Immunologically driven antibodies chemical engineering: Design and synthesis of a hapten aimed at nerve agent hydrolysis

Article abstract of DOI:10.1016/j.tetlet.2005.08.032

In this letter, we describe the design and the synthesis of an organophosphorus hapten aimed at a mixed biotechnological-chemical strategy for the mild decontamination of exceedingly toxic nerve agent VX. Hapten will be used to raise and select monoclonal antibodies (mAbs) able to bind both the nerve agent, and an oxime, derived from pyridinaldoxime, able to specifically hydrolyze its P-S bond. In order to significantly increase the hydrolysis reaction rate, the controlled respective positioning of the oxime and of the substrate will be achieved through the immunization, and in a second step, the oxime will be bound to the mAb in a reactive position towards the reactive thiophosphate functionality of the substrate.

Full text of DOI:10.1016/j.tetlet.2005.08.032

Tetrahedron
Letters
Tetrahedron Letters 46 (2005) 6809–6814
Immunologically driven antibodies chemical engineering: design
and synthesis of a hapten aimed at nerve agent hydrolysis
Florence Jovic,a Ludivine Louise,b Charles Mioskowskic and Pierre-Yves Renardb,*
aService de Marquage mole´culaire et de Chimie Bioorganique, CEA Saclay F-91191 Gif sur Yvette cedex, France
bInstitut de Recherche en Chimie Organique Fine, Laboratoire d’He´te´rochimie Organique, CNRS UMR 6014,
`
Universite´ de Rouen et INSA de Rouen, Rue Tesniere F-76131 Mont Saint Aignan cedex, France
c
`
´ ´
Laboratoire de Synthese Bioorganique, CNRS UMR 7514, Universite Louis Pasteur de Strasbourg, Faculte de Pharmacie, 74,
Route du Rhin BP 24, F-67401 Illkirch–Graffenstaden, France
Received 28 June 2005;revised 3 August 2005;accepted 5 August 2005
Abstract—In this letter, we describe the design and the synthesis of an organophosphorus hapten aimed at a mixed biotechnolog-
ical–chemical strategy for the mild decontamination of exceedingly toxic nerve agent VX. Hapten will be used to raise and select
monoclonal antibodies (mAbs) able to bind both the nerve agent, and an oxime, derived from pyridinaldoxime, able to specifically
hydrolyze its P–S bond. In order to significantly increase the hydrolysis reaction rate, the controlled respective positioning of the
oxime and of the substrate will be achieved through the immunization, and in a second step, the oxime will be bound to the
mAb in a reactive position towards the reactive thiophosphate functionality of the substrate.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Acetylcholinesterase irreversible inhibitors such as
organophosphorus derivatives Sarin (1a), Soman (1b),
Tabun (2) or exceedingly toxic VX (3a) (Scheme 1) are
nerve agents used as chemical warfare.1,2 The interna-
tional control of the proliferation of those weapons of
mass destruction3 is thwarted by the ease of their synthe-
sis, and by the similarity between their chemical precur-
sors and those of widely used pest-control agents.
mination and demilitarization of organophosphorus
nerve agents is therefore an urgent and topical problem.
Most of the decontamination means intended at neutral-
izing those nerve agents use strong alkaline and/or oxi-
dative media, and are thus incompatible with medical
use, but also skin treatment or sensitive material.4,5 In
the last few years, after September 11, considerable
amount of work has been undertaken on the biotechno-
logical approaches to decontamination of these toxic
substances.6 In this letter, we want to display a dual
biotechnological–chemical approach, aiming at an
affordable and environmentally friendly process for
decontamination of phosphonothioate VX.
The ease of production of these agents and their toxicity
make them attractive to terrorist groups. The deconta-
O
P
O
P
O
P
Among the nerve agents, VX, against which no satisfac-
tory mild decontamination mean has been proposed yet,
is by far the most difficult target to deal with, since (1) it
is exceedingly toxic (LD50 = 8 lg/kg, i.v., and 28 lg/kg,
percutaneous, rabbit),5 (2) its hydrolysis can occur
through at least four different pathways,5,7 and (3) con-
trary to the phosphorus–sulfur bond cleavage, phospho-
rus–oxygen bond cleavage8 gives rise to an equally toxic
compound.5 In order to have a less toxic VX mimic, but
which displays equivalent hydrolysis features, we pro-
posed the use of its aromatic equivalent PhX (3b)
(Scheme 1).9 Interestingly, PhX displays exactly the
same hydrolysis profile than VX, but its toxicity has
N
F
N
R
S
O
R
R
O
N
O
R
(1a) R = H : Sarin
(1b) R = Me : Soman
(3a) R = Me : VX
(3b) R = Ph : PhX
(2) Tabun
Scheme 1. Nerve agents structures.
Keywords: Catalytic antibodies;Bioorganic chemistry;Organophos-
phorus chemistry;Nerve agent.
Corresponding author. Tel.: +33 2 45 52 24 14;fax: +33 2 45 52 29
59;e-mail: pierre-yves.renard@univ-rouen.fr
*
0040-4039/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2005.08.032

6810
F. Jovic et al. / Tetrahedron Letters46 (2005) 6809–6814
been estimated to be two orders of magnitude lower
than VX, and in vitro AChE inhibition appears
reversible.10
to activate a water molecule. For strategic reasons, the
first solution was adopted. Among the a-nucleophiles,
oximes7,29 seemed to be the more suited since they cleave
specifically the P–S bond,30,31 and already shown great
efficiency (including in vivo)32,33 in reactivating poi-
soned cholinesterases. Other candidates such as peroxy-
acids,34 peroxides7,8,35 or iodosylcarboxylates,36 are less
adapted to an in vivo application, and might react with
the mAbs. 2-Pyridinealdoxime (4), pralidoxime (5) and
HI-6 (pyridinium, 1-[[[4-(aminocarbonyl) pyridi-
nio]methoxy]methyl]-2-[(hydroxyimino)methyl]-, dichlo-
ride (6) (Scheme 2)37,38 displaying both high P–S
cleavage efficiency and antigenic determinants were cho-
sen as structural bases for the catalytic residue. The
turn-over for the catalyst will be achieved, since the
intermediary nucleophilic addition product bears a par-
ticularly water labile P–O–N moiety. Synthetic dead
ends dealing with the pyridinium based haptens rapidly
led us to choose 2-pyridinaldoxime (4) as the target for
the catalytic residue.
In order to find efficient biocompatible decontamination
means against VX, we stressed our efforts on catalytic
antibodies11–15 which can be produced in high quantities
at very low costs. Our first efforts were led on monoclo-
nal antibodies selected for their ability to bind transition
state analogues.10,16 Catalytic but insufficiently active
antibodies were obtained, and our results led us to the
conclusion that a more efficient solution would be to
find a way to graft a residue capable of cleaving the
P–S bond in the binding site of an antibody. With this
goal in mind, we described a new strategy17 based on
chemical engineering of monoclonal antibodies in order
to covalently attach a targeted catalytic residue in the
binding pocket of an anti-PhX mAb. Our approach
can be considered as the merger of two already
described strategies for catalytic antibodies: the use of
cofactors,18–20 and the chemical introduction of a cata-
lytic function close to the antibody binding site.21,22
The hapten will be designed to include binding of a
nucleophile as well as the substrate in a reactive relative
position. Taking into account the limitations raised by a
previous study,17 the catalytic residue itself will be then
covalently attached onto the selected monoclonal anti-
body binding pocket.
A first hapten (H1, Fig. 1) was thus designed, displaying
the following features:
• A PhX analogue, with a noncleavable aminopropyl
side chain, methylene replacing the sulfur atom
in order to both lower the potential toxicity of this
hapten, and to ensure the structural integrity of the
hapten throughout the immunization procedure.
• An oxime analogue, mimicking the addition of the
oxygen atom onto the phosphorus, opposite to the
leaving group, in order to have a correct placement
of the two partners to enhance the SN2(P) character
of the reaction.7
The main advantages of our strategy rely on the facts
that (1) the monoclonal antibodies are screened and
selected through their binding abilities, which can be
done through well established techniques. (2) Any cata-
lytic residue can be grafted onto the antibody binding
site, widening thus the amount of catalytic functions
available. The residue yet requires strong antigenic
determinants in order to efficiently and specifically fit
into the mAb binding pocket. (3) The relative position
of the catalytic residue and the substrate are controlled
by the very structure of the hapten itself. Thus, through
binding to the antibody, both compounds will be in a
reactive position, lowering the required entropy of the
reaction, and orienting the reactivity towards the P–S
bond cleavage.
• The labile P–O–N moiety is replaced by a non cleav-
able P–CH2–N motive.
• An aliphatic linker for hapten binding to carrier pro-
tein via homobifunctional reagents such as glutaral-
dehyde or disuccinimidyl suberate.
Hapten (H1) retrosynthetic scheme is relatively straight-
forward, with formation of the imine bond as the key
step between aldehyde (7) and amine bearing phosphine
oxide (8) (Scheme 3).
The first point to be solved is the selection of the suitable
catalytic residue. With the selective P–S bond cleavage
as a goal, two possibilities are available: a nucleophile
displaying an a effect,23,24 or a metallic species25–28 able
Aldehyde (7) (R2 = phthalimide) was synthesized
through monoformylation of 2,6-dibromopyridine, fol-
lowed by introduction of the aminopropyl linker
(Scheme 4). Monoformylation of 2,6-dibromopyridine
N
N
N
N
N
N
HO
HO
HO
O
Cl
Cl
O
Pralidoxime (5)
HI-6 (6)
(4)
N
Cl
NH2
Scheme 2. Oxime structures.

F. Jovic et al. / Tetrahedron Letters46 (2005) 6809–6814
6811
O
H2N
Linker to
N
P
N
H1
N
N
carrier protein
Covalent linker
O
P
N
O
S
N
OH
PhX
oxime
Figure 1. Structure of first hapten (H1).
O
NR2
O
N
(7)
P
N
N
R2N
+
N
Ph
O
P
N
H2N
Ph
(8)
Scheme 3. Hapten (H1) retrosynthetic analysis.
was achieved via selective formation of 2-bromo-6-lithio-
pyridine (according to previously described proce-
dures),39 followed by electrophilic substitution with
DMF. In our hands, best yields (85%) were obtained
using diethyl ether as the solvent, with a careful monitor-
ing of the temperature (À60 ꢁC). Interestingly, the ali-
phatic linker could be readily introduced without
protection of the formyl moiety through Sonogashira
coupling between 6-bromo-pyridine-2-carbaldehyde
and N-prop-2-ynyl phthalimide (10) (prepared from
propargyl alcohol and phthalimide through Mistunobu
reaction). Yields for the Sonogashira coupling is rela-
tively modest (57%), but N-phthalimide moiety has al-
ready been described as poorly suitable for the
Sonogashira coupling process.40
side chains onto the phenyl phosphine oxide moiety.
First attempts to bind the aminopropyl side chain onto
the phosphorus atom through formation of organo-
metallic species showed that the hindered tertiary amine
moiety was poorly compatible even with Grignard or
organozinc reagent. This was achieved through two suc-
cessive Michaelis–Arbuzov reactions. The bromo amine
(14) was formed from the corresponding alcohol (13)43
through usual procedure (PPh3;Br 2 in dichloromethane,
98% yield). Heating at 100 ꢁC for 2 days a neat mixture
of O,O-diethyl phenyl phosphonite and bromo amine
(14) gave a satisfactory 78% yield of phosphinate (15)
(Scheme 5). In order to form the a amino phosphine
oxide moiety, the best method available is to perform
an Abramov–Pudovik like reaction through addition
of metal phosphites44–48 to aldehyde- and ketone-de-
rived imines, or as many imines are unstable or difficult
to purify, through the akin Lewis acid catalyzed one pot
three-components reaction (starting from carbonyl com-
pounds, amines and hydrogeno-phosphites).49–53 The
first step is thus to form the phosphinite (16) from phos-
phinate (15). Best results were obtained using Red-Al
(aluminium bis(2-methoxyethoxy)sodium hydride).54
1H and 31P NMR analysis of the crude material showed
that addition of 1.1 equiv hydride resulted in the com-
plete conversion of (15) to (16). Yet, the modest 53%
yield is related to the tedious purification of the phos-
phonite (Scheme 5).
Clean hydrogenation of the triple bond was also a tricky
task. When led using classical conditions, with platinum
oxide, Raney Nickel or palladium as catalysts, only
incomplete reduction of the alkyne to alkene was ob-
served. Complete reduction to alkane was only achieved
using higher hydrogen pressure, and resulted in the con-
secutive reduction of the conjugated aldehyde to the cor-
responding alcohol. Eventually, best and reproducible
yields of alcohol (12) (76–80%) were obtained under
drastic conditions: a concentrated 0.1 M solution of
(11) in glacial acetic acid, with 12.5% molar Pd/C, and
submitted to a 20 bar hydrogen pressure. Alcohol (12)
was then readily oxidized to aldehyde (7) using
IBX41,42 with a nearly quantitative yield. Aldehyde (7)
was thus synthesized in four steps and an overall 39%
yield (Scheme 4).
Whichever the base, we used (n-BuLi, NaOEt, i-Pr2Net
or even P4t-Bu phosphazene9), attempts to proceed to
the deprotonation of phosphonite (16) were yet unsuc-
cessful. On the other hand, trimethylsilylphosphinites
are described to be very reactive species for Michaelis–
Arbuzov reactions.55 Indeed, protected a amino phos-
Phosphine oxide (8) synthesis required attachment of
both the tertiary aminopropyl and the aminomethylene

6812
F. Jovic et al. / Tetrahedron Letters46 (2005) 6809–6814
1) n-BuLi, Et2O, -78˚C,
then DMF
2) MeOH/AcOHO
85%
Br
N
Br
N
Br
(9)
Pd(PPh3)4,
CuI, CH2Cl2 /Et3N
O
O
+
N
57%
NPht
PPh3, DIAD
THF
HN
(11)
O
80%
+
O
N
OH
(10)
O
IBX, AcOEt
Pd/C, AcOH, H2
80%
NPht
(11)
NPht
N
N
99%
(12)
(7)
O
OH
Scheme 4. Aldehyde (7) synthesis.
Red-Al
THF
O
PhP(OEt)2
O
100˚C neat
X
N
P
N
P
N
Ph
53%
78%
Ph
H
O
(16)
(15)
PPh3.Br2
CH2Cl2
98%
(13) : X = OH
(14) : X = Br
Scheme 5. Phosphonite (16) synthesis.
phine oxide (18) was formed in high yield when heating
at reflux a THF solution of phosphinite (16) with hex-
amethyl disilazane and bromomethyl phthalimide
through in situ formation of the trimethylsilylphosphi-
nite (17).56,57 Final deprotection of the phthalimide pro-
tective group afforded phosphine oxide (8), which was
obtained in four steps and 22% yield from O,O-diethyl
phenylphosphonite (Scheme 6).
water trap. Interestingly, imine (18) proved stable for
days in a 1/1 THF–H2O mixture and in phosphate buf-
1
fer (pH 7.4), as followed by H and 31P NMR, yet any
attempts to achieve the final deprotection of the N-
phthalimide failed (Scheme 7).
Indeed, when (18) was submitted to 10 equiv N-butyl-
amine in phosphate buffer (pH 7.4), imine slowly
decomposed. Within 3 days, all of (18) had disap-
peared, showing that despite the stabilization through
delocalization with the pyridine aromatic ring,
imine moiety of hapten (H1) will be incompatible with
Final coupling between aldehyde (7) and amine (8) was
easily achieved mixing the two partners in THF under
basic conditions (Et3N), and with MgSO4 acting as a
Me3Si
O
P
O
HMDS
Br
NPht
N
P
N
Ph
THF
∆
Ph
H
71%
(16)
(17)
O
P
O
P
H2N NH2
MeOH
N
N
Ph
Ph
O
75%
O
NH2
N
(18)
(8)
Scheme 6. Phosphine oxide (8) synthesis.

F. Jovic et al. / Tetrahedron Letters46 (2005) 6809–6814
6813
O
O
NPht
P
N
N
(7)
Ph
Et3N, MgSO4
THF
N
67%
O
P
N
NPht
N
Ph
NH2
(18)
(8)
Scheme 7. Imine formation.
12. Pollack, S. J.;Jacobs, J. W.;Schultz, P. G. Science 1986,
234, 1570–1573.
13. Schultz, P. G.;Yin, J.;Lerner, R. A. Angew. Chem., Int.
Ed. 2002, 41, 4427–4437.
14. Hasserodt, J. Synlett 1999, 12, 2007–2022.
15. Reymond, J.-L. Top. Curr. Chem. 1999, 200, 59–93.
16. Vayron, P.;Renard, P. Y.;Valleix, A.;Mioskowski, C.
Chem. Eur. J. 2000, 6, 1050–1063.
highly proteic media, and thus with the immunization
process.
Current efforts are stressed upon the formation of a new
hapten with the C–N double bond stabilized within an
oxazole or thiazole ring. Synthesis and results of the
immunization process will be reported in due time.
´
17. Dias, S.;Jovic, F.;Renard, P. Y.;Taran, F.;Cre
minon,
C.;Mioskowski, C.;Grassi, J. J. Immun. Methods 2002,
269, 81–98.
Acknowledgements
18. Ersoy, O.;Fleck, R.;Blanco, M.-J.;Masamune, S. Bioorg.
Med. Chem. 1999, 7, 279–286.
This work was financially supported by French ministry
19. Ersoy, O.;Fleck, R.;Sinskey, A.;Masamune, S.
J. Am.
´ ´
´ ´
of defence procurement agency (ÔDelegation Generale
pour lÕArmementÕ), through two Ph.D. fellowships
to F.J. and L.L. and the contract DGA/DSP/STTC/
DT-SH # 00-34-031.
Chem. Soc. 1998, 120, 817–818.
20. Mahy, J. P.;Desfosses, B.;de Lauzon, S.;Quilez, R.;
Desfosses, B.;Lion, L.;Mansuy, D.
Appl. Biochem.
Biotechnol. 1998, 75, 103–127.
21. Pollack, S. J.;Nakayama, G. R.;Schultz, P. G. Science
1988, 242, 1038–1040.
22. Pollack, S. J.;Schultz, P. G. J. Am. Chem. Soc. 1989, 111,
1929–1931.
References and notes
1. Taylor, P. In Goodman and GilmanÕsthe Pharmacological
Basis of Therapeutics, 10th ed.;Hardman, J. G., Limbird,
L. E., Goodman, A. G., Eds.;McGraw-Hill: New York,
2001;pp 175–192.
2. Langford, R. E. Introduction to Weaponsof Mass
Destruction, Radiological, Chemical and Biological;John
Wiley and Sons: Chichester, 2004.
3. Convention on the Prohibition of the Development, Produc-
tion, Stockpiling and Use of Chemical Weapons and on their
Destruction; The Technical Secretariat of the Organization
for the Prohibition of Chemical Weapons;Den Hague,
Netherlands, 1993.
23. Um, I. H.;Buncel, E. N. J. Org. Chem. 2000, 65, 577–582.
24. Um, I. K.;Hong, J.-Y.;Buncel, E. Chem. Commun. 2001,
27–28.
25. Hay, R. W.;Govan, N. Chem. Commun. 1990, 714–715.
26. Oh, S. J.;Yoon, C. W.;Park, J. W. J. Chem. Soc., Perkin
Trans. 2 1996, 329–331.
27. Hay, R. W.;Govan, N. Polyhedron 1998, 17, 2079–2085.
28. Kazankov, G. M.;Seergeva, V. S.;Efremenko, E. N.;
Alexandrova, L.;Varfolomeev, S. D.;Ryabov, A. D.
Angew. Chem., Int. Ed. 2000, 39, 3117–3119.
29. Epstein, J.;Callahan, J. J.;Bauer, V. E. Phosphorus 1974,
4, 157–163.
4. Yang, Y. C.;Baker, J. A.;Ward, J. R. Chem. Rev. 1992,
92, 1729–1743.
30. Boulet, C. A.;Hansen, A. S. Phosphorus, Sulfur Silicon
1991, 57, 147–161.
5. Yang, Y. C. Acc. Chem. Res. 1999, 32, 109–115.
6. Crabb, D.;DeFrank, J.;Penet, C. Chem. Ind. 2004, 14,
15.
7. Yang, Y. C.;Berg, F. J.;Szafraniec, L. L.;Beaudry, W. T.;
Bunton, C. A.;Kumar, A. J. Chem. Soc., Perkin Trans. 2
1997, 607–613.
8. Yang, Y. C.;Szafraniec, L. L.;Beaudry, W. T.;Rohr-
baugh, D. K.;Procell, L. R.;Samuel, J. B. J. Org. Chem.
1996, 61, 8407–8413.
9. Renard, P. Y.;Schwebel, H.;Vayron, P.;Josien, L.;
Valleix, A.;Mioskowski, C. Chem. Eur. J. 2002, 8, 2910–
2916.
31. Lundy, P. M.;Hansen, A. S.;Hand, B. T.;Boulet, C. A.
Toxicology 1992, 72, 99–105.
32. Dawson, R. M. J. Appl. Toxicol. 1994, 14, 317–331.
33. Leikin, J. B.;Thomas, R. G.;Walter, F. G.;Klein, R.;
Meislin, H. W. Crit. Care Med. 2002, 10, 2346–2354.
34. Cassagne, T.;Cristau, H. J.;Delmas, G.;Desgranges, M.;
Lion, C.;Magnaud, G.;Torreilles, E.;Virieux, D.
Heteroatom Chem. 2001, 12, 485–490.
35. Wagner, G. W.;Yang, Y. C. Ind. Eng. Chem. Res. 2002,
41, 1925–1928.
36. Morales-Rojas, H.;Moss, R. A. Chem. Rev. 2002, 102,
2497–2521.
´
10. Vayron, P.;Renard, P. Y.;Taran, F.;Cre
minon, C.;
37. Terrier, F.;MacCormack, P.;Kizilian, E.;Halle, J.-C.;
Demersemen, P.;Guir, F.;Lion, C. J. Chem. Soc., Perkin
Trans. 2 1991, 153–158, and references cited therein.
38. Koplovitz, I.;Stewart, J. R. Toxicol. Lett. 1994, 70, 269–
279.
Frobert, Y.;Grassi, J.;Mioskowski, C. Proc. Natl. Acad.
Sci. U.S.A. 2000, 97, 7058–7063.
11. Tramontano, A.;Janda, K. D.;Lerner, R. A.
Science
1986, 234, 1566–1570.

6814
F. Jovic et al. / Tetrahedron Letters46 (2005) 6809–6814
39. Cai, D.;Hughes, D. L.;Verhoeven, T. R. Tetrahedron
Lett. 1996, 37, 2537–2540, and references cited therein.
40. Toyota, M.;Komori, C.;Ihara, M. J. Org. Chem. 2000,
65, 7110–7113.
49. Qian, C. T.;Huang, T. S. J. Org. Chem. 1998, 63, 4125–
4128.
50. Lee, S.;Park, J. H.;Kang, J.;Lee, J. K. Chem. Commun.
2001, 1698–1699.
41. Frigerio, M.;Santagostino, M.;Sputore, S. J. Org. Chem.
1999, 64, 4537–4538.
51. Ranu, B. C.;Hajra, A.;Jana, U. Org. Lett. 1999, 1, 1141–
1143.
42. More, J. D.;Finney, N. S. Org. Lett. 2002, 4, 3001–3003.
43. Chini, M.;Crotti, L.;Favero, L.;Maccia, F. Tetrahedron
Lett. 1994, 35, 761–764.
44. Pudovik, A. N. Dokl. Akad. Nauk SSSR 1952, 83, 865–
868; Chem. Abstr. 1953, 47, 4300.
52. Manabe, K.;Kobayashi, S. Chem. Commun. 2000, 669–
670.
53. Xu, F.;Luo, Y.;Deng, M.;Shen, Q. Eur. J. Org. Chem.
2003, 4728–4730.
54. Hanaya, T.;Shigetoh, N.;Yamamoto, H. Bull. Chem. Soc.
Jpn. 1988, 61, 2499–2505.
55. Thottathil, J. K. In Handbook of Organophosphorus
Chemistry;Engel, R., Ed.;Marcel Dekker: New York,
1992, p 55.
45. Dhawan, B.;Redmore, D. Phosphorus, Sulfur Silicon
Relat. Elem. 1987, 32, 119–144.
46. Kukhar, V. P.;Soloshonok, V. A.;Solodenko, V. A.
Phosphorus, Sulfur Silicon Relat. Elem. 1994, 92, 239–264.
47. Boduszek, B. Tetrahedron 1996, 52, 12483–12494.
48. Kolodiazhnyi, O. I. Tetrahedron: Asymmetry 1998, 9,
1279–1332.
56. Hansen, H. I.;Kehler, J. Synthesis 1999, 11, 1925–1930.
57. Matziari, M.;Georgiadis, D.;Dive, V.;Yiotakis, A. Org.
Lett. 2001, 3, 659–662.