LABELLING OF ORGANIC MOLECULES

Abstract: A process for isotopically labelling certain chemical compounds, with a selected or desired isotopic label comprising first treating a precursor material with the isotopic material to exchange the hydrogen atoms of the precursor material with the isotopic material and then or simultaneously reacting the exchanged precursor with a chemical compound so as to label the chemical compound with the isotopic material at a desired preselected location within the molecule of the chemical compound. A typical example involves first forming deuterated Raney nickel and then reacting the deuterated Raney nickel with an organic compound under relatively mild conditions so that specific selective hydrogen-deuterium exchange occurs within the molecules of the chemical compound at a pre-selected location within the molecule.

Description: AEE I G- OF ORffABJilC MOIEC-DEES The present invention relates to chemical substances, and in particular to chemical substances having specific labels and to processes for making such substances. More particularly, the present invention relates to the labelling of organic molecules with isotopic hydrogen and optionally accompanied by reduction or deoxygenation of the chemical substance. Although the present invention will be described with particular reference to some types of organic molecules and to isotopic hydrogen exchange reactions, it is to be noted that labelling of any type of chemical species by isotopic hydrogen fall within the scope of the present invention. One aspect of the present invention is concerned with isotope exchange of chemical substances in which one or more hydrogen atoms of a molecule of a particular selected chemical substance are replaced by isotopic hydrogen atoms, i.e. deuterium or tritium, thereby labelling the molecule. The presence of a label may be used as a marker for the chemical substance i.e. as a means of always identifying the chemical substance, such as during or after subsequent reactions that the substance may undergo, by means of a suitable detector. However, in order to accurately follow the progress of the chemical substance it is necessary, at least in some cases to precisely identify the location of the isotopic marker within the molecule i«.e. the precise structural position within the molecule where the isotope exchange took place. In the past, it was not always possible to accurately predict where or select the location of this isotope exchange, and thus prior art attempts to label organic molecules were non-specific in regard to the location of the isotopic hydrogen exchange. Another aspect of the present invention is the reduction and/or deoxygenation of the chemical substance as well as the labelling of the substance by the isotopic atom. Thus, the present invention relates not only to labelling chemical substances but also to reducing and/or deoxygenating them as well. In the past, catalysts such as Raney nickel and its derivatives such as Raney Al alloy and the like, have been used to reduce certain selected chemical compounds. However, the Raney-Ni catalyst has always been used in the presence of a reducing agent, such as for example hydrogen, and often at temperatures of about 100 < '-200 o C and pressures of about 20 atmosphere. It was thought that in these processes the reduction was caused by the presence of the reducing agent, such as the hydrogen, with the Raney nickel acting as a catalyst. Further, it was thought that as the Raney-Ni was acting as a catalyst it required a hydrogen source, such as hydrogen or alcohol, to effect the reduction. Accordingly, the Raney-Ni had been used in catalytically small amounts of the order of about up to 1% by wt. of the total weight of reactants. In the case of isotopic hydrogen exchange reactions it has also been observed that deuteration of a chemical substance required the use of deuterium gas, and tritiation required the use of gaseous tritium. Further, in the past, such hydrogen-deuterium (or hydrogen-tritium) exchange reactions had been observed to be non-specific which meant that the exchange position in the molecule could not be predicted or selected with certainty. After due investigation it was discovered that the methods and processes of the present invention do not require the presence of an additional reducing agent in order to achieve reduction or deoxygenation of the selected chemical substance. It has been surprisingly found that Raney-Ni by itself is sufficient to promote reduction of the chemical substance. Furthermore, it has been discovered that the reactions may take place at convenient temperatures and pressures, such as between room temperature and 110°C, and at typically about 1 atmosphere, or at pressures less than about 5 atmospheres. In addition, the amount of Raney nickel required to promote the reduction and labelling may be considerably more than mere catalytical amounts previously used. The Raney-Ni may be present in an amount up to 1000% of the total amount of the reactants. It is thought that when large amounts of Raney nickel are used, it is the Raney nickel itself that supplies the hydrogen isotopes required for the reaction, and thus the co-presence of an additional reducing agent is not always required. Using the methods and processes of the present invention, preparative quantities of up to 100% labelled materials may be obtained in some examples. The reaction conditions are very mild relatively speaking and the labelled materials obtained are useful as marked biological substrates or solvents for n. .r spectroscopy or for theoretical studies such as for example where comparison of the effect of hydrogen or deuterium on a molecule is desired. According to one aspect of the present invention there is provided a process for isotopically labelling a chemical substance with a selected isotopic label comprising the steps of (1) treating a first material with a selective isotopic label so as to form a label-containing material, and (2) reacting the chemical substance with the label- containing material so as to label the chemical substance with the label wherein the chemical substance is specifically labelled at a specific pre-selected location. The treating of the first material with the label may be previous to or simultaneously with the reacting of the chemical substance. According to another aspect of the present invention there is provided a chemical substance specifically labelled at a desired preselected location with an isotopic label wherein the chemical substance has been treated with a label-containing material formed either simultaneously or previously by treating the material with isotopic label. Any chemical substance may be treated by the methods and processes of the present invention, however, the present invention finds particular application in treating chemical substances that can be hydrogenated. Preferably, the chemical substances are organic molecules that can be reduced or deoxygenated. More preferably, the chemical substances are aromatic or aliphatic compounds such as alkenes, alkynes, ketones, aldehydes and the like. Preferably, the label is deuterium or tritium. Preferably, the material forming the label containing material is Raney nickel or the like such as nickel-aluminium, nickel-copper and nickel-copper aluminium alloys, and other precursors of Raney-Ni. Other suitable materials are finely divided platinum, rhodium, iridiu , copper oxides, cobalt and the like. The present invention will now be more fully described by way of examples. The following reactions are examples of some of the reactions which may take place according to the process of the present invention. When the relevant chemical substance is heated with deuterated Raney nickel in deuterium oxide or any other suitable inert solvent (in the absence of deuterium gas) (1) In the case of compounds having an aromatic ring or rings structure, specific hydrogen-deuterium exchange in the aromatic ring of the molecule is achieved (2) In the case of organic compounds having functional groups adjacent to aliphatic hydrogens which are thus "activated", hydrogen-deuterium exchange of "activated" aliphatic hydrogens occurs (3) In the case of alkenes (and alkynes) these substances are reduced to the corresponding deuterated aliphatic molecule (4) Carbonyl compounds are deoxygenated to deuterated aliphatic products (5) Aromatic rings are fully reduced to the corresponding labelled aliphatic ring compound. DETAILED DESCRIPTION OF THE INVENTION . The hydrogen atoms on the surface of the Raney-Ni catalyst are exchanged with deuterium atoms by washing the catalyst with deuterium oxide (or tritium oxide) . By this process it is thought that the hydrogen atoms on the Raney-Ni are exchanged by the deuterium (or tritium) atoms of the deuterium (or tritium) oxide, and thereby the'Raney-Ni is deuterated (or tritiated) to form a label-containing material. The selected substance to be labelled is dissolved in deuterium oxide (or tritium) or other suitable inert solvent and heated with the deuterated (or tritiated) Raney-Ni catalyst for periods ranging from 1 to 20 hours at temperatures ranging from room temperature to 100°C. If the substance is insoluble in water, it is heated with the deuterated (or tritiated) Raney nickel i.e. one in which the hydrogen atoms have been replaced by deuterium (or tritium) atoms in a solution of tetrahydrofuran or dioxan or any other inert solvent. In particular the present invention provides for " the (1) exchange of activated aliphatic hydrogens for deuterium, (2) reduction (deuteration) of alkenes and carbonyl compounds, (3) aromatic hydrogen-deuterium exchange, and (4) reduction of aromatic rings to fully deuterated cyclohexanes. These reactions are performed under mild conditions and in the absence of deuterium gas or a deuterium donor. Similar reactions are possible with tritium. Reactions Conditions A stirred solution of the substrate (0.5 g) is refluxed for 18 hrs with deuterated Raney nickel (2 ml wet) . For aromatic hydrogen-deuterium exchange, the temperature is kept around 70°. The solvent (5 ml) is tetrahydrofuran or heavy water (carboxylic acids and phenols are deuterated as sodium salts) . The product is isolated in the usual manner after filtration to remove the Raney nickel. Yields are 70% or above. Deuterium Exchange of Benzylic Protons and Active Methylenes (i) Diphenylmethane gave 1,1-dideuterodiphenylmethane. However, some aromatic hydrogen-deuterium exchange took place. Reduction of trans-stilbene gave l,2-diphenyl-l,l,2,2-tetradeuteroethane (and not the expected dideuterated material) . Ethyl benzoylacetate was reduced to ethyl 2,2,3,3-tetradeutero-3-ρhenylpropionate. In the latter two cases, aromatic hydrogen-deuterium exchange was minimal. (ii) Glycine (in refluxing heavy water) gave 2,2-dideuteroglycine ( , -dideuteroglycine) . Similarly, 2-alanine gave 2-deutoroalanine (with racemisation) , 2-serine gave 2,3,3-trideuteroserine (with racemisation) and L-lysine hydrochloride gave 2,6,6-trideuterolysine hydrochloride (with racemisation) . In the amino acids, hydrogens adjacent to amino or hydroxyl functions are exchanged. Reduction of Alkenes As mentioned above, trans-stilbene gave l,2-diphenyl-l,l,2,2-tetradeuteroethane. Sodium cinnamate (in heavy water at 70°) gave 2,2,3,3,2' ,3* ,4' ,5' ,6'-nonadeuterophenyl- propanoic acid (I) . (The aromatic hydrogens were exchanged under these conditions) . Cinnamaldehyde gave a deuterated material (i.r., n.m.r.) the mass spectrum of which showed that it was polymeric. Maleic acid (at 101°) gave 2,2,3,3-tetradeuterosuccinic acid. Reduction of Carbonyl Compounds Benzophenone reacted smoothly to give 1,1-dideuterodiphenylmethane with some ring hydrogen-deuterium exchange. Ethyl benzoylacetate was reduced to- ethyl 2,2,3,3-tetradeuterσ-3-phenyl- propionate. The reaction of cyclohexanone with ("undeuterated") Raney nickel gave low yields of phenol while camphor was recovered unchanged even after 24 hours refluxing dioxan. Aromatic Hydrogen/Deuterium Exchange As mentioned above, the reaction of sodium cinnamate with deuterated Raney nickel at 70-80° gave nonadeuterophenylpropanoic acid (I) . Similarly, sodium benzoate gave pentadeuterobenzoic acid (II) and nicotinic acid gave 2,4,5,6-tetradeuteronicotinic acid. Reduction of Aromatic Nucleus The refluxing of sodium benzoate with "deuterated" Raney nickel in heavy water gave the fully deuterated (D»,) cyσlohexanecarboxylic acid (III) . Under the same conditions, sodium phenoxide gave fully deuterated • D ll- c Y c l° nexan °l. contaminated with some (approx. 10%) cyclohexanone. I) (ID (III) Class Starting Material Process Produce Reduction of maleic acid 1. Dissolve the di-sodiu salt in 20 butanedioic acid-2,2,3,3-d4 alkenes parts heavy water and heat for 18 hours with Raney nickel. 2. Filter, acidify and recrystallise from water. cinnamic acid 1. Dissolve the sodium salt in 20 3-phenylpropanoic-acid-2,2,3, parts heavy water and heat to 80 * 2',3\4\5\b--o 9 for 18 hours with Raney nickel. 2. Filter, acidify and recrystallise from light petroleum. stilbene 1. Dissolve in THF and heat for 18 l 8 2-diphenylethane-l,l, ,2-d4 18 hours with Raney nickel. 2. Filter, evaporate and recrystallise from ethanol . .Reduction benzoic acid 1. Dissolve sodium salt in 20 parts cyclohexanecarboxylic acid-1, of aroma ic heavy water and reflux for 18 hours 2,2,3,3,4,4,b,5,6,6-d n with Raney nickel 2. Filter, acidify, extract with chloroform and evaporate. phenol 1. Dissolve sodium salt in 20 parts eye 1 ohexano 1-1 ,2, 2, 3, 3,4,4 , 5 , heavy water and reflux for 18 hours 6,fa-dn with Raney nickel 2. Extract with chloroform evaporate and purify by column chroniatography. EXAMPLES Class Starting Material t Process Produce Al iphatic diphenylmethane 1. Dissolve in tetrahydrofuran (THF) diphenylmethane-l,l-d2 exchange and heat with 5 parts deuterated Raney nickel for 18 hours. 2. Filter and evaporate. 1 , 2-d i phen 1 e thane-1 ,1,2,2 1,2-diphenylethane 1. As above (for diphenylmethane). £-toluic acid SEE "AROMATIC EXCHANGE" glycine 1. Dissolve in heavy water and glycine-2,2-d 2 evaporate to exchange -COOH and - H 2 hydrogens. Dissolve in 20 parts heavy water and refiux 18 hours with Raney nickel. 3. Centrifuge, wash Raney nickel with dilute ammonia filter centrifugate and washing. Concentrate and apply to a column of chelex-100 (in ammonium form). Elute with water. Evaporate and recrystallise from aqueous methanol. L-alanine 1-5 as above for glycine. D,L-alanine-2-dι (D,L-alanine-«->*--dι) L-serine 1. 1-5 as above for glycine. D, -serine- 2 , 3 , 3- 3 (D,L-serine-o<, β,β- 3 ) s Starting Material Process Produce L-lysine hydrochloride 1. 1-5 as above for glycine , D,L-lysine-2,6,6-d3 hydrochlor (D,L-:Lysine-c_,__, --d3 hydrochlo _ — _ niat benzole acid 1. Dissolve sodium salt in 20 parts benzo le acid-2,3,4 ,b,fa-d5 hange heavy water and heat to 60° for 5 hours with Raney nickel. 2. Filter, acidify and recrystallise from water. 4-t-butylphenol 1, Dissolve sodium salt in 20 parts 4-t-butylphenol-2, 3,fa,6-d4 heavy water and heat to 60" for 1 with Raney nickel . 2. Filter, acidify and recrystallise from water. nicotinic acid 1. Dissolve sodium nicotinate in 20 nicotinic acid-2,4,5,6-d4 parts heavy water and heat to 60" for 18 hours with Raney nickel. 2. Filter, acidify and recrystallise from water. p-toluic acid 1. As above (nicotinic acid) £-toluic acid-ct,α,α,2,3, b, 6-d 7 Class Starting Material Process Produce Deoxygenation benzophenone 1. Dissolve in THF and heat at diphenylmethane-l.l-d reflux for 18 hours with Raney nickel . 2. Filter, evaporate. ethyl benzoylacetate 1. Dissolve in THF and heat at ethyl 3-phen lpropionate-2,2,3, reflux for 18 hours with Raney 3-d 4 nickel. 2. Fi Iter, evaporate. Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and mocifications which fall within its spirit and scope.

Claims: CLAIMS : 1. A process for isotopically labelling a chemical substance with a selected or desired isotopic label comprising the steps of (1) treating a first material with the selective isotope or isotopic label so as to isotopically labelled the first material, and (2) reacting the chemical substance to be labelled with the isotopically-labelled first material so as to isotopically label the chemical substance at a specific pre-selected location within the molecule of the chemical substance. 2. A process according to claim 1 in which the labelling of the first material occurs prior to the reaction of the chemical substance with the isotopically labelled first material. 3. A process according to claim 1 in which the labelling of the first material occurs substantially simultaneously with the reaction of the chemical substance with the isotopically labelled first material. 4. A process according to any preceding claim in which the chemical substance is a compound which is capable of being hydrogenated, reduced, deoxygenated or a combination thereof. 5. A process according to any preceding claim in which the chemical substance to be labelled is selected from the group comprising aromatic or aliphatic compounds such as alkenes, alkynes, ketones, aldehydes and the like. 6.. A process according to any preceding claim in which the isotopic label is deuterium or tritium. 7. A process of any preceding claim in which the first material is Raney nickel or a nickel containing material or precursor or derivative thereof or finely divided platinum, rhodium, iridium, copper oxides, cobalt, or derivatives thereof. 8. A process of any preceding claim in which the substance to be labelled has at least one aromatic ring and specific hydrogen-deuterium exchange occurs in the aromatic ring of the molecule. 9. A process of any preceding claim in which the substance to be labelled is an organic molecule having at least one functional group adjacent to an aliphatic hydrogen which are activated and the hydrogen-deuterium exchange of the activated aliphatic hydrogen occurs. 10. A process of any preceding claim in which the substance to be labelled is an alkene or an alkyne and is reduced to the corresponding deuterated aliphatic compound. 11. A process of any preceding claim in which the hydrogen atoms of the Raney nickel are exchanged with deuterium or tritium atoms by washing the Raney nickel with deuterium oxide or tritium oxide respectively. 12. A ' process of any preceding claim in which the chemical substance to be labelled is dissolved in deuterium oxide or tritium oxide and optionally an inert solvent and heated with deuterated or tritiated Raney nickel for a period from between about 1 and 20 hours at a temperature from about room temperature to about 110°C. 13. A process of any preceding claim in which the inert solvent is tetrahydrofuran or dioxan or a mixture of any other solvent containing one or both thereof. 14. A chemical substance specifically labelled at a desired pre-selected location with an isotopic label wherein the chemical substance has been treated with a label-containing material formed either simultaneously with or previous to the labelling reaction. 15. A chemical substance whenever prepared by the process of any one of claims 1 to 13. 16. A process substantially as hereinbefore described with reference to any one of the examples. 17. A chemical substance having a label substantially as hereinbefore described with reference to any one of the examples.

Inventors: POJER PETER MICHAEL, POJER, PETER, MICHAEL
Applicants: COMMW OF AUSTRALIA, THE COMMONWEALTH OF AUSTRALIA
Application year: 1985
Main classification: C07B59
IPC codes: C07B59