Inhibitor ionization as a determinant of binding to 3-dehydroquinate synthase

Article abstract of DOI:10.1021/jo960709h

Phosphinomethyl and carboxymethyl monoacids along with succinyl, malonyl ether, malonyl, and hydroxymalonyl diacids were substituted for phosphorylmethyl, phosphonoethyl, and phosphonomethyl groups in carbocyclic inhibitors of DHQ synthase. All but one of the carbocyclic inhibitors were synthesized via intermediacy of a 2,3-butane bisacetal-protected 3-dehydroquinic acid. Carbaphosphinate (K_i = 20 × 10^-6 M) was a modest competitive inhibitor of DHQ synthase, while carbaacetate was a linear mixed-type inhibitor (K_i = 3 × 10^-6 M, K′_i = 20 × 10^-6 M). Carbasuccinate (K_i = 5 × 10^-6 M), carbamalonate ether (K_i = 7 × 10^-6 M), carbamalonate (K_i = 0.7 × 10^-6 M), and carbahydroxymalonate (K_i = 0.3 × 10^-6 M) were all competitive inhibitors. Carbaacetate was the only inhibitor that was not oxidized by DHQ synthase. On the basis of these data, carbocyclic inhibitors with malonyl and hydroxymalonyl groups are apparently bound by DHQ synthase as tightly as carbocyclic inhibitors possessing phosphorylmethyl and phosphonoethyl moieties.

Full text of DOI:10.1021/jo960709h

J . Org. Chem. 1996, 61, 7373-7381
7373
In h ibitor Ion iza tion a s a Deter m in a n t of Bin d in g to
3-Deh yd r oqu in a te Syn th a se
Feng Tian, J ean-Luc Montchamp, and J . W. Frost*
Department of Chemistry, Michigan State University, East Lansing, Michigan 48824-1322
Received April 18, 1996X
Phosphinomethyl and carboxymethyl monoacids along with succinyl, malonyl ether, malonyl, and
hydroxymalonyl diacids were substituted for phosphorylmethyl, phosphonoethyl, and phospho-
nomethyl groups in carbocyclic inhibitors of DHQ synthase. All but one of the carbocyclic inhibitors
were synthesized via intermediacy of a 2,3-butane bisacetal-protected 3-dehydroquinic acid.
Carbaphosphinate (Ki ) 20 × 10-6 M) was a modest competitive inhibitor of DHQ synthase, while
carbaacetate was a linear mixed-type inhibitor (Ki ) 3 × 10-6 M, Ki′ ) 20 × 10-6 M). Carbasuccinate
(Ki ) 5 × 10-6 M), carbamalonate ether (Ki ) 7 × 10-6 M), carbamalonate (Ki ) 0.7 × 10-6 M), and
carbahydroxymalonate (Ki ) 0.3 × 10-6 M) were all competitive inhibitors. Carbaacetate was the
only inhibitor that was not oxidized by DHQ synthase. On the basis of these data, carbocyclic
inhibitors with malonyl and hydroxymalonyl groups are apparently bound by DHQ synthase as
tightly as carbocyclic inhibitors possessing phosphorylmethyl and phosphonoethyl moieties.
Comparatively little attention1 has been given to the
Sch em e 1
impact of inhibitor ionization on binding to the active site
of 3-dehydroquinate (DHQ) synthase (Scheme 1). To
explore the inhibitory potency of dianionic analogues of
substrate 3-deoxy-D-arabino-heptulosonic acid 7-phos-
phate (DAHP), carbaphosphinate 1 and carbaacetate 2
have been synthesized (Chart 1). Carbasuccinate 3,
carbamalonate ether 4, carbamalonate 5, and carbahy-
droxymalonate 6 were synthesized as trianionic carbocy-
clic substrate analogues (Chart 1). Carbacarboxylate 7
and spirocyclic carbaphosphodiester 12 are the only
examples (Chart 1) in the literature1b,2 of dianionic
carbocyclic DHQ synthase inhibitors. Trianionic car-
bocyclic inhibitors of DHQ synthase include (Chart 1)
carbocyclic phosphonates and phosphates having C-5
stereochemistry identical (8-11)1a,3 and epimeric (13-
6)4 to the stereochemistry at C-6 of DAHP. Yet another
example of a carbocyclic inhibitor having an accessible
trianionic ionization state is C-1 epimeric carbaphospho-
nate 17.5 Newly synthesized carbocyclic tricarboxylates
3-6 are the first examples of trianionic DHQ synthase
inhibitors possessing neither a phosphate monoester nor
phosphonic acid.
Ch a r t 1
A protected DHQ derivative proved to be an exception-
ally useful intermediate in the syntheses of carbaphos-
phinate 1, carbasuccinate 3, carbamalonate ether 4,
carbamalonate 5, and carbahydroxymalonate 6. The
utility of protected DHQ intermediacy was further dem-
onstrated in the shortest, highest-yielding synthesis yet
achieved of carbaphosphonate 11, which is the most
potent inhibitor known3a for DHQ synthase. Binding to
DHQ synthase was evaluated by the magnitude of the
measured inhibition constants, by the formation or
absence of enzyme-bound NADH during inhibition, and
X Abstract published in Advance ACS Abstracts, September 15, 1996.
(1) (a) Bender, S. L.; Widlanski, T.; Knowles, J . R. Biochemistry
1989, 28, 7560. (b) Montchamp, J .-L.; Frost, J . W. J . Org. Chem. 1994,
59, 7596.
(2) Nikolaides, N.; Ganem, B. Tetrahedron Lett. 1989, 30, 1461.
(3) (a) Widlanski, T.; Bender, S. L.; Knowles, J . R. J . Am. Chem.
Soc. 1989, 111, 2299. (b) Widlanski, T.; Bender, S. L.; Knowles, J . R.
Biochemistry 1989, 28, 7572.
(4) Montchamp, J .-L.; Peng, J .; Frost, J . W. J . Org. Chem. 1994,
59, 6999.
(5) (a) Montchamp, J .-L.; Piehler, L. T.; Frost, J . W. J . Am. Chem.
Soc. 1992, 114, 4453. (b) Piehler, L. T.; Montchamp, J .-L.; Frost, J .
W.; Manly, C. J . Tetrahedron 1991, 47, 2423.
by whether inhibition was competitive relative to sub-
strate DAHP binding. Of the newly synthesized carbocy-
clic inhibitors, carbamalonate 5 and carbahydroxyma-
lonate 6 were the most potent inhibitors with Ki values
in the submicromolar range. DHQ synthase inhibition
by carbamalonate 5 and carbahydroxymalonate 6 sug-
S0022-3263(96)00709-8 CCC: $12.00 © 1996 American Chemical Society

7374 J . Org. Chem., Vol. 61, No. 21, 1996
Tian et al.
Sch em e 2a
Sch em e 3a
a
Key: (a) (i) CH3OH, Dowex 50 (H+), reflux, (ii) 2,2,3,3-
tetramethoxybutane, CH(OCH3)3, CH3OH, CSA, reflux, 87%; (b)
KIO4, K2CO3, RuCl3, H2O, CHCl3, 77%; (c) see Schemes 3 and 4;
(d) allyltributyltin, AIBN, C6H6, reflux; (e) (i) NaIO4, RuCl3, CCl4/
CH3CN/H2O (2:2:3, v/v/v), (ii) CH2N2, Et2O, CH3OH, 65%; (f) (i)
0.2 N aqueous NaOH/THF (1:1, v/v), (ii) Dowex 50 (H+), 100%.
gests that C-5 malonyl and hydroxymalonyl moieties are
effective mimics of C-5 phosphorylmethyl and phospho-
noethyl groups in DHQ synthase inhibitors. This expan-
sion in the types of structures that lead to in vitro DHQ
synthase inhibition has ramifications important to ulti-
mately achieving in vivo inhibition of this enzyme in
pathogenic bacteria.
a
Key: (a) Ph3PdCHCO2Et, CH3CN, reflux, 90%; (b) Im2CO,
ClCH2CH2Cl, reflux, 82%; (c) PhSeH, ClCH2CH2Cl, reflux, 87%;
(d) Bu3SnH, AIBN, C6H6, reflux, 27%; (e) (i) TFA/H2O (20:1, v/v),
(ii) 0.2 N aqueous NaOH/THF (1:1, v/v), (iii) Dowex 50 (H+), 100%;
(f) NaBH(OAc)3, CH3CN/HOAc (1:1, v/v), 27: 86%, 30: 92%; (g)
N2dC(CO2Et)2, Rh2(OAc)4, C6H6, reflux, 75%; (h) CH2(CN)2,
NH4OAc, HOAc, C6H6, 90%; (i) (i) TFA/H2O (20:1, v/v), (ii) 6 N
aqueous HCl, reflux, (iii) 0.2 N aq NaOH/THF (1:1, v/v), (iv) Dowex
50 (H+), 95%; (j) (i) CH2N2, Et2O, CH3OH, (ii) 2,2,3,3-tetrameth-
oxybutane, CH(OCH3)3, CH3OH, CSA, reflux, 80% from 30; (k) (i)
(CH3)3COK, THF, 0 °C, (ii) 2-(benzenesulfonyl)-3-phenyloxaziri-
dine, THF, -78 °C, 75%.
Resu lts
Syn th etic Str a tegy Op tion s. Synthesis of carbocy-
clic DHQ synthase inhibitors 1 and 3-6 via protected
DHQ intermediacy (Scheme 2) constitutes a significant
departure from earlier syntheses of carbocyclic DHQ
synthase inhibitors. Previous syntheses of such inhibi-
tors have employed nucleophilic heteromethylation5a,6 of
oxiranes derived from quinic acid or routes strategically
related1a to the one (Scheme 2) used to assemble car-
baacetate 2 where bromo lactone 20 derived from quinic
acid underwent Keck reaction7 with allyltributyltin.
Oxidation of the resulting C-5 allyl substituent of 21 with
NaIO4 and catalytic, in situ-generated RuO4 was followed
by esterification to expedite purification of the protected
carbaacetate. Subsequent basic hydrolysis and neutral-
ization afforded carbaacetate 2.
yielding “backdoor” route into the desired protected DHQ
intermediate.
Starting material quinic acid was quantitatively con-
verted (Scheme 2) into its methyl ester by refluxing in
methanol with Dowex 50 (H+). Reaction of methyl
quinate with catalytic camphorsulfonic acid (CSA) and
2,2,3,3-tetramethoxybutane resulted in regioselective
protection of the 3,4-trans-diol over the 4,5-cis-diol and
afforded BBA-protected 18 in 87% yield. Oxidation of
the C-5 hydroxyl group in 18 with tetrapropylammonium
perruthenate10 (TPAP) and 4-methylmorpholine N-oxide
(NMO) furnished protected DHQ derivative 19 (Scheme
2). Large-scale (30 g) oxidations of 18 employed KIO4
and catalytic RuCl3 in CHCl3/H2O.
Tr ica r boxyla te Syn th eses. An essential feature of
all tricarboxylate syntheses was the use of the C-1 alcohol
to direct critical reactions. For example, the C-1 alcohol
was left unprotected during reaction of DHQ intermedi-
ate 19 with (carbethoxymethylene)triphenylphosphorane
(Scheme 3). Protection of the C-1 alcohol of DHQ
intermediate 19 as a TMS ether gave a slower condensa-
tion reaction that was accompanied by dehydration
involving elimination of the protected C-1 alcohol. Phos-
Previous attempts to use DHQ as a starting material
in the synthesis of carbocyclic DHQ synthase inhibitors
failed due to DHQ’s instability8 under a variety of the
conditions employed to selectively protect the molecule’s
hydroxyl groups. Access to protected DHQ has been
made possible by use of the recently elaborated9 2,3-
butane bisacetal (BBA) protecting group, which is highly
selective for the protection of diequatorial diols. By
selectively protecting the C-3 and C-4 hydroxyl groups
of stable methyl quinate (Scheme 2), oxidation of the
remaining secondary alcohol at C-5 provided a high-
(6) Montchamp, J .-L.; Migaud, M. E.; Frost, J . W. J . Org. Chem.
1993, 58, 7679.
(7) Keck, G. E.; Yates, J . B. J . Am. Chem. Soc. 1982, 104, 5829.
(8) Delfourne, E.; Despeyroux, P.; Gorrichon, L.; Ve´ronique, J . J .
Chem. Res. 1991, 56.
(9) Montchamp, J .-L.; Tian, F.; Hart, M.; Frost, J . W. J . Org. Chem.
1996, 61, 3897.
(10) Griffith, W. P.; Ley, S. V. Aldrichim. Acta 1990, 23, 13.

Inhibitor Ionization as a Determinant of Binding
J . Org. Chem., Vol. 61, No. 21, 1996 7375
Sch em e 4a
phorane complexation with the C-1 alcohol was likely
responsible for directing and accelerating the rate of the
condensation reaction.
The C-1 alcohol was also essential to establishing
carbasuccinate’s C-5 stereochemistry (Scheme 3) during
radical cyclization of the C-1 phenylselenocarbonate 25.
Because of its steric environment, functionalization of the
C-1 tertiary alcohol in dicarboxylate 23 required a two-
step sequence. Initial reaction with 1,1′-carbonyldiimi-
dazole was followed by interception of the imidazoyl
carbamate 24 with phenylselenol to afford phenylsele-
nocarbonate 25. Radical cyclization provided lactone 26
in 27% yield.11 Deoxygenation products epimeric at C-1
were also isolated which accounts for the modest yield
of 26 and likely reflects C-1 carboxylate stabilization of
a C-1 tertiary radical resulting from loss of CO2.12 The
BBA protecting group was easily removed in 95% aque-
ous trifluoroacetic acid at room temperature. Subsequent
hydrolysis of the esters and lactone under basic condi-
tions followed by protonation using Dowex 50 (H+)
provided carbasuccinate 3.
a
Key: (a) (i) CH2(OCH3)2, P2O5, CHCl3, (ii) CH2I2, Zn, TiCl4,
THF, 73%; (b) ethyl (diethoxymethyl)phosphinate, (PhCOO)2,
dioxane, reflux; (c) 6 N aqueous HCl, reflux, 1: 19% from 33, 11:
100% from 36; (d) n-BuLi, (CH3O)2P(O)CH2P(O)(OCH3)2, THF,
-78 °C to rt, 56%; (e) H2, 10% Pd/C, EtOAc, 82%.
The C-5 stereochemistry of carbamalonate ether 4,
carbamalonate 5, and carbahydroxymalonate 6 all de-
pended on C-1 alcohol direction of NaBH(OAc)3 reduc-
tions. For example (Scheme 3), reaction of DHQ inter-
mediate 19 with NaBH(OAc)3 exclusively gave epi-
quinate 27. The extremely high stereoselectivity of this
reaction can best be explained by delivery of a hydride
to the face of the carbonyl dictated by complexation of
the C-1 alcohol with NaBH(OAc)3.1a,13 Coupling of epi-
quinate 27 with diethyl diazomalonate to give protected
malonate ether 28 was easily accomplished using Rh2-
(OAc)4 catalysis. Subsequent ester and BBA removal
using the aforementioned reaction conditions yielded
carbamalonate ether 4.
Reduction of the activated olefin of R,â-unsaturated
carbamalonitrile 29 to give carbamalonitrile 29 (Scheme
3) is also consistent with C-1 alcohol complexation of
NaBH(OAc)3.13 R,â-Unsaturated carbamalonitrile 29 was
obtained by reaction of DHQ intermediate 19 with
malononitrile catalyzed by NH4OAc and HOAc. At-
tempts to react DHQ intermediate 19 with diethyl
malonate under basic conditions failed to yield a conden-
sation product. Removal of the BBA protection group of
30 was followed by hydrolysis of the nitrile groups in
refluxing 6 N hydrochloric acid, which also resulted in
lactonization of the C-4 hydroxyl and malonyl carboxyl-
ate. This lactone was hydrolyzed under basic conditions
prior to Dowex 50 (H+) treatment to give the carbama-
lonate 5 triacid. Carbamalonate 5 was also converted
into carbahydroxymalonate 6. Esterification of 5 with
CH2N2 and reprotection of the C-3,4 trans-diol provided
triester 31, which underwent oxaziridine hydroxylation14
yielding protected hydroxymalonate 32. TFA-catalyzed
removal of the BBA protecting group, base-catalyzed
hydrolysis of the methyl esters, and treatment with
Dowex 50 (H+) led to carbahydroxymalonate 6.
P h osp h in a te a n d P h osp h on a te Syn th eses. The
versatility of protected DHQ 19 as an intermediate in
the synthesis of carbocyclic DHQ synthase inhibitors was
further demonstrated (Scheme 4) with the synthesis of
carbaphosphinate 1 and carbaphosphonate 11. Protec-
tion of the C-1 alcohol of DHQ intermediate 19 as a
methoxymethyl (MOM) ether was followed by reaction
with TiCl4/Zn/CH2I2 using the Takai procedure15 to afford
exocyclic olefin 33. Radical addition of ethyl (diethoxy-
methyl)phosphinate16 to exocyclic olefin 33 yielded masked
phosphinate 34. Acid hydrolysis simultaneously removed
the BBA and MOM protecting groups, hydrolyzed the
methyl ester, and unmasked the phosphinic acid. Prod-
uct carbaphosphinate 1 was purified by anion exchange
chromatography.
Assignment of the C-5 stereochemistry in carbaphos-
phinate 1 followed from analysis of the 1H NMR coupling
constants associated with the C-4 methine at δ 3.15.
Coupling constants (J ) 9 Hz) diagnostic of trans-diaxial
relationships were observed between both the C-5 and
C-4 methine protons and the C-3 and C-4 methine
protons. The coupling constant (J ) 9 Hz) between the
C-5 and C-4 protons in 1 markedly differs from the
smaller coupling constant (J ) 3 Hz) observed in the C-5
epimeric series4 between the C-5 and C-4 methine
protons. Additional corroboration of the C-5 stereochem-
istry in carbaphosphinate 1 synthesized via Scheme 4
follows from independent synthesis of 1 from a C-5
bromomethyl intermediate with known C-5 stereochem-
istry. Arbuzov condensation of the in situ generated
product17 of ethyl (diethoxymethyl)phosphinate reaction
with hexamethyldisilazane and previously synthesized1a
methyl [1S-(1R,3â,4R,5â)]-5-(bromomethyl)-1,3,4-tris(ben-
zoyloxy)cyclohexane-1-carboxylate in refluxing mesityl-
ene followed by exhaustive hydrolysis afforded a product
that was spectroscopically identical to carbaphosphinate
1.
(11) (a) Singh, A. K.; Bakshi, R. K.; Corey, E. J . J . Am. Chem. Soc.
1987, 109, 6187. (b) Montchamp, J .-L. Ph.D. Dissertation, Purdue
University, 1992.
Carbaphosphonate 11 synthesis began with the con-
densation of DHQ intermediate 19 with tetramethyl
(12) Hartwig, W. Tetrahedron 1983, 39, 2609.
(13) (a) Saksena, A. K.; Mangiaracina, P. Tetrahedron Lett. 1983,
24, 273. (b) Turnbull, M. D.; Hatter, G.; Ledgerwood, D. E. Tetrahedron
Lett. 1984, 25, 5449.
(14) (a) Davis, F. A.; Lamendola, J ., J r.; Nadir, U.; Kluger, E. W.;
Sedergran, T. C.; Panunto, T. W.; Billmers, R.; J enkins, R., J r.; Turchi,
I. J .; Watson, W. H.; Chen, J . S.; Kimura, M. J . Am. Chem. Soc. 1980,
102, 2000. (b) Davis, F. A.; Vishwakarma, L. C.; Billmers, J . M.; Finn,
J . J . Org. Chem. 1984, 49, 3241.
(15) Hibino, J .; Okazoe, T.; Takai, K.; Nozaki, H. Tetrahedron Lett.
1985, 26, 5579.
(16) Gallagher, M. J .; Honegger, H. Aust. J . Chem. 1980, 33, 287.
(17) Dingwall, J . G.; Ehrenfreund, J .; Hall, R. G. Tetrahedron 1989,
45, 3787.

7376 J . Org. Chem., Vol. 61, No. 21, 1996
Tian et al.
Ta ble 1. In h ibition of DHQ Syn th a se by Ca r bocyclic
Sch em e 5
In h ibitor s 1-6a
E-NADH
inhibitor type of inhibiiton formation
Ki (M)
Ki/Km
1
2
3
4
5
6
competitive
linear mixed-type
competitive
competitive
competitive
competitive
+
-
+
+
+
+
20 × 10-6
3 × 10-6 a
5 × 10-6
5
0.8
1.3
1.8
0.2
0.08
7 × 10-6
0.7 × 10-6
0.3 × 10-6
Ki′ ) 20 × 10-6 M slopes and y-axis intercepts derived from
a
double reciprocal plots were plotted as a function of inhibitor
concentration. The base-line intercepts of the slope and y-axis
intercepts, respectively, provided inhibition constants Ki and Ki′.22
methylenediphosphonate (Scheme 4). After hydrogena-
tion of phosphonylmethylidene 35 using 10% Pd on C to
give phosphonomethyl 36, refluxing 6 N HCl was used
to remove the BBA protecting group, carbmethoxy ester,
and both phosphonate methyl esters in a single step. The
absence of clearly resolved, diagnostic 1H NMR reso-
nances precluded establishing the C-5 stereochemistry
of phosphonomethyl 36 obtained from the hydrogenation
of phosphonomethylidene 35. However, the final product
obtained after deprotection of 36 was identical, based on
1H and 13C NMR, to independently synthesized carba-
phosphonate 11. Synthesis (Scheme 4) of carbaphospho-
nate 11 from quinic acid via protected DHQ 19 compares
very favorably with two previously reported syntheses
of 11 from quinic acid. The first reported synthesis1a of
carbaphosphonate 11 required 14 steps and afforded a
12% overall yield while a later synthesis5a required 12
steps and gave a 5% overall yield. The synthesis of
Scheme 4 leads to carbaphosphonate 11 after only 5 steps
with an overall yield of 30%.
inhibition. Of the carbocyclic inhibitors 1-6 synthesized
in this account, carbaacetate’s inhibition of DHQ syn-
thase was the only instance where NADH formation was
not observed (Table 1). All of the tricarboxylates 3-6
were competitive inhibitors (Table 1) with carbamalonate
5 and carbahydroxymalonate 6 displaying the most
potent inhibition of DHQ synthase.
Discu ssion
Tr ia n ion ic Ion iza tion Sta tes. Considerable experi-
mental evidence now supports the proposal that the
phosphate monoester of intermediate A (Scheme 5)
mediates its own elimination.1a,4,20 Even though spiro-
cyclic carbaphosphodiester 12 is oxidized by DHQ syn-
thase, the putative transition state analogue (Scheme 5)
generated at the active site is a modest inhibitor with Ki
) 67 × 10-6 M.1b This relatively weak active site
interaction is consistent with a rather passive role for
DHQ synthase during phosphate monoester elimination
whereby the enzyme merely ensures that either DAHP
or intermediate A is in its trianionic ionization state.
Other indications of the possible importance of a trian-
ionic ionization state include the 560-fold weaker inhibi-
tion of DHQ synthase by spirocyclic carbaphosphodiester1b
12 relative to carbaDAHP2,3b 8 (Ki ) 0.12 × 10-6 M) and
the 25 000-fold weaker inhibition measured for carba-
phosphinate 1 relative to carbaphosphonate1a,3a 11 (Ki )
0.8 × 10-9 M). While carbaDAHP 8 and carbaphospho-
nate 11 can exist in the active site as trianions, spirocyclic
carbaphosphodiester 12 and carbaphosphinate 1 are
limited to dianionic ionization states.
Even though a dibasic phosphate monoester (and hence
an overall trianionic ionization state) at the active site
of DHQ synthase would seem to ensure the optimum
reaction environment for conversion of intermediate A
to intermediate B, elimination of a monobasic phosphate
monoester is also a mechanistic possibility. This leads
to an alternate explanation for the weaker inhibition of
DHQ synthase by carbaphosphinate 1 relative to carba-
phosphonate 11. A monobasic phosphonate hydroxyl
group in enzyme-bound, dianionic carbaphosphonate 11
can be viewed as having been replaced with a hydrogen
in the dominant tautomeric form of the phosphinate of
enzyme-bound, dianionic carbaphosphinate 1. Loss of
active site interactions with a hydroxyl group might then
explain the severely attenuated inhibitor potency of
carbaphosphinate 1. Active site interactions with hy-
droxyl groups in carbaphosphonate 11 can be approxi-
En zym ology. DHQ dehydratase18 was used as the
coupling enzyme for assay of DHQ synthase activity.
Dehydratase-catalyzed conversion of DHQ into 3-dehy-
droshikimate (DHS) and the increase in optical density
at 234 nm resulting from formation of DHS provided a
means for continuous quantitation of DHQ formation.
The measured value of the Michaelis constant (Km) for
DAHP binding to DHQ synthase has been reported to
depend on the assay method.19 For this work, the Km
for DAHP has been taken as 4 × 10-6 M. Inhibition
constants (Ki) were determined (Table 1) for carbocyclic
inhibitors 1-6. Interactions with DHQ synthase were
further evaluated by whether observed inhibition was
competitive, noncompetitive, or linear mixed-type relative
to substrate DAHP binding. Formation of NADH was
measured by increases in absorbance at 340 nm. This
measure of whether the carbocyclic inhibitor’s C-4 alcohol
was in close enough proximity to the bound NAD for C-4
alcohol oxidation to occur provided an indication of the
carbocyclic inhibitor’s alignment in the active site.
Carbocyclic diacids carbaphosphinate 1 and carbaac-
etate 2 were modest inhibitors of DHQ synthase. Car-
baacetate 2 was a linear mixed-type inhibitor (Table 1).
While carbaphosphinate 1 was a significantly weaker
inhibitor (Table 1) of DHQ synthase, its binding to the
active site was entirely competitive relative to substrate
DAHP. NADH formation was observed during carba-
phosphinate 1 inhibition but not during carbaacetate 2
(18) Chaudhuri, S.; Duncan, K.; Coggins, J . R. Methods Enzymol.
1987, 142, 320.
(19) Bender, S. L.; Mehdi, S.; Knowles, J . R. Biochemistry 1989, 28,
7555.
(20) Widlanski, T. S.; Bender, S. L.; Knowles, J . R. J . Am. Chem.
Soc. 1987, 109, 1873.

Inhibitor Ionization as a Determinant of Binding
J . Org. Chem., Vol. 61, No. 21, 1996 7377
mated by the 41-fold and 100-fold reduction in inhibitor
potency observed, respectively, for C-3 and C-4 monode-
oxycarbaphosphonates.21 This reduction in inhibitor
potency due to loss of active site interactions with a
hydroxyl group is substantially less than the 25 000-fold
loss of inhibition potency observed for carbaphosphinate
1 relative to carbaphosphonate 11. The lack of access to
a trianionic ionization state thus appears to be a better
explanation for weaker inhibition observed for carba-
phosphinate 1.
phosphonate 11, C-1 epimeric carbaphosphonate 17, C-5
epimeric phosphate 13, and C-5 epimeric phosphonate
16 are all nanomolar-level, competitive inhibitors. The
nonisosteric phosphonic acid moieties of carbaphospho-
nate 11 and C-1 epimeric carbaphosphonate 17 may well
be interacting with active site residues that bind to DHQ
synthase transition states and/or reactive intermediates
selectively over substrate DAHP (or phosphate monoester
of carbaDAHP 8 and isosteric phosphonoethyl group of
9). 5-[(Phosphonooxy)methyl]-5-deoxyquinate 13 and
5-(phosphonomethyl)-5-deoxyquinate 16 after oxidation
by DHQ synthase might be best viewed (Scheme 5) as
intermediate B covalently attached to inorganic phos-
phate. These C-5 epimers may be taking advantage of
active site residues that bind to intermediate B concur-
rently with separate active site residues that bind to
inorganic phosphate prior to its release from the enzyme.
The C-5 epimers 5-(phosphonoethyl)-5-deoxyquinate (14)
(Ki ) 3.0 × 10-5 M) and 3-(phosphonooxy)quinate (15)
(Ki ) 5.0 × 10-5 M) are much weaker inhibitors.4
Di- a n d Tr ica r boxyla te In h ibitor s. The carbocyclic
inhibitors 2-6 synthesized in this account can be viewed
as evolutionary extensions of the previously synthesized
carbacarboxylate 7, which was reported2 to be a competi-
tive inhibitor with Ki ) 100 × 10-6 M. By inserting a
methylene carbon between the carbocyclic ring and the
carboxylate, inhibition of DHQ synthase improves (Table
1) by a factor of approximately 30-fold based on the
competitive inhibition constant Ki for carbaacetate 2
relative to carbacarboxylate 7. The noncompetitive por-
tion (Ki′) of the linear mixed-type inhibition observed for
carbaacetate 2 suggests that this inhibitor’s interactions
with DHQ synthase are not restricted to the enzyme’s
active site.22 Providing access to a trianionic ionization
state led to competitive inhibition for carbasuccinate 3,
carbamalonate ether 4, carbamalonate 5, and carbahy-
droxymalonate 6. Employment of a malonate-type
charged appendage results in improved active site inter-
actions as indicated (Table 1) by the approximately 10-
fold improvement in Ki values observed for tricarboxyl-
ates 5 and 6 relative to tricarboxylates 3 and 4. Titration
of carbamalonate 5 indicated a single, large inflection
point indicative of multiple proton dissociation at an
apparent pKa ) 4.40. For comparison, two inflection
points indicating apparent acid dissociation constants at
pKa1 ) 2.55 and pKa2 ) 6.25 were observed during
titration of DAHP.
The hydroxymalonic acid and malonic acid moieties of,
respectively, carbahydroxymalonate 6 and carbamalonate
5 are excellent analogues of a phosphate monoester. This
follows from the competitive nature of these tricarboxyl-
ates’ inhibition relative to substrate DAHP, the magni-
tude of their inhibition constants relative to those of
carbaDAHP 8 and carbahomophosphonate 9, and forma-
tion of NADH during DHQ synthase inhibition. The
hydroxymalonic acid and malonic acid moieties are, not
surprisingly, poor analogues of a nonisosteric phosphonic
acid moiety of carbaphosphonate 11. How well a C-5
epimeric hydroxymalonic acid and a C-5 epimeric malonic
acid might approximate the inhibition of DHQ synthase
observed for C-5 epimeric phosphate 5-[(phosphonooxy)-
methyl]-5-deoxyquinate (13) and 5-(phosphonomethyl)-
5-deoxyquinate (16) remains for future investigation.
In Vivo In h ibition . The virulence of pathogenic
microbes is greatly attenuated upon introduction of
genomic mutations that eliminate the catalytic activity
of enzymes in the common pathway of aromatic amino
acid biosynthesis.23 Might chemical inhibition of a com-
mon pathway enzyme likewise attenuate the virulence
of pathogenic microbes? DHQ synthase is one possible
target given that some of the most potent inhibitors of
common pathway enzymes are available for this enzyme.
Carbaphosphonate 11, C-1 epimeric carbaphosphonate
17, 5-[(phosphonooxy)methyl]-5-deoxyquinate (13), and
5-(phosphonomethyl)-5-deoxyquinate (16) are appealing
candidates for “arobiotics” given their nanomolar, slowly-
reversible inhibition of DHQ synthase. However, the
ionization states of these inhibitors at physiologically
relevant pH are not compatible with effective penetration
into the bacterial cytosol, which is a prerequisite for in
vivo DHQ synthase inhibition.
Carbahydroxymalonate 6, the most potent of the newly
synthesized tricarboxylate inhibitors, is approximately
as potent a competitive inhibitor of DHQ synthase as
carbaDAHP2,3b 8 (Ki ) 0.12 × 10-6 M). Both carbama-
lonate 5 and carbahydroxymalonate 6 are better competi-
tive inhibitors than carbahomophosphonate1a,4 9 (Ki ) 1.7
× 10-6 M), which is the isosteric phosphonic acid ana-
logue of carbaDAHP 8. Other carbocyclic phosphate
esters such as carbaphosphate1a,4 10 (Ki ) 1.7 × 10-6 M)
and 3-(phosphonooxy)quinate4 15 (Ki ) 53 × 10-6 M) are
weaker competitive inhibitors of DHQ synthase. The
only phosphate monoester synthesized to date that is a
substantially more potent competitive inhibitor than
carbahydroxymalonate 6 is C-5 epimeric 5-[(phospho-
nooxy)methyl]-5-deoxyquinate4 13 (Ki ) 7.0 × 10-9 M).
Organophosphonic acid analogues such as carbaphos-
phonate1a,3a 11 (Ki ) 0.8 × 10-9 M), C-1 epimeric
carbaphosphonate5 17 (Ki ) 1.8 × 10-9 M), and C-5
epimeric 5-(phoshonomethyl)-5-deoxyquinate4 16 (Ki ) 13
× 10-9 M) are more potent inhibitors of DHQ synthase.
Prodrug strategies will need to be explored whereby
inhibitor 11, the most potent in vitro inhibitor of DHQ
synthase, is covalently modified to derivatives that can
penetrate into intact bacteria. Upon reaching the bacte-
rial cytosol, the covalently attached groups would be
enzymatically hydrolyzed, thereby releasing the DHQ
synthase inhibitor. Exploring such prodrug strategies
Designing inhibitors to structurally mimic substrate
DAHP does not appear to lead to the most potent DHQ
synthase inhibition. By contrast, nonisosteric carba-
(21) (a) Montchamp, J .-L.; Piehler, L. T.; Tolbert, T. J .; Frost, J . W.
Bioorg. Med. Chem. Lett. 1992, 2, 1349. (b) Montchamp, J .-L.; Piehler,
L. T.; Tolbert, T. J .; Frost, J . W. Bioorg. Med. Chem. Lett. 1993, 3,
1403.
(22) (a) Segel, I. H. Biochemical Calculations, 2nd ed.; Wiley: New
York, 1976; Chapter 4. (b) Galli, A.; Mori, F. J . Pharm. Pharmacol.
1996, 48, 71.
(23) (a) O’Callaghan, D.; Maskell, D.; Liew, F. Y.; Easmon, C. S. F.;
Dougan, G. Infect. Immun. 1988, 56, 419. (b) Dougan, G.; Chatfield,
S.; Pickard, D.; Bester, J .; O’Callaghan, D.; Maskell, D. J . Infect. Dis.
1988, 158, 1329. (c) Miller, I.; Maskell, D.; Hormaeche, C.; J ohnson,
K.; Pickard, D.; Dougan, G. Infect. Immun. 1989, 57, 2758. (d) Maskell,
D. Imperial College, personal communication, 1995.

7378 J . Org. Chem., Vol. 61, No. 21, 1996
Tian et al.
A solution containing a known concentration of an inhibitor
was added to the cuvette, and the change in absorbance at
340 nm was monitored.
has been complicated by the long syntheses and low
overall yields of carbaphosphonate 11. Simultaneous
pursuit of several different in vivo inhibition strategies
or examination of strategies that might require several
steps for covalent modification of carbaphosphonate 11
has not been possible. With synthesis via protected DHQ
intermediacy leading to a 30% yield in just five steps from
quinic acid, gram quantities of carbaphosphonate 11 can
now be conveniently obtained.
The availability of tricarboxylate inhibitors of DHQ
synthase such as carbamalonate 5 and carbahydroxy-
malonate 6 is also important given the wider spectrum
of prodrug strategies that are available for carboxylic
acids versus phosphate esters and phosphonic acids.
Carbamalonate 5 and carbahydroxymalonate 6 are al-
ready more potent inhibitors than R-C-(1,5-anhydro-7-
amino-2,7-dideoxy-D-manno-heptopyranosyl)carboxyl-
ate (Ki ) 4.0 × 10-6 M), a competitive inhibitor24 of CMP-
KDO synthetase that possesses chemotherapeutic activ-
ity against Gram-negative bacteria. A prodrug strategy
was used to drastically increase the concentrations of this
essentially nonpermeant ulosonic acid inhibitor that
penetrated into bacterial cytosols.24 Related prodrug
strategies can now be employed for DHQ synthase that
exploit the expanded structural diversity of inhibitory
molecules (tricarboxylates 5 and 6) and improved syn-
thetic accessiblity of the most potent in vitro inhibitor
(carbaphosphonate 11). In this fashion, the chemothera-
peutic utility of in vivo DHQ synthase inhibition can
ultimately be evaluated.
P r otected DHQ In ter m ed ia te 19. In the following order,
H2O (300 mL), KIO4 (43.0 g, 187 mmol), K2CO3 (3.38 g, 24.5
mmol), and finally RuCl3 (0.50 g, 1.9 mmol) were added to a
solution of 189 (30.2 g, 94.2 mmol) in CHCl3 (300 mL).
Vigorous stirring was continued at rt until the reaction was
complete. The mixture was filtered through Celite, and the
aqueous and organic phases were separated. After saturation
with NaCl, the aqueous layer was extracted with EtOAc (3×).
The combined organic layers were dried and concentrated to
give protected DHQ intermediate 19 as a white solid (23.0 g,
77%): 1H NMR (CDCl3) δ 4.43 (dd, J ) 10, 1 Hz, 1 H), 4.26
(ddd, J ) 13, 10, 4 Hz, 1 H), 3.85 (s, 3 H), 3.27 (s, 3 H), 3.24
(s, 3 H), 2.91 (dd, J ) 14, 1 Hz, 1 H), 2.52 (dd, J ) 14, 3 Hz,
1 H), 2.36 (dd, J ) 13, 13 Hz, 1 H), 2.13 (ddd, J ) 13, 4, 3 Hz,
1 H), 1.41 (s, 3 H), 1.31 (s, 3 H); 13C NMR (CDCl3) δ 199.4,
174.0, 100.4, 99.5, 77.1, 74.0, 66.9, 53.5, 48.9, 48.2, 47.9, 37.7,
17.6, 17.4. Anal. Calcd for C14H22O8: C, 52.82; H, 6.97.
Found: C, 52.65; H, 6.93.
Ca r ba a ceta te In ter m ed ia te 22. To a vigorously stirred
mixture of carbaacetate intermediate 213b (3.33 g, 11.0 mmol)
in CH3CN/CCl4/H2O (16 mL/16 mL/24 mL) were added NaIO4
(9.89 g, 46.2 mmol) and RuCl3‚7H2O (0.063 g, 0.24 mmol).
Stirring was continued overnight at rt. The biphasic reaction
mixture was filtered through Celite to remove suspended solids
and allow organic and aqueous layers to separate. After
extraction of the aqueous layer with CH2Cl2 (3×), the combined
organic layers were dried, concentrated, redissolved in CH3-
OH (20 mL), and treated with an ethereal CH2N2 solution at
-20 °C. The solvent was evaporated and the residue was
purified by flash chromatography (EtOAc/hexane, 1:1, v/v).
Carbaacetate intermediate 22 was obtained as a yellow oil
(2.39 g, 65%): 1H NMR (CDCl3) δ 8.02-8.05 (m, 2 H), 7.58-
7.64 (m, 1 H), 7.45-7.50 (m, 2 H), 5.14 -5.16 (m, 1 H), 4.91-
4.93 (m, 1 H), 3.83 (br, 1 H), 3.66 (s, 3 H), 2.82-2.88 (m, 1 H),
2.39-2.75 (m, 5 H), 1.87 (d, J ) 16 Hz, 1 H); 13C NMR (CDCl3)
δ 178.5, 171.9, 165.0, 133.6, 129.6, 129.5, 129.0, 128.6, 128.5,
75.3, 71.7, 70.4, 51.6, 38.4, 36.6, 36.5, 33.2; HRMS (FAB) calcd
for C17H18O7 (M + H+) 335.1130, found 335.1133.
Exp er im en ta l Section
Gen er a l Ch em istr y. Organic solutions of products were
dried over MgSO4. See ref 1b for general experimental
information. All air- and moisture-sensitive reactions were
conducted under Ar. 1,4-Dioxane and ClCH2CH2Cl were
distilled from calcium hydride under Ar. CH3CN was dried
over activated 3A molecular sieves. Melting points were
uncorrected and were determined using a Mel-Temp II melting
point apparatus. Spectrophotometric measurements were
made on a Hewlett-Packard 8452A diode array spectropho-
tometer.
Gen er a l En zym ology. To quantify the concentration of
a given dicarboxylate or tricarboxylate inhibitor, the carboxylic
acid solution was concentrated to dryness, exchanged twice
with D2O, and redissolved in a known volume of D2O. From
this stock solution, an aliquot was mixed with a known volume
of sodium 3-(trimethylsilyl)propionate-2,2,3,3-d4 (TSP) solution
(5.0 mM in D2O). The concentration of the carboxylic acid was
quantitated by comparison of the integration of selected proton
resonances of the carboxylic acid with the integrated resonance
of the internal TSP standard.
DHQ synthase activity was assayed in a 1.0 mL solution of
3-(N-morpholino)propanesulfonate (MOPS) buffer (50 mM, pH
7.7) containing CoCl2 (50 µM), NAD+ (10 µM), dehydroquinase
(1 unit), and varying amounts of DAHP and of the enzyme
inhibitor. After equilibration at rt, DHQ synthase (0.024
units) was added, and the increase in absorbance at 234 nm
was monitored over time. Initial rates were determined by
linear squares fits of the progress curves and were used to
determine inhibition constants.
To measure enzyme-bound NADH formation during enzyme
inhibition, a 0.8 mL solution of MOPS buffer (50 mM, pH 7.7)
containing CoCl2 (50 µM), NAD+ (10 µM), and DHQ synthase
(10 µM) was incubated at rt in a quartz cuvette. Spectra (190-
820 nm) were measured to establish the base-line absorbance.
Ca r ba a cet a t e 2. Carbaacetate intermediate 22 (0.23 g,
0.69 mmol) was stirred in a mixture of THF (10 mL) and
aqueous NaOH (0.2 N, 10 mL) for 24 h. The aqueous layer
was washed with EtOAc (1×), passed down a Dowex 50 (H+)
column, and concentrated in vacuo to yield carbaacetate 2 as
a colorless film (100% yield based on NMR analysis): 1H NMR
(D2O, pH 8.1) δ 3.77 (ddd, J ) 12, 10, 5 Hz, 1 H), 3.23 (dd, J
) 10, 10 Hz, 1 H), 2.08-2.34 (m, 4 H), 1.71-1.94 (m, 3 H); 13
C
NMR (D2O, pH 8.1) δ 181.1, 180.2, 80.1, 76.8, 73.1, 42.4, 40.5,
39.5, 37.7; HRMS (FAB) calcd for C9H12O7Na2 (M + H+)
279.0457, found 279.0450.
Ca r ba su ccin a te In ter m ed ia te 23. A solution of inter-
mediate 19 (0.661 g, 2.08 mmol) and (carbethoxymethylene)-
triphenylphosphorane (1.45 g, 4.16 mmol) in anhydrous CH3-
CN (10 mL) was refluxed for 15 min. After the solution was
cooled to rt, the solvent was removed and the residue was
purified by flash chromatography (EtOAc/hexane, 1:1, v/v) to
give carbasuccinate intermediate 23 as a yellow foam (0.727
g, 90%): 1H NMR (CDCl3) δ 6.25 (d, J ) 2 Hz, 1 H), 4.11-
4.20 (m, 3 H), 4.05 (dd, J ) 14, 3 Hz, 1 H), 3.97 (ddd, J ) 11,
10, 5 Hz, 1 H), 3.80 (s, 3 H), 3.25 (s, 3 H), 3.22 (s, 3 H), 3.21 (s,
1 H), 2.34 (d, J ) 14 Hz, 1 H), 2.12 (dd, J ) 12, 12 Hz, 1 H),
2.00 (ddd, J ) 13, 5, 3 Hz, 1 H), 1.36 (s, 3 H), 1.31 (s, 3 H),
1.28 (t, J ) 7 Hz, 3 H); 13C NMR (CDCl3) δ 174.8, 166.8, 150.6,
115.1, 100.1, 99.4, 74.8, 72.9, 68.5, 60.0, 53.0, 47.9 (2), 37.3,
36.9, 17.6 (2), 14.1; HRMS (FAB) calcd for C18H28O9 (M + H+)
389.1812, found 389.1829. Anal. Calcd for C18H28O9: C, 55.66;
H, 7.27. Found: C, 55.59; H, 7.32.
Ca r ba su ccin a te In ter m ed ia te 25. 1,1′-Carbonyldiimi-
dazole (0.415 g, 2.56 mmol) and carbasuccinate intermediate
23 (0.497 g, 1.28 mmol) were dissolved in anhydrous ClCH2-
CH2Cl (5 mL) and heated at reflux. Heating was stopped 5 h
later and the mixture cooled to rt. Ether was added, and the
organic layer was washed with water (1×) and brine (1×).
Concentration yielded a yellow oil that was purified by flash
(24) (a) Goldman, R.; Kohlbrenner, W.; Lartey, P.; Pernet, A. Nature
1987, 329, 162. (b) Hammond, S. M.; Claesson, A.; J ansson, A. M.;
Larsson, L.-G.; Pring, B. G.; Town, C. M.; Ekstro¨m, B. Nature 1987,
327, 730.

Inhibitor Ionization as a Determinant of Binding
J . Org. Chem., Vol. 61, No. 21, 1996 7379
chromatography (EtOAc/hexane, 1:1, v/v) to afford carbasuc-
cinate intermediate 24 as a white foam (0.506 g, 82%): 1H
NMR (CDCl3) δ 8.05 (d, J ) 1 Hz, 1 H), 7.33 (dd, J ) 2, 2 Hz,
1 H), 7.05 (dd, J ) 2, 1 Hz, 1 H), 6.27 (m, 1 H), 4.59 (dd, J )
15, 3 Hz, 1 H), 4.21 (dd, J ) 10, 2 Hz, 1 H), 4.08-4.16 (m, 2
H), 3.82 (s, 3 H), 3.75-3.84 (m, 1 H), 3.26 (s, 3 H), 3.21 (s, 3
H), 2.68 (ddd, J ) 14, 5, 3 Hz, 1 H), 2.47 (dd, J ) 15, 1 Hz, 1
H), 2.26 (dd, J ) 14, 12 Hz, 1 H), 1.37 (s, 3 H), 1.32 (s, 3 H),
1.22 (t, J ) 7 Hz, 3 H); 13C NMR (CDCl3) δ 169.4, 166.0, 147.4,
137.2, 130.7, 117.1, 116.8, 100.3, 99.7, 83.5, 72.5, 68.1, 60.3,
53.3, 48.2, 48.0, 35.3, 33.9, 17.6, 14.1; HRMS (FAB) calcd for
C22H30N2O10 (M + H+) 483.1979, found 483.1983.
Benzeneselenol (0.26 mL, 2.4 mmol) and carbasuccinate
intermediate 24 (0.567 g, 1.17 mmol) were dissolved in
anhydrous ClCH2CH2Cl (5 mL), and the resulting solution was
heated at reflux overnight. Solvent removal and purification
by flash chromatography (EtOAc/hexane, 5:1, v/v) yielded
carbasuccinate intermediate 25 as a white solid (0.582 g,
87%): mp 174-176 °C; 1H NMR (CDCl3) δ 7.57-7.60 (m, 2
H), 7.30-7.38 (m, 3 H), 6.25 (s, 3 H), 4.64 (dd, J ) 15, 3 Hz,
1 H), 4.10-4.22 (m, 3 H), 3.81 (ddd, J ) 12, 10, 5 Hz, 1 H),
3.74 (s, 3 H), 3.23 (s, 6 H), 2.44 (ddd, J ) 14, 4, 3 Hz, 1 H),
2.41 (dd, J ) 14, 12 Hz, 1 H), 1.38 (s, 3 H), 1.31 (s, 3 H), 2.02
(dd, J ) 14, 12 Hz, 1 H), 1.25 (t, J ) 7 Hz, 3 H); 13C NMR
(CDCl3) δ 169.8, 165.7, 165.2, 147.9, 135.4, 129.1, 129.0, 125.6,
116.1, 100.1, 99.5, 83.2, 72.3, 67.5, 59.9, 52.8, 47.9 (2), 35.8,
33.1, 17.5, 14.1; HRMS (EI) calcd for C25H32O10Se (M + H+)
573.1238, found 573.1260. Anal. Calcd for C25H32O10Se: C,
52.54; H, 5.65. Found: C, 52.45; H, 5.65.
Ca r ba su ccin a te In ter m ed ia te 26. A solution of AIBN
(0.035 g, 0.21 mmol) and Bu3SnH (0.86 mL, 3.2 mmol) in
benzene (50 mL) was slowly added via syringe pump (0.10
mmol/h) to a refluxing solution of carbasuccinate intermediate
25 (1.21 g, 2.12 mmol) in benzene (25 mL). After completion
of the addition, heating at reflux was continued for another 2
h. Solvent was removed under reduced pressure, and the
residue was purified by radial chromatography (2 mm thick-
ness, EtOAc/hexane, 5:1, v/v) to give carbasuccinate intermedi-
ate 26 as a white solid (0.23 g, 27%): mp 180-181 °C; 1H NMR
(CDCl3) δ 4.07-4.19 (m, 2 H), 3.90-3.96 (m, 2 H), 3.83 (s, 3
H), 3.21 (s, 3 H), 3.18 (s, 3 H), 2.89 (d, J ) 18 Hz, 1 H), 2.80
(d, J ) 18 Hz, 1 H), 2.76 (dd, J ) 12, 2 Hz, 1 H), 2.42-2.49
(m, 1 H), 2.34 (d, J ) 12 Hz, 1 H), 1.98-2.06 (m, 1 H), 1.28 (s,
6 H), 1.26 (t, J ) 7 Hz, 3 H); 13C NMR (CDCl3) δ 173.2, 170.0,
169.0, 101.1, 100.3, 81.6, 70.8, 66.8, 60.7, 52.9, 48.0, 47.8, 47.0,
42.9, 34.4, 31.7, 17.6, 17.5, 14.0; HRMS (FAB) calcd for
C19H28O10 (M - H+) 415.1604, found 415.1600. Anal. Calcd
for C19H28O10: C, 54.80; H, 6.78. Found: C, 54.70; H, 6.73.
Ca r ba su ccin a te 3. Carbasuccinate intermediate 26 (0.152
g, 0.360 mmol) was stirred in CF3CO2H/H2O (20:1, v/v, 3 mL)
for 20 min. Water and CF3CO2H were removed in vacuo. The
brown residue was stirred in a mixture of THF (10 mL) and
aqueous NaOH (0.2 N, 10 mL) for 24 h. The aqueous layer
was washed with EtOAc (1×), passed down a Dowex 50 (H+)
column, and concentrated to afford carbasuccinate 3 as a
colorless film (100% yield based on NMR analysis): 1H NMR
(D2O, pH 7.5) δ 3.87 (m, 1 H), 3.47 (d, J ) 10 Hz, 1 H), 2.67
(d, J ) 14 Hz, 1 H), 2.56 (d, J ) 14 Hz, 1 H), 2.36 (dd, J ) 15,
2 Hz, 1 H), 1.82-2.03 (m, 3 H); 13C NMR (D2O, pH 7.5) δ 185.5,
184.9, 182.1, 80.8, 78.1, 71.7, 52.4, 49.3, 44.3, 42.8; HRMS
(FAB) calcd for C10H11O9Na3 (M + H+) 345.0174, found
345.0171.
Ca r ba m a lon a te Eth er In ter m ed ia te 27. Intermediate
19 (2.01 g, 6.31 mmol) and NaBH(OAc)3 (5.35 g, 25.3 mmol)
were dissolved in CH3CN (10 mL) and HOAc (10 mL). The
mixture was stirred at rt for 8 h. Solvents were removed in
vacuo, and the residue was dissolved in ether and washed with
aqueous NaHSO4 (0.1 N). The aqueous layer was back-
extracted with ether (3×). The combined organic layers were
then washed with aqueous phosphate buffer (2 N, pH 7), dried,
and concentrated to a yellow solid. Purification by flash
chromatography (EtOAc/hexane, 2:1, v/v) gave white, crystal-
line diol 27 (1.74 g, 86%): m.p. 164-166 °C; 1H NMR (CDCl3)
δ 3.90-4.07 (m, 2 H), 3.80 (s, 3 H), 3.48 (dd, J ) 10, 10 Hz, 1
H), 3.30 (s, 3 H), 3.26 (s, 3 H), 3.24 (s, 1 H), 2.58 (d, J ) 2 Hz,
1 H), 1.99-2.06 (m, 1 H), 1.98 (dd, J ) 12, 12 Hz, 1 H), 1.82-
1.90 (m, 2 H), 1.35 (s, 3 H), 1.30 (s, 3 H); 13C NMR (CDCl3) δ
175.7, 99.6, 99.5, 76.3, 73.3, 66.9, 65.1, 53.2, 47.9, 47.8, 40.5,
37.9, 17.7 (2). Anal. Calcd for C14H24O8: C, 52.49; H, 7.55.
Found: C, 52.43; H, 7.51.
Ca r ba m a lon a te Eth er In ter m ed ia te 28. A solution of
diethyl diazomalonate (0.239 g, 1.29 mmol) in benzene (2 mL)
was slowly added via syringe pump (2.4 mmol/h) to a solution
of carbamalonate ether intermediate 27 (0.206 g, 0.643 mmol)
and Rh2(OAc)4 (0.004 g, 0.01 mmol) in refluxing benzene (10
mL). After completion of the addition, the mixture was
refluxed for another 2 h. Removal of the solvent gave a green
residue, which was purified by flash chromatography (EtOAc/
hexane, 1:1, v/v) to yield carbamalonate ether intermediate
28 as a colorless oil (0.220 g, 72%): 1H NMR (CDCl3) δ 4.18-
4.31 (m, 4 H), 3.98 (ddd, J ) 12, 10, 5 Hz, 1 H), 3.89 (ddd, J
) 11, 10, 5 Hz, 1 H), 3.79 (s, 3 H), 3.71 (dd, J ) 10, 10 Hz, 1
H), 3.29 (s, 3 H), 3.28 (s, 1 H), 3.25 (s, 3 H), 2.23 (ddd, J ) 13,
5, 2 Hz, 1 H), 1.95-2.05 (m, 1 H), 1.93 (dd, J ) 12, 12 Hz, 1
H), 1.80 (ddd, J ) 13, 5, 2 Hz, 1 H), 1.32 (s, 3 H), 1.29 (s, 3 H),
1.25-1.31 (m, 6 H); 13C NMR (CDCl3) δ 175.4, 167.6, 166.2,
99.5, 99.3, 80.0, 76.7, 75.6, 73.1, 65.3, 61.7, 61.6, 53.1, 47.9,
47.7, 39.9, 37.3, 17.6, 14.0, 13.9 Anal. Calcd for C21H34O12
C, 52.60; H, 7.15. Found: C, 52.74; H, 7.20.
:
Ca r ba m a lon a te Eth er 4. Carbamalonate ether interme-
diate 28 (0.22 g, 0.48 mmol) was deprotected as described for
carbasuccinate intermediate 26 to give carbamalonate ether
4 as a colorless film (100% yield based on NMR analysis): 1H
NMR (D2O, pH 7.7) δ 4.98 (s, 1 H), 3.70-3.83 (m, 2 H), 3.54
(dd, J ) 9, 9 Hz, 1 H), 2.25-2.31 (m, 1 H), 2.08-2.15 (m, 1 H),
1.97 (dd, J ) 13, 12 Hz, 1 H), 1.90 (dd, J ) 13, 12 Hz, 1 H);
13C NMR (D2O, pH 7.7) δ 180.2, 173.0, 172.9, 82.2, 81.4, 81.0,
76.0, 71.8, 42.2, 40.1; HRMS (FAB) calcd for C10H11O10Na3 (M
+ H+) 361.0123, found 361.0122.
Ca r ba m a lon a te In ter m ed ia te 29. To a solution of in-
termediate 19 (1.03 g, 3.23 mmol) in benzene (15 mL) was
added malononitrile (0.235 g, 3.56 mmol), ammonium acetate
(0.025 g, 0.32 mmol), and one drop of HOAc. The resulting
mixture was stirred overnight at rt. Ether was added, and
the organic layer was washed with aqueous NaHCO3 (1×) and
brine (1×). Drying and concentration afforded a yellow oil that
was purified by flash chromatography (EtOAc/hexane, 1:1, v/v)
to give carbamalonate intermediate 29 as a white crystalline
1
solid (1.07 g, 90%): mp 189-190 °C; H NMR (CDCl3) δ 4.54
(d, J ) 9 Hz, 1 H), 4.16 (ddd, J ) 11, 9, 5 Hz, 1 H), 3.87 (s, 3
H), 3.62 (s, 1 H), 3.31 (s, 3 H), 3.24 (s, 3 H), 3.09 (dd, J ) 14,
2 Hz, 1 H), 2.81 (d, J ) 14 Hz, 1 H), 2.11 (dd, J ) 14, 11 Hz,
1 H), 2.02 (ddd, J ) 13, 5, 2 Hz, 1 H), 1.41 (s, 3 H), 1.29 (s, 3
H); 13C NMR (CDCl3) δ 173.5, 169.9, 112.3, 110.8, 101.1, 99.8,
85.3, 74.6, 74.1, 67.9, 53.8, 48.6, 48.1, 42.5, 37.5, 17.4, 16.7;
HRMS (EI) calcd for C17H22N2O7 (M + H+) 367.1505, found
367.1502. Anal. Calcd for C17H22N2O7: C, 55.73; H, 6.05; N,
7.65. Found: C, 55.71; H, 6.04; N, 7.68.
Ca r ba m a lon a te In ter m ed ia te 30. NaBH(OAc)3 (2.31 g,
10.9 mmol) was dissolved in CH3CN (10 mL) and HOAc (10
mL). After the mixture was stirred at rt for 10 min, carba-
malonate intermediate 29 (1.00 g, 2.73 mmol) was added as a
solid in one portion. Stirring was continued overnight at rt.
Solvents were removed in vacuo, and the residue was dissolved
in ether and washed with aqueous NaHSO4 (0.1 N). The
aqueous layer was back-extracted with ether (3×). The
combined organic layers were then washed with aqueous
phosphate buffer (2 N, pH 7), dried, and concentrated to a
yellow oil. Flash chromatography (EtOAc/hexane, 1:1, v/v)
yielded carbamalonate intermediate 30 as a white crystalline
solid (0.925 g, 92%): mp 158-159 °C; 1H NMR (CDCl3) δ 4.44
(d, J ) 4 Hz, 1 H), 4.04 (ddd, J ) 11, 10, 5 Hz, 1 H), 3.84 (s,
3 H), 3.66 (dd, J ) 11, 10 Hz, 1 H), 3.52 (s, 1 H), 3.31 (s, 3 H),
3.26 (s, 3 H), 2.60-2.71 (m, 1 H), 2.11-1.89 (m, 4 H); 13C NMR
(CDCl3) δ 174.9, 111.7, 110.7, 100.2, 99.7, 72.9, 70.4, 66.1, 53.4,
48.4, 48.0, 37.5, 37.3, 35.8, 23.2, 17.5 (2); HRMS (EI) calcd for
C17H24N2O7 (M+) 368.1583, found 368.1585. Anal. Calcd for
C17H24N2O7: C, 55.42; H, 6.57; N, 7.61. Found: C, 55.28; H,
6.53; N, 7.50.
Ca r ba m a lon a te 5. Carbamalonate intermediate 30 (2.0
g, 5.4 mmol) was stirred in CF3CO2H/H2O (20:1, v/v, 5 mL)
for 20 min. After concentration, the residue was dissolved in

7380 J . Org. Chem., Vol. 61, No. 21, 1996
Tian et al.
aqueous HCl (6 N, 20 mL) and heated at reflux for 2 h. After
being cooled to rt, the mixture was evaporated to dryness,
dissolved in water, and the solution basicified by addition of
aqueous NaOH to pH 12. The resulting solution was stirred
overnight. The aqueous layer was washed with EtOAc (1×),
passed down Dowex 50 (H+), and concentrated to afford
carbamalonate 5 as a colorless film (95% yield based on NMR
analysis): 1H NMR (D2O, pH 7.9) δ 3.86 (d, J ) 5 Hz, 1 H),
3.79 (ddd, J ) 12, 10, 5 Hz, 1 H), 3.47 (dd, J ) 11, 10 Hz, 1
H), 2.50-2.65 (m, 1 H), 2.09-2.19 (m, 1 H), 1.88-1.99 (m, 1
H), 1.86 (dd, J ) 13, 12 Hz, 1 H); 13C NMR (D2O, pH 7.9) δ
180.8, 175.4, 174.8, 78.4, 76.6, 73.3, 55.0, 42.4, 40.8, 37.7;
HRMS (FAB) calcd for C10H14O9 (M + H+) 279.0716, found
279.0711.
rt with vigorous stirring. After completion of the reaction, the
reaction mixture was poured into saturated aqueous Na2CO3
and then extracted with ether. The organic layer was washed
with brine (1×), dried, and concentrated to a white solid (8.40
g, 91%) that could be used directly in the next experiment:
1H NMR (CDCl3) δ 4.76 (d, J ) 7 Hz, 1 H), 4.64 (d, J ) 7 Hz,
1 H), 4.41 (d, J ) 10 Hz, 1 H), 4.19 (ddd, J ) 10, 10, 4 Hz, 1
H), 3.78 (s, 3 H), 3.34 (s, 3 H), 3.26 (s, 3 H), 3.24 (s, 3 H), 2.96
(dd, J ) 15, 3 Hz, 1 H), 2.84 (d, J ) 15 Hz, 1 H), 2.51 (ddd, J
) 13, 4, 3 Hz, 1 H), 2.22 (dd, J ) 13, 10 Hz, 1 H), 1.39 (s, 3 H),
1.31 (s, 3 H); 13C NMR (CDCl3) δ 199.7, 171.4, 100.2, 99.4, 92.8,
78.4, 76.7, 66.4, 56.5, 52.6, 48.1, 47.7, 45.5, 35.8, 17.4, 17.3.
Anal. Calcd for C16H26O9: C, 53.03; H, 7.23. Found: C, 53.11;
H, 7.20.
Neat CH2I2 (5.30 mL, 65.6 mmol) was added to a suspension
of activated Zn dust (7.72 g, 118 mmol) in THF (60 mL) at
rt.15 After 30 min, TiCl4 (1.0 M in CH2Cl2, 14.4 mL, 14.4 mmol)
was added slowly in portions at 0 °C to avoid boiling of the
solvent. Removal of the ice bath was followed by stirring at
rt for 30 min. The dark suspension was then cooled to 0 °C,
and a solution of MOM-protected ketol (4.75 g, 13.1 mmol) in
THF (40 mL) was added. The mixture was vigorously stirred
at rt for 2 h, ether was added, and the mixture was carefully
poured into ice-cold aqueous HCl (1 N). After extraction of
the aqueous layer with ether (2×), the combined ether extracts
were washed with saturated aqueous NaHCO3 (1×). Drying
and concentration afforded a yellow oil. Purification by flash
chromatography (EtOAc/hexane, 1:5, 1:1, v/v) afforded carba-
phosphinate intermediate 33 as a colorless oil (3.80 g, 73%):
1H NMR (CDCl3) δ 5.29 (dd, J ) 4, 1 Hz, 1 H), 4.95 (dd, J )
4, 1 Hz, 1 H), 4.77 (d, J ) 7 Hz, 1 H), 4.62 (d, J ) 7 Hz, 1 H),
4.06 (dd, J ) 10, 1 Hz, 1 H), 3.86 (ddd, J ) 12, 10, 4 Hz, 1 H),
3.75 (s, 3 H), 3.36 (s, 3 H), 3.26 (s, 3 H), 3.24 (s, 3 H), 2.81 (dd,
J ) 15, 3 Hz, 1 H), 2.50 (dd, J ) 15, 1 Hz, 1 H), 2.32 (ddd, J
) 14, 4, 3 Hz, 1 H) 1.94 (dd, J ) 14, 12 Hz, 1 H), 1.37 (s, 3 H),
1.31 (s, 3 H); 13C NMR (CDCl3) δ 173.1, 138.9, 109.8, 99.8, 99.5,
92.7, 78.7, 72.3, 67.5, 56.6, 52.3, 47.8, 47.7, 39.2, 36.0, 17.7
(2). Anal. Calcd for C17H28O8: C, 56.65; H, 7.83. Found: C,
56.64; H, 7.78.
Ca r ba p h osp h in a te 1. A solution of carbaphosphinate
intermediate 33 (0.657 g, 1.82 mmol), ethyl (diethoxymethyl)-
phosphinate16 (1.07 g, 5.43 mmol), and benzoyl peroxide (0.10
g, 0.42 mmol) in anhydrous dioxane (5 mL) was heated at
reflux. After 14 h, more benzoyl peroxide (0.11 g, 0.44 mmol)
was added, and refluxing was continued for an additional 10
h. Concentration in vacuo afforded a yellow oil that was
purified by radial chromatography (4 mm thickness, EtOAc,
EtOAc/MeOH, 9:1, v/v) to afford 34 as a colorless oil: 1H NMR
(CDCl3) δ 4.75-4.85 (m, 1 H), 4.60-4.70 (m, 2 H), 4.05-4.30
(m, 3 H), 3.75-3.95 (m, 3 H), 3.65-3.75 (m, 5 H), 3.41 (s, 3
H), 3.20-3.25 (m, 6 H), 2.20-2.75 (m, 5 H), 1.75-1.95 (m, 1
H), 1.35-1.65 (m, 1 H), 1.20-1.40 (m, 15 H); 13C NMR (CDCl3)
δ 173.2, 173.1, 101.8, 99.9, 99.6, 99.2, 92.7, 92.6, 78.2, 78.1,
74.4 (2), 74.2 (2), 66.4, 65.5, 65.4, 65.2, 61.5, 61.4, 61.3, 56.4,
52.1, 47.6, 36.5, 36.1, 36.0, 35.9, 29.7, 29.6 (2), 26.3, 25.9, 25.0,
24.7, 17.6, 17.5, 16.5 (2), 16.3, 16.2, 15.0, 14.9.
Ca r ba h yd r oxym a lon a te In ter m ed ia te 31. Carbama-
lonate intermediate 30 (1.6 g, 4.3 mmol) was stirred in CF3-
CO2H/H2O (20:1, v/v, 5 mL) for 20 min. CF3CO2H and water
were removed in vacuo. The residue was refluxed in aqueous
HCl (6 N, 20 mL) for 2 h. After being cooled to rt, the mixture
was evaporated to dryness. The residue was dissolved in CH3-
OH (100 mL), treated with an ethereal CH2N2 solution at -20
°C, and allowed to stand overnight. Evaporation gave a brown
oil that was redissolved in CH3OH (10 mL). To this solution
were added 2,2,3,3-tetramethoxybutane9 (0.918 g, 5.15 mmol),
(CH3O)3CH (4.8 mL, 43 mmol), and (()-10-camphorsulfonic
acid (0.05 g, 0.2 mmol), and the solution was refluxed for 18
h. The resulting dark brown solution was treated with
powdered NaHCO3 (2 g) and concentrated. The resulting
residue was purified by flash chromatography (EtOAc/hexane,
1:1, v/v) to afford carbahydroxymalonate intermediate 31 as
a colorless oil (1.49 g, 80%): 1H NMR (CDCl3) δ 3.99 (ddd, J
) 12, 10, 5 Hz, 1 H), 3.95 (d, J ) 4 Hz, 1 H), 3.77 (s, 3 H), 3.73
(s, 3 H), 3.72 (s, 3 H), 3.68 (dd, J ) 10, 10 Hz, 1 H), 2.69-2.80
(m, 1 H), 1.82-2.01 (m, 4 H); 13C NMR (CDCl3) δ 175.7, 169.1,
168.6, 99.8, 99.4, 73.4, 71.3, 67.1, 53.0, 52.3, 52.0, 49.5, 47.9,
47.8, 37.5, 36.0, 35.4, 17.7, 17.6. Anal. Calcd for C19H30O11
C, 52.53; H, 6.96. Found: C, 52.72; H, 7.01.
:
Ca r ba h yd r oxym a lon a te In ter m ed ia te 32. To a solution
of carbahydroxymalonate intermediate 31 (0.266 g, 0.613
mmol) in THF (10 mL) at 0 °C was added solid t-BuOK (0.151
g, 1.35 mmol) in one portion. After 20 min, the reaction
mixture was cooled to -78 °C and stirring was continued at
-78 °C for 15 min. A solution of 2-(benzenesulfonyl)-3-
phenyloxaziridine14 (0.320 g, 1.23 mmol) in THF (2 mL) was
then cannulated into the reaction flask. The resulting mixture
was stirred at -78 °C for 1 h and quenched with saturated
aqueous NH4Cl (10 mL). After addition of ether, the organic
layer was separated and the aqueous layer was then extracted
with ether (3×). The combined organic layers were dried,
filtered, and evaporated. The resulting yellow oil was purified
by flash chromatography (EtOAc/hexane, 2:1, v/v) to give
carbahydroxymalonate intermediate 32 as white crystals
(0.208 g, 75%): mp 206-207 °C; 1H NMR (CDCl3) δ 4.01 (ddd,
J ) 12, 10, 5 Hz, 1 H), 3.95 (s, 1 H), 3.81 (s, 3 H), 3.78 (s, 3 H),
3.77 (s, 3 H), 3.76 (dd, J ) 11, 10 Hz, 1 H), 3.26 (s, 3 H), 3.21
(s, 3 H), 3.12 (ddd, J ) 13, 11, 3 Hz, 1 H), 1.94 (dd, J ) 12, 12
Hz, 1 H), 1.92 (dd, J ) 13, 13 Hz, 1 H), 1.83 (ddd, J ) 13, 5,
3 Hz, 1 H), 1.72 (br, 1 H), 1.55 (dt, J ) 13, 3, 3 Hz, 1 H), 1.25
(s, 3 H), 1.18 (s, 3 H); 13C NMR (CDCl3) δ 176.1, 171.3, 170.2,
99.6, 99.4, 78.7, 73.6, 70.0, 66.7, 53.4, 53.3, 53.1, 48.5, 47.8,
40.5, 37.7, 35.0, 17.9, 17.5. Anal. Calcd for C19H30O12: C,
50.66; H, 6.71. Found: C, 50.72; H, 6.73.
Carbaphosphinate intermediate 34 was directly treated with
aqueous HCl (6 N, 5 mL), and the mixture was refluxed for 3
h. After removal of the solvent in vacuo, the residue was
dissolved in water and the solution was neutralized to pH 7.4.
The solution was applied to AG-1 X8 anion exchange resin (20
-
mL) that had been equilibrated with 200 mM Et3NH+HCO3
(pH 7.3). The column was washed with water (40 mL) and
Ca r ba h yd r oxym a lon a te 6. Carbahydroxymalonate in-
termediate 32 (0.21 g, 0.47 mmol) was deprotected as described
for carbasuccinate intermediate 26 to afford carbahydroxy-
malonate 6 as a colorless film (100% yield based on NMR
analysis): 1H NMR (D2O, pH 8.5) δ 3.84 (ddd, J ) 12, 10, 5
Hz, 1 H), 3.53 (dd, J ) 10, 10 Hz, 1 H), 2.93 (ddd, J ) 13, 10,
3 Hz, 1 H), 2.14 (ddd, J ) 13, 5, 3 Hz, 1 H), 1.89 (dd, J ) 13,
13 Hz, 1 H), 1.82 (dd, J ) 13, 12 Hz, 1 H), 1.72 (ddd, J ) 14,
3, 3 Hz, 1 H); 13C NMR (D2O, pH 8.5) δ 180.7, 176.3, 175.4,
82.7, 76.7, 76.5, 73.2, 46.3, 42.5, 36.3; HRMS (FAB) calcd for
C10H11O10Na3 (M + H+) 361.0123, found 361.0129.
eluted with a linear gradient (200 mL + 200 mL, 200-500
-
mM) of Et3NH+HCO3 (pH 7.3). Fractions containing phos-
phorus were identified by the methods of Avila and Ames25
and then concentrated to dryness. The resulting white residue
was azeotroped six times with 2-propanol, dissolved in water,
and passed down a short column of Dowex 50 (H+). The
filtrate was neutralized to pH 7.7 with 0.1 N aqueous NaOH.
Concentration in vacuo afforded carbaphosphinate 1 as a white
foam (19%, from intermediate 33): 1H NMR (D2O, pH 7.7) δ
6.74 (d, J PH ) 572 Hz, 1 H), 3.73 (ddd, J ) 11, 9, 5 Hz, 1 H),
Ca r ba p h osp h in a te In ter m ed ia te 33. Powdered P2O5
was added in portions to a solution of intermediate 19 (8.10
g, 25.4 mmol) and (CH3O)2CH2 (50 mL) in CHCl3 (200 mL) at
(25) (a) Avila, L. Z. Ph.D. Dissertation, Stanford University, 1990.
(b) Ames, B. N. Methods Enzymol. 1966, 8, 115.

Inhibitor Ionization as a Determinant of Binding
J . Org. Chem., Vol. 61, No. 21, 1996 7381
3.15 (dd, J ) 9, 9 Hz, 1 H), 1.65-2.05 (m, 6 H), 1.35-1.50 (m,
1 H); 13C NMR (D2O, pH 7.75) δ 184.7, 81.2 (J PCCC ) 10 Hz),
78.3, 73.8, 43.3, 42.6 (J PCC ) 5 Hz), 37.2 (J PC ) 10 Hz), 36.1;
31P NMR (D2O, pH 7.7) δ 29.3; HRMS (FAB) calcd for C8H13O7-
Na2P (M + H+) 299.0272, found 299.0267.
mixture was filtered through Celite, and the filtrate was
concentrated in vacuo. Carbaphosphonate intermediate 36
was obtained as a white foam (0.776 g, 82%): 1H NMR (CDCl3)
δ 3.98 (ddd, J ) 10, 10, 6 Hz, 1 H), 3.70-3.85 (m, 1 H), 3.65-
3.80 (m, 9 H), 3.25 (s, 3 H), 3.23 (s, 3 H), 2.40-2.60 (m, 1 H),
2.20-2.40 (m, 2 H), 1.85-2.00 (m, 2 H), 1.59 (dd, J ) 13, 13
Hz, 1 H), 1.30-1.50 (m, 1 H), 1.30 (s, 3 H), 1.28 (s, 3 H); 13C
NMR (CDCl3) δ 175.7, 99.7, 99.2, 74.3 (J PCCC ) 17 Hz), 73.4,
66.6, 52.7, 52.4 (J POC ) 7 Hz), 52.1 (J POC ) 7 Hz), 47.7, 47.6,
39.3, 37.5, 30.3 (J PCC ) 3 Hz), 25.1 (J PC ) 141 Hz), 17.6 (2).
Anal. Calcd for C17H31O10P: C, 47.88; H, 7.33. Found: C,
47.74; H, 7.36. Carbaphosphonate intermediate 36 was depro-
tected under acidic conditions as described for carbaphosphi-
nate 1 to give carbaphosphonate 11 which was spectroscopi-
cally identical to previously synthesized material.1a,5a
Ca r ba p h osp h on a te In ter m ed ia te 35. Tetramethyl me-
thylenediphosphonate (2.12 g, 9.15 mmol) in THF (20 mL) was
deprotonated with n-BuLi (1.6 M in hexane, 5.8 mL, 9.3 mmol)
at -78 °C. The resulting solution was cannulated into a
solution of intermediate 19 (2.92 g, 9.17 mmol) in THF (20
mL) at -78 °C. After 10 min at -78 °C the reaction mixture
was warmed to rt and was stirred at rt overnight. Aqueous
NH4Cl was added, and the aqueous layer was saturated with
NaCl and extracted with EtOAc (3×). The combined organic
layers were dried and concentrated to an off-white foam.
Purification by radial chromatography (4 mm thickness,
EtOAc, EtOAc/MeOH, 9:1, v/v) afforded carbaphosphonate
intermediate 35 as a white foam (2.18 g, 56%): 1H NMR
(CDCl3) δ 5.99 (d, J PCH ) 18 Hz), 4.18 (d, J ) 10 Hz, 1 H),
3.95 (ddd, J ) 11, 10, 5 Hz, 1 H), 3.65-3.80 (m, 9 H), 3.58 (d,
J ) 14 Hz, 1 H), 3.24 (s, 3 H), 3.23 (s, 3 H), 2.51 (dd, J ) 14,
Ack n ow led gm en t. Research was supported by a
grant from the National Institutes of Health. Mass
spectral data were obtained at the Michigan State
University Mass Spectrometry Facility, which is sup-
ported, in part, by a grant (DRR-00480) from the
Biotechnology Research Technology Program, National
Center for Research Resources, National Institutes of
Health, or by Washington University Mass Spectrom-
etry Resource, an NIH Research Resource (Grant No.
P41RR0954). The NMR data were obtained on instru-
mentation that was purchased in part with funds from
NIH (1-S10-RR04750) and NSF (CHE-88-00770 and
92-13241).
3 Hz, 1 H), 2.00-2.15 (m, 2 H), 1.36 (s, 3 H), 1.31 (s, 3 H); 13
C
NMR (CDCl3) δ 174.5, 155.7 (J PCC ) 8 Hz), 109.6 (J PC ) 189
Hz), 100.1, 99.3, 74.4, 73.3 (J PCCC ) 18 Hz), 68.5, 52.8, 52.3
(J POC ) 6 Hz), 51.9 (J POC ) 6 Hz), 47.9, 47.8, 38.9 (J PCCC ) 8
Hz), 37.4, 17.5 (2). Anal. Calcd for C17H29O10P: C, 48.11; H,
6.89. Found: C, 48.13; H, 6.83.
Ca r ba p h osp h on a te In ter m ed ia te 36. Carbaphospho-
nate intermediate 35 (0.940 g, 2.21 mmol) was dissolved in
EtOAc (13 mL) and hydrogenolyzed over 10% Pd on C (0.250
g) at 50 psi H2 pressure for 54 h. After 30 and 48 h, the
catalyst was replaced with fresh 10% Pd on C (0.250 g). The
J O960709H