Use of the dehydrophos biosynthetic enzymes to prepare antimicrobial analogs of alaphosphin

Article abstract of DOI:10.1039/c8ob02860e

The C-terminal domain of the dehydrophos biosynthetic enzyme DhpH (DhpH-C) catalyzes the condensation of Leu-tRNA^Leu with (R)-1-aminoethylphosphonate, the aminophosphonate analog of alanine called Ala(P). The product of this reaction, Leu-Ala(P), is a phosphonodipeptide, a class of compounds that have previously been investigated for use as clinical antibiotics. In this study, we show that DhpH-C is highly substrate tolerant and can condense various aminophosphonates (Gly(P), Ser(P), Val(P), 1-amino-propylphosphonate, and phenylglycine(P)) to Leu. Moreover, the enzyme is also tolerant with respect to the amino acid attached to tRNA^Leu. Using a mutant of leucyl tRNA synthetase that is deficient in its proofreading ability allowed the preparation of a series of aminoacyl-tRNA^Leu derivatives (Ile, Ala, Val, Met, norvaline, and norleucine). DhpH-C accepted these aminoacyl-tRNA derivatives and condensed the amino acid with l-Ala(P) to form the corresponding phosphonodipeptides. A subset of these peptides displayed antimicrobial activities demonstrating that the enzyme is a versatile biocatalyst for the preparation of antimicrobial peptides. We also investigated another enzyme from the dehydrophos biosynthetic pathway, the 2-oxoglutarate dependent enzyme DhpA. This enzyme oxidizes 2-hydroxyethylphosphonate to 1,2-dihydroxyethylphosphonate en route to l-Ala(P), but longer incubation results in overoxidation to 1-oxo-2-hydroxyethylphosphonate. This α-ketophosphonate was converted by the pyridoxal phosphate dependent enzyme DhpD into l-Ser(P). Thus, the dehydrophos biosynthetic enzymes can generate not only l-Ala(P) but also l-Ser(P).

Full text of DOI:10.1039/c8ob02860e

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Use of the dehydrophos biosynthetic enzymes to
prepare antimicrobial analogs of alaphosphin†
Cite this: DOI: 10.1039/c8ob02860e
Despina J. Bougioukou,‡a Chi P. Ting, ‡a Spencer C. Peck,a,b Subha Mukherjeeb
a,b,c
and Wilfred A. van der Donk
*
The C-terminal domain of the dehydrophos biosynthetic enzyme DhpH (DhpH-C) catalyzes the conden-
sation of Leu-tRNALeu with (R)-1-aminoethylphosphonate, the aminophosphonate analog of alanine
called Ala(P). The product of this reaction, Leu-Ala(P), is a phosphonodipeptide, a class of compounds
that have previously been investigated for use as clinical antibiotics. In this study, we show that DhpH-C is
highly substrate tolerant and can condense various aminophosphonates (Gly(P), Ser(P), Val(P), 1-amino-
propylphosphonate, and phenylglycine(P)) to Leu. Moreover, the enzyme is also tolerant with respect to
the amino acid attached to tRNALeu. Using a mutant of leucyl tRNA synthetase that is deficient in its proof-
reading ability allowed the preparation of a series of aminoacyl-tRNALeu derivatives (Ile, Ala, Val, Met,
norvaline, and norleucine). DhpH-C accepted these aminoacyl-tRNA derivatives and condensed the
amino acid with L-Ala(P) to form the corresponding phosphonodipeptides. A subset of these peptides
displayed antimicrobial activities demonstrating that the enzyme is a versatile biocatalyst for the prepa-
ration of antimicrobial peptides. We also investigated another enzyme from the dehydrophos biosynthetic
pathway, the 2-oxoglutarate dependent enzyme DhpA. This enzyme oxidizes 2-hydroxyethylphosphonate
to 1,2-dihydroxyethylphosphonate en route to L-Ala(P), but longer incubation results in overoxidation to
1-oxo-2-hydroxyethylphosphonate. This α-ketophosphonate was converted by the pyridoxal phosphate
dependent enzyme DhpD into L-Ser(P). Thus, the dehydrophos biosynthetic enzymes can generate not
only L-Ala(P) but also L-Ser(P).
Received 16th November 2018,
Accepted 20th December 2018
DOI: 10.1039/c8ob02860e
rsc.li/obc
1-Aminoalkylphosphonates are structural analogues of α-amino peptidyl prodrug depends on the chemical structure of the
acids in which the carboxylic acid group has been replaced C–P containing warhead, and on the length and nature of the
by a phosphonic acid. They often inhibit enzymes involved in peptide.5
primary metabolic processes and thus they have been used
Efforts towards understanding the biosynthesis of natural
successfully as drugs or pesticides in medicine and agricul- phosphonopeptides6,7 have shown that Nature uses different
ture.1 Unmodified aminophosphonates cannot easily penetrate mechanisms8–10 to attach amino acids to an otherwise non-
cell membranes. Therefore, Nature and chemists have used bioavailable aminophosphonate. For example, in the case of
phosphonopeptide derivatives as prodrugs to release the phosphinothricin tripeptide (PTT), the warhead is a phosphi-
potential biological activity of a simple aminophosphonate nic acid mimic of glutamic acid and a potent inhibitor of glut-
inside the targeted cell, an example of a Trojan horse strategy.2 amine synthetase that is ligated to the dipeptide Ala–Ala.11
Several distinct permeases can transport phosphonopeptides The assembly of PTT is accomplished by three stand-alone
into a cell, where the phosphonate warhead is released upon non-ribosomal peptide synthetase modules (Fig. 1a).12–15 In
the action of cytoplasmic peptidases.3,4 The in vivo activity of a the biosynthesis of rhizocticins and plumbemycins the attach-
ment of different proteinogenic amino acids to the threonine
synthase antagonist (Z)-L-2-amino-5-phosphono-3-pentenoic
acid (APPA) defines the anti-fungal or anti-bacterial activity of
these phosphonopeptides.16–18 In this case, regardless of the
exact nature of the side chain, the amino acids are converted
to aminoacyl phosphates by ATP-dependent L-amino acid
ligases of the ATP-Grasp family19 before the formation of the
desired amide bonds (Fig. 1b).20
aInstitute for Genomic Biology, University of Illinois at Urbana-Champaign,
1206 West Gregory Drive, Urbana, IL 61801, USA. E-mail: vddonk@illinois.edu;
Fax: +1 (217) 244 8533; Tel: +1 (217) 244 5360
bDepartment of Chemistry, University of Illinois at Urbana-Champaign,
600 South Mathews Ave, Urbana, IL 61801, USA
cHoward Hughes Medical Institute, USA
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c8ob02860e
Interestingly, in the biosynthesis of dehydrophos in
Streptomyces luridus, the two enzymes involved in the for-
‡These authors contributed equally to this study.
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Fig. 1 Different strategies for generating amide bonds in natural phosphonopeptides. a. Activation of amino-acids via formation of an aminoacyl-
adenosine monophosphate intermediate during the biosynthesis of PTT. b. Activation of amino acids via formation of an aminoacyl phosphate inter-
mediate during biosynthesis of rhizocticin B. c. Activation of amino acids via formation of an aminoacyl-tRNA intermediate in the biosynthesis of
dehydrophos. d. Other examples of bioactive phosphonopeptides and aminophosphonates.
mation of the two peptide bonds (DhpH and DhpK) do not of fosfazinomycin in which the Arg-phosphonate linkage
interact directly with ATP. Instead, aminoacyl-tRNA intermedi- appears to be generated by an ATP-GRASP enzyme, but the Val-
ates are the co-substrates of these biocatalysts (Fig. 1c).21,22 Arg linkage is formed in a valinyl-tRNA dependent process.23,24
Perhaps this case should still be considered as an ATP-depen-
One of the most extensively studied phosphonopeptides,
dent example as the involvement of leucyl- and glycyl-tRNA alaphosphin (Fig. 1d), also known as alafosfalin, is
a
synthetase requires ATP to charge amino acids to tRNA. A final manmade compound with significant antibacterial pro-
strategy to generate amide bonds in phosphonopeptides is a perties25 that has been investigated as a prodrug for treatment
combination of different mechanisms such as the biosynthesis of urinary tract infections.26 The L-form of alaninephos-
Fig. 2 Dehydrophos biosynthetic pathway. a. Enzymatic steps involved in biosynthesis of dehydrophos.21 b. Enzymatic pathway for formation of
L-Leu-Ser(P) from DHEP. c. Structure of rac-1-HEP.
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phonate (L-Ala(P)), the warhead of alafosfalin, is a potent
inhibitor of alanine racemases isolated from Gram-positive
bacteria.3 However, the compound cannot penetrate the cell
membrane. During our investigations of the biosynthesis of
the phosphonopeptide dehydrophos we concluded that the
generation of L-Ala(P) is necessary for the formation of the
final product, because it acts as the acceptor of Leu during
DhpH-mediated tRNA-dependent peptide bond formation
(Fig. 1c). The generated dipeptide L-Leu-L-Ala(P) is then modi-
fied further by other enzymes encoded by genes downstream
of dhpH. Thus, while L-Leu-L-Ala(P) does not appear to be the
final product, this phosphonopeptide is generated as an inter-
mediate and is a natural analogue of synthetic alaphosphin.
Interestingly, the extensive structure–activity relationship
studies carried out by Roche during the development of ala-
phosphin3,27 showed that the intracellular amounts of Ala(P)
found in six-different microorganisms was much higher when
Leu-Ala(P) was administered instead of Ala–Ala(P), although
Fig. 3 31P NMR spectra of DhpA oxidation of rac-DHEP and DhpD
transamination to produce Ser(P). a. 31P NMR spectrum after conversion
■
▼
of rac-DHEP ( ) into OH-EP ( ) by DhpA in the presence of Fe(II)/α-KG/
O2 and L-ascorbic acid. b. 31P NMR spectrum of the reaction in panel a
●
with conversion of OH-EP into Ser(P) ( ) by DhpD in the presence of
L-alanine. c. 31P NMR spectrum after incubation of rac-Ser(P) (peak
marked with × corresponds to unknown impurity present in Ser(P)) with
DhpD in the absence of pyruvate. d. 31P NMR spectrum after partial con-
version of rac-Ser(P) into OH-EP by DhpD in the presence of pyruvate.
The unlabeled peak between ∼2–3 ppm is orthophosphate. Phosphonate
and orthophosphate chemical shifts vary with small changes in pH.
Fig. 5 Determination of the enantioselectivity of the reaction of DhpA
with 1-HEP. a. Design of experiments to determine the preference of
DhpA for the two enantiomers of 1-HEP using the known stereo-
selectivity upon reaction with HEPD. b. 31P NMR spectrum of reaction of
1.2 mM rac-1-HEP with 75 µM of Fe(II)-reconstituted DhpA in the pres-
ence of 5 mM α-KG, 1 mM L-ascorbic acid in 50 mM Na-HEPES, pH 7.5.
c. 1-HEP remaining after treatment with HEPD exposed to 75 µM of
Fe(II)-reconstituted DhpA, 5 mM α-KG, 1 mM L-ascorbic acid in 50 mM
Na-HEPES, pH 7.5. d. Assay of 1-HEP remaining after treatment with
HEPD and then spiked with 1.2 mM rac-1-HEP (0.6 mM total) and
treated with 75 µM of Fe(II)-reconstituted DhpA, 5 mM α-KG, 1 mM
L-ascorbic acid in 50 mM Na-HEPES, pH 7.5. Signal at ∼19.5 ppm corre-
Fig. 4 Kinetics of DhpA-mediated oxidation of various substrates at
25 °C. a. The assay with 2-HEP contained 0.01–0.2 mM 2-HEP, 2 mM
α-KG, 0.65 µM DhpA, and 50 mM Na-HEPES, pH 8.0 (slower rates were
observed when concentrations higher than 0.25 mM of 2-HEP were
used suggesting substrate inhibition). b. The assay with rac-1-HEP con-
tained: 0.01–0.7 mM rac-1-HEP, 2 mM α-KG, 3.25 µM DhpA, in 50 mM
Na-HEPES, pH 8.0. c. The assay with rac-DHEP contained: 0.01–1.0 mM
rac-DHEP,
2 mM α-KG, 3.25 µM DhpA, in 50 mM Na-HEPES,
pH 8.0. d. Summary of kinetic parameters for DhpA (data are not cor-
rected to account for the racemic nature of 1-HEP and DHEP; Km values
for the (S) enantiomers would be expected to be two-fold lower).
●
■
sponds to 1-HEP ( ); signal at ∼0.5 ppm corresponds to AP ( ); the
unlabeled signal at ∼3 ppm is orthophosphate.
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the former was eliminated from plasma faster that Ala–Ala(P).5 dation of the secondary alcohol of DHEP to the corresponding
The documented bioactivity of phosphonodipeptides as well carbonyl, we tested if we could produce the same signal by
as the unexplored possibility to generate these molecules enzy- incubating pyruvate and rac-Ser(P), with DhpD, a PLP-depen-
matically by using the tRNA-dependent ligase DhpH as catalyst dent aminotransferase from the dehydrophos pathway that
prompted us to investigate its substrate scope.
normally interconverts acetylphosphonate and Ala(P).21
Indeed, a 31P NMR signal with the same chemical shift was
observed (Fig. 3c & d). Thus, we infer from this set of experi-
ments that the 31P NMR signal at a −1.4 ppm is due to for-
mation of the unstable acylphosphonate 1-oxo-2-hydroxyethyl-
Results and discussion
Before investigating the condensation of α-amino acids with phosphonate (OH-EP). Therefore, DhpA not only can hydroxy-
aminophosphonates using DhpH, we first reexamined some of late 2-HEP at the alpha carbon but it can further oxidize the
the early steps of dehydrophos biosynthesis as we recognized secondary hydroxyl group to the corresponding carbonyl
an opportunity to potentially make 1-aminoalkylphosphonates (Fig. 2b). DhpD can then convert this intermediate to the
other than Ala(P). The biosynthesis of dehydrophos begins like known compound Ser(P).29
most phosphonate biosynthetic pathways7 with the rearrange-
We also incubated rac-1-HEP instead of DHEP with DhpA
ment of phosphoenolpyruvate (PEP) to phosphonopyruvate and observed formation of acetylphosphonate (AP) as verified
(PnPy) catalyzed by PEP phosphomutase (DhpE) and the sub- with authentic standard. By using an oxygen electrode probe,
sequent DhpF-mediated decarboxylation of PnPy to phospho- the steady-state kinetics of hydroxylation of 2-HEP and the
noacetaldehyde (PnAA).21 An iron-dependent alcohol dehydro- overoxidation of rac-DHEP and rac-1-HEP by DhpA in the pres-
genase DhpG then reduces PnAA to 2-hydroxyethyl-phospho- ence of 2-oxoglutarate and atmospheric molecular oxygen were
nate (2-HEP) (Fig. 2a). Subsequently, DhpA, a 2-oxoglutarate/ determined. DhpA exhibits a kcat/Km,2-HEP = 8.4 × 104 M−1 s−1
Fe(II)-dependent oxygenase, oxidizes 2-HEP28 to 1,2-dihydroxy- whereas the catalytic efficiency of DhpA for the oxidation of
ethylphosphonate (DHEP). When we examined the reaction racemic DHEP and 1-HEP was ca. 10-fold lower (Fig. 4), con-
mixture upon incubation of 2-HEP with DhpA and sistent with the hydroxylation of 2-HEP as the physiological
α-ketoglutarate (α-KG) for a longer period of time (i.e. 5 h reaction for DhpA.
instead of 0.5 h), we observed by 31P NMR spectroscopy the for-
When using rac-1-HEP as substrate, we observed oxidation
mation of a new signal at −1.4 ppm in the 31P NMR spectrum of roughly half of the substrate to AP by DhpA (Fig. 5b),
(Fig. 3a). Furthermore, when DHEP was incubated directly suggesting that the enzyme accepts only one of the enantio-
with DhpA followed by DhpD and alanine, the phosphonic mers. The stereochemical preference of DhpA for the oxidation
acid analog of serine, Ser(P),29 was formed (Fig. 3b). Reasoning of 1-HEP was determined as illustrated in Fig. 5a. We incu-
that the signal could be an acyl phosphonate by further oxi- bated rac-1-HEP in the presence of atmospheric oxygen with
Fig. 6 NMR analysis of L-Leu-L-Ser(P) ( ). a. 31P NMR spectrum of L-Leu-L-Ser(P) prepared under the following reaction conditions: 10 mM rac-Ser(P)
▼
●
(
, peak marked with × corresponds to an impurity of the starting material), 2 mM L-Leu, 4 mM ATP, 2 mg total tRNA (from E. coli), 250 µg BSA,
5 µM LeuRS, 20 U TIPP, and 50 µM DhpH-C. b. Enzymatic product in panel a spiked with synthetic L-Leu-rac-Ser(P) of panel c. 31P NMR spectrum of
L-Leu-rac-Ser(P). The products are diastereomeric at the α-carbon of Ser(P). d. 1H NMR spectrum of synthetic L-Leu-rac-Ser(P).
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the S-specific non-heme-iron dependent enzyme HEPD.30 As tially reacts with the S-enantiomer of 1-HEP. Indirectly, this
expected based on previous studies, 50% of 1-HEP (i.e. only also suggests that the DHEP product of DhpA has the
the S enantiomer) was converted to acetyl phosphate, an S-configuration, which was not investigated in previous studies
unstable intermediate prone to hydrolysis to form phosphate on dehydrophos biosynthesis.
and acetate (Fig. 5a). Thus, the product of this process is
In prior work, several other phosphonodipeptides with
mainly (R)-1-HEP. Subsequently, after removal of HEPD and structural changes in the phosphonic acid part have been
lyophilization of the reaction mixture, half of the lyophilized synthesized chemically and tested for their biological activity.
material was re-dissolved in buffer and incubated with excess In general replacement of Ala(P) with various phosphono
α-KG and Fe(II)-reconstituted DhpA (Fig. 5c). The other half of amino acids Xxx(P) lead to loss of antibacterial activity with
the lyophilized material was spiked with rac-1-HEP and treated the exception of L-Ser(P), Gly(P),31 L-Phe(P)32 and phosphonate
with DhpA in the same manner as the unspiked sample analogues of Glu and α-methylalanine.33 We tested herein the
(Fig. 5d). No formation of AP was observed in the non-spiked enzymatic formation by DhpH of L-Leu-L-Xxx(P) peptides using
sample when the DhpA was removed and the 31P NMR spec- commercial available aminophosphonates.34 We first observed
trum was recorded. Thus, we conclude that DhpA preferen- that Ser(P) was accepted as substrate by DhpH-C (Fig. 6). We
Fig. 7 31P NMR analysis of DhpH-C activity on various aminophosphonates. For rac-1-amino-propylphosphonate and aminomethyl phosphonate
each reaction contained: 10 mM aminophosphonate, 4 mM L-Leu, 4 mM ATP, 2 mg total tRNA from E. coli, 250 µg BSA, 5 µM LeuRS, 20 U TIPP, and
an aliquot of 50 µM DhpH-C in 100 mM Na-HEPES, 10 mM KCl, 20 mM MgCl2, pH 7.5. For rac-Val(P), rac-Phg(P) and rac-1-aminoethylphosphinic
acid each reaction contained: 5 mM aminophosphonate, 6 mM L-Leu, 6 mM ATP, 1.5 mg total tRNA from E. coli, 5 µM LeuRS, 2 U TIPP, and 50 µM
DhpH-C in 100 mM Na-HEPES, 10 mM KCl, 20 mM MgCl2, pH 7.5. a. Reaction with aminomethyl phosphonate [Gly(P)]. b. Reaction with
rac-1-amino-propylphosphonate. c. Reaction with rac-Val(P). d. Reaction with rac-Phg(P). e. Reaction with rac-1-aminoethylphosphinic
acid. f. Reaction with monomethyl Ala(P). Arrow denotes the 31P signal that belongs to phosphonodipeptide product. The chemical shifts of the
dipeptides are all downfield of the starting aminophosphonate (for spectral data, see ESI†). The peaks at ∼2 and 3 ppm are phosphate and AMP,
respectively.
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then evaluated whether Gly(P), 1-amino-propylphosphonate, presence of L-Ala(P). Assuming at this point that the integrity
Val(P) and phenylglycine(P) (Phg(P)) could act as Leu acceptors of the tRNALeu is important for recognition of the donor
in a leucyl-tRNA dependent process (Fig. 7) in comparison amino acid covalently attached to it, we employed a different
with synthetic standards of the products (see ESI†). In most strategy in order to mischarge tRNALeu with standard and non-
cases, the reaction went to partial completion when a racemic standard amino acids. Martinis and coworkers reported
α-amino phosphonic acid was employed and only one mutants of E. coli leucyl-tRNA synthetase with impaired
diastereomer of the product was formed presumably due editing activity which do not hydrolyze misacylated
to the stereochemical preference of DhpH-C for tRNALeu
molecules.37
One
of
these
mutants,
L-aminophosphonates as shown by its selectivity for L-Ala(P).21 LeuRS-Y330A/D342A/D345A from E. coli, was used in this study
Thus, enzymatic resolution of racemic α-amino phosphonic due to its demonstrated high level misacylation activity.37
acids can in principle be accomplished by this method. Indeed, by using the LeuRS triple mutant for charging tRNALeu
Another biocatalyzed kinetic resolution of racemic mixtures of instead of wt LeuRS, DhpH-C was able to accept several acti-
chiral α-amino phosphonic acids has been reported recently, vated amino acids (Fig. 8). Perhaps the second most interest-
but the enzyme responsible for the transformation is unknown ing dipeptide formed after alaphosphin (Fig. 8e) is norvalyl-1-
and only whole-cells were used.35 In accordance to previous aminoethylphosphonic acid (Nva-Ala(P), Fig. 8b), which when
studies,21 pSer(P) and 1-hydroxy-2-amino phosphonate were combined with nocardicin A has been shown to possess
not accepted by DhpH-C. The dianionic form of a phosphonate potent synergistic antibacterial activity.38 In these assays, the
does not seem to be required, because both the phosphinic conversion varied from 90% for norvaline and norleucine to
form of Ala(P) (Fig. 7e) and the monomethyl ester of Ala(P) less than 10% for alanine. We note that the overall efficiency
were tolerated by DhpH-C (Fig. 7f). However, the tetrahedral of these reactions dependents on both the efficiency of the
geometry of phosphonic acids seems to be important for reco- triple LeuRS mutant to recognize non-canonical amino acids
gnition because Ala(P) could not be replaced by Ala.
and the ability of DhpH-C to recognize the mischarged
We also investigated the tolerance of DhpH-C towards aminoacyl-tRNALeu. Given the precedent of 1-aminoalkylphos-
amino acid donors other than Leu. We initially tested if phonic acids as antimicrobial agents, the phosphonodipeptides
DhpH-C can tolerate a minimal substrate of Leu attached to prepared in this study were tested against Escherichia coli
the 3′ hydroxyl group of adenosine as was reported recently for ATCC 25922.39 Amino acyl analogs of alaphosphin, such as
the Escherichia coli aminoacyl tRNA transferase.36 Therefore, L-norvaline-L-Ala(P), L-leucine-L-Ala(P), L-norleucine-L-Ala(P)
we synthesized a mixture of 2′- and 3′-leucyl adenosine follow- and L-methionine-L-Ala(P), showed antimicrobial activity with
ing the reaction scheme and purification method reported in clear zones of inhibition by agar disk diffusion assay (see ESI,
the literature.36 Unfortunately, neither of the two consti- Fig. S2†). However, compounds containing other phosphonic
tutional isomers was a substrate at 2 mM for DhpH-C in the acids instead of Ala(P) were inactive against this strain.
Fig. 8 31P NMR analysis of DhpH-C activity on various amino acids with L-Ala(P). Each reaction contained 5 mM Ala(P), 6 mM amino acid, 6 mM
ATP, 1.5 mg total tRNA from E. coli, 6 µM LeuRS (triple mutant), 10 U TIPP, and DhpH-C (50 µM) in 100 mM Na-HEPES, 10 mM KCl, 20 mM MgCl2,
pH 7.5 (500 µL total volume). Arrow indicates the expected product. The identity of the products was confirmed by MS. For spectral data, see ESI.†
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(1.0 equiv.). The reaction vessel was evacuated and backfilled
with nitrogen (three times in total) followed by the addition of
Conclusions
We demonstrate in this study that DhpH-C has broad substrate freshly distilled hexamethyldisilazane (5 mL per mmol amino-
tolerance with respect to its two substrates, leucyl-tRNA and phosphonate). The reaction mixture was heated to 150 °C and
aminophosphonates, and can be used to generate a range of kept at this temperature for 5 h or until the aminophospho-
phosphonodipeptides. Other amino acids attached to tRNALeu nate dissolved. The reaction vessel was cooled to room temp-
are accepted by the enzyme and various L-aminophosphonates erature and concentration in vacuo. The crude residue was
could be condensed with Leu. The enzyme only accepts the used in the following step without purification. In a separate
L-enantiomer of the 1-aminoalkylphosphonate and hence can reaction vessel, an oven-dried round bottom flask was charged
be used to resolve racemic mixtures. DhpH-C can therefore with N-benzyloxycarbonyl L-amino acid (1 equiv.). The reaction
serve as a biocatalyst for the preparation of phosphonodipep- vessel was evacuated and backfilled with nitrogen (three times
tides that have been shown to be efficient antimicrobial agents in total), cooled to 0 °C followed by the addition of ethyl
in previous studies. This work provides a blueprint for investi- acetate (5 mL per mmol amino acid), freshly distilled triethyl-
gating the substrate selectivity of other tRNA-dependent bio- amine (1 equiv.) and then ethyl chloroformate (1 equiv.). The
synthetic systems.40–43
reaction mixture was stirred for 30 minutes at 0 °C followed by
the dropwise addition of the crude residue containing TMS-
protected aminophosphonate in EtOAc. The reaction vessel
was maintained at this temperature for 20 minutes, warmed to
room temperature for 20 minutes, and finally heated to 80 °C
for 1 h. The reaction mixture was quenched with saturated aq.
NaHCO3, and the resulting suspension was acidified with 3 M
HCl until pH 1. The crude mixture was extracted with EtOAc
(3 × 5 mL) and the combined organic layers were washed with
brine, dried over NaSO4, and concentrated in vacuo. The crude
residue was added glacial acetic acid (5 mL per mmol amino-
phosphonate) followed by 48% hydrobromic acid (5 mL per
mmol aminophosphonate) and stirred for 2 h at room tem-
perature. The reaction mixture was concentrated in vacuo and
the crude residue was purified by CombiFlash® Rf+ equipped
with a 15.5 g RediSep Rf Gold C18Aq column. Acetonitrile and
H2O were used as the mobile phases. The purification method
began with 100% H2O for 5 min and then a gradient of
0–100% aq. MeCN was applied over 5 min at 30 mL min−1 flow
rate. Fractions containing the product were concentrated
in vacuo to afford the pure phosphonodipeptide. For spectro-
scopic data of synthetic compounds, see the ESI.†
Experimental section
General procedures
DhpH and DhpA were obtained by heterologous expression in
E. coli of the corresponding genes from Streptomyces luridus
and the corresponding activity assays were carried out as
described previously.21,22 Unless otherwise stated, all reactions
to produce authentic standards were performed in oven-dried
or flame-dried glassware under an atmosphere of dry nitrogen
or argon. Triethylamine and ethyl acetate were distilled from
calcium hydride prior to use. Ethyl chloroformate was freshly
distilled immediately before use. Reactions were monitored by
thin layer chromatography (TLC) on Silicycle Siliaplate™
G TLC plates (250 μm thickness, 60 Å porosity, F-254 indicator)
and visualized by UV irradiation and staining with p-anis-
aldehyde, phosphomolybdic acid, or potassium permanganate
developing agents. Volatile solvents were removed under
reduced pressure using a rotary evaporator. Flash column
chromatography was performed using Silicycle F60 silica gel
(60 Å, 230–400 mesh, 40–63 μm). Phosphonates were purified
using CombiFlash® Rf+ equipped with a 15.5 g RediSep Rf Gold
C18Aq column using acetonitrile and water as mobile phases.
Proton nuclear magnetic resonance (1H NMR) and carbon
nuclear magnetic resonance (13C NMR) spectra were recorded
on a Agilent-600 MHz NMR spectrometer operating respectively
Conflicts of interest
The authors declare no competing financial interest.
1
at 600 MHz for H, and 150 MHz for 13C. Chemical shifts are
Acknowledgements
reported in parts per million (ppm) with respect to the residual
solvent signal CDCl3 (1H NMR: δ = 7.26; 13C NMR: δ = 77.16)
and D2O (1H NMR: δ = 4.79). Peak multiplicities are reported as
follows: s = singlet, bs = broad singlet, d = doublet, t = triplet,
dd = doublet of doublets, td = triplet of doublets, m = multiplet,
app = apparent. High-resolution mass spectra (HRMS) were
obtained using a Waters SynApt mass spectrometer.
This work was supported by the National Institutes of Health
(GM 077596 to W. A. V.; F32 GM129944 to C. P. T.). W. A. V. is a
Howard Hughes Medical Institute Investigator. An Agilent
600 MHz NMR spectrometer used in this work was purchased
in part with a grant from the National Institutes of Health
(S10-RR028833).
General procedure for chemical synthesis of
phosphonodipeptides
Notes and references
Phosphonates were prepared using a modified procedure
reported by Solodenko and co-workers.44 A 10 mL flame-dried
reaction tube was charged with aminophosphonate
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