Regiospecific syntheses of 6a-(1 R-hydroxyoctyl)penicillanic acid and 6β(1 R-hydroxyoctyl)penicillanic acid as mechanistic probes of class D β-lactamases

Article abstract of DOI:10.1021/ol900668k

The unique hydrophobic surface patches in class D β-Mactamases presented an opportunity for designing two compounds, 6α-(1R-hydroxyoctyl) penicillanic acid and 6β-(1R-hydroxyoctyl)penicillanic acid, as mechanistic probes of these enzymes. In a sequence of

Full text of DOI:10.1021/ol900668k

ORGANIC
LETTERS
2009
Vol. 11, No. 12
2515-2518
Regiospecific Syntheses of
6r-(1R-Hydroxyoctyl)penicillanic Acid
and 6ꢀ-(1R-Hydroxyoctyl)penicillanic
Acid as Mechanistic Probes of Class D
ꢀ-Lactamases
Sebastian A. Testero, Peter I. O’Daniel, Qicun Shi, Mijoon Lee, Dusan Hesek,
Akihiro Ishiwata, Bruce C. Noll, and Shahriar Mobashery*
Department of Chemistry and Biochemistry, UniVersity of Notre Dame,
Notre Dame, Indiana 46556
mobashery@nd.edu
Received March 31, 2009
ABSTRACT
The unique hydrophobic surface patches in class D ꢀ-lactamases presented an opportunity for designing two compounds, 6r-(1R-
hydroxyoctyl)penicillanic acid and 6ꢀ-(1R-hydroxyoctyl)penicillanic acid, as mechanistic probes of these enzymes. In a sequence of three
synthetic steps from benzhydryl 6,6-dibromopenicillanate, the targeted compounds were prepared in a stereospecific manner.
ꢀ-Lactamases are important resistance determinants for
ꢀ-lactam antibiotics (penicillins, cephalosporins, carbapen-
ems, etc.).1 Bacteria have evolved four classes of these
enzymes, of which three (classes A, C, and D) are serine-
dependent enzymes.1,2 The antibiotic acylates an active site
serine, and the acyl-enzyme species subsequently experiences
hydrolysis to complete the catalytic process. Because evolu-
tion of these ꢀ-lactamases has been independent of each
other, nature has devised the deacylation step on three
separate occasions.1,2 As such, the trajectory that the
hydrolytic water uses for approach to the acyl-enzyme
carbonyl has been either from one face of the carbonyl entity
or the other.
We have described a class of 6-hydroxyalkylpenicillanate
inhibitors for these enzymes.3-5 These compounds acylate
the active site serine by the virtue of the fact that they are
penicillins. However, if the stereochemistry of the 6-hy-
droxyalkyl moiety (R or ꢀ) predisposes the functionality in
the direction of the approach of the water molecule, the
hydroxyl moiety would present a steric impediment to the
(3) Miyashita, K.; Massova, I.; Taibi, P.; Mobashery, S. J. Am. Chem.
Soc. 1995, 117, 11055–11059
.
(4) Maveyraud, L; Massova, I.; Birck, C.; Miyashita, K.; Samama, J. P.;
(1) Fisher, J. F.; Meroueh, S. O.; Mobashery, S. Chem. ReV. 2005, 105,
395–424.
Mobashery, S. J. Am. Chem. Soc. 1996, 118, 7435–7440.
(5) Golemi, D.; Maveyraud, L.; Ishiwata, A.; Tranier, S.; Miyashita,
(2) Massova, I; Mobashery, S. Antimicrob. Agents Chemother. 1998,
K.; Nagase, T.; Massova, I.; Mourey, L.; Samama, J. P.; Mobashery, S. J.
Antibiot. 2000, 53, 1022–1027.
42, 1–17
.
10.1021/ol900668k CCC: $40.75
Published on Web 05/15/2009
 2009 American Chemical Society

travel of the hydrolytic water molecule to the acyl-enzyme
species.5 The opposite stereochemistry at position 6 would
render the molecule a substrate, as the approach of the
hydrolytic water to the acyl-enzyme species would be
unencumbered.
cation of the C-3 carboxylic acid by diphenyldiazomethane
gave the key intermediary compound 1 (Scheme 1), which
Scheme 1
The work that has been performed with these penicillanate
inhibitors has been limited to classes A and C of ꢀ-lacta-
mases. However, in the past five years, the members of the
class D of these enzymes have become prominent, with >140
members having been identified to date. These enzymes are
distinctly different from the other two classes by having an
N-carboxylated lysine within their active sites.6-8 Further-
more, the environments surrounding the lysine and im-
mediately adjacent to the active site are highly hydrophobic.
This presented an opportunity for design of variants of these
6-hydroxyalkylpenicillanates that would show preference for
class D enzymes. This task was undertaken by computational
methods, building on the knowledge of interactions of these
penicillanates with the active sites of the enzymes (Figure
1). The exercise led us to 6R- and 6ꢀ-hydroxyloctylpenicil-
was crystallized from ethyl acetate and the structure was
confirmed by X-ray crystallography (Figure 2). Transmeta-
Figure 1. Stereoview representation of the acyl-enzyme species of
the class D OXA-10 ꢀ-lactamase and 6-ꢀ-(1-hydroxyloctyl)-
penicillanate from molecular dynamics (MD) equilibration. The
penicillanate covalently bound to Ser67 (in capped sticks, carbon
in gray, nitrogen in blue, sulfur in yellow and oxygen in red) is
depicted within the active site, which is shown as a solvent-
accessible Connolly surface (blue). N-Carboxylate lysine (KCX70)
is at 7 o’clock and the octyl moiety is pointing to 4 o’clock. Note
that the hydroxyl moiety of the C-6 functionality is hydrogen-
bonded to the N-carboxylated lysine.
lanic acids as probes targeting class D ꢀ-lactamases. The
synthetic work was also performed for 6R- and 6ꢀ-(hydroxyl-
1-methylethyl)penicillanic acids with the intention of the
optimization of the syntheses of these compounds, which
we had reported earlier.
Figure 2. ORTEP diagram of X-ray structure of compounds 1, 2,
and 7.
The synthesis began with the preparation of 6,6-dibro-
mopenicillanic acid (1) from 6-aminopenicillanic acid (6-
APA) by the procedure of Volkman.9 Subsequent esterifi-
lation with the use of the Grignard reagent, followed by the
addition of acetone, gave a mixture of 2 and 3, a method
that has been reported for alkylation of penicillins.3,10 As
observed in the present study, a freshly prepared anhydrous
THF was critical for this reaction in the presence of
stoichiometric amount of the Grignard reagent. The yield of
the carbon-carbon bond formation step improved over the
earlier reports by nearly 3-fold by simply preparing the
(6) Maveyraud, L.; Golemi, D.; Kotra, L. P.; Tranier, S.; Vakulenko,
S.; Mobashery, S.; Samama, J. P. Structure 2000, 8, 1289–1298
(7) Golemi, D.; Maveyraud, L.; Vakulenko, S.; Samama, J. P.; Mobash-
ery, S. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 14280–14285
(8) Maveyraud, L.; Golemi, D.; Ishiwata, A.; Meroueh, O.; Mobashery,
.
.
S.; Samama, J. P. J. Am. Chem. Soc. 2002, 124, 2461–2465
.
(9) Volkmann, R. A; Carroll, R. D; Drolet, R. B.; Elliot, M. L.; Moore,
B. S. J. Org. Chem. 1982, 47, 3344–3345.
2516
Org. Lett., Vol. 11, No. 12, 2009

anhydrous THF prior to carrying out the reaction. After
purification, the yields of isomers 2 (ꢀ) and 3 (R) were 54
and 30%, respectively. The assignment of the stereochemistry
was greatly helped by the determination of the crystal
structure for compound 2 (Figure 2).
hydroxyoctylpenicillanate 8 in 76% yield, along with the
undesired mixture of two isomers 9a,b (24%; Scheme 3).
Scheme 3
The reduction of the carbon-halide bond could be
achieved by the use of tributyltin hydride,11 zinc-mediated
chemistry,12 or hydrogenolysis over Pd-C.13 We have
previously reported the stereoselective synthesis of 6R-
substituted penicillanate ester by the use of tributylphosphine,
but we have noted that the stereoselectivity can be quite
variable from run to run and was not reliable in its
reproducibility.14 The use of this procedure on either a
mixture of 2 and 3 or on pure isomer as starting material
afforded an inseparable mixture of the R and ꢀ isomers (4
1
and 5) in the ratio of 2.8/1 (determined by H NMR of the
crude) in 69%. The stereochemical assignments for C-6 of
1
compounds 4 and 5 were based on their H NMR coupling
constants JH5-H6 ) 1.8 and 4.6 Hz, respectively, which are
in agreement with the Karplus equation.
Several methods for the removal of the benzhydryl group
in the mixture of compounds 4 and 5 were tried (hydro-
genolysis over palladium, TFA/anisole, formic acid, Lewis
acids with anisole or m-cresol). Of these, hydrogenolysis gave
good yields, but more importantly, the reaction products were
clean at the end of the workup.
The configuration at C-6 and the stereochemistry of the
stereogenic carbon at the C-6 side chain in compounds 8
and 9a,b were assigned by NMR according to DiNinno et
al.15
Treatment of bromide 8 with tributylphosphine in methanol
afforded solely the 6R-substituted penicillanate 10 in 79%
yield (Scheme 4). A coupling constant of 1.7 Hz between
Fractional crystallization from chloroform-hexane afforded
colorless crystals of the ꢀ-isomer 7, whose structure was
confirmed by X-ray crystallography (Figure 2). The R-isomer
6 was isolated in pure form from the solution.
Scheme 4
Whereas the reductive step in the previous procedure was
useful in that we obtained the two isomers in pure forms
(after deprotection), we observed that the use of tributyltin
hydride (with 1,1′ azobis(cyanocyclohexane) as initiator) with
the mixture of 2 and 3 produced the ꢀ-isomer 5 as the
exclusive product in 98% yield. This allowed us to access
compound 7 after hydrogenolysis by an additional route
(Scheme 2).
Scheme 2
the vicinal protons H-5 and H-6 established a trans relation-
ship. This reaction would appear to proceed through a
tributylphosphonium ꢀ-lactam enolate, followed by diaster-
oselective protonation from the ꢀ-face.14 Finally, removal
of the benzhydryl group furnished compound 11.
(10) (a) Buynak, J. D.; Chen, H.; Vogeti, L.; Gadhachanda, V. R.;
Buchanan, C. A.; Palzkill, T.; Shaw, R. W.; Spencer, J.; Walsh, T. R. Bioorg.
Med. Chem. Lett. 2004, 14, 1299–1304. (b) Norris, T.; Ripin, D. H. B.;
Ahlijanian, P.; Andersen, B. M.; Barrila, M. T.; Colon-Cruz, R.; Couturier,
M.; Hawkins, J. M.; Loubkina, I. V.; Rutherford, J.; Stickley, K.; Wei, L.;
Vollinga, R.; de Pater, R.; Maas, P.; de Lange, B.; Callant, D.; Konigs, J.;
Andrien, J.; Versleijen, J.; Hulshof, J.; Daia, E.; Johnson, N.; Sung, D. W. L.
Org. Process Res. DeV. 2005, 9, 432–439.
The computational design procedure with the recently
emerged structural information of the class D ꢀ-lactamases
revealed the opportunity for the design of a novel family of
hydroxyalkylpenicillanate compounds for their inhibition.
The methodology that we have described above was applied
to the preparation of these new penicillanates for mechanistic
studies of class D ꢀ-lactamases. The reaction of the dibro-
mide 1 in the presence of the Grignard reagent in freshly
prepared anhydrous THF and octyl aldehyde produced 6ꢀ-
(11) For examples, see: (a) Ziegler, C. B., Jr.; Fields, T. L. Tetrahedron
1993, 49, 3919–3932. (b) Hanessian, S.; Alpegiani, M. Tetrahedron 1989,
45, 941–950. (c) Foulds, C. D.; Kosmirak, M.; Sammes, P. G. J. Chem.
Soc., Perkin Trans. 1 1985, 763–768. (d) Hirai, K.; Iwano, Y.; Fujimoto,
K. Tetrahedron Lett. 1982, 23, 4021–4024. (e) Bitha, P.; Li, Z.; Francisco,
G. D.; Rasmussen, B. A.; Lin, Y.-I. Bioorg. Med. Chem. Lett. 1999, 9,
991–996.
Org. Lett., Vol. 11, No. 12, 2009
2517

On the other hand, reduction of the bromide 8 by tributyltin
hydride provided the opposite configuration at C-6, to result
in compound 12 as the exclusive product. The establishment
of the stereochemistry at C-6 was based on the coupling
constant between H-5 and H-6 (J ) 4.5 Hz). In this case,
the reaction would take place through an intermediary
penicillin radical that would be quenched on the R-face of
the penicillin to yield the 6-ꢀ-hydroxyoctyl product 12.16
Deprotection of the benzhydryl group of 12 afforded the 6ꢀ-
hydroxyoctylpenicillanic acid 13 in an acceptable yield
(54%).
In summary, we have described short and regiospecific
methodology into preparation of penicillanate derivatives
11 and 13, which will be valuable tools in investigations
of mechanistic and structural details of class D ꢀ-lacta-
mases.
(12) For examples, see: (a) Yoshida, A.; Hayashi, T.; Takeda, N.; Oida,
S.; Ohki, E. Chem. Pharm. Bull. 1981, 10, 2899–2909. (b) Leanza, W. J.;
DiNinno, F.; Muthard, D. A.; Wilening, R. R.; Wildonger, K. J.; Ratcliffe,
R. W.; Christensen, B. G. Tetrahedron 1983, 39, 2505–2513. (c) Fujimoto,
K.; Iwano, Y.; Hirai, K.; Sugawara, S. Chem. Pharm. Bull. 1986, 34, 999–
1014.
Acknowledgment. This work was supported by the NIH.
(13) For examples, see: (a) Girijavallabhan, V. M.; Ganguly, A. K.;
McCombie, S. W.; Pinto, P.; Rizvi, R. Tetrahedron Lett. 1981, 22, 3485–
3488. (b) Rossi, R. L.; Kapilani, L. V.; Morrissey, P.; Retsema, J. A. J. Med.
Chem. 1990, 33, 291–297.
Supporting Information Available: General experimental
procedures, compound characterization data, including copies
of 1D NMR spectra (1H, 13C NMR) and X-ray data of
compounds 1, 2, and 7. This material is available free of
charge via the Internet at http://pubs.acs.org.
(14) Ishiwata, A.; Kotra, L. P.; Miyashita, K.; Nagase, T.; Mobashery,
S. Org. Lett. 2000, 2, 2889–2892.
(15) DiNinno, F; Beattie, T. R.; Christensen, B. G. J. Org. Chem. 1977,
42, 2960–2965.
(16) Norris, T.; Dowdeswell, C.; Johnson, N.; Daia, D. Org. Process
Res. DeV. 2005, 9, 792–799.
OL900668K
2518
Org. Lett., Vol. 11, No. 12, 2009