How human neuroblastoma cells make morphine

Abstract

Recently, our laboratory demonstrated that human neuroblastoma cells (SH-SY5Y) are capable of synthesizing morphine, the major active metabolite of opium poppy. Now our experiments are further substantiated by extending the biochemical studies to the entire morphine pathway in this human cell line. L-[1,2,3-13C3]- and [ring-2',5',6'-2H3]dopa showed high isotopic enrichment and incorporation in both the isoquinoline and the benzyl moiety of the endogenous morphine. [2,2-2H2]Dopamine, however, was exclusively incorporated only into the isoquinoline moiety. Neither the trioxygenated (R,S)-[1,3-13C2]norcoclaurine, the precursor of morphine in the poppy plant, nor (R)-[1,3,4-2H3]norlaudanosoline showed incorporation into endogenous morphine. However, (S)-[1,3,4-2H3]norlaudanosoline furnished a good isotopic enrichment and the loss of a single deuterium atom at the C-9 position of the morphine molecule, indicating that the change of configuration from (S)- to (R)-reticuline occurs via the intermediacy of 1,2-dehydroreticuline. Additional feeding experiments with potential morphinan precursors demonstrated substantial incorporation of [7-2H]salutaridinol, but not 7-[7-2H]episalutaridinol, and [7-2H,N-C2H3]oripavine, and [6-2H]codeine into morphine. Human morphine biosynthesis involves at least 19 chemical steps. For the most part, it is a reflection of the biosynthesis in opium poppy; however, there is a fundamental difference in the formation of the key intermediate (S)-reticuline: it proceeds via the tetraoxygenated initial isoquinoline alkaloid (S)-norlaudanosoline, whereas the plant morphine biosynthesis proceeds via the trioxygenated (S)-norcoclaurine. Following the plant biosynthetic pathway, (S)-reticuline undergoes a change of configuration at C-1 during its transformation to salutaridinol and thebaine. From thebaine, there is a bifurcate pathway leading to morphine proceeding via codeine or oripavine, in both plants and mammals.

Fig. 1.

Structure of the alkaloid morphine (the isoquinoline moiety is shown by bold lines and the nitrogen atom).

Fig. 2.

GC/MS/MS chromatograms of morphine derivatives. (A) The m/z 429 [M]^•+ of TMS-morphine isolated from SH-SY5Y cells cultured under standard conditions without isotope-labeled precursor. (B) The m/z 431 [M]^•+ of TMS-[15,15-²H₂]morphine isolated from SH-SY5Y cells cultured in the presence of 20 μM [2,2-²H₂]dopamine. (C) The m/z 359 [M - H-CH₃NCH₂C²H₂] of TMS-[15,15-²H₂]morphine isolated from SH-SY5Y cells cultured in the presence of 20 μM [2,2-²H₂]dopamine. This fragment ion showed loss of two ²H atoms at position C-15 of the morphine molecule.

Fig. 3.

GC/MS/MS chromatograms of morphine derivatives. (A) The m/z 429 [M]^•+ of TMS-morphine isolated from SH-SY5Y cells cultured under standard conditions without isotope-labeled precursor. (B) The m/z 433 [M]^•+ of TMS-[9,10,15,16-¹³C₄]morphine isolated from SH-SY5Y cells cultured in the presence of 20 μM l-[1,2,3-¹³C₃]dopa (¹³C-enriched carbon atoms are indicated by •). (C) The m/z 361 [M - H-CH₃N¹³CH₂¹³CH₂]⁺ of TMS-[9,10,15,16-¹³C₄]morphine isolated from SH-SY5Y cells cultured in the presence of 20 μM l-[1,2,3-¹³C₃]dopa. This fragment ion showed loss of two ¹³C atoms at positions C-15 and C-16 of the morphine molecule and retention of two ¹³C atoms at positions C-9 and C-10 (¹³C-enriched carbon atoms are indicated by •). (D) The m/z 432 [M]^•+ of TMS-[1,2,8-²H₃]morphine isolated from SH-SY5Y cells cultured in the presence of 20 μM l-[ring-2′,5′,6′-²H₃]dopa. (E) The m/z 362 [M - H-CH₃NCH₂CH₂]⁺ of TMS-[1,2,8-²H₃]morphine isolated from SH-SY5Y cells cultured in the presence of 20 μM l-[ring-2′,5′,6′-²H₃]dopa. This fragment ion showed retention of all three ²H atoms at positions C-1, C-2, and C-8.

Fig. 4.

MS/MS product ion spectra of morphine derivatives. (A) The m/z 429 [M]^•+ ion of TMS-morphine isolated from SH-SY5Y cells cultured under standard conditions without isotope-labeled precursor. (B) The m/z 431 [M]^•+ of TMS-[15,16-²H₂]morphine isolated from SH-SY5Y cells cultured in the presence of 20 μM(S)-[1,3,4-²H₃]norlaudanosoline. This product ion spectrum showed that the C-9 position, originating from C-1 of (S)-[1,3,4-²H₃]norlaudanosoline, of the mammalian morphine did not carry a ²H label, demonstrating a loss of one deuterium atom during mammalian morphine biosynthesis.

Fig. 5.

An abbreviated biosynthetic pathway leading from l-tyrosine to morphine showing the initial isoquinoline alkaloid, (S)-norlaudanosoline, the change of configuration from (S)- to (R)-reticuline and the bifurcate pathway from thebaine to morphine.