Analytical method development for directed enzyme evolution research: a high throughput high-performance liquid chromatography method for analysis of ribose and ribitol and a capillary electrophoresis method for the separation of ribose enantiomers.

Analytical methods were developed for a directed enzyme evolution research programme, which pursued high performance enzymes to produce high quality L-ribose using large scale biocatalytic reaction. A high throughput HPLC method with evaporative light-scattering detection was developed to test ribose and ribitol in the enzymatic reaction, a ß-cyclobond 2000 analytical column separated ribose and ribitol in 2.3 min, a C(18) guard column was used as an on-line filter to clean up the enzyme sample matrix and a short gradient was applied to wash the column, the enzymatic reaction solution can be directly injected after quenching. Total run time of each sample was approx. 4 min which provided capability of screening 4×96-well plates/day/instrument. Meanwhile, a capillary electrophoresis method was developed for the separation of ribose enantiomers, while 7-aminonaphthalene-1,3-disulfonic acid was used as derivatisation reagent and 25 mM tetraborate with 5 mM ß-cyclodextrin was used as electrolyte. 0.35%of D-ribose in L-ribose can be detected which can be translated into 99.3% ee of L-ribose. Derivatisation reagent and sample matrix did not interfere with the measurement.

The search for a potentially prebiotic synthesis of nucleotides via arabinose-3-phosphate and its cyanamide derivative.

For the RNA world hypothesis to be accepted, the constitutional self-assembly of RNA will have to be demonstrated. Conceptually, the simplest route to RNA involves nucleotide polymerisation. Activated pyrimidine nucleotides can be derived from arabinose-3-phosphate under potentially prebiotic conditions, but the prebiotic synthesis of this sugar phosphate has not hitherto been investigated. The results of synthetic approaches involving phosphorylation, phosphate migration and 2,3-C--C bond construction are described herein.

On the prebiotic synthesis of ribonucleotides: photoanomerisation of cytosine nucleosides and nucleotides revisited.

Recent work has emphasised the importance of D-ribose aminooxazoline 1 in the synthesis of cytidine ribonucleosides under potentially prebiotic conditions. Upon treatment with cyanoacetylene, 1 is transformed into alpha-D-cytidine (alpha-2), and if an efficient means of anomerising this nucleoside or a derivative thereof were to be found, then the synthesis of one of the key beta-D-nucleosides required to make RNA would be realised. Photoanomerisation of alpha-2 has previously been described, but the yield was extremely low. Therefore, the present study was initiated to determine whether this low yield was the result of a low conversion or competing reaction pathways.

RNA: prebiotic product, or biotic invention?

Spectacular advances in structural and molecular biology have added support to the 'RNA world' hypothesis, and provide a mandate for chemistry to explain how RNA might have been generated prebiotically on the early earth. Difficulties in achieving a prebiotically plausible synthesis of RNA, however, have led many to ponder the question posed in the title of this paper. Herein, we review recent experimental work on the assembly of potential RNA precursors, focusing on methods for stereoselective C-C bond construction by aldolisation and related processes. This chemistry is presented in the context of a broader picture of the potential constitutional self-assembly of RNA. Finally, the relative accessibility of RNA and alternative nucleic acids is considered.

Reaction of cytidine nucleotides with cyanoacetylene: support for the intermediacy of nucleoside-2',3'-cyclic phosphates in the prebiotic synthesis of RNA.

A robust and prebiotically plausible synthesis of RNA is a key requirement of the "RNA World" hypothesis, but, to date, no such synthesis has been demonstrated. Monomer synthesis strategies involving attachment of preformed nucleobases to sugars have failed, and, even if activated 5'-nucleotides could be made, the hydrolysis of these intermediates in water makes their efficient oligomerisation appear unlikely. We recently reported a synthesis of cytidine-2',3'-cyclic phosphate 1 (C>p) in which the nucleobase was assembled in stages on a sugar-phosphate template. However, 2',3'-cyclic nucleotides (N>p's) also undergo hydrolysis, in this case giving a mixture of the 2'- and 3'-monophosphates. This hydrolysis has previously been seen as making the, otherwise promising, oligomerisation of N>p's seem as unlikely as that of the 5'-activated nucleotides. We now find that cyanoacetylene, the reagent used for the second stage of nucleobase assembly in the synthesis of C>p, also reverses the effect of the hydrolysis by driving efficient cyclisation of C2'p and C3'p back to C>p. Excess cyanoacetylene also derivatises the nucleobase, but this modification is reversible at neutral pH. These findings significantly strengthen the case for N>p's in a prebiotic synthesis of RNA.

Exploratory studies to investigate a linked prebiotic origin of RNA and coded peptides. 4th Communication. Further observations concerning pyrimidine nucleoside synthesis by stepwise nucleobase assembly.

As part of an ongoing investigation into a possible linked origin of RNA and coded peptides, we have (further) investigated the potentially prebiotic synthesis of pyrimidine ribonucleosides (ribonucleotides), by stepwise nucleobase assembly on sugar (sugar phosphate) scaffolds. In this paper, we complete our description of this assembly process on aldopentose scaffolds. Treatment of D-ribose (14) and D-xylose (20) with cyanamide smoothly produces the furanosylamino-oxazolines 15 and 21, respectively (Schemes 1 and 3). Similar treatment of D-lyxose (26) also produces the furanosylamino-oxazoline 29, but as a minor component of an equilibrating mixture in which the pyranosylamino-oxazoline 28 predominates (Scheme 4). Treatment of the various amino-oxazolines with cyanoacetylene gives furanosylcytidines 16, 25, and 31, and the lyxopyranosylcytidine 30 (Schemes 1-4). Minor by-products of the cyanoacetylene reaction include imino lactone 17, acetals 19, 22, and 24, and cyanovinyl adducts 22 and 23 resulting from initial addition of nucleoside OH groups to the terminal acetylenic C-atom of cyanoacetylene (Schemes 2-4).

Exploratory studies to investigate a linked prebiotic origin of RNA and coded peptides.

An introduction to the premise that RNA and genetically coded proteins should not be viewed as etiologically discrete entities in the origin of life is presented. This premise follows from the mutual interdependence of RNA and coded proteins in biology and the lack of prebiotically plausible constitutional self-assembly processes leading to either polymeric species. The RNA:coded peptides subsystem and its informational core, the genetic code, are then analysed retrosynthetically to suggest a (replicative) synthesis involving the intermediacy of aminoacyl-RNA trimers (cf. Scheme 5). A number of potential candidate aminoacyl-RNA trimers are identified (23-26; Scheme 6) and a chemical strategy to assess their validity is outlined. Experimental investigation of potential aminoacylation chemistry, nucleobase assembly and phosphate activation rules out three of the trimers but suggests that 26 is worthy of further investigation.