418 Extended Abstracts / Chemico-Biological Interactions 157–158 (2005) 353–434

Scheme 1. The stereochemical course of the irreversible inhibition of serine hydrolases.

(40)The stereochemistry of the inhibition of acetyl-cholinesterase with acetylcholine-mimetic 7-aza-2,4-dioxaphosphadecalins

Stefan Furegati, Oliver Zerbe, Peter Ruedi∗

Institute of Organic Chemistry, University ofZurich, Winterthurerstrasse 190, CH-8057 Zurich,Switzerland

Abstract

The irreversible inhibition of acetylcholinesterasewith the decalin-typecis- andtrans-3-fluoro-2,4-dioxa-7-aza-3-phosphadecalins was investigated by31P-NMRspectroscopy. The stereochemical outcome (inversion orretention at the P-atom) is dependent on the structure ofthe inhibitors.

1. Introduction

31P-NMR spectroscopic investigations with the enan-tiomerically purecis- andtrans-3-(2,4-dinitrophenoxy)-2,4-dioxa-3-phosphabicyclo[4.4.0]decane 3-oxides(Z = CH2) evidenced that the irreversible inhibitionof �-chymotrypsin follows different stereochemicalpathways[1,2]. Depending on the isomer, neat inversion(trans-axial), inversion and retention (trans-equatorial),and neat retention (cis-equatorial) of the configuration at

ratesmicale ofare

based on the fact that the31P-NMR resonance of theaxial epimers is shifted upfield with respect to that ofthe equatorial ones and that the3JP,H is indicative ofthe conformation of the heterocyclic ring (Table 1).The covalent nature of the Ser195 O P bond wasproven by preparing 1,5,5-trideuterated inhibitors andconducting1H-correlated31P{2H}-NMR experimentsthat verified a vicinal coupling between the P-atomof the inhibitors and the Ser195-H2 of the enzyme[3] (Scheme 1).

2. Results and discussion

2.1. Spectroscopic data of the inhibitors and themodel compounds (Table 1)

2.2. Inhibition experiments

Non-specific, peripheric phosphorylations result in31P-NMR spectra yielding uninterpretable signals thatare only visible after long accumulation times. In order toobtain reliable active-site specific information about thephosphorylated enzyme, a thorough elaboration of thesample handling, especially the denaturation procedurewas required: incubation of AChE (5 mg, 9× 10−8 mol)with the inhibitor (ca. 1000 eq) in 0.1 M phosphatebuffer, pH 7.5 (1 d), followed by dialysis (2 d) in0.01 M TRIS, pH 7.7 and lyophilisation. The31P{1H}-NMR were run at 202.5 MHz; sample in 240�l D2O,

d

in

the P-atom was found. This result clearly demonstthe unexpected dependence of the stereochecourse of an enzymatic reaction on the structurthe inhibitor. The spectroscopic investigations

∗ Corresponding author. Tel.: +41 1 635 42 41.E-mail address: peru@oci.unizh.ch (P. Ruedi).

6 M guanidine-d5 × DCl, 50 mM NaOAc, pH 4.0, 27◦;reference 5× 10−8 mol POPh3, calibrated with 85%H3PO4.

According to the observed31P chemical shifts ancomparison with the model compounds (Table 1), thereactions of the racemic inhibitors1ax, 1eq, and 3axwith AchE[5] yield the diastereomeric adducts5, 6 and7 (Schemes 2 and 3). The mechanistic explication

Extended Abstracts / Chemico-Biological Interactions 157–158 (2005) 353–434 419

Table 131P-NMR data of the inhibitors1 and3 [4], and the model compounds2 and4.

Scheme 2. Stereochemistry and mechanism of the inhibition of AChE as exemplified with1eq.

terms of adjacent or in-line attack of the nucleophile isexemplified with1eq (Scheme 2).

The results are similar to those obtained with�-chymotrypsin[1,2]: Inhibitor 1eq with the predominanttwist boat conformation (TB)[4,5] reacts with inver-

sion and retention (Scheme 2), whereas neat retention isobserved with3ax (Scheme 3). The outcome of1ax can-not be explained in simple terms since an in-line attackof the enzyme at1ax with a double chair conforma-tion would be preferred, and inversion at P(3) would

Scheme 3. Stereochemistry of the inhibition of AChE with1ax and3ax.

420 Extended Abstracts / Chemico-Biological Interactions 157–158 (2005) 353–434

be expected. As the spectra represent the final state ofthe inhibition reaction, the results might be influencedby post-inhibitory equilibrations.

As evidenced with�-chymotrypsin, the findingsclearly show the dependence of the stereochemicalcourse of an enzymatic reaction on the structure of theinhibitor.

References

[1] W. Ganci, E.J.M. Meier, F.A. Merckling, G. Przibille, U. Ringeisen,P. Ruedi, Stereochemistry of the inhibition of�-chymotrypsinwith optically active bicyclic organophosphates:31P-NMR stud-ies, Helv. Chim. Acta 80 (1997) 421–435.

[2] S. Furegati, W. Ganci, G. Przibille, P. Ruedi, Stereochemistry of theinhibition of �-chymotrypsin with optically activecis-decalin-typeorganophosphates:31P-NMR studies, Helv. Chim. Acta 81 (1998)1127–1138.

[3] M.J. Stockli, P. Ruedi, Covalent-bond formation in the course of theinhibition of �-chymotrypsin withtrans-decalin-type organophos-phates:31P-NMR evidence, Helv. Chim. Acta 84 (2001) 106–116.

[4] S. Furegati, W. Ganci, F. Gorla, U. Ringeisen, P. Ruedi, 2,4-Dioxa-7-aza-, 2,4-dioxa-8-aza-, and 2,4-dioxa-9-aza-3-phosphadecalinsas rigid acetylcholine mimetics: syntheses and characterization,Helv. Chim. Acta 87 (2004) 2629–2661.

[5] S. Furegati, F. Gorla, A. Linden, P. Ruedi, Decalin-typeacetylcholine mimetic organophosphates as inhibitors of acetyl-cholinesterase, Chem. Biol. Interact. 151 (2005) (preceding paper).

doi:10.1016/j.cbi.2005.10.085

(41)

ndigh

ivehe

oxime compounds after being inhibited by chiralorganophosphates. The aim of this work was to studythe stereoselectivity of AChE and BChE in reactionswith enantiomers of quinuclidin-3-yl acetates as sub-strates as well as in the reversible inhibition of enzymesby enantiomers of quinuclidin-3-ol derivatives. For thatpurpose (R)- and (S)-alcohols and their acetates wereprepared and characterized.

2. Experimental procedure

The (R)- and (S)-enantiomers of quinuclidin-3-ol,(R)- and (S)-QOH, were prepared by the resolution ofcommercially available racemic quinuclidin-3-ol usingd- andl-tartaric acid. The quinuclidin-3-acetates, (R)-and (S)-QA were synthesized by esterification of the(R)-and (S)-QOH with acetic anhydride. Subsequently,both enantiomers of quaternaryN-methyl alcohols andacetates (MeQOH and MeQOA) were prepared usingmethyl iodide as a quaternization agent. Enantiomersof quaternaryN-benzyl alcohols and acetates (BzQOHand BzQA) were synthesized in the same manner byusing benzyl bromide as a quaternization agent. Syn-thesized compounds were characterized, identified, andtheir purity was established by1H NMR, 13C NMR andMS spectroscopies, elemental analyses and by determin-ing the melting points and optical rotation values.

anymeu-thehEandtylth-

rved

Preparation of enantiomers of quinuclidin-3-Olderivatives and their interactions with humancholinesterases

Vera Simeon-Rudolfa,∗, Sr–danka Tomicb,Anita Bosaka, Ines Primozicb, Mislav Orsulicb

a Institute for Medical Research and OccupationalHealth, Ksaverska cesta 2, POB 291, 10001 Zagreb,Croatiab Department of Chemistry, Faculty of Science, Univer-sity of Zagreb, Strossmayerov trg 14, 10000 Zagreb,Croatia

1. Introduction

Acetylcholinesterase (AChE; EC 3.1.1.7) abutyrylcholinesterase (BChE; EC 3.1.1.8) reveal hstereoselectivity in inhibition by chiral progressinhibitors as well as in their reactivation by t

∗ Corresponding author.E-mail address: vesimeon@imi.hr (V. Simeon-Rudolf).

The source of AChE and BChE were native humerythrocyte and human plasma, respectively. Enzhydrolysis of the (R) -and (S)-enantiomers of the quinclidine acetates was followed by pH-stat titration ofliberated acetic acid. The inhibition of AChE and BCby the enantiomers of QOH, MeQOH and BzQOHby the respective acetates was measured with aceiocholine as substrate with the thiol reagent DTNB.

3. Results and discussion

Stereoselectivity of the cholinesterases was obsein the hydrolysis of the acetates. All (R)-derivativeswere hydrolyzed by both AChE and BChE while (S)-