Observation of an acryloyl-thiamin diphosphate adduct in the first step of clavulanic acid biosynthesis

Article abstract of DOI:10.1021/ja076704r

The first committed biosynthetic step toward clavulanic acid, the clinically important β-lactamase inhibitor, is catalyzed by the thiamin diphosphate (ThDP)-dependent enzyme N²-(2-carboxyethyl)arginine synthase (CEAS). This protein carries out

Full text of DOI:10.1021/ja076704r

Published on Web 12/05/2007
Observation of an Acryloyl-Thiamin Diphosphate Adduct in the First Step of
Clavulanic Acid Biosynthesis
Matthew Merski and Craig A. Townsend*
Departments of Biophysics and Chemistry, The Johns Hopkins UniVersity, Baltimore, Maryland 21218
Received September 5, 2007; E-mail: ctownsend@jhu.edu
Scheme 1
With the rise in resistance to penicillins and cephalosporins, the
â-lactamase inhibitor clavulanic acid (1) has grown in importance
for the treatment of bacterial infections.1 Several mechanistically
intriguing reactions take place in the biosynthesis of 1,2 the first of
which is mediated by N2-(2-carboxyethyl)arginine synthase (CEAS).
The primary metabolites D-glyceraldehyde-3-phosphate (G3P) and
L-arginine are recruited by this enzyme and reacted in a thiamine
diphosphate (ThDP)-dependent internal redox process to yield N2-
(2-carboxyethyl)arginine (6, CEA; Scheme 1).3 The mechanism
shown in Scheme 1 was proposed in accord with extensive isotopic
labeling and stereochemical experiments.3 A key feature of this
mechanism was the suggested intermediacy of an acryloyl-ThDP
adduct 4. The observation of overall retention of configuration at
the G3P â-carbon is consistent with a â-elimination/addition process
proceeding by way of this highly electrophilic4 partner for reaction
with the incoming L-arginine. This is a unique reaction cycle among
ThDP-dependent enzymes where typically intermediates formed are
nucleophilic rather than electrophilic, and N-C bond formation
has not been seen previously. In this Communication, we demon-
strate the formation of the acryloyl species 4 and report the
observation of an unexpectedly long-wavelength chromophore,
which owes to the presence of the acryloyl intermediate.
Recombinant CEAS3 was purified to near homogeneity on a large
scale using two-phase aqueous separation,5 followed by chromato-
graphic steps involving ion exchange and particle size to remove
Figure 1. (A) Synthesis of G3P. (B) Spectroscopic observation of the CEAS
intermediate at 25 °C, pH 7.8 (CEAS ( G3P).
aggregated protein (in preparation). Synthesis of the co-substrate
G3P in high optical purity began with acrolein diethylacetal (7)
spectroscopy. Multiple turnovers of G3P to acrylate were found,
and proceeded according to the literature6 to the diethylacetal of
and controls without CEAS gave no spontaneous generation of
G3P (8, Figure 1A). Removal of unwanted salts and unreacted
acrylate.
organic starting materials and byproducts was achieved by chro-
matography on microcrystalline cellulose followed by a short C-18
Quite unexpected during these experiments, however, was the
observation of a yellow chromophore (λmax ) 433 nm, ꢀM ) ∼4000
column eluted with water. Analysis of an intermediate alcohol as
M-1 cm-1, Figure 1B) thought to arise from the acryloyl intermedi-
its Mosher ester showed an enantiomeric purity of >95%. Dowex-
ate. Addition of sodium dodecylsulfate (SDS, 0.1% w/v) to mildly
denature the protein destroyed the chromophore, suggesting that
its formation is intimately tied to the native state of the protein
and not a released byproduct of reaction. Last, extended incubation
consumed all G3P with loss of the chromophore, but it was restored
by addition of fresh substrate. The cycle could be repeated several
50 deprotection of the acetal afforded G3P, which could be
quantified in solution by assay with G3P dehydrogenase.7
It was anticipated that incubation of CEAS with G3P alone would
generate the acryloyl-ThDP intermediate 4, which, it was hoped,
could be dislodged from the protein under acidic conditions as
precedented for other enzymes of this family.8 Failure of this
times.
approach was not surprising given the intrinsic reactivity of the
Models of intermediates proposed to be bound to ThDP-
dependent enzymes have been synthesized over the years,12-16 and
comparison of their UV-visible behavior allows confident predic-
tion of the λmax for acryloyl-ThDP (4) at 310-320 nm in water,
a far shorter wavelength than observed for the CEAS intermediate.
To confirm this expectation, a synthetic model of 4 was prepared,
2-acryloyl-4-methyl thiazole (11, Scheme 2). 4-Methylthiazole (9)
was lithiated at -78 °C with n-BuLi and reacted with acrolein.12
The sensitive allylic alcohol 10 could be oxidized to 11 in 87%
yield with the Dess-Martin periodinane.17 Its lowest energy
absorption band in acetonitrile (λmax ) 316 nm, ꢀM ) 7150 M-1
acryloyl-ThDP adduct under the conditions of the experiment.
Turnover of G3P, however, was detected at pH 7.8 by phosphate
release9 relative to controls. We expected concomitant hydrolytic
release of free acrylate, which, while not volatile at this pH, is prone
to â-addition, perhaps with CEAS itself, and polymerization. The
incubation mixture was lyophilized and separately derivatized with
p-nitrobenzylchloride10 or tert-butyldimethylsilylchloride11 in DMF.
Isolations of the corresponding esters of acrylic acid were co-
incident with authentic standards by TLC and HPLC, and their
1
distinctive vinyl resonance patterns were observed by H NMR
9
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J. AM. CHEM. SOC. 2007, 129, 15750-15751
10.1021/ja076704r CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
Scheme 2
is typically altered by changes in solvent composition or temper-
ature. This behavior was not observed.
We have isolated acrylic acid from the first half-reaction of
CEAS, supporting the proposed existence of the acryloyl-ThDP
adduct 4 (Scheme 1). The thiazolium ring of this reactive
intermediate bears a formal positive charge irrespective of pH. There
are well-known examples of charged molecules that exhibit
significantly red-shifted UV-visible spectra compared to their
neutral forms as, for example, polyene cations23 and dyes such as
indigo and the cyanines.24 Rhodopsin is the classic biochemical
example where the protein (opsin)-bound protonated Schiff base
(PSB) of retinal displays a remarkable range of red-shifted
absorptions modulated by the protein environment and a highly
conjugated protonated iminium ion. Extensive dipole and electro-
static effects imparted by the protein are thought to “tune” a
collective Stark effect for each rhodopsin chromophore.25 The extent
to which a similar model can be applied to CEAS will be addressed
in due course.
cm-1) was fully within expectations. Addition of trifluoroacetic acid
(TFA) allowed measurement of the transient protonated species,
blue-shifted in accord with the literature (λmax ) 312 nm),12,15 before
rapid conversion to the TFA addition product 12.
Acknowledgment. We thank Dr. J. M. McFadden and K. A.
Moshos for advice with synthetic procedures, and we are grateful
to the National Institutes of Health for financial support (AI
014937).
The high electrophilicity of the acryloyl-ThDP intermediate 4
raised the possibility that intramolecular imine formation with the
aminopyrimidine portion of the cofactor could occur to extend the
conjugation of the chromophore (13, Scheme 2) and thereby account
for its 433 nm absorption. This condensation notwithstanding,
reversible hydrolysis can be readily visualized to give acrylate
isolated above. Formation of a seven-membered ring was expected
to be disfavored, particularly one containing three double bonds.
A careful study by Gruys et al. of a related system, acetyl-ThDP
(18, Scheme 2), showed that, while imine formation could not be
detected, cyclization to the carbinolamine (19, Scheme 2) was
clearly evident.12 To examine the UV spectroscopic behavior of
such a possible conjugated π-system, a second model, N-(1-(4-
methylthiazol-2-yl)allylidene)benzenamine (17, Scheme 2), was
constructed. Once again, 4-methylthiazole (9) was lithiated and
carbonated to carboxylic acid 14.18 Reaction of the corresponding
acid chloride 15 proceeded simply to the anilide 16, which
underwent Von Braun reaction19 to the imidoylchloride and Stille
coupling20 to the desired through-conjugated model 17. Its UV
Supporting Information Available: Syntheses of model com-
pounds 11 and 17 and the isolation of acrylate are described. This
material is available free of charge via the Internet at http://pubs.acs.org.
References
(1) Elander, R. P. Appl. Microbiol. Biotechnol. 2003, 61, 385-392.
(2) Townsend, C. A. Curr. Opin. Chem. Biol. 2002, 6, 583-589.
(3) Khaleeli, N.; Li, R. F.; Townsend, C. A. J. Am. Chem. Soc. 1999, 121,
9223-9224 and refs cited therein.
(4) Evans, D. A.; Fandrick, K. R.; Song, H. J. J. Am. Chem. Soc. 2005, 127,
8942-8943.
(5) Engel, S.; Barak, Z.; Chipman, D. M.; Merchuk, J. C. J. Chromatogr., B:
Biomed. Sci. Appl. 2000, 743, 281-286.
(6) Pederson, R. L.; Liu, K. K. C.; Rutan, J. F.; Chen, L.; Wong, C. H. J.
Org. Chem. 1990, 55, 4897-4901.
(7) Sigma-Aldrich, Technical Bulletin.
(8) Tittmann, K.; Golbik, R.; Uhlemann, K.; Khailova, L.; Schneider, G.;
Patel, M.; Jordan, F.; Chipman, D. M.; Duggleby, R. G.; Hubner, G.
Biochemistry 2003, 42, 7885-7891.
(9) Webb, M. R. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 4884-4887.
(10) Li, R. F.; Stapon, A.; Blanchfield, J. T.; Townsend, C. A. J. Am. Chem.
Soc. 2000, 122, 9296-9297.
(11) Mawhinney, T. P.; Madson, M. A. J. Org. Chem. 1982, 47, 3336-3339.
(12) Gruys, K. J.; Halkides, C. J.; Frey, P. A. Biochemistry 1987, 26, 7575-
7585.
(13) Daigo, K.; Reed, L. J. J. Am. Chem. Soc. 1962, 84, 659-662.
(14) Oka, Y.; Kishimot, S.; Hirano, H. Chem. Pharm. Bull. 1970, 18, 527-
533.
spectrum in acetonitrile of both the neutral [λmax ) 311 nm, ꢀM
)
6250 M-1 cm-1; 360 nm (sh), ꢀM ) 2440 M-1 cm-1] and its
protonated form (λmax ) 377 nm, ꢀM ) 12 600 M-1 cm-1) fell
considerably short of that of the acryloyl intermediate on CEAS.
Charge-transfer (CT) phenomena are often invoked to explain
bathochromic shifts seen in protein chromophores, and indeed, they
have been proposed for other ThDP-dependent enzymes.21 Three
lines of evidence, however, discount CT as a probable cause for
the appearance of the 433 nm band in CEAS. First, the ∼120 nm
(∼25 kcal/mol) difference between model compounds and the
observed intermediate is unusually large to be rationalized by charge
transfer.22 Second, features in the circular dichroism (CD) spectrum
are frequently, although not always, seen as a consequence of CT.21
No changes in the CD spectrum of CEAS were detected upon
formation of the chromophore. Third, the appearance of CT bands
(15) Lienhard, G. E. J. Am. Chem. Soc. 1966, 88, 5642-5649.
(16) White, F. G.; Ingraham, L. L. J. Am. Chem. Soc. 1962, 84, 3109-3111.
(17) Ireland, R. E.; Liu, L. B. J. Org. Chem. 1993, 58, 2899-2899.
(18) Iversen, P. E. Acta Chem. Scand. 1968, 22, 694-695.
(19) Vaughan, W. R.; Carlson, R. D. J. Am. Chem. Soc. 1961, 83, 769-774.
(20) Kosugi, M.; Koshiba, M.; Atoh, A.; Sano, H.; Migita, T. Bull. Chem.
Soc., Jpn. 1985, 59, 677-679.
(21) Nemeria, N.; Baykal, A.; Joseph, E.; Zhang, S.; Yan, Y.; Furey, W.; Jordan,
F. Biochemistry 2004, 43, 6565-6575.
(22) Mataga, N.; Kubota, T. Molecular Interactions and Electronic Spectra;
Marcel Dekker Inc.: New York, 1970; pp 371-484.
(23) Sorensen, T. S. J. Am. Chem. Soc. 1965, 87, 5075-5084.
(24) Hamer, F. M. The Cyanine Dyes and Related Compounds. In The
Chemistry of Heterocyclic Compounds; Weissberger, A., Ed.; John Wiley
& Sons: New York, 1969.
(25) Kochendoerfer, G. G.; Lin, S. W.; Sakmar, T. P.; Mathies, R. A. Trends
Biochem. Sci. 1999, 24, 300-305.
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