Tautomerism, Oxidation of Xenobiotics and of Macrolide Immunosuppressive Drugs                                                        

 Dr. Lhoëst G.J.J.        

 

The word tautomerism was proposed by LAAR (1886) to describe the mobile equilibrium between two compounds containing weekly bonded hydrogen atoms.

Considerably later, recognition was given to the possibility of a related reversible isomeric change, ring-chain tautomerism, in which one of the tautomeric forms was cyclic (MEYER and JACOBSON , 1913). A very peculiar example of ring-chain tautomerism effects, attributed to the hemiketal and carbinolamine functions, is examplified by the case of an indole alkaloid voamonine.

 The structural requirements for ring-chain tautomerism can be stated in very general terms with regard to the chain tautomer.  It must possess at least two functional groups, one containing a multiple bond and the other capable of effecting an additive reaction at the multiple bond.  For example if YZ is a function containing a double bond which will react additively with X, the corresponding cyclic tautomer could possess one of the two structures depending of the direction of addition. 

Types of ring-chain tautomers can be grouped according to the nature of X.  If X tends to be electron-deficient, it will become attached to the more electronegative atom Z.  This type of inter-conversion might be called “electrophilic tautomerism” .  When X, is electron-rich, it becomes attached to Y and might be called “ nucleophilic tautomerism”.

 Electrophilic and Nucleophilic tautomerism are exemplified by the case of O-phthalaldehydic acid (Wheeler, Young and Erley) and O-Phthaloyl chloride (OTT, 1943).  A recent example of electrophilic tautomerism was observed for a peculiar metabolite of the dihydroindole alkaloid  Malagashanine. 

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When incubated in the presence of human liver microsomes, this drug is mainly metabolized under the influence of the cytochrome P-450  dependent mixed oxygenase enzymic system  to two metabolites, a N-demethylated metabolite and a hydroxylated metabolite in the a position of the N-CH3 group leading to the possibility of ring and open chain tautomerism effects.

The electrospray mass spectra of malagashanine metabolite hydroxylated in the a position of the N-methyl group revealed the presence of a quasi-molecular ion of mass m/z = 415, of sodium and potassium adducts of mass m/z = 437 and 453.  The main fragmentation pathway leading to the loss of acetaldehyde and methylamine were  demonstrated on a Jeol Lcmate and on a Bruker Quistor ion trap instrument by MS/MS mass spectrometry

 

 

 

 

 

 

Resulting from the opening of the carbinolamine group giving rise to an aldehydic group ,  a dimerization process between two chain tautomers may be observed, leading to different molecular adducts, and mainly to a fragment ion  of mass m/z = 793 as shown in the ESI+ mass spectrum of this compound.

 

                                                                                                                                                                                             

 

 

 

Are ring-chain tautomerism effects observed in the mode of action and metabolism of immunosuppressive drugs ?

In order to mediate their effects,  FK506, CsA  must each bind with high affinity to a cytosolic target protein known as immunophilins.  FK506 or SDZ-RAD bind to FKBP as a result of their identical binding domain.

It has been demonstrated that the immunophilin –drug complex was the active moiety interacting with intracellular molecules involved in signal transduction.  The CYP-CsA and FKBP-FK506 complexes interfere with Ca2+ -dependent signalling pathways, whereas SDZ-RAD interferes with a distinct set of Ca2+ independent pathways most probably because the structures of the respective effector sectors are different.

The target of the immunophilin-drug complexes is the calcium and calmodulin  -dependent protein phosphatase, calcineurin.  The formation of this ternary complex inhibits dephosphorylation by calcineurin of the nucleic factor of activated T-cell (NFATc) preventing its translocation into the nucleus, a step which is necessary to start IL2 transcription.

Let us come back to the formation of the binary complex and a second question  may be addressed:

Is it  possible to identify an important site of inter-molecular interaction of the immunophilin FKBP-12 with FK506?

To my opinion a transient tetrahedral covalent adduct is formed at the C8 position by inter-molecular interaction of a nucleophile residue on the enzyme, such as an amino group giving rise to a carbinolamine function which is submitted to ring-and chain tautomerism effects. The piperidine  free N-H group is also most probably engaged in hydrogen bonding with other polar amino acids side chains of the FK506 binding protein ( FKBP-12).

If this occurs, it may be foreseen that the modulation of the binding affinity between the binary complex and calcineurin at the effector site of FK506 has a great chance to occur.

What did we learn from intra-molecular interactions observed with specific metabolites of FK506?

 

 

Intra-molecular interactions were observed for certain specific metabolites of FK-506 and were detected by FAB and electrospray mass spectrometry.  Before introducing some FAB and electrospray mass spectrometric data corresponding to oxidation compounds of FK506, the electrospray mass spectrum of  FK506 will be discussed. The electrospray mass spectrum of FK506 obtained with a JEOL Lcmate instrument reveals the presence of ammonium, sodium and potassium adducts at m/z = 821, 826 and 842, respectively. Two main fragmentation pathways were observed, the first one corresponding to the loss of 111 Da plus additional elimination of water and CO2 and the other corresponding to the loss of 210 Da with subsequent eliminaton of 111 Da.

This figure illustrates mainly a fragmentation pathway of FK506 where  losses of 111 Da  and 210 Da as detailed in the subsequent fragmentation pathways.

 

 

What was observed is that the FK506 C36-C37 dihydrodiol exists under of different tautomeric forms separated by HPLC and specific tautomers result from the  interaction of the C37 hydroxy group with the C8 FK506 carbonyl function.

 

 

 

The FAB mass spectrum of the FK506 C36-C37 dihydrodiol isolated from erythromycin-induced rabbit liver microsomes reveals the presence of a sodium and potassium adduct at m/z = 860 and 876, respectively. Also fragment ions are observed at m/z = 820 and 802 corresponding to the elimination of water molecules and at 772 corresponding to the loss of formaldehyde proving that a FK506 C36-C37 FK506 dihydrodiol was formed. Significant fragment ions corresponding to the loss of pipecolic acid and water were observed proving that direct interactions between the C37 hydroxy group and the C8 carbonyl function did occur without the intermediate elimination of 2 formyl piperidine -2-ene (111 Da).

 Intra-molecular interactions of this type were also described for allyl 5 (hydroxy-2-propyl) 5 barbituric acid where ring and open chain tautomers of this compound where observed by a French laboratory.

 

The observations made on the FK506 dihydrodiol were also confirmed by the electrospray mass spectrum of the FK506 C36-C37 epoxide (slide 30) , metabolic precursor of the FK506 dihydrodiol which was obtained by chemical synthesis, extraction and finally was isolated by reverse phase HPLC.

The electrospray mass spectrum  of the FK506 epoxide reveals the presence of an ammonium adduct at m/z = 837, of a sodium  adduct at m/z = 842 and of a potassium adduct at m/z = 858 indicating that one atom of oxygen has been introduced in the molecule. Moreover fragment ions are observed at m/z = 560 ( M - 282)Na+, 544 (M - 282 - O)Na+ and 433 resulting from the loss of 111 Da . As shown in the next fragmentation pathways, the product ions of m/z = 842 were found to be 560, 544 and 433 proving the existence of an epoxide and the elimination of 2-formyl-piperidine 2-ene (111 Da).  For the FK506 C36-C37 epoxide, interaction with the C8 FK506 carbonyl group does not occur and the direct loss of pipecolic acid (129 Da) is no more observed and is replaced by the elimination of 2-formyl piperidine 2-ene (111 Da) . 

The same observations were made for the FK506 C19-C20 epoxide.

 

 

The nanospray mass spectrum of the FK506 binding protein (FKBP-12) obtained with the new Jeol nanospray source demonstratres that the molecular weight  is 11821 and according to Sigma the molecular weight calculated from the known sequence is 11820.  The first results acquired on the FKBP-12-FK506 complex  reveals unabundant peaks at 943 ,941 and 1007 corresponding to adducted FK506 fragments to FKBP12   as  illustrated in the next slide. The adducted fragments of FK506 to FKBP12 resulting from the loss of pipecolic acid , 210 Da and 44 Da are expected to be found either at 11821 + 443 (826 – 129 – 210 – 44) = 12264/13 =  943.3 or at 12241/13 = 941.6 Da.  When fragmentation occurs in this part of the molecule, the FK506 adducted fragment is smaller and the expected molecular weight for a 12 charges species is 1007.

 

 

 

In vitro Immunosuppressive activity of some metabolites

nThe in vitro immunosuppressive activity, as measured by the mixed lymphocyte reaction (MLR), indicates that the FK506 C36-C37 dihydrodiol and C36-C37 dihydrodiol tris- epoxide do not exhibit significant in vitro immunosuppressive activity compared to the parent compound most probably because   intra-molecular competition for the same binding site exists at the C8 position  of FK506. 

 

 

Conclusions

 

Data we have shown seem to confirm that some reversible covalent intra

or inter-molecular interaction between a nucleophile and the C8 FK506

position do exist .

 

The FK506 C36-C37 dihydrodiol and the C36-C37 dihydrodiol tris-

epoxide do not exhibit significant in vitro immunosuppressive activity

compared to the parent compound most probably because intra-

molecular competition exist for the same binding site.