Identification of ACE pharmacophore in the phosphonopeptide metabolite K-26

Article abstract of DOI:10.1016/j.bmcl.2007.11.130

The naturally occurring phosphonotripeptide K-26 is a potent angiotensin converting enzyme (ACE) inhibitor containing an α-amino phosphonic acid analogue of tyrosine. Previous studies have demonstrated that canonical peptide analogues of K-26 are micromolar inhibitors of ACE. To ascertain the structure-activity relationships in this class of ACE inhibitory natural products, K-26 and eight analogues were chemically synthesized and evaluated. Phosphonyl substitution was found to be the critical determinant of activity, resulting in a 1500-fold increase in ACE inhibition versus carboxyl analogues. Secondarily, the absolute configuration of the terminal α-amino phosphonate and N-acetylation were found to significantly modulate ACE inhibitory activity.

Full text of DOI:10.1016/j.bmcl.2007.11.130

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 3068–3071
Identification of ACE pharmacophore in the
phosphonopeptide metabolite K-26
Ioanna Ntai and Brian O. Bachmann*
Department of Chemistry, Vanderbilt University, Nashville, TN 37235, USA
Received 16 October 2007; revised 28 November 2007; accepted 30 November 2007
Available online 5 December 2007
Abstract—The naturally occurring phosphonotripeptide K-26 is a potent angiotensin converting enzyme (ACE) inhibitor containing
an a-amino phosphonic acid analogue of tyrosine. Previous studies have demonstrated that canonical peptide analogues of K-26 are
micromolar inhibitors of ACE. To ascertain the structure–activity relationships in this class of ACE inhibitory natural products,
K-26 and eight analogues were chemically synthesized and evaluated. Phosphonyl substitution was found to be the critical deter-
minant of activity, resulting in a 1500-fold increase in ACE inhibition versus carboxyl analogues. Secondarily, the absolute
configuration of the terminal a-amino phosphonate and N-acetylation were found to significantly modulate ACE inhibitory activity.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Phosphonic acids are important pharmacophores of sig-
nificant relevance to human health. Under physiological
conditions, the phosphonate moiety (R–PO3H2,
pKa ꢀ 2.5, 8.01) can serve as an isosteric replacement
for phosphate or carboxylate functional groups, which
are ubiquitous ligands in the active sites of many en-
zymes. The carbon–phosphorus bond renders phospho-
nates hydrolytically stable relative to their phosphate
congeners and the mode of action of phosphonates is of-
ten either through competitive interaction with substrate
binding regions or as an analogue of tetrahedral transi-
tion states. As a result, synthetic phosphonates find real
world application in medicine as antibiotics, antivirals,
antiosteoclastics, and as environmentally benign herbi-
cides.2 The phosphonate moiety is also well represented
Figure 1. Bioactive natural C–P containing metabolites.
in biologically active natural products and can be found
in such diverse molecular entities as bialaphos (5; anti-
fungal, antibiotic, herbicide), fosfomycin (4; antibiotic),
fosmidomycin (3, antimalarial), and K-26 (1; ACE
inhibitor) (Fig. 1).2
sess ACE inhibitory activity comparable to the widely
prescribed antihypertensive drug Captopril in both
in vitro assays and in intravenously administered animal
models. NMR, mass spectrometry, degradation, and syn-
thetic studies have demonstrated that K-26 is comprised
of N-acetylated L-isoleucine, L-tyrosine, and the nonpro-
teinogenic amino acid, (R)-1-amino-2-(4-hydroxy-
phenyl)ethylphosphonic acid (AHEP, 2). This
K-26 was initially discovered via ACE bioactivity guided
fractionation of extracts of a soil dwelling prokaryote,
‘actinomycete strain K-26’.3–5 It has been reported to pos-
‘phosphonotyrosine’ functional group is shared among
several ACE inhibitor peptide analogues of K-26 pro-
duced by various Streptosporangium6 and Actinomadura7
species. The stereochemistry of AHEP has been estab-
lished by X-ray analysis of the ethyl ester of K-26 (1c).
Keywords: Natural product; Phosphonate; K-26; Angiotensin; ACE;
Inhibitor.
*
Corresponding author. Tel.: +1 615 322 8865; e-mail:
brian.bachmann@vanderbilt.edu
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.11.130

I. Ntai, B. O. Bachmann / Bioorg. Med. Chem. Lett. 18 (2008) 3068–3071
3069
Scheme 1. Synthesis of two diastereomers of K-26. Reagents and conditions: (a) P(OEt)3, THF, 0 ! 70 ꢁC; (b) NH2OH–HCl, pyridine, ethanol,
25 ꢁC; (c) Zn/HCOOH, 25 ꢁC; (d) EDC–HCl, HOBt, 2,4,6-trimethylpyridine, DMF, 25 ꢁC; (e) H2, Pd/C, 25 ꢁC; (f) TMSI, thioanisole, MeCN, 0 ꢁC.
Synthetic ACE inhibitors are widely prescribed for car-
diovascular diseases, including high blood pressure,
heart failure, heart attack, and kidney failure, and have
combined annual sales in excess of six billion dollars.8
ACE has a critical role in the regulation of blood pres-
sure by catalyzing the hydrolytic cleavage of His-Leu
dipeptide from decapeptide angiotensin I to yield angio-
tensin II, a potent vasoconstrictor. ACE also cleaves
bradykinin resulting in antagonistic effects to angioten-
sin II. Typical ACE inhibitors consist of peptide or pep-
tidomimetic structures incorporating a functional group
that interacts with the active site zinc, such as a thiol, a
carboxylate, a phosphonic acid (Fig. 2).
ucts, we have synthesized K-26 and several peptide ana-
logues (Table 1).
Production levels of K-26 in the producing organism are
low (est. <10 lg/L in our hands), necessitating the devel-
opment of a synthetic route for K-26 and analogues.
AHEP was synthesized as previously described10–14 with
minor modifications (Scheme 1). Commercially avail-
able 4-benzyloxyphenylacetyl chloride (9) was reacted
with triethyl phosphite in an Arbuzov type reaction to
yield the corresponding a-keto phosphonate (not iso-
lated). Subsequent conversion to the oxime (10) with
hydroxylamine and reduction with zinc/formic acid re-
sulted in racemic AHEP diethyl ester (11). Standard
peptide coupling conditions15 were employed in cou-
pling the free base of AHEP diethyl ester to
Ac-Ile-Tyr(Bzl)-COOH (12). In a two-step deprotection
process, benzyl groups were removed from the resultant
tripeptide by catalytic hydrogenation and ethyl groups
were removed by reaction with iodotrimethylsilane. This
synthetic route (Scheme 1) resulted in the preparation of
a mixture of two major diastereomers of K-26 (1a and
1b), which were readily separated via C18 chromatogra-
phy using a gradient of 5–40% acetonitrile containing
0.1% trifluoroacetic acid. Stereochemistry was assigned
by comparison of chemical shift values and NOESY
cross-peak correlations to peptide analogue Ac-Ile-
Tyr-Tyr (15a) and Ac-Ile-Tyr-D-Tyr (15b). Des-acetyl
K-26 (14a and 14b) was synthesized in an analogous
fashion to K-26 with the exception that Cbz-Ile-
Tyr(Bzl)-COOH was coupled to AHEP in place of Ac-
Ile-Tyr(Bzl)-COOH. Stereochemical assignments of the
two des-acetyl K-26 diastereomers (14a and 14b) were
also established based on chemical shift values and
NOESY correlations in comparison to synthetic Ile-
Tyr-Tyr (16a) and Ile-Tyr-D-Tyr (16b). Of note, while
the structure of K-26 has been previously unambigu-
ously determined, this work provides the first high-reso-
lution NMR spectroscopic data for this series of
compounds.
It has been previously suggested that the phosphonate
moiety of K-26 may interact with the active site zinc
atom and contribute to the potent inhibitory activity
of this class of compounds.4 However, small proteino-
genic amino acid containing di- and tri-peptides have
also been reported to possess significant ACE activity.
For instance L-Ile-L-Tyr and other analogues of K-26
have been reported to be low micromolar inhibitors of
mammalian ACE9 and it has remained unclear how
the ACE activity of K-26 is modulated by the substitu-
tion of carboxyl group functionality and N-acetylation.
To identify the structural determinants of ACE activity
in the class of chiral a-amino phosphonyl natural prod-
NH2
O
HS
N
O
OH
N
HOOC
N
H
O
6, captopril
OH
O
7, lisinopril
COOH
O
OH CH3
P
O
H
H
N
N
H3C
N
H
NH2
CH3
O
The method outlined by Holmquist and coworkers16
for measuring ACE activity was adapted for assay in
96-well format (see Supplementary data). In short,
ACE activity was extracted from rabbit lung acetone
O
O
8, RXP-407
Figure 2. Representative ACE inhibitors.

3070
I. Ntai, B. O. Bachmann / Bioorg. Med. Chem. Lett. 18 (2008) 3068–3071
Table 1. Measured IC50 values of K-26 and analogues
of FAPGG hydrolysis was obtained by measuring the
change in absorbance at 340 nm versus time. IC50 val-
ues were obtained by fitting triplicate measurements
to a sigmoidal dose response curve. As a benchmark,
the IC50 of Captopril (6) was determined in parallel
to ensure the accuracy of the assay. The IC50 values
of the peptide inhibitors and Captopril are summarized
in Table 1.
Compound
IC50 (nM)
OH
O
P
O
H
H
OH
OH
R
N
N
N
H
O
O
OH
Measurement of ACE inhibition by the natural tripep-
tide analogue of K-26, L-Ile-L-Tyr-L-Tyr (16a), con-
firmed the previously reported micromolar range
activities of related small natural peptides. N-Acetyla-
tion of this compound to (15a) improved its activity
by approximately 10-fold. However, the potentiating ef-
fect of phosphonyl substitution in this compound class
was more pronounced. K-26 (1a) was found to be
1500-fold more potent than Ac-Ile-Tyr-Tyr (15a). In-
deed, all analogues containing AHEP were found to
be markedly improved inhibitors relative to their car-
boxylate congeners.
1a, (K-26) Ac-L-lle-L-Tyr-R-AHEP
1b, Ac-L-lle-L-Tyr-S-AHEP
14.4
139
3.83 · 10ꢁ5
1c, Ac-L-lle-L-Tyr-R-AHEP (OEt)2
OH
O
O
H
OH
OH
H2N
N
P
N
H
O
OH
14a, L-lle-L-Tyr-R-AHEP
14b, L-lle-L-Tyr-S-AHEP
234.3
5.46 · 104
Other general trends emerge from these data. Separation
of the synthetic diastereomers permitted the evaluation
of the effect of stereochemistry of AHEP on ACE inhi-
bition. We observed that the cost of inverting the stereo-
chemistry of the phosphonate from the natural
configuration (R-AHEP) was an approximately 10-fold
decrease in activity and similar trends for acetylated
and des-acetyl analogues in all series were observed.
Additionally, alkylation of the phosphonate group
(ethyl esters) decreased activity over 25,000-fold, as
demonstrated by the comparison of 1a and 1c. The influ-
ence of N-acetylation on ACE activity in K-26 was also
consistent, with an across the board improvement of 10-
to 20-fold for most acetylated analogues.
OH
O
O
H
H
N
N
N
OH
H
O
O
OH
15a, Ac-L-lle-L-Tyr-L-Tyr
15b, Ac-L-lle-L-Tyr-D-Tyr
2.1 · 104
2.0 · 106
OH
O
O
H
H2N
N
These data provide an additional basis for understand-
ing the improvement in ACE inhibition by K-26 versus
the carboxylate analogues. The ACE potentiating ef-
fects of phosphonate substitution may originate from
a stronger ionic interaction of the phosphonate cen-
tered anion with the enzyme, either through side chain
interactions or by direct interaction with Zn2+ ligand.
Previous X-ray structural studies5 have also suggested
that phosphonyl substitution may limit the conforma-
tional flexibility of the peptide backbone relative to
the carboxyl analogue. This notion is supported by
the observation of weak long range coupling between
the c-methyl of isoleucine (0.5 ppm) and the AHEP
phenyl ring (6.7 ppm) in K-26 NOESY experiments.
No such distal interactions were observed in Ac-Ile-
Tyr-Tyr.
N
OH
H
O
OH
16a, L-lle-L-Tyr-L-Tyr
16b, L-lle-L-Tyr-D-Tyr
2.36 · 105
>106
O
HS
N
HO2C
Captopril
7.7
powder by soaking with 100 mM borate buffer followed
by ultracentrifugation.17 Furanacryloyl-L-phenylalanyl-
glycylglycine (FAPGG) was used as the chromogenic
substrate for measuring initial velocities. The progress
of the reaction can be monitored in a continuous fash-
ion based on the hypsochromic shift of the absorption
spectra that occurs upon hydrolysis of FAPGG to FAP
and GG. In a typical reaction, ACE extract was prein-
cubated with a range of inhibitor concentrations in the
responsive concentration region for 5 min followed by
the addition of a 1 mM solution of FAPGG. The rate
The isolation of a mammalian ACE modulating sub-
stance from a bacterium may appear somewhat surpris-
ing. Recently however, bacterial ACE homologues have
been identified in Xanthomonas,18 suggesting that the
bacterial homologues of eukaryotic ACE may play
important roles in bacterial physiology. While the natu-
ral target of these terminal phosphonopeptides remains
unidentified, the potent inhibitory activity of this entire
class of compounds toward ACE suggests the possibility

I. Ntai, B. O. Bachmann / Bioorg. Med. Chem. Lett. 18 (2008) 3068–3071
3071
that these compounds may target analogous metallopro-
References and notes
teases, either endogenously or metalloproteases of con-
sequence in the ecological niche of the producing
organisms. Indeed, the specific functionalities that dis-
tinguish K-26 from its natural peptide congener, N-acet-
ylation and a-phosphonylation, are shown here to direct
markedly improved activity against the zinc metallopro-
tease ACE. It is therefore possible that the natural target
of K-26 is a catalytically similar metalloprotease and
these results suggest that the nature’s strategy of C-ter-
minal phosphonyl substitution may find meritorious
application in the discovery of new C-terminal phospho-
nate inhibitors.
1. Quin, L. D. Organophosphorus Chemistry; Wiley-Inter-
science: New York, 2000.
2. Seto, H.; Kuzuyama, T. Nat. Prod. Rep. 1999, 16, 589.
3. Kase, H.; Yamato, M.; Koguchi, T.; Okachi, R.; Kasai,
M.; Shirahata, K.; Kawamoto, I.; Shuto, K.; Karasawa,
A. EP 61172, 1982, 60 pp.
4. Yamato, M.; Koguchi, T.; Okachi, R.; Yamada, K.;
Nakayama, K.; Kase, H.; Karasawa, A.; Shuto, K.
J. Antibiot. 1986, 39, 44.
5. Hirayama, N.; Kasai, M.; Shirahata, K. Int. J. Pept. Prot.
Res. 1991, 38, 20.
6. Ohuchio, S.; Kurihara, K.; Shinohara, A.; Takei, T.;
Yoshida, J.; Amano, S.; Miyadoh, S.; Matsushida, Y.;
Somura, T.; Sezaki, M. Sci. Rep. Meiji Seika Kaisha 1988,
27, 46.
7. Koguchi, T.; Yamada, K.; Yamato, M.; Okachi,
R.; Nakayama, K.; Kase, H. J. Antibiot. 1986, 39,
364.
8. Acharya, K. R.; Sturrock, E. D.; Riordan, J. F.; Ehlers,
M. R. Nat. Rev. 2003, 2, 891.
9. Wu, J.; Aluko, R. E.; Nakai, S. J. Agric. Food Chem. 2006,
54, 732.
Recent studies13,19 have determined that the biosynthe-
sis of K-26 is distinct from most other C–P bond con-
taining metabolites20,21 in that the precursor for
AHEP ‘phosphonotyrosine’ is derived from tyrosine, in-
stead of phosphoenolpyruvate. Labeling studies have
demonstrated that AHEP is a discrete amino acid pre-
cursor of K-26 in the producing organism and that the
transformation of tyrosine to AHEP occurs with reten-
tion of stereochemical configuration at the a-carbon of
tyrosine and retention of nitrogen. The demonstration
of the potency of the phosphonate pharmacophore in
this class of compounds underlines the utility of further
studies aimed at identifying the biosynthetic gene(s) re-
quired for assembly of a-aminophosphonates.
10. Burk, M. J.; Stammers, T. A.; Straub, J. A. Org. Lett.
1999, 1, 387.
11. Kowalik, J.; Kupczyk-Subotkowska, L.; Mastalerz, P.
Synthesis 1981, 57.
12. Maier, L.; Diel, P. J. Phosphorus Sulfur 1991, 62, 15.
13. Ntai, I.; Phelan, V. V.; Bachmann, B. O. Chem. Commun.
2006, 4518.
14. Tam, C. C.; Mattocks, K. L.; Tishler, M. Proc. Natl. Acad.
Sci. U.S.A. 1981, 78, 3301.
Acknowledgments
15. Carpino, L. A.; El-Faham, A.; Albericio, F. J. Org. Chem.
1995, 60, 3561.
16. Holmquist, B.; Bunning, P.; Riordan, J. F. Anal. Biochem.
1979, 95, 540.
17. Murray, B. A.; Walsh, D. J.; FitzGerald, R. J. J. Biochem.
Biophys. Methods 2004, 59, 127.
18. Riviere, G.; Michaud, A.; Corradi, H.; Sturrock, E.;
Acharya, K.; Cogez, V.; Bohin, J.; Vieau, D.; Corvol, P.
Gene 2007, 399, 81.
19. Ntai, I.; Manier, M. L.; Hachey, D. L.; Bachmann, B. O.
Org. Lett. 2005, 7, 2763.
This work was supported by the National Institutes of
Health (RO1GM077189-01), the Petroleum Research
Fund (ACS PRF#42064-G4), and the Vanderbilt Insti-
tute of Chemical Biology.
Supplementary data
Supplementary data include additional information and
data concerning synthesis and spectroscopic character-
ization of synthesized compounds and ACE inhibition
assay. Supplementary data associated with this article
can be found, in the online version, at doi:10.1016/
j.bmcl.2007.11.130.
20. Woodyer, R. D.; Shao, Z.; Thomas, P. M.; Kelleher, N.
L.; Blodgett, J. A.; Metcalf, W. W.; van der Donk, W. A.;
Zhao, H. Chem. Biol. 2006, 13, 1171.
21. Blodgett, J. A.; Thomas, P. M.; Li, G.; Velasquez, J. E.;
van der Donk, W. A.; Kelleher, N. L.; Metcalf, W. W.
Nat. Chem. Biol. 2007, 3, 480.