Synthesis and evaluation of novel substrates and inhibitors of N-succinyl-LL-diaminopimelate aminotransferase (DAP-AT) from Escherichia coli

Article abstract of DOI:10.1021/ja960640v

N-Succinyl-LL-diaminopimelate aminotransferase (DAP-AT) (EC 2.6.1.17), a key enzyme in the bacterial pathway to L-lysine, was purified to near homogeneity (1500-fold) in five steps from wild type Escherichia coli ATCC 9637. This pyridoxal phosphate (PLP)

Full text of DOI:10.1021/ja960640v

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J. Am. Chem. Soc. 1996, 118, 7449-7460
7449
Synthesis and Evaluation of Novel Substrates and Inhibitors of
N-Succinyl-LL-diaminopimelate Aminotransferase (DAP-AT)
from Escherichia coli
Russell J. Cox, William A. Sherwin, Lister K. P. Lam, and John C. Vederas*
Contribution from the Department of Chemistry, UniVersity of Alberta,
Edmonton, Alberta, Canada T6G 2G2
ReceiVed February 27, 1996X
Abstract: N-Succinyl-LL-diaminopimelate aminotransferase (DAP-AT) (EC 2.6.1.17), a key enzyme in the bacterial
pathway to L-lysine, was purified to near homogeneity (1500-fold) in five steps from wild type Escherichia coli
ATCC 9637. This pyridoxal phosphate (PLP) dependent enzyme has a molecular weight of 39.9 kDa, appears to
form an active homodimer, and uses L-glutamate as the amino group donor for its substrate, N-succinyl-R-amino-
ꢀ-ketopimelic acid (1a) (Km ) 0.18 ( 0.04 mM, kcat ) 86 ( 5 s-1). Progress of the reaction is monitored by
spectrophotometric observation of decrease in NADPH concentration at 340 nm in a coupled enzyme assay with
L-glutamate dehydrogenase (EC 1.4.1.4). Stereochemically pure 1a was synthesized as its trilithium salt by ene
reaction of methyl glyoxylate with methyl N-succinyl-L-allylglycinate (4a) followed by hydrogenation of the double
bond, Dess-Martin oxidation of the alcohol, and careful lithium hydroxide hydrolysis. Similar approaches allowed
synthesis of a series of substrate analogues 1b-g having different N-acyl substituents, as well as derivatives missing
the carboxyl group or the amide functionality (13 and 17, respectively). Compounds lacking the keto functionality
(18a, 18c, and 19) were also prepared. Assay of DAP-AT shows that the enzyme has quite strict requirements for
substrate recognition, but it will accept compounds with an aromatic ring in place of the terminal succinyl carboxyl
group in 1a (e.g. N-Cbz-R-amino-ꢀ-ketopimelic acid (1c)). Reaction of substrates 1a,c with hydrazine hydrate followed
by NaCNBH3 reduction gives 2-(N-(succinylamino))- (20a) and 2-(N-Cbz-amino)-6-hydrazinoheptane-1,7-dioic acids
(20c), respectively. These are the most potent slow-binding inhibitors of any DAP-metabolyzing enzyme reported
so far (Ki* for DAP-AT: 20a is 22 ( 4 nM; 20c is 54 ( 9 nM).
Introduction
The increased incidence of pathogenic bacteria which are
high specificity of some enzymes for the structural features
inherent in the substrates,5b the ability of key enzymes to
transform certain inhibitors into normal metabolic intermediates,6
ineffective transport into cells, and the presence of alternative
routes to meso-DAP.
L-Lysine is formed in bacteria via three main avenues from
L-tetrahydrodipicolinic acid (L-THDP) (Figure 1).4 In E. coli,
and many other bacteria, succinylation of L-THDP affords
N-succinyl-R-amino-ꢀ-ketopimelic acid,7,8 which after transami-
nation by N-succinyl-LL-DAP aminotransferase (EC 2.6.1.17),9
gives N-succinyl-LL-DAP. Desuccinylation yields LL-DAP,10
resistant to conventional antibiotics currently used in treatment
of infectious disease continues to stimulate interest in alternative
strategies to disrupt microbial cell wall synthesis and thereby
inhibit their growth.1 The biosynthesis of the peptidoglycan
layer in bacterial cells provides numerous targets for further
antibiotic development.2 The key cross-linking amino acid in
this essential structural polymer can be variable but is usually
meso-diaminopimelic acid (meso-DAP) in Gram negative
bacteria and its biosynthetic product, L-lysine, in Gram positive
organisms. Since mammals lack the DAP biosynthetic pathway
and require L-lysine in their diet,3 there has been considerable
interest in specific inhibition of enzymes enroute to these
metabolites.4,5 Some of the major obstacles to antibiotic
development emerging from these studies include unexpectedly
(5) For leading references see: (a) Abbott, S. D.; Lane-Bell, P.; Sidhu,
K. P. S.; Vederas, J. C. J. Am. Chem. Soc. 1994, 116, 6513-6520. (b)
Song, Y. H.; Niederer, D.; Lane-Bell, P.; Lam, L. K. P.; Crawley, S.; Palcic,
M. M.; Pickard, M. A.; Pruess, D. L.; Vederas, J. C. J. Org. Chem. 1994,
59, 5784-5793. (c) Williams, R. M.; Yuan, C. J. Org. Chem. 1992, 57,
6519-6527. (d) Bold, G.; Allmendinger, T.; Herold, P.; Moesch, L.; Scha¨r,
H.-P.; Duthaler, R. O. HelV. Chim. Acta 1992, 75, 865-882. (e) Jurgens,
A. R. Tetrahedron Lett. 1992, 33, 4727-4730. (f) Mengin-Lecreulx, D.;
Blanot, D.; Van Heijenoort, J. J. Bacteriol. 1994, 176, 4321-4327. (g)
Williams, R. M.; Fegley, G. J.; Gallegos, R.; Schaefer, F.; Pruess, D. L.
Tetrahedron 1996, 52, 1149-1164.
X Abstract published in AdVance ACS Abstracts, July 15, 1996.
(1) Ama´bile-Cuevas, C. F.; Ca´rdenas-Garcia, M.; Ludgar, M. Am.
Scientist 1995, 83, 320-329.
(2) (a) Ward, J. B. In Antibiotic Inhibitors of Bacterial Cell Wall
Biosynthesis; Tipper, D. J., Ed.; Pergamon Press: New York, 1987; pp
1-43. (b) Bugg, T. D. H.; Walsh, C. T. Nat. Prod. Rep. 1992, 9, 199-
215. (c) Cooper, S.; Metzger, N. FEMS Microbiol. Lett. 1988, 36, 191-
194.
(6) Gelb, M. H.; Lin, Y.; Pickard, M. A.; Song, Y.; Vederas, J. C., J.
Am. Chem. Soc. 1990, 112, 4932-4942.
(7) For a summary of early work on the E. coli L-lysine pathway see:
(a) Gilvarg, C. Meth. Enzymol. 1962, 5, 848-850. (b) Kindler, S. H. Meth.
Enzymol. 1962, 5, 851-853. (c) Peterkofsky, B. Meth. Enzymol. 1962, 5,
853-858. (d) Work, E. Meth. Enzymol. 1962, 5, 858-864. (e) Work, E.
Meth. Enzymol. 1962, 5, 864-870. (f) Berges, D. A.; DeWolf, W. E.; Dunn,
G. L.; Newman, S. J.; Schmidt, J.; Taggart, J.; Gilvarg, C. J. Biol. Chem.
1986, 261, 6160-6167.
(3) (a) Patte, J.-C. In Amino Acids; Biosynthesis and Genetic Regulation;
Herrmann, K. M., Somerville, R. L., Eds.; Addison-Wesley: Reading, MA,
1983; pp 213-228. Saqib, K. M.; Hay, S. M.; Rees, W. D. Biochim.
Biophys. Acta 1994, 1219, 398-404. (b) For recent purification of a plant
enzyme (dihydrodipicolinate synthase) in the DAP pathway see: Dereppe,
C.; Bold, G.; Ghisalba, O.; Ebert, E.; Scha¨r, H.-P. Plant Physiol. 1992, 98,
813-821. (c) For recent crystal structures of dihydrodipicolinate synthase
from E. coli see: Mirwaldt, C.; Korndo¨rfer, I.; Huber, R. J. Mol. Biol.
1995, 246, 227-239. Scapin, G.; Blanchard, J. S.; Sacchettini, J. C.
Biochemistry 1995, 34, 3502-3512.
(8) Simms, S. A.; Voige, W. H.; Gilvarg, C. J. Biol. Chem. 1984, 259,
2734-2741.
(9) (a) Gilvarg, C. J. Biol. Chem. 1961, 236, 1429-1431. (b) Peterkofsky,
B.; Gilvarg, C. J. Biol. Chem. 1961, 236, 1432-1438. (c) Literature values
for DAP-AT conversion of substrate: for 1a, Km ) 0.50 mM; for
L-glutamate, Km ) 5.20 mM (Tris, pH 8.0, 30 °C).
(4) For a review see: Cox, R. J. Nat. Prod. Rep. 1996, 13, 29-43.
S0002-7863(96)00640-3 CCC: $12 00 © 1996 American Chemical Society

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7450 J. Am. Chem. Soc., Vol. 118, No. 32, 1996
Cox et al.
Figure 1. Biochemical routes to L-lysine in bacteria.
and epimerization then generates meso-DAP.11 Certain bacteria
such as Bacillus megaterium and B. subtilis utilize N-acetylated
intermediates instead of the N-succinylated versions,12 whereas
others such as Bacillus sphaericus reductively aminate L-
THDPA directly in a single NADPH dependent step catalyzed
by DAP dehydrogenase to generate meso-DAP directly.13 Dual
pathways are known to occur in B. macerans , which uses both
the N-acetylated route and DAP dehydrogenase,14 and also in
Mycobacterium boVis15 and Corynebacterium glutamicum,16
both of which have the enzymes required for the N-succinylated
route as well as the DAP dehydrogenase.
Figure 2. Syntheses of DAP-AT substrates 1a-g. See Table 1 for
yields.
to apparent homogeneity, and substrate specificity studies, as
well as the synthesis and testing of potent inhibitors of this
enzyme.18
Results and Discussion
Synthesis of (S)-N-Succinyl-r-amino-ꢀ-ketopimelic Acid
(1a) and Substrate Analogues. The intermolecular ene reac-
tion provides a convenient route to functionalized R-ami-
nopimelic acid derivatives by condensation of protected allylg-
lycines with methyl glyoxylate.19 We initially sought to
synthesize the pure L enantiomers (S configuration) of both
N-succinyl and N-acetyl substrates 1a,b in order to compare
their activities with purified N-succinyl-LL-DAP aminotrans-
ferase (DAP-AT). Racemic N-acetyl allylglycine (2) is readily
resolved with pork kidney acylase by the literature procedure,20
with modified purification using cation exchange chromatog-
raphy to generate L-allylglycine (3) in >99% enantiomeric
excess and superior yield (>90%) (Figure 2). Acylation with
either succinic or acetic anhydride in aqueous base, followed
by methyl ester formation with diazomethane, and ene reaction
of the resulting protected derivatives 4a,b with methyl glyoxy-
late (obtained by oxidation of dimethyl tartrate) under Lewis
acid conditions (FeCl3) at room temperature affords the ene
adducts 5a,b (1:1 mixture of diastereomers) (Table 1). Catalytic
hydrogenation proceeds smoothly to give the saturated hydroxy
Despite extensive study of the enzymes and genes involved
in DAP biosynthesis in bacteria and higher plants,3,4,17 detailed
examination of N-succinyl-LL-DAP aminotransferase has not
been described since Gilvarg and co-workers achieved partial
purification in the 1960s.9 One of the difficulties has been the
lack of an efficient chemical synthesis of pure substrate,
N-succinyl-R-amino-ꢀ-ketopimelic acid (1a), and its analogues.
As part of our continuing effort to develop antimicrobial
inhibitors of the DAP pathway,5a,b,6 we now describe the
chemical synthesis of pure substrates, the purification of
N-succinyl-LL-DAP aminotransferase (EC 2.6.1.17) (DAP-AT)
(10) (a) Kindler, S. H.; Gilvarg, C. J. Biol. Chem. 1960, 235, 3532-
3535. (b) Lin, Y.; Myhrman, R.; Schrag, M. L.; Gelb, M. H. J. Biol. Chem.
1988, 263, 1622-1627.
(11) Wiseman, J. S.; Nichols, J. S. J. Biol. Chem. 1984, 259, 8907-
8914.
esters 6a,b. Although many standard oxidation methods (e.g.
(12) (a) Sundharadas, G.; Gilvarg, C. J. Biol. Chem. 1967, 242, 3983-
3984. (b) Chen, N.-Y.; Jiang, S.-Q.; Klein, D. A.; Paulus, H. J. Biol. Chem.
1993, 268, 9448-9465.
(13) (a) Misono, H.; Soda, K. J. Biol. Chem. 1980, 255, 10599-10605.
(b) Misono, H.; Soda, K. Agric. Biol. Chem. 1980, 44, 227-229.
(14) Bartlett, A. T. M.; White, P. J. J. Gen. Microbiol. 1985, 131, 2145-
2152.
(15) Cirillo, J. D.; Weisbrod, T. R.; Banerjee, A.; Bloom, B. R.; Jacobs,
W. R., Jr. J. Bacteriol. 1994, 176, 4424-4429.
(16) Eggeling, L. Amino Acids 1994, 6, 261-272.
(17) Couper, L.; McKendrick, J. E.; Robins, D. J.; Chrystal, E. J. T.
Bioorg. Med. Chem. Lett. 1994, 4, 2267-2272.
24
Swern,21 H2CrO4/SiO2,22 (nPr)4RuO4/NMO,23 MnO4/CuSO4
)
(18) For an example of a substrate analog inhibitor of another ami-
notransferase as a useful drug see: Casara, P. Tetrahedron Lett. 1994, 35,
3049-3050.
(19) Agouridas, K.; Girodeau, J. M.; Pineau, R. Tetrahedron Lett. 1985,
26, 3115-3118.
(20) Black, S.; Wright, N. G. J. Biol. Chem. 1955, 213, 39-50.
(21) Mancuso, A. J.; Huang, S.-L.; Swern, D. J. Org. Chem. 1978, 43,
2480-2482.
(22) Santaniello, E.; Ponti, F.; Manzocchi, A. Synthesis 1978, 534-535.

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Substrates and Inhibitors of DAP-AT
J. Am. Chem. Soc., Vol. 118, No. 32, 1996 7451
Table 1. Isolated % Yields of Reaction Productsa
reacn, product
ene reacn, 5 redn, 6 acylation, 6 oxidn, 8 hydrolysis, 1
a
b
c
d
e
f
47
46
65
91
95
79
70
56
13c
64
60
63
99
92
98
99
99
99
95
99 (7)b
84
80
56
83
58
g
a See Figure 2 for structures and Experimental Section for procedures.
b Reduction removes Cbz group. c Not optimized.
proceed in unexpectedly low yield, a 1.2-1.5 fold excess of
the Dess-Martin periodinane25 readily affords the desired
R-keto esters 8a,b. Careful hydrolysis with 1.0 equiv/ester
moiety of lithium hydroxide hydrate in 50/50 water/acetonitrile
quantitatively generates the pure lithium salts of 1a,b as stable
compounds. Acidification of 1a (e.g. pH 4) results in immediate
decomposition.
Slight modification of this procedure permits preparation of
a variety of N-acyl derivatives 1c-g. Ene reaction of the N-Cbz
analogue 4c requires use of SnCl4 instead of FeCl3, and
hydrogenation of the adduct 5c in the presence of Boc anhydride
gives the Boc-protected compound 6d in 80% yield. Alterna-
tively, hydrogenation of 4c in ca. 10% CHCl3 in methanol
without an acylating agent present produces the expected amino
alcohol as its hydrochloride salt 7 in quantitative yield. Selective
N-acylation using standard conditions in the presence of pyridine
followed by oxidation yields the amides 8c-g, which are
transformed to the corresponding substrate analogues (as lithium
salts) 1c-g as above.
To test the optical purity of these compounds, the lithium
salts of the R-keto acids 1a,c were reductively aminated26 using
ammonium acetate and sodium cyanoborohydride in methanol
to produce N-protected DAP derivatives (Figure 3). Depro-
tection by hydrogenolysis or acid-catalyzed hydrolysis afforded
mixtures of DAP stereoisomers which were converted to bis-
Marfey derivatives27 and analyzed by reverse phase HPLC.
Comparison of samples obtained from 1a,c with those derived
from commercial DAP (statistical mixture of stereoisomers)
shows the presence of only LL- and meso-DAP isomers in equal
amounts. The lack of a detectable level of DD-DAP indicates
that the optical purity at the 2-position is >99% and that there
is no significant diastereoselectivity during the reductive ami-
nation.
In order to determine structural requirements for substrate
recognition by the aminotransferase (DAP-AT), analogues
missing the 2-carboxyl group (i.e. 13), the 2-amido group (i.e.
17), or the 6-keto group (i.e. 18a, 18c, 19) were also synthesized
(Figure 4). Treatment of 3-butenylamine with succinic anhy-
dride followed by diazomethane generates 9, which is readily
transformed by the ene procedure with functional group
manipulation to 13. Similarly, ene condensation of diethyl
2-allyl-1,7-heptanedioate with methyl glyoxylate followed by
hydrogenation in methanol affords a partially transesterified
mixture 15, which after oxidation and basic hydrolysis gives
Figure 3. Stereochemical analysis of 1a,c by formation of Marfey
derivatives for HPLC.
17. Direct acylation of racemic R-aminopimelic acid provides
access to 18a,c, whereas hydrolysis of 6a gives 19.
Assay, Purification, and Substrate Specificity of DAP
Aminotransferase. The availability of the natural substrate 1a
allows utilization of an efficient continuous assay for the
pyridoxal phosphate (PLP) dependent DAP-AT through cou-
pling of the “forward” reaction with L-glutamate dehydrogenase
(EC 1.4.1.4) (Figure 5).28 The assay mixture contains the
substrate 1a, PLP, L-glutamate as the amino group donor for
the transamination, L-glutamate dehydrogenase, NH4Cl, and
NADPH. The R-ketoglutarate formed in the DAP-AT reaction
is rapidly transformed back to L-glutamate by the dehydrogenase
with the concomitant consumption of 1 equiv of NADPH. The
progress of the reaction can be monitored by spectrophotometric
observation of decrease in NADPH concentration at 340 nm.
Purification of DAP-AT from wild type E. coli requires five
chromatographic steps (Table 2). Precipitation methods are
relatively ineffective for concentrating the enzyme, but dye
affinity chromatography in the “negative” mode to extract out
unwanted proteins followed by “positive” mode dye affinity to
bind the aminotransferase affords efficient partial purification.
Hydroxylapatite chromatography, gel filtration, and HPLC anion
exchange on Mono-Q gives nearly pure DAP-AT (by SDS
PAGE) with an overall purification of 1500-fold.29 MALDI
TOF mass spectra and SDS PAGE indicate a subunit molecular
weight of 39.9 kDa, whereas calibrated gel filtration chroma-
tography suggests an approximate molecular weight of 82 kDa.
The enzyme is therefore likely to be dimeric in its active form.
Michaelis and kinetic constants obtained for the pure enzyme
(for 1a, Km ) 0.18 ( 0.04 mM, kcat ) 85 ( 5 s-1, (cf. Table
3); for L-glutamate (Tris buffer), Km ) 1.21 ( 0.14 mM)
compare favorably with those obtained by Gilvarg and Pe-
(28) The procedure is a modification of that initially developed by Gilvarg
and Peterkofsky: see refs 7 and 9b.
(23) Griffith, W. P.; Ley, S. V.; Whitcombe, G. P.; White, A. D. J. Chem.
Soc., Chem. Commun. 1987, 1625-1627.
(29) Literature values for other DAP pathway enzyme purifications: (a)
L-THDPA succinyltransferase 1900-fold from E. coli; see ref 8. (b) DAP
desuccinylase (EC 3.5.1.18) 7100-fold from E. coli; see ref 10b. (c) DAP
epimerase (EC 5.1.1.7) 6000-fold from E. coli; see ref 11. (d) DAP
dehydrogenase (EC 1.4.1.16) 290-fold from B. sphaericus; see ref 13b. (d)
DAP decarboxylase (EC4.1.1.20) 500-fold from B. sphaericus: Asada, Y.;
Tanizawa, K.; Kawabata, Y.; Misono, H.; Soda, K. Agric. Biol. Chem. 1981,
45, 1513-1514. Andersen, Å. B.; Hansen, E. B. Gene 1993, 124, 105-
109.
(24) Menger, F. M.; Lee, C. J. Org. Chem. 1979, 44, 3446-3448.
(25) (a) Dess, D. B.; Martin, J. C. J. Am. Chem. Soc. 1991, 113, 7277-
7287. (b) Ireland, R. E.; Liu, L. J. Org. Chem. 1993, 58, 2899. (c) Burkhart,
J. P.; Peet, N. P.; Bey, P. Tetrahedron Lett. 1988, 29, 3433-3436.
(26) Williams, R. M.; Ehrlich, P. P.; Zhai, W.; Hendrix, J. J. Org. Chem.
1987, 52, 2615-2617.
(27) (a) Marfey, P. Carlsberg Res. Commun. 1984, 49, 591-596. (b)
Bruckner, H.; Keller-Hoehl, C. Chromatographia 1990, 30, 621-629.

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7452 J. Am. Chem. Soc., Vol. 118, No. 32, 1996
Cox et al.
Figure 4. Syntheses of DAP-AT substrate analogues 13 and 17 and structures of 18a,c and 19.
Figure 5. Assay of DAP-AT by coupled reaction with L-glutamate dehydrogenase.
Table 2. Purification of DAP-AT from E. coli ATCC 9637
% activity specific fold
In the initial studies by Gilvarg and co-workers, the relative
abilities of a number of common amino acids to act as amine
donors were examined for the “forward” aminotransferase
reaction (generation of N-succinyl DAP).9 Only L-glutamate
showed activity in their studies. In the reverse direction, amino
acids resembling LL-N-succinyl DAP were tested. No activity
could be detected when meso-N-succinyl-DAP, LL-DAP, L-lysine
or L-N-Cbz-lysine were utilized. Clearly the enzyme has high
specificity for the correct stereoisomer, and the lack of reactivity
of L-lysine derivatives indicates a requirement for the terminal
C-7 carboxyl group.
Kinetic parameters for the substrate analogues synthesized
in the current study (Table 3) reveal that only certain structural
variations are tolerated by DAP-AT. The presence of the C-1
carboxyl is crucial for substrate turnover, and 13 is not accepted
by the aminotransferase. Similarly, the 2-amido group is also
of key importance, and even though 17 can act as a substrate,
it is so poor that reliable kinetic parameters could not be
obtained. The N-acetyl derivative 1b is also quite ineffective
with a specificity only 4.3% that of the natural substrate 1a.
The N-Boc derivative 1d shows very similar behavior. Since
recovery activity, purification cumulative
step
for step units/mga
for step
purification
crude E. Coli
prep
brown-10
affinity column
blue 3-GA
affinity column
hydroxyl apatite
column
Sephadex G-75
column
Mono-Q anion
exchange
-
49%
33%
90%
86%
95%
0.089
0.121
0.282
1.60
-
1.0
1.4
2.3
1.4
3.3
5.7
18.6
39.4
1508
3.37
2.1
129
38.3
a Enzyme unit defined as 1.0 µmol of L-N-succinyl-R-amino-ꢀ-
ketopimelate (1a) transformed/min at 30 °C.
terkofsky utilizing less pure enzyme and substrates under
somewhat different experimental conditions.9b,c The physical
characteristics of N-succinyl-LL-DAP aminotransferase (EC
2.6.1.17) are similar to aspartate aminotransferase (EC 2.6.1.1)
which is also dimeric and has a subunit molecular weight of
ca. 45 kDa.30
(30) Kirsch, J. F.; Eichele, G.; Ford, G. C.; Vincent, M. G.; Jansonius,
J. N.; Gehring, H.; Christen, P. J. Mol. Biol. 1984, 174, 497-525.

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Substrates and Inhibitors of DAP-AT
J. Am. Chem. Soc., Vol. 118, No. 32, 1996 7453
Table 3. Conversion of Substrate Analogues by DAP-AT
substratea
app
entry
1
no.
structure
Kmapp, mMb,c
kcat, s-1
kcat/Kmapp, s-1 M-1
rel to 1a, % (kcat/Km )
1a
O
1.2
86
73
100
R
OH
OH
O
HN
OH
O
O
2
1b
6.9
23
3.1
4.3
R
HN
O
3
4
5
13
17
1c
R
O
O
HN
OH
OH
O
O
>10
<5
<0.5
<0.7
R
OH
O
3.6
62
17
23
R
OH
O
HN
O
O
6
7
8
9
1d
1e
1f
8.2
1.2
3.5
3.5
26
61
20.
76
3.1
51.
5.6
22
4.3
70.
7.7
30
R
OH
O
HN
O
O
R
OH
OH
HN
O
O
R
HN
O
O
1g
OMe
R
OH
O
HN
OMe
O
a R ) (CH2)3(CO)CO2H. b At 10 mM L-Glu; Km for 1a independent of L-Glu is 0.18 mM. c (10%.
1b is presumably the natural substrate for the N-acetyl-LL-DAP
aminotransferase from B. megaterium, the relatively poor
interaction of 1b with N-succinyl-LL-DAP aminotransferase
supports earlier observations31 that the two acylated pathways
to meso-DAP are quite specific.
electrostatic interaction with a cation (e.g. protonated basic
amino acid side chain) on the protein surface; this enzyme cation
would presumably stack against and recognize the aromatic
residue of the substrate analogues. However, the conforma-
tionally flexible hydrocinnamoyl derivative 1f shows a specific-
ity of only 7.6% that of 1a. This suggests that increased electron
donation from either carbamate oxygen (1c,g) or cinnamoyl
olefin moiety (1e) may also be important for binding and
recognition and that hydrophobic interactions of the aromatic
ring could also play a critical role. PLP-dependent aminotrans-
ferases show considerable homology,34 and enlargement of a
cluster of hydrophobic residues near the active site entrance of
aspartate aminotransferase35 has been suggested36 to be the cause
of the enhanced specificity toward both hydrophobic and acidic
Interestingly, replacement of the terminal carboxyl group of
the succinate moiety in 1a with an aromatic ring produces good
app 32
substrates. The specificity (kcat/Km
)
calculated for each
compound and expressed as a percentage of that of the natural
substrate 1a indicates that the N-cinnamoyl derivative 1e is the
best of this group (specificity 70% of N-succinyl). The N-Cbz
derivative 1c and its dimethoxy-substituted analogue 1g are also
quite effective. We initially rationalized that these aromatic
groups could substitute for the natural succinyl moiety by
replacing a carboxylate-cation interaction with an aromatic
π-cation interaction.33 Thus the terminal carboxylate moiety
of the succinyl residue of 1a could bind to DAP-AT by an
(34) For examples see: (a) Mehta, P. K.; Hale, T. I.; Christen, P. Eur.
J. Biochem. 1989, 186, 249-253. (b) Mehta, P. K.; Hale, T. I.; Christen,
P. Eur. J. Biochem. 1993, 214, 549-561. (c) Alexander, F. W.; Sandmeier,
E.; Mehta, P. K.; Christen, P. Eur. J. Biochem. 1994, 219, 953-960.
(35) (a) Ja¨ger, J.; Pauptit, R. A.; Sauder, U.; Jansonius, J. N. Protein
Eng. 1994, 7, 605-612. (b) Ja¨ger, J.; Moser, M.; Sauder, U.; Jansonius, J.
N. J. Mol. Biol. 1994, 239, 285-305.
(31) Studies with the desuccinylase enzyme from E. coli10a demonstrate
that acetylated DAP analogues are not substrates for this hydrolytic enzyme.
(32) Fersht, A. Enzyme Structure and Mechanism, 2nd ed.; W. H.
Freeman and Co.: New York, 1984; pp 105-106.
(33) For a recent review of π-cation interactions see: Dougherty, D. A.
Science 1996, 271, 163-168.
(36) Hayashi, H.; Inoue, K.; Nagata, T.; Kuramitsu, S.; Kagamiyama,
H. Biochemistry 1993, 32, 12229-12239.

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7454 J. Am. Chem. Soc., Vol. 118, No. 32, 1996
Cox et al.
Figure 6. Syntheses of DAP-AT slow-binding inhibitors 20a,c.
substrates by aromatic amino acid aminotransferase, an enzyme
which shares 41% sequence identity with aspartate aminotrans-
ferase.37
Figure 7. Possible mechanism of inhibition of DAP-AT by hydrazino
analogues 20a,c.
Inhibition of DAP Aminotransferase by Analogues of 1a.
Since L-glutamate and R-ketoglutarate are utilized extensively
by mammalian systems, inhibitors of DAP-AT based on these
compounds may be toxic, and analogues of 1a or N-succinyl
DAP are more attractive targets. Inhibition experiments with
N-succinyl- and N-Cbz-R-aminopimelic acids (18a,c) and also
with the N-succinyl-2-hydroxy-6-aminopimelic acid (19) show
no detectable effect on DAP-AT at concentrations up to 10 mM.
These results further support the common observation that DAP-
metabolizing enzymes usually recognize the complete substrate
skeleton as well as all polar functional groups (i.e. amino and
carboxyl groups).4,5b The substrate specificity studies described
above also support this concept. The R-hydrazino analogues
20a,c would appear to fulfill the skeletal and functional group
requirements since 1c is a substrate. A number of R-hydrazino
analogues of R-amino acids are known to inhibit PLP-dependent
enzymes, presumably by reaction with the enzyme-cofactor
aldimine to generate very stable hydrazones in the active site.38
Indeed, the mono-R-hydrazino analogue of DAP is a good
inhibitor of the PLP-dependent DAP decarboxylase.39
Compounds 20a,c are readily accessible as a 1:1 mixture of
diastereomers by treatment of aqueous solutions of the lithium
salts of R-keto acids 1a,c with hydrazine followed by removal
of volatiles and immediate reduction of the crude hydrazone in
methanol with sodium cyanoborohydride (Figure 6). A variety
of alternative approaches, including in situ reduction of the
aqueous hydrazone solution with sodium amalgam, fail due to
competing hydrolysis or, in the case of 20c, cleavage of the
Cbz group. Both compounds are potent inhibitors of DAP-AT
which display time-dependent behavior. Detailed analyses of
interactions of 20a or 20c with DAP-AT show the usual
characteristics of a slow-binding mechanism in addition to time
dependence, namely substrate protection, reversibility upon 100-
fold dilution, and progressive onset of inhibition.40 Progress
curves obtained from absorbance measurements in the standard
assay were fitted using nonlinear least-squares regression
analysis.40b The Ki* determined for 20a is 22 ( 4 nM and for
20c is 54 ( 9 nM, making these compounds the most potent
reversible substrate analogue inhibitors of any DAP enzyme
reported thus far. The t1/2 values for release of these inhibitors
from the active site of DAP-AT are 79 ( 10 and 45 ( 5 min,
respectively. The actual chemical mechanism of inhibition is
uncertain, but it probably involves reaction of the terminal amino
group of the hydrazine with the PLP cofactor held in the
aminotransferase active site (Figure 7). Although the amino
acid sequence of the enzyme is still unknown, the homology of
most PLP-dependent aminotransferases34-37 suggests that the
ꢀ-amino group of an active site lysine forms an imine with PLP.
This species would presumably undergo transimination with the
terminal NH2 group of 20a or 20c to form the inhibitor-PLP
hydrazone held by noncovalent interactions in the DAP-AT
active site. Slow release of the complex and/or hydrolysis of
the hydrazone double bond would regenerate active enzyme.
Initial antimicrobial tests with these inhibitors against a
limited array of bacteria as described previously5a,b showed
negligible activity at concentrations up to 100 µg/mL, possibly
due to problems of transport into cells, as has been previously
observed for DAP analogues.5b
Conclusions
The present work describes the isolation and purification of
N-succinyl-LL-DAP aminotransferase (DAP-AT) (EC 2.6.1.17)
from wild type E. coli to apparent homogeneity and studies the
interaction of this enzyme with a series of substrate analogues
and inhibitors. The sensitive substrate of this pyridoxal
phosphate-dependent enzyme, (L)-N-succinyl-R-amino-ꢀ-ke-
topimelic acid (1a), is synthesized in stereochemically pure form
using an ene reaction between methyl N-succinyl-L-allylglyci-
nate and methyl glyoxylate as a key step. Analogous approaches
afford efficient access to a series of analogues missing the
carboxyl or amido functionality or having other N-acyl groups
in place of succinyl. The results of substrate specificity studies
confirm that, like other DAP-metabolizing enzymes, this ami-
notransferase recognizes all polar functional groups as well as
the overall skeleton of its substrates. However, replacement
of the terminal carboxyl in the succinyl moiety by an aromatic
ring is permitted, possibly because an aromatic π-cation
interaction may partly mimic the substrate carboxylate-enzyme
cation recognition. Alternatively, additional hydrophobic in-
teractions could contribute to the binding of the aromatic ring.
Derivatives 20a,c in which an R-hydrazino group replaces the
R-amino of the enzyme product are the most potent reversible
inhibitors of any DAP enzyme reported thus far. Current studies
to sequence and further characterize DAP-AT as well as to apply
ene methodology to stereospecific syntheses of other DAP
analogues will be reported in the future.
Experimental Section
(37) Miyazawa, K.; Kawaguchi, S.; Okamoto, A.; Kato, R.; Ogawa, T.;
Kuramitsu, S. J. Biochem. 1994, 115, 568-577.
(38) For examples see references in: (a) Scaman, C. H.; Palcic, M. M.;
McPhalen, C.; Gore, M. P.; Lam, L. K. P.; Vederas, J. C. J. Biol. Chem.
1991, 266, 5525-5533. (b) Lacoste, A-M.; Dumora, C.; Zon, J. J. Enzyme
Inhib. 1993, 7, 237-248.
(39) Kelland, J. G.; Arnold, L. D.; Palcic, M. M.; Pickard, M. A.;
Vederas, J. C. J. Biol. Chem. 1986, 261, 13216-13223.
(40) (a) Morrison, J. F. Trends Biochem. Sci. 1982, 7, 102-105. (b)
Morrison, J. F.; Stone, S. R. Comments Mol. Cell. Biophys. 1985, 2, 347-
368. (c) Morrison, J. F.; Walsh, C. T. AdV. Enzymol. 1988, 61, 201-301.
General. General procedures and instrumentation have been previ-
ously described.41 Ion exchange resins were obtained from BioRad
(Mississauga, ON, Canada) and were washed before use according to
the manufacturer’s instructions. Diazomethane42 and methyl glyoxyl-
(41) Harris, J. M.; Bolessa, E. A.; Mendonca, A. J.; Feng, S.-C.; Vederas,
J. C. J. Chem. Soc., Perkin Trans. 1 1995, 1945-1950.
(42) Furniss, B. S.; Hannaford, A. J.; Smith, P. W. G.; Tatchell, A. R.
Vogel’s Textbook of Organic Chemistry, 5th ed.; Longman: New York,
1989; pp 430-433.

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Substrates and Inhibitors of DAP-AT
J. Am. Chem. Soc., Vol. 118, No. 32, 1996 7455
ate43 were generated by standard literature procedures. The Cleland
matrix for positive ion fast atom bombardment (POSFAB) mass spectra
consists of a 5:1 mixture of dithiothreitol and dithioerythritol.
(2S)-2-(N-(Cinnamoyl)amino)-6-oxoheptane-1,7-dioic Acid, Di-
lithium Salt Monohydrate (1e). The procedure used for preparation
of 1a was employed to convert 8e to 1e in 99% yield: mp 198 °C dec;
IR (KBr) 3397, 3083, 3061, 2973, 2934, 2870, 1709, 1656, 1600, 1449,
1415 cm-1; 1H NMR (300 MHz, D2O) δ 7.49-7.46 (m, 2H), 7.36 (d,
1H, J ) 15.8 Hz), 7.38-7.29 (m, 3H), 6.54 (d, 1H, J ) 15.8 Hz),
4.15-4.11 (m, 1H), 2.65 (t, 2H, J ) 7.2 Hz, D2O exchanged), 1.74-
1.20 (m, 4H); 13C NMR (100 MHz, CDCl3) δ 207.0, 179.2, 170.9,
168.4, 141.4, 134.7, 130.4, 129.3, 128.2, 120.4, 55.6, 38.0, 31.4, 19.5;
MS (POSFAB, nitrobenzyl alcohol) m/z 338 (MLi+, 3.3%), 332 (MH+,
2.3%), 326 ((M - Li + H)H+, 1.0%). Anal. Calcd for C16H15NO6Li2‚
H2O: C, 55.03; H, 4.91; N, 4.01. Found: C, 55.22; H, 4.52; N, 3.80.
(2S)-2-(N-(Succinyl)amino)-6-oxoheptane-1,7-dioic Acid, Trilith-
ium Salt Trihydrate (1a). To a stirred solution of oxo diester 8a (338
mg, 1.02 mmol) in 1:1 MeCN:H2O (10 mL) was added LiOH‚H2O
(129 mg, 3.06 mmol). After 90 min the solvent was removed in Vacuo,
and the residue was dissolved in water (2 mL). The solution was
lyophilized to afford 1a as a powder (310 mg, 99%): mp 125 °C dec;
[R]D ) +6.4° (c ) 0.68, H2O); IR (KBr) 3482, 2970, 1675, 1671,
1
1596, 1420 cm-1; H NMR (360 MHz, D2O) δ 4.13-4.05 (m, 1H),
2.71-2.64 (m, 2H, D2O exchanged), 2.55-2.39 (m, 4H), 1.82-1.45
(m, 4H); 13C NMR (90 MHz, D2O) δ 190.0, 180.0, 174.4, 174.3, 54.4,
38.8, 32.4, 31.6, 30.4, 18.5; MS (POSFAB, glycerol/HCl) m/z 307 (M+,
3.4%), 308 (MH+, 1.2%). Anal. Calcd for C11H12NO8Li3‚3H2O: C,
36.59; H, 5.02; N, 3.88. Found: C, 36.30; H, 4.71; N, 3.74.
(2S)-2-(N-(Hydrocinnamoyl)amino)-6-oxoheptane-1,7-dioic Acid,
Dilithium Salt Hemihydrate (1f). The procedure used for preparation
of 1a was employed to convert 8f to 1f in 99% yield: mp 99-103 °C;
IR (KBr) 3406, 3086, 3063, 3028, 2934, 1709, 1605, 1496, 1453, 1417
1
cm-1; H NMR (400 MHz, D2O) δ 7.23-7.03 (m, 5H), 3.88-3.84
Analysis of Enantiomeric Excess of 1a. To a suspension of 1a
(19.0 mg, 61.9 µmol) in absolute MeOH (1 mL) was added NH4OAc
(47.7 mg, 620 µmol). After 30 min, NaCNBH3 (39 mg, 620 µmol)
was added and the mixture was stirred for 16 h. Excess NaCNBH3
was destroyed by the addition of 1 N HCl (1 mL). Solvent was
removed in Vacuo, and the residue was dissolved in concentrated HCl.
The solution was stirred for 2 h at 80 °C and then evaporated to dryness.
The residue was purified by cation exchange chromatography to afford
2.5 mg of a white solid. The solid was derivatized by the method of
Marfey,27 and the resultant solution was analyzed by HPLC. (Col-
umn: Chemcosorb 5-ODS-UH, 4.6 × 150 mm; solvent A 12 mM
ammonium phosphate buffer + 4% DMF, pH 6.5, solvent B 70%
MeCN in milli-Q water. Method: gradient 15%-35% B in 35 min, at
1 mL/min, detection at 340 nM. Retention times for Marfey deriva-
tives: meso-DAP 22.0 min, LL-DAP 26.6 min, DD-DAP 31.6 min.)
Less than 1% DD-DAP was observed confirming that 1a had exclusively
2S configuration within experimental error.
(m, 1H), 2.81-2.72 (m, 2H), 2.48-2.41 (m, 4H, (2H D2O exchanged)),
1.55-1.27 (m, 3H), 1.15-0.98 (m, 1H); 13C NMR (100 MHz, D2O +
CH3CN) δ 206.8, 179.0, 175.1, 170.6, 140.8, 129.1, 129.0, 126.8, 55.1,
39.1, 37.8, 33.5, 31.7, 19.2; MS (POSFAB, Cleland) m/z 340 (MLi+,
11.0%), 334 (MH+, 6.8%), 333 (M+, 1.5%). Anal. Calcd for C16H17-
NO6Li2‚0.5H2O: C, 56.16; H, 5.30; N, 4.09. Found: C, 55.99; H,
5.25; N, 4.08.
(2S)-2-(N-((3,5-Dimethoxybenzyl)oxycarbonyl)amino)-6-oxohep-
tane-1,7-dioic Acid, Dilithium Salt (1g). The procedure used for
preparation of 1a was employed to convert 8g to 1g in 95% yield: mp
189-193 °C dec; IR (KBr) 3423, 2942, 1703, 1600, 1530, 1460, 1428
cm-1; 1H NMR (300 MHz, D2O) δ 6.46 (br, 2H), 6.32 (br s, 1H), 4.91-
4.83 (m, 2H), 3.66 (s, 6H), 3.64-3.59 (m, 1H), 2.64-2.61 (m, 2H,
D2O exchanged), 1.72-1.35 (m, 4H); 13C NMR (100 MHz, D2O +
CH3CN) δ 206.9, 179.6, 170.2, 160.8, 157.9, 139.9, 106.1, 100.4, 66.7,
56.8, 55.9, 39.1, 31.8, 19.7; MS (POSFAB, Cleland) m/z 402 (MLi+,
6.3%), 396 (MH+, 5.9%), 395 (M+, 1.4%).
(2S)-2-(N-Acetylamino)-6-oxoheptane-1,7-dioic Acid, Dilithium
Salt Monohydrate (1b). The procedure used for preparation of 1a
was employed to convert 8b to 1b in 92% yield: mp 218 °C dec; IR
(S)-Allylglycine (3). A stirred solution of (RS)-N-acetylallylglycine
(2) (30.0 g, 191 mmol) in water (1.4 L) was adjusted to pH 7.8 with
LiOH. Porcine kidney acylase I (Sigma, 350 mg) was added, and the
solution was stirred 12 h at 38 °C. Norit A charcoal (5 g) was added,
and the suspension was filtered through Celite. The solid was washed
with water (200 mL) and the combined aqueous filtrate applied to
BioRad AG-50 WX-8 cation exchange resin (H+, 600 mL). The
column was washed with water until the eluent pH was neutral and
then with 1 M aqueous ammonia. Amine-containing fractions (nin-
hydrin) were evaporated in Vacuo, and the residue was recrystallised
from water/ethanol to afford pure (S)-allylglycine (3) (10.4 g 95%):
mp 282-283 °C; [R]D -37.3° (4% H2O, lit.44 -37.1°); IR (KBr) 3420,
2900, 1643, 1612, 1586, 1561, 1512, 1405 cm-1; 1H NMR (360 MHz,
D2O) δ 5.86-5.72 (m, 1H), 5.36-5.22 (m, 2H), 3.83 (dd, 1H, J )
7.0, 5.2 Hz), 2.74-2.55 (m, 2H); 13C NMR (100 MHz, D2O) δ 173.1,
130.5, 119.5, 53.0, 33.9; MS (POSFAB, glycerol/HCO2H) m/z 116
(MH+). Anal. Calcd for C5H9NO2: C, 52.17; H, 7.88; N, 12.17.
Found: C, 52.12; H, 7.73; N, 12.23.
(KBr) 3427, 1716, 1617, 1418 cm-1 1H NMR (400 MHz, D2O +
;
CD3CN) δ 4.00-3.92 (m, 1H), 1.89 (s, 3H), 1.68-1.35 (m, 4H); 13C
NMR (90 MHz, H2O + CD3CN) δ 206.4, 177.4, 172.0, 169.7, 54.3,
36.6, 30.7, 21.3, 18.6; MS (POSFAB, glycerol/HCl) m/z 244 (MH+,
2.7%), 243 (M+, 4.1%), 238 ((M - Li + H)H+, 2.7%). Anal. Calcd
for C9H11NO6Li2‚H2O: C, 41.40; H, 5.02; N, 5.36. Found: C, 41.55;
H, 4.87; N, 5.25.
(2S)-2-(N-(Benzyloxycarbonyl)amino)-6-oxoheptane-1,7-dioic Acid,
Dilithium Salt Dihydrate (1c). The procedure used for preparation
of 1a was employed to convert 8c to 1c in 98% yield: mp 67-69°C;
1
IR (KBr) 3423, 2933, 1697, 1615, 1522, 1454, 1420 cm-1; H NMR
(360 MHz, D2O) δ 7.51-7.35 (m, 5H), 5.23-5.05 (m, 2H), 4.00-
3.85 (m, 1H), 2.81-2.73 (m, 2H), 1.95-1.40 (m, 4H); 13C NMR (90
MHz, D2O + CH3CN) δ 203.5, 181.6, 174.5, 161.8, 140.4, 132.8, 132.2,
129.9, 70.6, 60.0, 41.0, 34.8, 22.5; MS (POSFAB, glycerol/HCl) m/z
335 (M+, 0.6%), 330 ((M - Li + H)H+, 1.33%), 324 ((M - 2Li +
2H)H+, 1.1%). Anal. Calcd for C15H15NO7Li2‚2H2O: C, 48.54; H,
5.16; N, 3.77. Found: C, 48.48; H, 4.91; N, 3.71.
Dimethyl N-(Succinyl)-(S)-allylglycinate (4a). A stirred solution
of 3 (2.80 g, 24.3 mmol) in aqueous K2CO3 (1.2M, 50mL) was treated
with succinic anhydride (3.65 g, 36.5 mmol) in portions over a period
of 20 min. After 40 min, the solution was acidified to pH 2.0 with
concentrated HCl and extracted with EtOAc (4 × 50 mL). The
combined extracts were dried (MgSO4) and concentrated in Vacuo. The
residue was treated with excess ethereal diazomethane. After 10 min
excess diazomethane was destroyed by the addition of acetic acid. The
solution was evaporated in Vacuo to afford an oil which was purified
by flash chromatography (70% EtOAc in hexane, Rf 0.22) to afford
the product 4a as an oil (5.03 g, 85%): [R]D ) +36.2° (c ) 4.35,
CHCl3); IR (neat) 3314, 3003, 2982, 2954, 1741, 1678, 1658, 1536,
The stereochemical analysis of 1c was accomplished analogously
to the method for 1a described above using reductive amination and
derivatization. The Marfey derivative27 of DD-DAP was not observed,
indicating pure 2S configuration for 1c.
(2S)-2-(N-(tert-butyloxycarbonyl)amino)-6-oxoheptane-1,7-di-
oic Acid, Dilithium Salt (1d). The procedure used for preparation of
1a was employed to convert 8d to 1d in 99% yield: mp 87-90 °C; IR
(KBr) 3429, 2978, 2934, 1700, 1617, 1512, 1416 cm-1; 1H NMR (360
MHz, CDCl3) δ 3.72-3.58 (m, 1H), 2.62-2.54 (m, 2H, D2O
exchanged), 1.70-1.33 (m, 4H), 1.25 (s, 9H); 13C NMR (90 MHz,
D2O + CH3CN) δ 199.9, 180.0, 171.5, 157.6, 81.2, 56.0, 39.1, 31.5,
27.9, 19.5; MS (POSFAB, glycerol/HCl) m/z 302 (MH+, 1.2%), 297
((M - Li + H)H+, 3.36%), 296 ((M - Li + H)+, 2.32%).
1
1438 cm-1; H NMR (400 MHz, CDCl3) δ 6.29 (d, 1H, J ) 6.7 Hz),
5.75-5.62 (m, 1H), 5.17-5.09 (m, 2H), 4.72-4.66 (m, 1H), 3.80 (s,
3H), 3.65 (s, 3H), 2.75-2.47 (m, 6H); 13C NMR (75.5 MHz, CDCl3)
δ 172.9, 171.9, 170.9, 132.1, 118.7, 52.0, 51.5, 51.4, 36.1, 30.4, 28.9;
exact mass 243.1104 (M+) (243.1107 calcd for C11H17NO5). Anal.
Calcd for C11H17NO5: C, 54.31; H, 7.04; N, 5.76. Found: C, 54.17;
H, 6.99; N, 5.78.
(43) (a) Kelly, T. R.; Schmidt, T. E.; Haggerty, J. G. Synthesis 1972,
544-545. (b) Jung, M. E.; Shishido, K.; Davis, L. H. J. Org. Chem. 1982,
47, 891-892.

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7456 J. Am. Chem. Soc., Vol. 118, No. 32, 1996
Cox et al.
Methyl N-(Acetyl)allylglycinate (4b). Reaction of 3 and acetic
anhydride analogous to preparation of 4a gave known44 4b in 91%
yield (Rf 0.37, EtOAc on SiO2): [R]D ) +45.4° (c ) 3.57, CHCl3); IR
(neat) 3285, 3079, 2954, 1747, 1658, 1544, 1438 cm-1; 1H NMR (360
MHz, CDCl3) δ 6.22 (s, 1H), 5.67-5.56 (m, 1H), 5.08-5.02 (m, 2H),
4.64-4.59 (m, 1H), 3.67 (s, 3H), 2.51-2.40 (m, 2H), 1.95 (s, 3H);
13C NMR (100 MHz, CDCl3) δ 172.8, 170.1, 132.6, 119.7, 52.8, 52.1,
37.0, 26.4, 23.7; exact mass 171.08987 (M+) (171.08954 calcd for
C8H13NO3).
1738, 1656, 1531, 1437 cm-1; 1H NMR (360 MHz, CDCl3) δ 6.24 (t,
1H, J ) 7.0 Hz), 4.63-4.58 (m, 1H), 4.20-4.15 (m, 1H), 3.78 (s,
3H), 3.75 (s, 3H), 3.69 (s, 3H), 2.90 (s, 1H), 2.76-2.55 (m, 4H), 1.89-
1.66 (m, 4H), 1.50-1.20 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 175.1,
173.2, 172.8, 171.3, 70.0, 52.4, 52.2, 51.8, 51.7, 33.4, 31.7, 30.5, 29.0,
20.5; exact mass 333.1414 (M+) (333.1424 calcd for C14H23NO8).
Dimethyl (2S,6RS)-2-(N-(Acetylamino))-6-hydroxyheptane-1,7-
dioate (6b). Alkene 5b was hydrogenated by the procedure used for
5a to give 6b as an oil in 95% yield (EtOAc, Rf 0.13): IR (CHCl3
1
cast) 3303, 2955, 1742, 1655, 1541, 1437 cm-1; H NMR (360 MHz,
Methyl N-(Benzyloxycarbonyl)allylglycinate (4c). Reaction of 3
and CbzCl analogous to preparation of 4a gave known 4c in 90% yield
(Rf 0.25, 30% EtOAc/Hexane on SiO2). All spectral properties agreed
with those previously reported.5a
CDCl3) δ 6.34 (d, 1H, J ) 7.9 Hz), 4.59-4.53 (m, 1H), 4.15-4.10
(m, 1H), 3.73 (s, 3H), 3.69 (s, 3H), 3.06 (s, 1H), 1.97 (s, 3H), 1.81-
1.37 (m, 6H); 13C NMR (90 MHz, CDCl3) δ 175.5, 173.2, 170.4, 70.5,
52.6, 52.4, 52.3, 33.9, 32.1, 23.0, 21.1; exact mass 262.12872 (MH+)
(262.12906 calcd for C11H20NO6).
Ene Reaction with FeCl3. Dimethyl (2S,6RS)-2-(N-(Methyl
succinyl)amino)-6-hydroxyhept-3-ene-1,7-dioate (5a). A stirred ice-
cooled solution of freshly distilled methyl glyoxylate (1.10 g, 12.5
mmol) in CH2Cl2 (20 mL) under Ar was treated with anhydrous FeCl3
(4.00 g, 24.6 mmol). The suspension was stirred at 0 °C for 30 min,
and then a solution of alkene ester 4a (1.0 g, 4.1 mmol) in CH2Cl2 (30
mL) was added dropwise over 10 min. The suspension was warmed
to 20 °C and stirred 24 h. The black suspension was poured onto
crushed ice (100 mL), and the mixture was extracted into CH2Cl2 (4 ×
100 mL). The combined organic extracts were washed with 1 N HCl
(100 mL) followed by saturated aqueous NaHCO3 (100 mL) and then
dried (MgSO4) and evaporated in Vacuo. The residue was purified by
flash chromatography (EtOAc, Rf 0.33) to afford 5a as an oil (646 mg,
47%): IR (CHCl3 cast) 3140, 2950, 1738, 1656, 1531, 1437 cm-1; 1H
NMR (360 MHz, CDCl3) δ 6.22 (s, 1H), 5.82-5.72 (m, 1H), 5.62
(dd, 1H, J ) 6.0, 15.5 Hz), 5.05 (dd, 1H, J ) 12.0, 6.0 Hz), 4.23 (dd,
1H, J ) 6.6, 4.5 Hz), 3.74 (s, 3H), 3.73 (s, 3H), 3.68 (s, 3H), 3.08 (s,
1H), 2.76-2.38 (m, 6H); 13C NMR (100 MHz, CDCl3) δ 174.2, 173.2,
171.3, 171.0, 128.3, 127.4, 69.8, 53.8, 52.4, 52.1, 51.6, 36.8, 30.3, 28.9;
exact mass 332.1347 (MH+) (332.1345 calcd for C14H22NO8).
Acylation of 7. Dimethyl (2S,6RS)-2-(N-(Benzyloxycarbonyl)-
amino)-6-hydroxyheptane-1,7-dioate (6c). A stirred solution of amine
hydrochloride 7 (455 mg, 1.78 mmol) in CH2Cl2 (20 mL) at 20 °C
under Ar was treated with pyridine (288 µL, 282 mg, 3.56 mmol).
Benzyl chloroformate (280 µL, 334 mg, 1.96 mmol) was added
dropwise, and the solution was stirred 90 min before 1 N HCl (50 mL)
was added. The mixture was extracted into CH2Cl2 (3 × 100 mL),
and the combined extracts were dried (MgSO4) and evaporated in Vacuo.
The residue was purified by flash chromatography (70% EtOAc in
hexane, Rf 0.17) to give 6c as an oil (527 mg, 84%): IR (CH2Cl2 cast)
3356, 2954, 1735, 1528, 1455, 1438 cm-1; 1H NMR (360 MHz, CDCl3)
δ 7.35-7.28 (m, 5H), 5.39 (d, 1H, J ) 7.9 Hz), 5.05 (s, 2H), 4.36-
4.28 (m, 1H), 4.14-4.08 (m, 1H), 3.75 (s, 3H), 3.70 (s, 3H), 3.05 (s,
1H), 1.88-1.35 (m, 6H); 13C NMR (90 MHz, CDCl3) δ 175.8, 173.3,
156.4, 136.7, 129.0, 128.6, 128.5, 70.5, 67.4, 54.1, 52.9, 52.8, 34.1,
32.6, 21.1; exact mass 353.14775 (M+) (353.14746 calcd for C17H23-
NO7). Anal. Calcd for C17H23NO7: C, 57.77; H, 6.56; N, 3.97.
Found: C, 57.88; H, 6.89; N, 3.84.
Dimethyl (2S,6RS)-2-(N-(tert-Butyloxycarbonyl)amino)-6-hydrox-
yheptane-1,7-dioate (6d). To a solution of N-Cbz-alkene 5c (1.42 g,
4.05 mmol) in MeOH (30 mL) was added tert-butyl pyrocarbonate (1.32
g, 6.0 mmol) and 10% Pd/C (200 mg). The suspension was stirred
under 1 atm of H2 for 15 h and then vacuum filtered through Celite,
which was washed with MeOH. The solution was evaporated in Vacuo
and the residue was purified by flash chromatography (25% EtOAc in
CH2Cl2, Rf 0.30) to afford 6d as an oil (1.03 g, 80%): IR (CH2Cl2
cast) 3375, 2976, 2955, 2935, 1741, 1715, 1518, 1455, 1438 cm-1; 1H
NMR (360 MHz, CDCl3) δ 5.12 (s, 1H), 4.21-4.12 (m, 1H), 4.10-
4.04 (m, 1H), 3.65 (s, 3H), 3.60 (s, 3H), 3.11 (s, 1H), 1.78-1.62 (m,
2H), 1.60-1.48 (m, 2H), 1.46-1.30 (m, 2H), 1.29 (s, 9H); 13C NMR
(90 MHz, CDCl3) δ 175.7, 173.6, 155.8, 80.1, 70.5, 53.6, 52.7, 52.5,
34.0, 32.6, 28.6, 21.2; MS (CI, NH3) m/z 337 (16.5%, MNH4+), 320
(17.6%, MH+). Anal. Calcd for C14H25NO7: C, 52.65; H, 7.89; N,
4.39. Found: C, 52.40; H, 7.98; N, 4.19.
Dimethyl (2S,6RS)-2-(N-(Cinnamoyl)amino)-6-hydroxyheptane-
1,7-dioate (6e). Amine hydrochloride 7 was acylated with cinnamoyl
chloride as described for preparation of 6c to give 6e as an oil in 56%
yield (50% EtOAc in hexane, Rf 0.17): IR (CH2Cl2 cast) 3305, 2953,
1742, 1658, 1621, 1577, 1538, 1497, 1450, 1436 cm-1; 1H NMR (400
MHz, CDCl3) δ 7.60 (d, 1H, J ) 15.6 Hz), 7.46-7.44 (m, 2H), 7.32-
7.30 (m, 3H), 6.65 (d, 1H, J ) 7.6 Hz), 6.48 (d, 1H, J ) 15.6 Hz)
4.75-4.72 (m, 1H), 4.17-4.14 (m, 1H), 3.72 (s, 6H), 2.00-1.43 (m,
6H); 13C NMR (100 MHz, CDCl3) δ 173.8, 171.6, 140.2, 133.1, 128.3,
127.3, 126.4, 118.5, 68.4, 51.4, 51.1, 50.8, 32.0, 30.5, 19.2; exact mass
349.15275 (M+) (349.15253 calcd for C18H23NO6).
Dimethyl (2S,6RS)-2-(N-(Hydrocinnamoyl)amino)-6-hydroxyhep-
tane-1,7-dioate (6f). Amine hydrochloride 7 was acylated with
hydrocinnamoyl chloride as described for preparation of 6c to give 6f
as an oil in 83% yield (60% EtOAc in hexane, Rf 0.20): IR (CH2Cl2
cast) 3308, 3085, 3028, 2953, 2865, 1740, 1651, 1604, 1541, 1497,
1454, 1438 cm-1; 1H NMR (300 MHz, CDCl3) δ 7.26-7.15 (m, 5H),
6.23 (d, 1H, J ) 6.2 Hz), 4.59-4.52 (m, 1H), 4.12-4.08 (m, 1H),
3.72 (s, 3H), 3.67 (s, 3H), 3.11 (s, 1H), 2.94-2.69 (m, 2H), 2.55-
2.44 (m, 2H), 1.83-1.50 (m, 4H), 1.37-1.32 (m, 2H); 13C NMR (75.5
MHz, CDCl3) δ 175.7, 173.3, 172.6, 141.0, 128.8, 128.6, 126.5, 70.4,
52.7, 52.6, 52.2, 38.2, 33.8, 32.1, 31.7, 21.0; exact mass 351.1691 (M+)
(351.16818 calcd for C18H25NO6).
Dimethyl (2S,6RS)-2-(N-Acetylamino)-6-hydroxyhept-3-ene-1,7-
dioate (5b). Ene reaction of 4b following the procedure for 5a gave
5b as an oil in 46% yield (EtOAc, Rf 0.38): IR (CH2Cl2 cast) 3448,
3309, 3293, 2956, 1742, 1737, 1655, 1649, 1541, 1534, 1509, 1457,
1
1438 cm-1; H NMR (360 MHz, CDCl3) δ 6.22 (s, 1H), 5.79-5.69
(m, 1H), 5.64-5.58 (m, 1H), 5.09-5.04 (m, 1H), 4.26-4.20 (m, 1H),
3.76 (s, 3H), 3.75 (s, 3H), 2.99 (s, 1H), 2.59-2.52 (m, 1H), 2.46-
2.36 (m, 1H); 13C NMR (90 MHz, CDCl3) δ 174.5, 171.5, 169.6, 128.3,
128.0, 69.5, 53.5, 52.4, 52.2, 36.8, 22.5; exact mass 259.10534 (M+)
(259.10559 calcd for C11H17NO6).
Ene Reaction with SnCl4. Dimethyl (2S,6RS)-2-(N-(Benzyloxy-
carbonyl)amino)-6-hydroxyhept-3-ene-1,7-dioate (5c). To a solution
of freshly distilled methyl glyoxylate (4.2 g, 48 mmol) in CH2Cl2 (65
mL) stirred under Ar and cooled to -55 °C was added freshly distilled
SnCl4 (25.0 mL, 214 mmol) over a period of 10 min. The solution
was stirred a further 10 min and then cooled to -78 °C. A solution of
alkene ester 4c (3.20 g, 12.2 mmol) in CH2Cl2 (20 mL) was added
dropwise over 10 min. The suspension was warmed to -23 °C and
stirred for 4 h and then poured onto crushed ice (200 mL) (caution:
Vigorous reaction). The mixture was extracted into CH2Cl2 (4 × 200
mL), and the combined organic extracts were dried (MgSO4) and
evaporated in Vacuo to afford an oil which was purified by flash
chromatography (70% EtOAc in hexane, Rf 0.30) to afford 5c as an oil
(2.79 g, 65%): IR (CHCl3 cast) 3462, 3364, 2955, 1743, 1523, 1455,
1438, 1411 cm-1; 1H NMR (360 MHz, CDCl3) δ 7.30 (m, 5H), 5.82-
5.72 (m, 1H), 5.65-5.50 (m, 2H), 5.10 (s, 2H), 4.85 (m, 1H), 4.22 (m,
1H), 3.72 (s, 6H), 2.99 (s (br), 1H), 2.60-2.50 (m, 1H), 2.45-2.38
(m, 1H); 13C NMR (100 MHz, CDCl3) δ 174.4, 171.0, 155.5, 136.1,
129.0, 128.9, 128.8, 128.1, 128.0, 69.8, 67.0, 55.4, 52.6, 52.4, 39.9;
exact mass 351.13162 (M+) (351.13181 calcd for C17H21NO7).
Dimethyl (2S,6RS)-2-(N-(Methyl succinyl)amino)-6-hydroxyhep-
tane-1,7-dioate (6a). To a solution of 5a (430 mg, 1.30 mmol) in
MeOH (20 mL) was added 10% Pd/C (50 mg). The suspension was
stirred under 1 atm of H2 for 19 h and then vacuum filtered through a
bed of Celite, which was washed with MeOH. Evaporation in Vacuo
afforded 6a as an oil (395 mg, 91%): IR (CHCl3 cast) 3140, 2950,
(44) Black, S.; Wright, N. G. J. Biol. Chem. 1955, 213, 39-50.

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Substrates and Inhibitors of DAP-AT
J. Am. Chem. Soc., Vol. 118, No. 32, 1996 7457
Dimethyl (2S,6RS)-2-(N-((3,5-Dimethoxybenzyl)oxycarbonyl))-
amino-6-hydroxyheptane-1,7-dioate (6g). Amine hydrochloride 7 was
acylated with p-nitrophenyl 3,5-dimethoxybenzyl carbonate45 as de-
scribed for preparation of 6c to give 6g as an oil in 58% yield (50%
EtOAc in hexane, Rf 0.15): IR (CH2Cl2 cast) 3356, 3002, 2954, 2842,
1740, 1609, 1598, 1525, 1489, 1460, 1434, 1404 cm-1; 1H NMR (360
MHz, CDCl3) δ 6.48 (s, 2H), 6.39 (s, 2H), 5.65-5.62 (m, 1H), 5.03
(s, 2H), 4.46-4.33 (m, 1H), 4.22-4.15 (m, 1H), 3.77 (s, 6H), 3.74 (s,
3H), 3.72 (s, 3H), 3.19 (s, 1H), 1.89-1.46 (m, 6H); 13C NMR (90
MHz, CDCl3) δ 175.5, 173.1, 161.3, 156.3, 138.9, 106.1, 100.5, 70.5,
67.1, 55.6, 54.1, 52.6, 52.1, 33.9, 32.4, 21.1; exact mass 413.16835
(M+) (413.16858 calcd for C19H27NO9).
2H), 1.89-1.70 (m, 1H), 1.70-1.58 (m, 3H), 1.35 (s, 9H); 13C NMR
(90 MHz, CDCl3) δ 193.9, 173.4, 161.7, 155.5, 80.4, 53.4, 53.3, 52.7,
38.9, 32.2, 28.7, 19.1; MS (CI, NH3) m/z 335 (MNH4+, 21.1%), 318
(MH+, 3.4%), 317 (M+, 7.4%).
Dimethyl (2S)-2-(N-(Cinnamoyl)amino)-6-oxoheptane-1,7-dioate
(8e). Hydroxy ester 6e was oxidized by the procedure used to form
8a to give 8e as an oil in 64% yield (50% EtOAc in hexane, Rf 0.29):
IR (CH2Cl2 cast) 3283, 2953, 1733, 1658, 1624, 1577, 1534, 1449,
1
1437, 1400 cm-1; H NMR (360 MHz, CDCl3) δ 7.59 (d, 1H, J )
15.6 Hz), 7-45-7.42 (m, 2H), 7.31-7.26 (m, 3H), 6.67 (d, 1H, J )
8.0 Hz), 6.50 (d, 1H, J ) 15.6 Hz), 4.77-4.72 (m, 1H), 3.81 (s, 3H),
3.74 (s, 3H), 2.87 (t, 2H, J ) 7.0 Hz), 1.94-1.91 (m, 1H), 1.77-1.65
(m, 3H); 13C NMR (100 MHz, CDCl3) δ 193.9, 173.2, 166.1, 161.6,
142.1, 135.0, 130.2, 129.2, 128.3, 120.4, 53.4, 52.9, 52.3, 38.9, 31.8,
19.0; exact mass 347.13736 (M+) (347.13690 calcd for C18H21NO6).
Dimethyl (2S)-2-(N-(Hydrocinnamoyl)amino)-6-oxoheptane-1,7-
dioate (8f). Hydroxy ester 6f was oxidized by the procedure used to
form 8a to give 8f as an oil in 60% yield (50% EtOAc in hexane, Rf
0.15): IR (CH2Cl2 cast) 3302, 2953, 1731, 1652, 1537, 1497, 1454,
1437, 1400 cm-1; 1H NMR (300 MHz, CDCl3) δ 7.25-7.11 (m, 5H),
6.16 (d, 1H, J ) 7.8 Hz), 4.60-4.53 (m, 1H), 3.82 (s, 3H), 3.66 (s,
3H), 2.93 (t, 2H, J ) 6.6 Hz), 2.78 (t, 2H, J ) 7.0 Hz), 2.54-2.48 (m,
2H), 1.80-1.71 (m, 1H), 1.62-1.45 (m, 3H); 13C NMR (75.5 MHz,
CDCl3) δ 194.0, 172.9, 172.3, 161.6, 140.9, 128.8, 128.7, 126.5, 53.3,
52.8, 51.9, 38.6, 38.4, 31.8, 31.7, 18.7; exact mass 349.15237 (M+)
(349.15253 calcd for C18H23NO6). Anal. Calcd for C18H23NO6: C,
61.88; H, 6.64; N, 4.01. Found: C, 61.71; H, 6.54; N, 4.00.
Dimethyl (2S,6RS)-2-Amino-6-hydroxyheptane-1,7-dioate Hy-
drochloride (7). A suspension of N-Cbz-alkene 5c (2.01 g, 5.73 mmol)
and 10% Pd/C (100 mg) in MeOH (25 mL) and CHCl3 (5 mL) was
hydrogenated under 1 atm of H2 for 24 h. The mixture was vacuum
filtered through Celite, which was then washed with MeOH. The filtrate
was evaporated in Vacuo to afford a gum which was dissolved in CH2Cl2
(10 mL). Evaporation of this solution in Vacuo gave 7 as a wax (1.46
g, 99%): IR (CH2Cl2 cast) 2954, 2630, 1744, 1506, 1456, 1439 cm-1
;
1H NMR (360 MHz, D2O) δ 4.21-4.15 (m, 1H), 4.10-4.01 (m, 1H),
3.95 (s, 3H), 3.83 (s, 3H), 2.05-1.43 (m, 6H); 13C NMR (90 MHz,
CD3OD) δ 176.1, 170.8, 71.3, 54,1, 53.6, 52.4, 34.4, 31.2, 21.8; MS
(CI, NH3) m/z 220 (MH+, 100%).
Dess-Martin Oxidation of r-Hydroxy Esters. Dimethyl (2S)-
2-(N-(Methyl succinyl)amino)-6-oxoheptane-1,7-dioate (8a). To a
stirred solution of 6a (140 mg, 420 µmol) in dry CH2Cl2 (15 mL) under
Ar at 20 °C was added a solution of freshly prepared Dess-Martin
periodinane25 (356 mg, 840 µmol) in dry CH2Cl2 (5.0 mL). After 90
min the solution was added to a saturated aqueous solution of NaHCO3
(20 mL) containing Na2S2O3 (2.0 g). The mixture was stirred
vigorously for 5 min and then extracted into CH2Cl2 (3 × 100 mL).
The dried (MgSO4) extracts were evaporated in Vacuo, and the residue
was purified by flash chromatography (EtOAc, Rf 0.49) to afford 8a
as an oil (110 mg, 79%): [R]D ) +18.4° (c ) 4.76, CHCl3); IR (CH2Cl2
cast) 3350, 2956, 1734, 1678, 1660 cm-1; 1H NMR (360 MHz, CDCl3)
δ 6.26 (d, 1H, J ) 7.6Hz), 4.61-4.55 (m, 1H), 3.83 (s, 3H), 3.71 (s,
3H), 3.65 (s, 3H), 2.85 (t, 2H, J ) 6.9 Hz), 2.67-2.59 (m, 2H), 2.54-
2.57 (m, 2H), 1.92-1.82 (m, 1H), 1.76-1.62 (m, 3H); 13C NMR (75.5
MHz, CDCl3) δ 193.4, 173.2, 172.5, 171.2, 161.2, 52.7, 52.1, 51.5,
51.4, 38.3, 31.4, 30.4, 28.9, 18.3; exact mass 331.1262 (M+) (331.1267
calcd for C14H21NO8). Anal. Calcd for C14H21NO8: C, 50.75; H, 6.39;
N, 4.23. Found: C, 50.34; H, 6.46; N, 4.47.
Dimethyl (2S)-2-(N-Acetylamino)-6-oxoheptane-1,7-dioate (8b).
Hydroxy ester 6b was oxidized by the procedure used to form 8a to
give 8b as an oil in 70% yield (EtOAc, Rf 0.22): IR (CH2Cl2 cast)
3291, 2933, 2955, 1733, 1654, 1539, 1437, 1401 cm-1; 1H NMR (360
MHz, CDCl3) δ 6.35 (s, 1H), 4.60 (m, 1H), 3.82 (s, 3H), 3.65 (s, 3H),
2.83 (m, 2H), 1.97 (s, 3H), 1.85-1.76 (m, 1H), 1.70-1.50 (m, 3H);
13C NMR (90 MHz, CDCl3) δ 193.7, 172.9, 170.4, 161.5, 53.0, 52.5,
51.9, 38.7, 31.5, 23.0, 18.9; exact mass 259.10571 (M+) (259.10559
calcd for C11H17NO6). Anal. Calcd for C11H17NO6: C, 50.96; H, 6.61;
N, 5.40. Found: C, 50.76; H, 6.59; N, 5.27.
Dimethyl (2S)-2-(N-(Benzyloxycarbonyl)amino)-6-oxoheptane-
1,7-dioate (8c). Hydroxy ester 6c was oxidized by the procedure used
to form 8a to give 8c as an oil in 56% yield (30% EtOAc in hexane,
Rf 0.15): IR (CH2Cl2 cast) 3365, 2954, 1730, 1524, 1454, 1437, 1400
cm-1; 1H NMR (360 MHz, CDCl3) δ 7.26-7.15 (m, 5H), 5.26 (d, 1H,
J ) 7.2 Hz), 4.99 (s, 2H), 4.28-4.22 (m, 1H), 3.72 (s, 3H), 3.62 (s,
3H), 2.75 (m, 2H), 1.86-1.65 (m, 1H), 1.65-1.45 (m, 3H); 13C NMR
(90 MHz, CDCl3) δ 193.5, 172.2, 161.0, 157.9, 137.9, 128.5, 128.3,
128.2, 65.5, 53.0, 52.5, 51.7, 37.8, 31.0, 17.6; exact mass 351.13203
(M+) (351.13181 calcd for C17H21NO7). Anal. Calcd for C17H21NO7:
C, 58.10; H, 6.03; N, 3.99. Found: C, 57.93; H, 6.00; N, 3.91.
Dimethyl (2S)-2-(N-(tert-Butyloxycarbonyl)amino)-6-oxoheptane-
1,7-dioate (8d). Hydroxy ester 6d was oxidized by the procedure used
to form 8a to give 8d as an oil in 13% yield (10% EtOAc in CH2Cl2,
Rf 0.25): IR (CHCl3 cast) 3383, 2976, 2955, 2934, 1733, 1714, 1513,
1454, 1437 cm-1; 1H NMR (360 MHz, CDCl3) δ 5.05 (d, 1H, J ) 7.0
Hz), 4.28-4.20 (m, 1H), 3.77 (s, 3H), 3.66 (s, 3H), 2.84-2.75 (m,
Dimethyl (2S)-2-(N-((3,5-Dimethoxybenzyl)oxycarbonyl))amino)-
6-oxoheptane-1,7-dioate (8g). Hydroxy ester 6g was oxidized by the
procedure used to form 8a to give 8g as an oil in 63% yield (50%
EtOAc in hexane, Rf 0.40): IR (CH2Cl2 cast) 3366, 2954, 1731, 1610,
1
1598, 1523, 1458, 1434, 1398 cm-1; H NMR (300 MHz, CDCl3) δ
6.47-6.42 (m, 2H), 6.39-6.37 (m, 1H), 5.40 (d, 1H, J ) 8.0 Hz),
5.02 (s, 2H), 3.82 (s, 3H), 3.76 (s, 6H), 3.73 (s, 3H), 2.88-2.84 (m,
2H), 1.92-1.80 (m, 1H), 1.77-1.61 (m, 3H); 13C NMR (75.5 MHz,
CDCl3) δ 194.0, 173.1, 162.5, 162.3, 156.0, 138.6, 107.0, 101.3, 67.1,
56.0, 54.1, 53.2, 52.9, 39.0, 31.7, 19.6; exact mass 411.15226 (M+)
(411.15292 calcd for C19H25NO9). Anal. Calcd for C19H25NO9: C,
55.47; H, 6.12; N, 3.40. Found C, 55.30; H, 5.89; N, 3.54.
Methyl N-(3-Butenyl)succinamide (9). To a solution of 3-buten-
ylamine hydrochloride46 (3.0 g, 28 mmol) in aqueous KHCO3 (6.98 g,
69.8 mmol, 50 mL) cooled to 4 °C was added succinic anhydride (5.58
g, 55.8 mmol) in portions over 20 min. After a further 40 min, the
solution was acidified with concentrated HCl to pH 2.0 and extracted
into EtOAc (4 × 150 mL). The combined organic extracts were dried
(MgSO4) and evaporated in Vacuo. The solid residue was treated with
an excess of ethereal diazomethane. Excess diazomethane was
destroyed by addition of glacial acetic acid. Evaporation of solvent in
Vacuo gave an oil which was purified by flash chromatography (50%
EtOAc in hexane, Rf 0.22) to yield 9 as an oil (3.61 g, 77%): IR (CHCl3
cast) 3310, 3078, 3002, 2980, 2955, 1740, 1649, 1550, 1435 cm-1; 1H
NMR (300 MHz, CDCl3) δ 6.15 (s, 1H), 5.83-5.69 (m, 1H), 5.12-
5.05 (m, 2H), 3.68 (s, 3H), 3.34-3.28 (m, 2H), 2.66 (t, 2H, J ) 6.8
Hz), 2.48 (t, 2H, J ) 6.8 Hz), 2.29-2.22 (m, 2H); 13C NMR (75.5
MHz, CDCl3) δ 173.3, 171.2, 135.1, 116.8, 51.6, 38.4, 33.5, 30.7, 29.2;
MS (CI, NH3) m/z 186 (MH+, 100%). Anal. Calcd for C9H15NO3:
C, 58.36; H, 8.16; N, 7.56. Found: C, 58.20; H, 8.34; N, 7.64.
Methyl 2-Hydroxy-6-(N-(methyl succinyl)amino)hex-4-enoate
(10). The ene reaction (FeCl3 catalyzed) used to prepare 5a was
employed to convert 9 to 10 as an oil in 52% yield (EtOAc, Rf 0.25):
bp 185-190 °C, 0.7 mmHg; IR (CH2Cl2 cast) 3298, 2953, 1737, 1650,
1544, 1438 cm-1; 1H NMR (300 MHz, CDCl3) δ 6.87 (t, 1H, J ) 5.4
Hz), 5.67-5.52 (m, 1H), 4.27-4.23 (m, 1H), 4.04 (d, 1H, J ) 5.6
Hz), 3.81-3.76 (m, 2H), 3.75 (s, 3H), 3.67 (s, 3H), 2.65 (t, 2H, J )
6.9 Hz), 2.55-2.48 (m, 3H), 2.44-2.35 (m, 1H); 13C NMR (75.5 MHz,
CDCl3) δ 174.1, 173.0, 171.1, 129.5, 126.3, 69.8, 51.8, 51.3, 40.7, 36.6,
30.2, 28.7; MS (CI, NH3) m/z 291 (MNH4+, 22.7%), 274 (MH+, 100%).
Anal. Calcd for C12H19NO6: C, 52.74; H, 7.01; N, 5.13. Found: C,
52.49; H, 7.17; N, 5.17.
(46) Abd El Samii, Z. K. M.; Al Ashmawy, M. I.; Mellor, J. M. J. Chem.
Soc., Perkin Trans. 1 1988, 2517-2522.
(45) Chamberlin, J. W. J. Org. Chem. 1966, 31, 1658-1659.

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7458 J. Am. Chem. Soc., Vol. 118, No. 32, 1996
Cox et al.
Methyl 2-Hydroxy-6-(N-(methyl succinyl)amino)hexanoate (11).
Hydrogenation of 10 for 3 h using the procedure for preparation of 6a
gave 11 as an oil in 96% yield: IR (CHCl3 cast) 3320, 2952, 1737,
(m, 1H), 1.63-1.52 (m, 3H), 1.34-1.20 (m, 2H); 13C NMR (90 MHz,
CDCl3) δ 174.2, 173.6, 173.3, 171.6, 52.8, 52.4, 52.3, 51.9, 34.1, 32.5,
31.3, 29.6, 25.1, 24.8; exact mass 317.1475 (M+) (317.14746 calcd
for C14H23NO7).
1
1651, 1550, 1437 cm-1; H NMR (300 MHz, CDCl3) δ 6.32 (s, 1H),
4.22-4.17 (m, 1H), 3.77 (s, 3H), 3.68 (s, 3H), 3.47 (d, 1H, J ) 5.7
Hz), 3.24 (q, 2H, J ) 6.3 Hz), 2.66 (t, 2H, J ) 7.1 Hz), 2.47 (t, 2H,
J ) 7.1 Hz), 1.81-1.41 (m, 6H); 13C NMR (75.5 MHz, CDCl3) δ 175.2,
173.4, 171.3, 70.1, 52.1, 51.6, 39.1, 33.6, 30.7, 29.2, 28.9, 21.9; MS
(CI, NH3) m/z 293 (MNH4+, 4.5%), 276 (MH+, 100%).
Data for 18a: mp 89-91 °C; IR (KBr) 3420, 2920, 1650, 1610,
1600, 1410 cm-1; 1H NMR (360 MHz, D2O) δ 3.94 (dd, 1H, J ) 4.8,
8.6 Hz), 2.37-2.25 (m, 4H), 2.01 (t, 2H, J ) 7.6 Hz), 1.60-1.36 (m,
4H), 1.20-1.14 (m, 2H); 13C NMR (90 MHz, D2O + CH3CN) δ 182.4,
179.9, 178.3, 174.0, 54.3, 36.5, 32.2, 31.5, 30.7, 24.8, 24.3; MS
(POSFAB, Cleland) m/z 310 ((M - Li + Na)H+, 2.1%), 309 ((M -
Li + Na)+, 11.8%), 294 (MH+, 0.5%), 293 (M+, 0.3%), 288 ((M - Li
+ H)H+, 1.5%), 275 ((M - 3Li + 3H)+, 1.4%). Anal. Calcd for
C11H14NO7Li3‚2H2O: C, 40.15; H, 5.51; N, 4.26. Found: C, 40.41;
H, 5.26; N, 4.31.
Methyl 2-Oxo-6-(N-(methyl succinyl)amino)hexanoate (12). Dess-
Martin oxidation as described for preparation of 8a was employed to
convert 11 to 12 as a solid in 15% yield (EtOAc, Rf 0.33): mp 61-66
1
°C; IR (CHCl3 cast) 3300, 2955, 1733, 1650, 1400 cm-1; H NMR
(300 MHz, CDCl3) δ 5.76 (br s, 1H), 3.87 (s, 3H), 3.69 (s, 3H), 3.27
(q, 2H, J ) 6.9 Hz), 2.88 (t, 2H, J ) 6.9 Hz), 2.68 (t, 2H, J ) 6.6 Hz),
2-(N-(Benzyloxycarbonyl)amino)heptane-1,7-dioic Acid, Dilith-
ium Salt Hemihydrate (18b). The 2-step procedure described to
prepare 18a was employed to convert R-aminopimelic acid using CbzCl
2.47 (t, 2H, J ) 6.9 Hz), 1.69-1.62 (m, 2H), 1.59-1.51 (m, 2H); 13
C
NMR (75.5 MHz, CDCl3) δ 193.9, 173.5, 171.4, 161.4, 52.9, 51.8,
+
1
39.0, 38.8, 31.0, 29.4, 28.8, 20.0; MS (CI, NH3) m/z 291 (MNH4
,
to 18b (85% over 2 steps): mp 185-186.5 °C; H NMR (360 MHz,
4.7%), 274 (MH+, 100%).
D2O) δ 7.29-7.21 (m, 5H), 4.99-4.89 (m, 2H), 3.76-3.72 (m, 1H),
1.98 (t, 2H, J ) 7.5 Hz), 1.61-1.55 (m, 1H), 1.48-1.33 (m, 3H), 1.20-
1.14 (m, 2H); 13C NMR (90 MHz, D2O + CH3CN) δ 183.8, 180.2,
158.5, 129.2, 128.7, 128.1, 67.2, 57.0, 37.9, 32.2, 26.1, 25.7; IR (KBr)
3568, 3402, 3090, 2946, 1701, 1578, 1500, 1421 cm-1; MS (POSFAB,
Cleland) m/z 322 (MH+, 19.6%), 321 (M+, 4.3%). Anal. Calcd for
C15H17NO6Li2‚0.5H2O: C, 54.54; H, 5.45; N, 4.24. Found: C, 54.75;
H, 5.26; N, 4.28.
2-Oxo-6-(N-(succinyl)amino)hexanoic Acid, Dilithium Salt (13).
Careful LiOH hydrolysis as described for preparation of 1a was
employed to convert 12 to solid 13 in 99% yield: mp 146 °C dec; IR
(KBr) 3430, 2931, 1636, 1417 cm-1; 1H NMR (400 MHz, D2O) δ 3.13
(t, 2H, J ) 6.7 Hz), 2.71 (t, 2H, J ) 7.1 Hz), 2.39-2.33 (m, 4H),
1.58-1.42 (m, 4H); 13C NMR (100 MHz, D2O + 1,4-dioxane) δ 195.6,
170.7, 165.3, 160.4, 28.8, 22.7, 23.0, 17.9, 17.8, 11.9; MS (POSFAB,
Cleland) m/z 258 (MH+, 6.8%), 257 (M+, 2.2%), 252 ((M - Li +
H)H+, 4.3%), 251 ((M - Li + H)+, 1.1%).
1-Methyl 11-Ethyl 6-(Carboethoxy)-2-hydroxy-4-undecene-1,11-
dioate (14). The ene reaction (FeCl3 catalyzed) used to prepare 5a
was employed to convert diethyl 2-allylheptane-1,7-dioate47 to 14 as
an oil in 65% yield (33% EtOAc in hexane, Rf 0.30): IR (CHCl3 cast)
3480, 2936, 1732 cm-1; 1H NMR (400 MHz, CDCl3) δ 5.56-5.53 (m,
2H), 4.28-4.24 (m, 1H), 4.15-4.09 (m, 4H), 3.76 (s, 3H), 3.06 (d,
1H, J ) 6.2 Hz), 2.99-2.93 (m, 1H), 2.56-2.52 (m, 1H), 2.45-2.38
(m, 1H), 2.29 (t, 2H, J ) 7.5 Hz), 1.75-1.69 (m, 1H), 1.68-1.49 (m,
2H), 1.47-1.35 (m, 1H), 1.35-1.28 (m, 2H), 1.25 (t, 6H, J ) 6.7
Hz); 13C NMR (100 MHz, CDCl3) δ 174.5, 173.9, 173.4, 131.8, 126.8,
70.1, 60.4, 60.0, 52.2, 48.8, 37.2, 33.9, 31.8, 26.3, 24.4, 14.1, 14.0;
MS (CI, NH3) m/z 362 (MNH4+, 100%), 345 (MH+, 15.2%).
6-Carboxy-2-oxo-undecane-1,11-dioic Acid (17). Hydrogenation
of 14 in MeOH for 24 h using the procedure for preparation of 6a
gave 15 as an inseparable mixture of methyl and ethyl esters at the
C-11 carboxyl group in 98% yield. Dess-Martin oxidation of this
mixture as described for preparation of 8a converted it to the
corresponding 2-oxo mixture of esters 16 (30% EtOAc in hexane, Rf
0.25). The mixture of esters 16 (150.5 mg) was hydrolyzed by reflux
in 1 N HCl (25 mL) for 2 h. Evaporation in Vacuo afforded a solid
which was dissolved in saturated NaHCO3 (25 mL). The solution was
washed with CH2Cl2 (2 × 25 mL). The aqueous layer was acidified
to pH 1.0 with 1 N HCl and extracted with diethyl ether (3 × 100
mL). The combined ether extracts were dried (MgSO4) and evaporated
in Vacuo to give 17 as a waxy solid (70.0 mg, 40% over 2 steps): IR
(KBr) 3430, 2931, 1636, 1417 cm-1; 1H NMR (300 MHz, acetone-d6)
δ 9.71 (s , 3H), 2.91-2.87 (m, 2H), 2.51-2.24 (m, 3H), 1.90-1.20
(m, 10H); 13C NMR (75.5 MHz, acetone-d6) δ 196.0, 172.3, 176.0,
175.0, 45.6, 38.8, 34.0, 32.7, 32.1, 27.5, 25.5, 21.5; MS (POSFAB,
Cleland) m/z 274 (M+).
(2S,6RS)-2-(N-(Succinyl)amino)-6-hydroxyheptane-1,7-dioic Acid,
Trilithium Salt (19). Hydrolysis of 6a with LiOH as described for
preparation of 1a gave 19 in 92% yield: mp 114-116 °C dec; IR (KBr)
3536, 3410, 1663, 1608, 1413 cm-1; 1H NMR (360 MHz, D2O) δ 4.05
(dd, 1H, J ) 8.3, 5.0 Hz), 3.93 (dd, 1H, J ) 7.6, 4.7 Hz), 2.50-2.37
(m, 4H), 1.75-1.50 (m, 4H), 1.38-1.28 (m, 2H); 13C NMR (90 MHz,
D2O + 1,4-dioxane) δ 176.9, 176.6, 170.6, 61.9, 50.6, 29.1, 28.3, 27.6,
26.8, 16.5; MS (POSFAB, Cleland) m/z 310 (MH+, 3.6%), 309 (M+,
9.1%).
(2S,6RS)-2-(N-(Succinyl)amino)-6-hydrazinoheptane-1,7-dioic Acid
(20a). To a stirred suspension of trilithium salt 1a (21.1 mg, 68.7 µmol)
in anhydrous MeOH (1 mL) was added hydrazine hydrate (688 µmol,
34.5 mg, 33.5 µL). After 60 min, NaCNBH3 (43.2 mg, 688 µmol)
was added, and the white suspension was stirred for 16 h at 20 °C.
Excess NaCNBH3 was destroyed by the addition of 1 N HCl followed
by stirring for 30 min. The clear solution was applied to BioRAD
AG-50-WX-8 cation exchange resin (H+ form, 10 mL). The column
was eluted with water until washings were neutral and then with 0.5 N
aqueous ammonia. Amino acid-containing fractions (ninhydrin) were
pooled and evaporated in Vacuo. The residue was suspended in water
(2 mL) and lyophilized to give 20a as a solid (20.4 mg, 97%): mp
1
92-94 °C; IR (KBr) 3416, 3329, 2956, 1577, 1401 cm-1; H NMR
(360 MHz, D2O) δ 4.08-4.04 (m, 1H), 3.41-3.32 (m, 1H), 2.52-
2.38 (m, 4H), 1.90-1.55 (m, 4H), 1.40-1.25 (m, 2H); 13C NMR (100
MHz, D2O + CH3CN) δ 181.6, 179.8, 175.7, 154.5, 66.5, 55.4, 33.5,
32.0, 31.5, 29.8, 21.9; MS (POSFAB, glycerol/HCl) m/z 306 (MH+,
4.4%), 305 (M+, 1.0%).
(2S,6RS)-2-(N-(Benzyloxycarbonyl)amino)-6-hydrazino-1,7-di-
oic Acid (20c). The procedure for the preparation of 20a was employed
to convert dilithium salt 1c to 20c. After cation exchange and
concentration, the residue was purified by reverse-phase HPLC
(Column: 2× Waters RadialPak Resolve 0.5 m (8 × 100 mm); solvent
A 0.1% aqueous trifluoroacetic acid, solvent B 70% aqueous MeCN
+ 0.1% trifluoroacetic acid. Method: ∼1.0 mg injection, gradient 30%
to 56% B over 13.0 min at 2.0 mL/min, detecting at 254 nm, retention
time 10.4 min.) Product-containing fractions were lyophilized to afford
20c as a hygroscopic powder (29.8 mg, 69%): mp 53-55 °C; IR (KBr)
2-(N-(Succinyl)amino)heptane-1,7-dioic Acid, Trilithium Salt
Dihydrate (18a). Although this could be prepared by direct acylation
of R-aminopimelic acid, a more pure product was obtained by a 2-step
procedure involving formation of the corresponding N-succinyl trimeth-
yl ester and LiOH hydrolysis as described above for generation of 1a.
Data for the intermediate dimethyl 2-(N-(methyl succinyl)amino)-
heptane-1,7-dioate isolated as an oil (50% EtOAc in hexane, Rf 0.11):
IR (CHCl3 cast) 3323, 2997, 2953, 2865, 1739, 1679, 1676, 1654, 1539,
3424, 3150, 3049, 2856, 1701, 1618, 1534, 1499, 1439, 1404 cm-1
;
1H NMR (360 MHz, D2O + CD3CN) δ 7.34-7.28 (m, 5H), 5.03 (m,
2H), 4.09-4.03 (m, 1H), 3.68-3.64 (m, 1H), 1.79-1.63 (m, 4H), 1.45-
1.41 (m, 2H); 13C NMR (75.5 MHz, D2O + CD3CN) δ 175.0, 173.3,
158.5, 137.2, 127.8, 127.4, 126.7, 66.1, 59.6, 52.8, 29.2, 27.8, 20.2;
MS (POSFAB, glycerol/HCl) m/z 362.2 (MNa+, 2.5%), 340.2 (MH+,
87.1%), 339.1 (M+, 6.3%).
1
1437 cm-1; H NMR (360 MHz, CDCl3) δ 6.36 (d, 1H, J ) 7.9 Hz),
4.55-4.50 (m, 1H), 3.67 (s, 3H), 3.62 (s, 3H), 3.59 (s, 3H), 2.66-
2.57 (m, 2H), 2.53-2.46 (m, 2H), 2.43 (t, 2H, J ) 7.4 Hz), 1.79-1.73
N-Succinyl-LL-DAP Aminotransferase (DAP-AT) Assay. Stock
solution (100 mM Tris buffer pH 8.0 containing EDTA tetrasodium
(47) (a) Grewe, R. Chem. Ber. 1943, 76, 1072-1076. (b) Colonge, J.;
Collomb, F. Bull. Soc. Chim. Fr. 1951, 241-243.

+
+
Substrates and Inhibitors of DAP-AT
J. Am. Chem. Soc., Vol. 118, No. 32, 1996 7459
salt (0.1 mM), NaN3 (5 mM), bovine serum albumin (BSA, Sigma,
1.0 mg/mL), and NH4Cl (100 mM)) was used to prepare the assay
solution (pyridoxal phosphate (PLP), 3.0 mg, and â-NADPH, 6.0 mg,
made up to 40.0 mL with stock solution). The solutions were made
using ACS grade reagents and Milli-Q water. Assays were performed
at 30 °C and contained sufficient N-succinyl-LL-DAP aminotransferase
to give ∆-OD340 of 20-100 mAU340/min, 1 mM substrate 1a, 10 mM
L-glutamate, 10 units of L-glutamate dehydrogenase (EC 1.4.1.4, Sigma),
and assay solution to give a final volume of 1000 µL. The decrease in
â-NADPH concentration was observed at 340 nm (with a reference
value observed at 520 nm) over 300 s using a Hewlett Packard 8452A
diode array spectrophotometer equipped with a cuvette having a water
jacket. Stock solution was kept refrigerated at 4 °C for up to 6 months;
assay solution was made up fresh daily. The assay conditions were
varied as outlined below for kinetic analyses.
solution could be stored indefinitely at -20 °C without appreciable
loss of activity.
Step 4. Gel Filtration Chromatography. A portion (32 mL) of
the solution from step 3 was concentrated using Amicon Centriprep
ultrafiltration-10 units to a final volume of 2.5 mL. The concentrated
solution was applied to a Sephadex G-75 column (Pharmacia, 15 ×
300 mm) and eluted at 100 µL/min with 200 mM phosphate buffer pH
6.8; 1.5 mL fractions were collected. Active fractions (tubes 20-25,
total volume 7.5 mL, calibrated molecular weight 81.5 kDa) were
pooled. Glycerol (750 µL) was added and the solution frozen.
Step 5. Mono-Q Anion Exchange Chromatography. A portion
(4.0 mL) of the soution from step 4 was desalted using Amicon
centricon-30 filters and the residue made up to 4.0 mL with 20 mM
phosphate buffer, pH 7.0. This solution was purified by HPLC
chromatography using a BioRAD gradient module and UV detector
(218 nm) and a Pharmacia Resource mono-Q anion exchange column
(1 mL of bed volume) eluted at 1 mL/min: Eluent A, 20 mM phosphate
buffer, pH 7.0; eluent B, 20 mM phosphate buffer plus 500 mM NaCl,
pH 7.0. The buffers were cooled to 0 °C and did not contain PLP.
Injections of 200 µL were made, then 0.0-2.0 min 0% B, and then
gradient 2.0-26.0 min 0-80% B, then 26.0-28.0 min 80-0% B. Peaks
were collected manually as they were eluted into ice cooled polypro-
pylene test tubes. Activity was eluted in a single peak, retention time
15.3 min. To this enzyme solution was added glycerol to 10%, and
the solution was frozen at -20 °C. Thawed samples of this solution
were used for kinetic analyses. The solution was desalted utilizing
Amicon centricon-30 ultrafiltration units to provide samples suitable
for SDS PAGE.
Purification of DAP-AT. All procedures were done at 4 °C, all
buffers contained 10 mg/L PLP, and protein elution during chromato-
graphic steps was monitored at 280 nm unless otherwise stated. Buffers
were made using ACS grade reagents and Milli-Q water. Centrifuga-
tions were performed using a DuPont Sorvall RC-5B centrifuge
equipped with a Sorvall GSA rotor (6 × 250 mL tubes). Protein
determinations were done using the BioRAD reagent kit, standardized
against BSA, and activity assays were performed as described above.
Wild type E. coli (ATCC 9637) glycerol stock solution (2 × 1 mL)
was added to 2 × 500 mL of a sterilized solution containing yeast
extract (3 g/L), peptone (5 g/L), and glucose (5 g/L) in 2 L Erlenmeyer
flasks. The solution was shaken at 200 rpm, 37 °C, for 15.5 h and
then used to innoculate 50 L of modified Davis and Mingioli medium48
(KH2PO4, 13.6 g/L; (NH4)2SO4, 2.0 g/L; MgSO4‚2H2O, 200 mg/L;
FeSO4‚7H2O, 0.5 mg/L; glycerol, 2.0 g/L; PLP, 10 mg/L; yeast extract,
3.0 g/L; peptone, 5.0 g/L; glucose, 5.0 g/L). Fermentation was done
at 37 °C, pH 7.0, with 200 rpm stirring, using a 76 L LH2000 fermentor
until the end of logarithmic growth as determined by OD600 (3 h). Cells
were harvested by ultrafiltration (0.3 µ filter) and washed with 30 mM
phosphate buffer pH 7.5. The resultant concentrate (2 L) was
centrifuged (2 × 10 000 rpm, 10 min). The cells were resuspended in
30 mM phosphate buffer, pH 7.5, and the pooled cell concentrate (600
mL) was passed six times through a continuous-flow sonicator cooled
to -20 °C. The sonicated cell suspension was centrifuged (8000 rpm,
15 min) to remove cell debris and the clear supernatant (450 mL)
containing soluble protein decanted.
Kinetic Analyses and Inhibition Studies. Absolute Km values for
the natural substrate 1a and L-glutamate were obtained by measurement
of initial rates (10-30 s) for a matrix of 7 substrate 1a concentrations
by five L-glutamate concentrations. Assays were performed in a 1 mL
quartz cuvette containing substrate 1a, L-glutamate, L-glutamate de-
hydrogenase (10 units), and assay solution to make up 1000 µL.
Reactions were initiated by the addition of N-succinyl-LL-DAP ami-
notransferase (17.9 nM) and were followed at 340 nm over 300 s. The
array of data points was analyzed using the computer program Enzyme
app
Kinetics (Trinity Software, Compton, NH). Values of Km and Vmax
were measured for the natural substrate 1a and substrate analogues
1b-g at fixed L-glutamate concentration (10 mM). Initial reaction rates
(10-30 s) were determined for reactions performed in a 1 mL quartz
cuvette containing substrates 1a-g at seven concentrations (0.1-10.0
mM), L-glutamate (10 mM), L-glutamate dehydrogenase (10 units), and
assay solution to make 1000 µL. Reactions were initiated by the
addition of N-succinyl-LL-DAP aminotransferase (17.9 nM) and were
followed at 340 nm. Initial rates (10-30 s) were plotted vs substrate
concentration and kinetic parameters (Kmapp, Vmax) were calculated using
the EnzFitter computer program (Elsevier Biosoft, Cambridge, U.K.).
Step 1. Reactive Brown Affinity Chromatography. A portion
of the soluble protein solution (230 mL, 2 g protein) was applied to a
column (60 × 300 mm) of Reactive Brown-10 (Sigma) immobilized49
on Sepharose-6CL (Sigma), which was then eluted with 30 mM
phosphate buffer, pH 7.5, at a flow rate of 2 mL/min; 20 mL fractions
were collected. Active fractions (tubes 31-50, total volume 380 mL)
were pooled and concentrated to a volume of 120 mL using an Amicon
ultrafiltration cell fitted with an Amicon YM-10 ultrafiltration mem-
brane under N2 (50 psi).
Step 2. Blue-3GA Affinity Chromatography. The concentrated
solution from step 1 was applied to a column (60 × 300 mm) of
Cibacron Blue-3GA immobilized on cross-linked 4% Agarose (Sigma),
which was then eluted with 30 mM phosphate buffer pH 6.0 at a flow
rate of 2 mL/min; 20 mL fractions were collected. After 900 mL of
elution a pH gradient was run to 30 mM phosphate buffer, pH 8.0,
over 1000 mL. Active fractions (tubes 87-119, total volume 640 mL)
were pooled and concentrated to a volume of 70 mL. This solution
was dialyzed three times vs 1 L of 1.0 mM phosphate buffer pH 6.8 (2
h, 15 h, and then 2 h).
For preliminary inhibition studies, reactions were initiated by the
addition of N-succinyl-LL-DAP aminotransferase to standard assay
mixtures containing natural substrate 1a (1 mM), L-glutamate (10mM),
L-glutamate dehydrogenase (10 units), and varying concentrations (0-
10 mM) of potential inhibitor made up to 1000 µL with assay solution.
No significant reduction of reaction rates could be observed for 18a,
18c, or 19. For detailed examination of slow-binding inhibitors 20a,c,
stock solution was used to prepare assay solution as described above,
except that PLP was deleted from the mixture. No inhibition of the
coupling enzyme, L-glutamate dehydrogenase, was observed for 20a
or 20c. Absorbance, time pairs were collected every 15 s over 3600 s
for reactions performed in a 1 mL quartz cuvette containing substrate
1a (1 mM), L-glutamate (10 mM), L-glutamate dehydrogenase (10
units), inhibitor at seven concentrations (0.1-20.0 mM), and assay
solution to make 1000 µL. Reactions were initiated by the addition of
N-succinyl-LL-DAP aminotransferase (1.3 nM), and reaction progress
was observed at 340 nm over 3600 s. The data points were fitted to
the integrated rate equation
Step 3. Hydroxylapatite Chromatography. The dialyzed solution
from step 2 was applied to a hydroxylapatite column (BioRAD, 25 ×
170 mm) which was eluted with 1 mM phosphate buffer pH 6.8 at 600
µL/min; 6 mL fractions were collected. After initial protein elution
had ceased, a concentration gradient was run to 300 mM phosphate
buffer, pH 6.8, over 600 mL. Active fractions (tubes 96-110, total
volume 84 mL) were pooled, and glycerol (8.5 mL) was added. This
(48) Davis, B. D.; Mingioli, E. S. J. Bacteriol. 1950, 60, 17-28.
(49) (a) Stellwagen, E. Meth. Enzymol. 1990, 182, 343-3547. (b) Scopes,
R. K. Protein Purification Principles and Practice, 2nd ed.; Springer-
Verlag: New York, 1987.
P ) Vst + (V0 - Vs)(1 - exp(-kobst))/k obs + d

+
+
7460 J. Am. Chem. Soc., Vol. 118, No. 32, 1996
Cox et al.
using the program MacCurveFit50 to obtain estimates for the parameters
V0 (initial velocity), Vs (steady-state velocity), kobs (pseudo-first-order
rate constant), and d (displacement from x axis). In each case the
goodness of fit (residual) of the theoretical curve to the experimental
data points was >99.5%. Data analysis was performed according to
the methods of Morrison.40 For preincubation studies, inhibitor (20a
or 20c) at varying concentrations (0-1000 µM) was added to enzyme
(660 nM) in stock solution containing 10 mM L-glutamate at time zero.
Aliquots of this solution were tested for residual activity at intervals
over 60 min using the standard activity assay. The experiments were
repeated at 0.0 mM L-glutamate concentration for varying inhibitor 20a
or 20c concentrations (0-5 µM). Results were plotted as % residual
acivity Vs time or as % residual activity after 60 min Vs inhibitor
concentration. For reversal of inhibition studies, a solution of enzyme
was treated with 10 µM inhibitor 20a or 20c for 60 min in 0.0 mM
L-glutamate stock solution. An aliquot of this solution was then diluted
100-fold into a standard activity assay and reaction progress observed
at 340 nm over 3600 s.
Acknowledgment. The authors are grateful to Drs. Joanna
Harris and Christine Scaman for valuable discussions, Ms.
Lesley Caswell and Mr. Will Cheng for technical assistance,
and Professor Michael Pickard (Department of Microbiology,
University of Alberta) for the provision of large quantities of
E. coli cells. These investigations were supported by the Natural
Sciences and Engineering Research Council of Canada and the
Alberta Heritage Foundation for Medical Research. This paper
is dedicated to Professor George Bu¨chi on the occasion of his
birthday.
Supporting Information Available: A table giving details
of enzyme purification, figures showing enzyme mass spectra
and an SDS PAGE gel, and plots of kinetic data for inhibition
studies (11 pages). Ordering information is given on any current
masthead page.
(50) Shareware: Kevin Raner, 77 Therese Ave, Mt. Waverly 3149,
Australia.
JA960640V