the adjacent ring nitrogen of adenosine, cytidine, adenine nucleotides and poly(A) by addition across the triple bond and formation of a lactam with the ester group.

The new bifunctional reagent, N-(4-chloromercuriphenyl)-4-chloro-3,5-dinitrobenzamide (I) was used to investigate the quaternary structure of yeast alcohol dehydrogenase. The four essential – SH groups of the enzyme were substituted by the mercuriphenyl moiety of compound I in the course of the reaction of one mole of protein with four moles of the reagent (one molecule of compound I incorporated by yeast alcohol dehydrogenase monomer). In a second step only two of the four chlorodinitrophenyl fragments bound to the protein established intermonomeric cross-links with non-essential – NH2 groups. The resulting dimers could be re-dissociated with mercaptoethanol. This result suggests that the four protomers of the enzyme could be arranged as a dimer of dimers.

Analogues of adenine nucleotides, containing an additional chloromethyl-pyrimidone ring fused to the purine base, were obtained by treatment of AMP, ADP and ATP with an alpha-acetylenic ester, methyl 4-chlorobut-2-ynoate. These compounds were tested for their ability to substitute for the natural substrates or cofactors of several enzymes. With the ADP analogue, pyruvate kinase showed a significant increase of the Km value and a moderate decrease of V, while the reverse was observed when hexokinase was tested with modified ATP. Adenylate kinase was active with the ATP analogue but not with the AMP derivative. Myosin accepted the ATP analogue as a substrate, but was irreversibly modified. Among the dehydrogenases tested, only glucose-6-phosphate dehydrogenase was inhibited by the nucleotide analogue. The structure--activity relationship of these nucleotide derivatives, which represent the largest dimensional deviation known from natural nucleotides, is discussed in comparison with some earlier described dimensional probes of enzyme-nucleotide binding sites.

The reaction of chlorotetrolic (4-chloro-2-butynoic) esters with adenine and cytosine derivatives, in which a new heterocycle bearing an alkylating chloromethyl side chain is fused to the purine or pyrimidine ring, was extended to poly(A) and poly(C) used as models of nucleic acids. The derivatization proceeds under mild conditions and its extent can be controlled by the reaction time. The additional rings can exist in two isomeric forms and the nature of the isomer formed depends on steric factors in the vicinity of the reacting base. The reaction with chlorotetrolic esters discriminates between the single-stranded (reactive) and double-stranded (unreactive) forms, between the exposed an hidden adenine and cytosine bases and even between the exposed and sterically hindered fragments of the base moiety and thus allows structural investigations of these nucleic acids. The chloromethyl group of the derivatized nucleobases can be used to bind the modified polymers to other molecules.

The new compound 4-chloro-3,5-dinitrophenacyl bromide (CNPB) was studied as a potential polyfunctional protein reagent. Under mild experimental conditions the bromine atom, more reactive than the chlorine, is easily substituted by thiol groups. The amino groups in amino acids are not attacked, with the exception of phenylalanine, tyrosine and tryptophan, where a charge-transfer complex formed between the reagent and the aromatic nuclei of the amino acids facilitates the substitution of chlorine or bromine by the α-NH 2 groups. A reaction between the bromoacetyl moiety of the reagent and carboxylic acids is also possible. CNPB inactivates ribonuclease by substitution of approximately two histidyl residues accompanied by the formation of a π-complex, presumably with a vicinal aromatic residue.

Es wird die Cyclisierung der Athinylamine (III) und ihrer Carbathoxyderivate (V) untersucht.

Treatment of propargyl chloride with methyl-lithium and an excess of alkyl chloroformate leads to chlorotetrolic esters in an average yield of 65%, in contrast with the previously reported three-step, low-yield synthesis of these biochemical reagents.