Ribonuclease chemical synthesis

Chemical synthesis has provided an additional route to peptides containing halogenat-ed amino acids. Early 19F-NMR studies of proteins were performed on semi-synthetic polypeptides prepared by attachment of fluorinated probes to the polypeptide. For example, Heustis and Raftery modified ribonuclease by trifluoroacetylation of Lys residues 1 and 7. They then used 19F-NMR to study conformational changes brought about by the presence of inhibitors200. In his review, Gerig provides several other examples of this strategy187. 

Moore, S. and W. Stein. 1972. The chemical synthesis of pancreatic ribonuclease and deoxyribonuclease. Nobel Lecture 80-93. 

Prepn. 3, 9 (1953). Bovine pancreatic RNase A is a single-chain peptide of 124 amino add residues. Primary structure C. H. W. Hits et al J. Biol. Chem. 235, 633 (I960). Amino acid sequence D. H. Spackman et al.. Ibid. 648. Pictorial representation of entire structure Stein, Moore, Sci. Am. 204, no. 2, 81-92 (Feb. 1961). Ribonuclease from plant leaves has slightly different characteristics Markham, Strominger, Biochem. J. 64, 46p (1956). Can be obtained as a by-product of microbial erythromydn production Japan, pat. 263>38( 63) (to Shionogi). Chemical synthesis of materials possessing partial RNase enzyme activity Gutte,... 

Modark, A. S., Gard, J. K., Merriman, M. C., Winkeler, K. A., Bashkin, J. K., Stem, M. K. (1991). Toward chemical ribonucleases. 2. Synthesis and characterization of nucle-oside-bipyridine conjugates. Hydrolytic cleavage of RNAby their Copper(II) complexes, J. Am. Chem. Soc., 113 283... 

Any chemist knows that the more steps there are in a chemical synthesis, the lower the final yield. For example, if each step in a 10-step synthesis furnishes a 90 percent yield of product, the yield of the final product will be only about 35 percent. That is why it is not possible to extend Du Vigneaud s masterful syntheses (see chapter 6) of the hormones oxytocin and vasopressin (9 amino acid residues each) to proteins, even small ones such as ribonuclease A (124 amino acid residues). In order to pursue this daunting challenge Robert Bruce Merrifield (1921-2006), at Rockefeller University, devised a new concept solid-phase synthesis. The idea is disarmingly simple covalently attach an amino acid to a macroscopic particle that can be exposed to the reaction, washed, and then separated by simple filtration. Each reaction step requires no chromatography and no crystallization, just washing and filtering. At the end, completed peptide chains are chemically released from the particles. 

H. Yajima, N. Fujii, Chemical synthesis of ribonuclease A with full enzymatic activity in Chemical Synthesis and Sequencing of Peptides and Proteins (t-Y. Liu, A.N. Schechter, R.L. Heinrikson, P.G. Condliffe eds.) Elseview/North Holland, New York, 1981 p. 21-39... 

RNA probes RNA probes bind tighter to their complementary strands than do DNA probes. Poor stability due to ubiquitous ribonucleases has hampered more widespread use of short RNA probes, as has the difficulty of efficient chemical synthesis of long RNA oligomers. Recent advances in RNA synthetic chemistry have solved the latter problem. 

As the workup of each coupling reaction is so simple, it has been possible to automate the procedure, thus allowing for an even more rapid polypeptide synthesis. As such, the first chemical synthesis of an enzyme (bovine pancreatic ribonuclease, 124 amino acid residues) was accomplished by this method. 

Unlike most proteins, where precise control of the caging reaction is difficult to achieve, chemically synthesized peptides can be caged relatively easily at specific amino acids.In one case, this approach was used to generate photoactivatable ribonuclease S. This enzyme is composed of two units S-peptide (Residues 1 to 20) and S-protein (Residues 21 to 124). A series of S-peptides carrying a 2-nitrobenzyl caging group on either Asp, Glu, Gin, or Lys side chains was prepared by chemical synthesis and chimeric RNAse S variants composed of these caged peptides assembled. In one case, a complete inactivation of RNAse S was achieved with 37% activity recovered upon illumination. 

The reaction of hexahydropyrroloindole (HPI) (46) with thiols to give the corresponding 2-thioether-tryptophan compounds has been further investigated (464). Reaction of cysteine with HPI (1.2 equiv) in 25% tri-fluoroacetic acid produces quantitatively tryptathionine, an amino acid contained in the toxic peptides of Amanita phalloides (see Section VI.2.4.). Reduced ribonuclease, a protein containing 8 cysteine residues per molecule, was treated with HPI, and the modified protein purified by gel filtration. The completeness of the reaction was confirmed by hydrolysis with /7-toluenesulfonic acid (233) and analysis of the hydrolyzate. A value of 7.6 (theory 8) residues per mole of protein of oxindolylalanine, the product of hydrolysis of the tryptathionine residues (431) (see Section III.4.2.), was obtained. This new reaction of cysteine residues should be of value in peptide synthesis, providing a simple method for linking tryptophan and cysteine as a basic step in the chemical synthesis of the peptides of Amanita phalloides. 

To probe for one-dimensional diffusion, we synthesized DNA/RNA chimeric oligonucleotides. Special precautions were taken to avoid ribonuclease contamination during synthesis, purification, and use of these chimeras. For example, all water was treated with diethylpyrocarbonate before exposure to the chimeras. Ribonucleotide 2 -hydroxyl groups were deprotected with 1 M tetrabutyl ammonium fluoride in dimethyl formamide (Aldrich Chemical Milwaukee, WI). Purified oligonucleotides were labeled on the 5 end with [y-32p]ATP (duPont Wilmington, DE) by T4 kinase (Promega Madison, WI), and desalted with a Nick gel filtration column (Pharmacia Uppsala, Sweden). 

The enzymatic method was extended using chemically modified serine proteases like thiolsubtilisin for the synthesis of the ribonuclease Ti (12-23) fragment. 

A straight-forward approach to the synthesis of pure substances, which can be extended to pure copolymers such as the ribonuclease, is the stepwise addition of one monomer, A, to a monomer, B, followed by separation from the excess chemicals. A third step can then add the next monomer. Again, separation from the excess chemicals has to follow this reaction step. This separation becomes more difficult as the chain gets longer, since the differences in properties between the successively made molecules become smaller as one approaches the length of polymer molecules. A yield of 90% for each of the 123 steps needed to make ribonuclease, for example, would produce only 0.002% of the polymer. In order to make a reasonable amount, the enormous, practically impossible yield of 99% must be achieved for each combined reaction and separation step. Only then can one count on converting 29% of the monomers into the proper polymer. 

All operations in solid-phase peptide synthesis have been automated. The reactions occur in a single reaction vessel, with reagents and wash solvents automatically added from reservoirs by mechanical pumps. Merrifield synthesized the nonapeptide bradykinin (page 503) in just 27 hours using this technique. And, in 1969, he used the automated synthesizer to prepare the enzyme ribonuclease (124 amino acid residues), the first enzyme to be prepared synthetically from its amino acid components. The synthesis, which required 369 chemical reactions and 11,391 steps, was completed in only six weeks. Automated computerized peptide synthesis, though still not without occasional problems, is now a fairly routine matter. 

Merrifield used this procedure to prepare a number of peptides. For example, he synthesized the nonapeptide bradykinin in 68% yield in only eight days, a remarkable feat at the time.The biological activity of the synthetic peptide was identical with that of the natural peptide. Merrifield was ultimately able to automate all the steps in his technique for solid-phase peptide synthesis and demonstrated its power by using a homemade machine to prepare bovine pancreatic ribonuclease, an enzyme that contains 124 amino acids. This synthesis proceeded in 17% overall yield and required 369 chemical reactions and 11,931 individual operations The synthetic ribonuclease had a specific activity that was 13-24% that of the native enzyme. The lower activity of the synthetic enzyme can probably be attributed to the fact that each coupling step did not proceed with 100% efficiency, so some polypeptides lacking one or more individual amino acids in the sequence were also produced. Because of their close similarity to ribonuclease, it was not possible to separate these polypeptides from the synthetic enzyme. 

Automated peptide synthesizers are available that can complete one cycle in 40 min and carry out 45 cycles of unattended operation. Though not as efficient as protein synthesis in the body, where enzymes directed by DNA can catalyze assembly of a protein with 150 amino acids in about 1 min, automated peptide synthesis is a far cry from the tedious process of manually synthesizing a peptide step after step. A hallmark example of automated peptide synthesis was the synthesis of ribonuclease, a protein with 124 amino acid residues. The synthesis involved 369 chemical reactions and 11,930 automated steps—aU carried out without isolating an intermediate. The synthetic ribonuclease not only had the same physical characteristics as the natural enzyme, it possessed the identical biological activity as well. The overall yield was 17%, which means that the average yield of each individual step was greater than 99%. 

From the scheme, we see immediately that the presence of sRNA is essential, since here we have a RNA molecule in actual chemical combination with an activated amino acid, presumably on its way to protein. Furthermore, as we have seen, the formation of the RNA-amino acid compound is extremely sensitive to ribonuclease. In principle, therefore, this step could account for the fact that protein synthesis does not take place in the absence of RNA, and that it is inhibited by ribonuclease. Nevertheless, all evidence to date strongly indicates that the cytoplasmic... 

---