Structure-activity relationships of the phosphonate antibiotic dehydrophos

Article abstract of DOI:10.1039/c0cc02958k

Synthetic derivatives of the phosphonate antibiotic dehydrophos were tested for antimicrobial activity. Both the phosphonate monomethyl ester and the vinyl phosphonate moiety proved to be important for bacteriocidal activity of the natural product. The Ro

Full text of DOI:10.1039/c0cc02958k

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Structure–activity relationships of the phosphonate antibiotic
dehydrophoswz
Michael Kueminab and Wilfred A. van der Donk*ab
Received 1st August 2010, Accepted 2nd September 2010
DOI: 10.1039/c0cc02958k
Synthetic derivatives of the phosphonate antibiotic dehydrophos
were tested for antimicrobial activity. Both the phosphonate
monomethyl ester and the vinyl phosphonate moiety proved to be
important for bacteriocidal activity of the natural product.
Many naturally occurring phosphonates are produced
as peptides to facilitate uptake by the target organism via
nonspecific oligopeptide permeases.6,7 Hydrolysis of the
peptide linkages inside the organism then releases the active
moiety. Alternatively, for some phosphonate-containing
peptides such as K-26, the entire peptide is important for
enzyme inhibition.8,9 Dehydrophos has several unusual charac-
teristics. The vinylphosphonate moiety may be important for
its mode of action as it represents an electrophilic group that
could covalently modify its target. This electrophile would be
lost upon peptidase action, because the resulting enamine
would hydrolyze. Another unique aspect of the dehydrophos
structure is its methyl ester; no other examples have been
reported of esterified phosphonate natural products. Methyla-
tion of a phosphonate reduces its negative charge and results
in a charge distribution closer to that of a tetrahedral inter-
mediate in carboxylate metabolism. Therefore, the phospho-
nate methyl ester group in dehydrophos may mimic such
an intermediate resulting in inhibition of a target enzyme
comparable to synthetic phosphonate/phosphonamide containing
peptides that were identified as transition state analogue
inhibitors of proteases.10 To investigate the importance of
the various structural motifs found in dehydrophos, several
analogs were prepared in this study.
The phosphonate tripeptide dehydrophos (1) produced by
Streptomycis luridus is a broad spectrum antibiotic against
both Gram-positive and Gram-negative bacteria.1 Initially
discovered in 1984, the correct structure of dehydrophos was
determined in 2007 by comparison with a synthetic sample.
The C-terminal residue, which is attached to a glycine–leucine
dipeptide, was demonstrated to be the monomethyl ester of
1-aminovinylphosphonate (Fig. 1).2 This unique structure
attracted interest in the biosynthesis of dehydrophos3,4 and
also raised questions about its mode of action. As part of a
program to provide more insight into phosphonate natural
products,5 we present structure activity relationship studies on
dehydrophos and synthetic analogs to address these questions.
To test the importance of the methyl ester, the phosphonic
acid derivative 2 and its dimethyl analog 3 (Fig. 1) were
prepared. N-Cbz protected serine with a tert-butyldimethyl-
silyl (TBDMS) protected side chain was decarboxylated with
lead tetraacetate (Scheme 1).11 The N,O-acetal product was
then reacted with trimethylphosphite in the presence of titanium
tetrachloride.12 Hydrogenation of the Cbz protecting group
Fig. 1 Structures of synthetic compounds 1–6. Note that in aqueous
solutions, the anions of 4 and 6 are not chiral at phosphorus.
a Department of Chemistry and Howard Hughes Medical Institute,
University of Illinois at Urbana-Champaign, 600 S. Mathews Ave.,
Urbana, IL, 61801, USA. E-mail: vddonk@illinois.edu;
Fax: +1 217 244 8533; Tel: +1 217 244 5360
b Institute for Genomic Biology, University of Illinois at
Urbana-Champaign, 1206 West Gregory Drive, Urbana, IL,
61801, USA.
w This article is part of the ‘Enzymes and Proteins’ web-theme issue for
ChemComm.
z Electronic supplementary information (ESI) available: Experimental
details on the syntheses, agar diffusion assays and liquid broth studies
are presented. See DOI: 10.1039/c0cc02958k
Scheme 1 Synthesis of desmethyl dehydrophos 2: (a) Pb(OAc)4,
DMF, 0 1C to 25 1C; (b) TiCl4, P(OMe)3, CH2Cl2, ꢀ78 1C to 25 1C;
(c) Pd/C, H2(g), MeOH, 25 1C; (d) Cbz-Leu-OH, EDC, CH2Cl2, 25 1C; (e)
Pd/C, H2(g), MeOH, 25 1C; (f) Cbz-Gly-OH, EDC, CH2Cl2, 25 1C;
(g) TBAF, THF, 25 1C; (h) MsCl, NEt3, CH2Cl2, 0 1C to 25 1C;
(i) DBU, CH2ClCH2Cl, 84 1C; (k) BBr3, hexanes, toluene 0 1C to 70 1C.
c
7694 Chem. Commun., 2010, 46, 7694–7696
This journal is The Royal Society of Chemistry 2010

Scheme 2 Synthesis of the derivative 4. (a) Toluene, 110 1C;
(b) HPO(OMe)2, 100 1C, (c) HCl in MeOH, 25 1C; (d) Boc-Leu-OH,
EDC, NMM, CH2Cl2, 25 1C; (e) HCl in MeOH, 65 1C; (f) Boc-
Gly-OH, EDC, NMM, CH2Cl2, 25 1C; (g) HCl in MeOH, 65 1C;
(h) NaOH, H2O, 25 1C.
Fig. 2 Bioassay against E. coli: (ꢀ) negative control (water), (+)
positive control (ampicillin), left: compounds 1, ent-1, 2, 3; right:
compound 4, 4b, 5 and 5b. See also ESIz.
followed by solution phase peptide synthesis provided the
protected tripeptide. Treatment with TBAF resulted in the
alcohol that was activated as the methylsulfonate to introduce
the vinyl phosphonate after elimination. Global deprotec-
tion with boron tribromide then resulted in desmethyl
dehydrophos 2. The dimethyl derivative 3 was prepared
similarly (see ESIz).
on M9 media resulted in zones of growth inhibition for com-
pounds 1–5 against E. coli (Fig. 2, Table 1). The diastereomer
of 4 (4b) showed no activity and 5b demonstrated only a small
zone of inhibition. With B. subtilis compounds 1 and 3–5
resulted in a zone of inhibition but not the phosphonic acid 2
(see ESIz). D-Stereochemistry in the alanine derivatives 4b and
5b resulted in inactive compounds. Bioactivity was also not
detected for either diastereomer of the serine derivatives 6
against both E. coli and B. subtilis, and on rich LB media none
of the compounds 1–6 showed antimicrobial activity under the
tested conditions. This latter observation suggests that some
components in LB can overcome the inhibition of the target(s)
of these compounds thereby rescuing bacterial growth. IC50
values (growth inhibition) were determined for E. coli in liquid
broth (90% M9 and 10% LB media) to quantitatively com-
pare the activities (Table 1).
To test the importance of the electrophilic vinylphosphonate,
compound 4 (Fig. 1) lacking the vinyl functionality was
prepared resulting in a methylated phosphonate analog of
L-alanine as the C-terminal residue. N-(Triphenylmethyl)
protected dimethyl 1-aminoethyl-phosphonate was synthesised
as a racemic mixture as illustrated in Scheme 2.13 After peptide
synthesis, the N-Boc protecting group was removed under
acidic conditions, and the monomethyl phosphonate ester was
isolated after treatment with 10% aqueous NaOH.4 Compound
4 and its diastereomer with the opposite stereochemistry of
the aminophosphonate (4b) were subsequently separated by
RP-HPLC.
(1R)-Aminoethylphosphonate (R-AlaP), the compound that
would result from proteolysis of the C-terminal amide bond of
5 has been studied in-depth as the active component of
alaphosphin.14–18 It inhibits alanine racemase and therefore
bacterial cell wall synthesis. The growth inhibition can be
rescued by providing D-alanine and it is known that alapho-
sphin peptide analogs with an S-AlaP residue are less potent.16
The growth inhibition by compound 5 tested in this study was
also rescued by D-alanine and since the diastereomer 5b was
less active (E. coli) or not active (B. subtilis), it is very likely
that alanine analog 5 of dehydrophos is also hydrolysed inside
the cell and inhibits alanine racemase by a mechanism similar
to that of alaphosphin. The effect of compounds 1–4 was not
In addition, peptide 5 and its epimer at C1 of residue 3 (5b)
that contain neither the methyl ester nor the vinyl moiety of
the natural product were prepared using boron tribromide to
deprotect the tripeptide.4 The resulting two diastereomers
were isolated by RP-HPLC, and a crystal structure of stereo-
isomer 5 was used to assign the stereochemistry of both
compounds.4 Derivatives containing the phosphonate analog
of serine (6a and 6b, Fig. 1, epimers at C1 of the methyl
phosphonate) were also prepared as they are potential
precursors in the biosynthesis of the vinyl group (see ESIz).
Finally, the enantiomer of dehydrophos (ent-1) was synthesised
as described previously for 12 to analyse the effect of a D-leucine
as the second residue. The introduction of a D-amino acid in
dehydrophos was anticipated to affect its bioactivity if the
entire peptide interacts with its target. If not, (i.e. if the peptide
requires proteolysis for bioactivity) then the D-Leu could still
affect bioactivity because the uptake and proteolysis processes
may be less effective. Therefore, another diastereomer of 5
with a D-Leu as the second residue (ent-5b) was also prepared.
Because compound 5 requires proteolysis for activity (vide infra),
introduction of a D-Leu was envisioned to independently report
on the importance of stereochemistry in the 2nd position for
uptake and proteolysis.
Table 1 Comparison of the dehydrophos analogs 1–6
Compound
E. colia
B. subtilisa
IC50/mM, E. coli
1
ent-1
2
3
4
4b
5
5b
ent-5
ent-5b
6
++
++
+
+
+
—
++
+
++
+
—
++
++
—
181 ꢁ 6
299 ꢁ 18
>1000
>1000
>1000
ndb
42 ꢁ 5
121 ꢁ 17
34 ꢁ 22
182 ꢁ 17
ndb
+
++
—
+
—
+
—
—
Compounds 1–6 and their stereoisomers were tested for
antimicrobial activity with Escherichia coli and Bacillus subtilis
ATCC 6633 as indicator strains. Solid agar diffusion bioassays
a
Solid agar diffusion assays: + zone of inhibition, — no activity
b
detected. nd: not determined.
c
This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 7694–7696 7695

rescued by D-alanine, suggesting that their inhibition mecha-
nism is different.
At any rate, we cannot make any conclusions regarding the
need for proteolysis of dehydrophos on the basis of these SAR
studies.
If proteolysis is also required for compounds 1–4, then
hydrolysis of the C-terminal amide bond of compound 4
would release the methyl ester of R-AlaP. Previous studies
with alanine racemase of B. stearothermophilus showed
no inhibition by methyl R-AlaP.19 Nevertheless, activity is
detected for tripeptide 4 consistent with a different target for
this analog, which is also consistent with the lack of rescue by
D-Ala. Compared to dehydrophos, saturation of the double
bond in 4 reduces the antimicrobial activity against E. coli
substantially (Table 1) demonstrating the importance of the
vinyl phosphonate functionality (assuming as in any SAR
studies that 4 has the same target as 1). The inactive serine
analogs 6 also support this conclusion.
In summary, the structure–activity comparisons presented
here demonstrate that both the vinyl and methyl ester func-
tionalities are key for dehydrophos bioactivity, and these
findings are consistent with pyruvate dehydrogenase/oxidase
as its target.
This work was supported by the NIH (PO1 GM077596 to
WAV). MK was supported by a postdoctoral fellowship from
the Swiss National Science Foundation.
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against B. subtilis, and the activity of 2 against E. coli was
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groups are important for bioactivity. If intracellular hydrolysis
of the C-terminal amide bond of dehydrophos is important for
activity, this process would release methyl acetylphosphonate,
a phosphonate analog of pyruvate. This compound is known
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acetylphosphonate is a stronger inhibitor of pyruvate oxidase
at neutral pH than acetylphosphonate, suggesting that the
methyl ester might indeed be beneficial to reduce the charge
of the phosphonate. Neither acetylphosphonate nor methyl
acetylphosphonate showed any antimicrobial activity in our
assays, presumably, because they did not enter into the cells.
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7696 Chem. Commun., 2010, 46, 7694–7696
This journal is The Royal Society of Chemistry 2010