Simple Methods for Preparing Oligonucleotides Containing 1) Thiol Groups at their 5′-Termini, 2) 5′-Phosphates

A simple method has been developed for synthesising oligonucleotides containing a thiol group at their 5′-termini. The sequence required is prepared using standard solid phase phosphoramidite methods and an extra round of synthesis is then performed with S-triphenylmethyl O-methoxymorpholino-phosphinyl 3-mercaptopropan (1) ol. After normal deblocking this gives an oligonucleotide containing a tritylthiol group attached to the 5′-phosphate of an oligonucleotide via a 3-carbon spacer arm. The trityl group can be removed with AgNO3 at pH 5 to give the free thiol. This compound is stable at pH 8 and reacts cleanly and rapidly with sulphydryl specific probes (eg fluorescent iodoacetates) at this pH value. This method can be used to prepare a wide variety of usefully labelled oligonucleotides and it is envisaged that fluorescent oligonucleotides will be useful in the study of protein nucleic acid interactions and to replace 32P labelled hybridisation probes.