4,4-Difluorinated analogues of l-arginine and NG-hydroxy-l-arginine as mechanistic probes for nitric oxide synthase

Article abstract of DOI:10.1016/j.bmcl.2009.01.076

4,4-Difluoro-l-arginine and 4,4-difluoro-N^G-hydroxy-l-arginine were synthesized and shown to be substrates for the inducible isoform of nitric oxide synthase (iNOS). Binding of both fluorinated analogues to the NOS active site was also investigated using a spectral binding assay employing a heme domain construct of the inducible NOS isoform (iNOS_heme). 4,4-Difluoro-N^G-hydroxy-arginine was found to bind at the NOS active site in a unique manner consistent with a model involving ligation of the Fe^III heme center by the oxygen atom of the N^G-hydroxy moiety.

Full text of DOI:10.1016/j.bmcl.2009.01.076

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1758–1762
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
4,4-Difluorinated analogues of L-arginine and NG-hydroxy-L-arginine
as mechanistic probes for nitric oxide synthase
Nathaniel I. Martin a, Joshua J. Woodward b, Michael B. Winter b, Michael A. Marletta b,c,d,
*
a Department of Medicinal Chemistry and Chemical Biology, University of Utrecht, 3584 CA, The Netherlands
b Department of Chemistry, University of California, 556 Stanley Hall, Berkeley, CA 94720-3220, USA
c Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA
d Division of Physical Biosciences, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA
a r t i c l e i n f o
a b s t r a c t
4,4-Difluoro-L L-arginine were synthesized and shown to be sub-
-arginine and 4,4-difluoro-NG-hydroxy-
Article history:
Received 26 December 2008
Revised 21 January 2009
Accepted 22 January 2009
Available online 18 February 2009
strates for the inducible isoform of nitric oxide synthase (iNOS). Binding of both fluorinated analogues
to the NOS active site was also investigated using a spectral binding assay employing a heme domain con-
struct of the inducible NOS isoform (iNOSheme). 4,4-Difluoro-NG-hydroxy-arginine was found to bind at
the NOS active site in a unique manner consistent with a model involving ligation of the FeIII heme center
by the oxygen atom of the NG-hydroxy moiety.
Keywords:
Ó 2009 Elsevier Ltd. All rights reserved.
Nitric oxide synthase
Fluorinated substrate analogues
Mechanistic probes
The nitric oxide synthases (NOSs) carry out a complex series of
istry remains to be fully elucidated.5,6 The first NOS reaction, in
which -arginine, is often con-
-arginine is oxidized to NG-hydroxy-
redox reactions that convert
L
-arginine to NO, an important signal-
L
L
ing molecule involved in a wide range of biological functions.1–3
NOSs are comprised of both a reductase domain (binding NADPH,
FAD, and FMN) and a catalytic oxygenase domain containing the
sidered to proceed via an oxygen-rebound mechanism analogous
to that of the cytochrome P450-type enzymes7–9 (Silverman and
co-workers have recently suggested an alternate mechanistic
explanation for this step).6 The second NOS reaction, however, is
without enzymatic precedent. Requiring the net input of a single
electron, NOS converts a NG-hydroxyguanidine into a urea and
NO. While the precise mechanism of the second NOS reaction re-
mains unclear, a proposed10,11 explanation involves nucleophilic
heme prosthetic group. The NOS substrate
essential redox cofactor (6R)-5,6,7,8 tetrahydro-
L
-arginine, and the
-biopterin (H4B)
L
also bind in the oxygenase domain with a specific orientation rel-
ative to the heme.4 The three NOS isoforms identified in various
mammalian cells (neuronal, inducible, and endothelial NOS) all
catalyze the oxidation of
L
-arginine to
L
-citrulline and NO via the
addition of a FeIII-peroxo species to NG-hydroxy-
a tetrahedral intermediate. Collapse of this intermediate then leads
to formation of -citrulline and NO, along with one-electron reduc-
tion of the tetrahydro- -biopterin cofactor, returning NOS to its
L-arginine forming
intermediate NG-hydroxy-
L
-arginine (NHA) (Fig. 1).2,3
L
L
H2N NH2
NH
H2N N OH
NH
H2N
O
resting state (Fig. 2). Experimental approaches to investigating
the NOS reaction mechanism(s) have most often relied upon muta-
genesis of the enzyme,12–14 the use of structurally altered sub-
strates,6,15–20 and electron paramagnetic resonance (EPR)
spectroscopy to identify radical reaction intermediates.1,21,22 Given
that fluorinated substrates have been successfully used to probe
the mechanistic properties of numerous other enzymes23–25 we
were interested to see what effects substrate fluorination might
NH
NOS
NOS
+
NO
O2
O2
1
NADPH
NADPH
2
O
O
O
H3N
H3N
H3N
O
O
O
L-arginine
N
G-hydroxy-L-arginine
L-citrulline nitric oxide
Figure 1. Two-step reaction catalyzed by NOS.
have with NOS. While fluorinated analogues of
reported as moderately selective inhibitors for iNOS,26 the effect of
fluorination of the native substrates
-arginine and NG-hydroxy-
arginine (Fig. 3) on NOS catalysis has not been thoroughly investi-
gated. In this regard, we recently reported the unique product
distribution obtained when reacting a fluorinated substrate ana-
L-lysine have been
While physiological signaling roles for NO are well established,
a thorough understanding of NO biosynthesis including the mech-
anistic details by which NOS performs its unique oxidative chem-
L
L-
* Corresponding author. Tel.: +1 510 666 2763; fax: +1 510 666 2764.
E-mail address: marletta@berkeley.edu (M.A. Marletta).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.01.076

N. I. Martin et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1758–1762
1759
N
OH
H2N
HN
H2N
HN
OH
H2N
HN
OH
Proposed tetrahedral
intermediate.
N
N
O
O H
O
O
H
e, O2
H3B
FeIII
FeIII
FeIII
S
H4B
NHA
S
S
N O
O
H2N
HN
H2N
HN
O
Formation of L-citrulline and NO
with return of enzyme to resting state.
N
O
H
OH
O
FeIII
FeIII
O
OH
H
N
H3B H4B
HN
H4B =
S
S
One of many proposed mechanistic
routes for collapse of intermediate.
OH
H2N
N
N
H
(6R)-5,6,7,8 tetrahydro-L-biopterin
Figure 2. Proposed mechanism for the oxidation of NG-hydroxy-
L
-arginine to
L-citrulline and NO.
logue with the iNOS catalytic domain (iNOSheme) in the presence of
hydrogen peroxide.14 We here further describe the syntheses of
S
N
N
O
4,4-difluoro-L L-arginine
-arginine 1 and 4,4-difluoro-NG-hydroxy-
O
O
O
a
b
2 and the effects observed in the interaction of both analogues with
full-length iNOS.
OEt
O
HO
c
OCH3
H
O
F
F
NH3Cl
NCbz
NBoc
The synthesis of 4,4-difluoro-L-arginine 1 (Scheme 1) was
3
4
5
accomplished in a manner similar to that previously described by
Kim and co-workers.27 Incorporation of the C4 difluoromethylene
unit was accomplished via the zinc mediated addition of ethyl bro-
modifluoroacetate to the appropriately protected Garner aldehyde.
Standard functional group manipulations were then employed to
elaborate the amino acid side chain and install the guanidine moi-
O
O
NH2
OEt
d
e
O
O
F
F
F
F
NCbz
NCbz
6
7
NCbz
NHCbz
NCbz
NHCbz
ety prior to global deprotection. 4,4-Difluoro-L-arginine 1 (12 lin-
f
N
H
N
H
HO
O
g
ear steps overall yield 23%) was then tested as a substrate for
iNOS. Incubation of full-length iNOS with 1, H4B, and NADPH, fol-
lowed by a LC–MS-based amino acid product assay, showed that
the difluorinated analogue was converted into the same pattern
F
F
F
F
NCbz
HO
NHCbz
8
9
NCbz
NHCbz
NH
O
O
of products (the intermediate NG-hydroxy-
line) as the native substrate
L
-arginine and
L-citrul-
h
L
-arginine (data provided in the
N
H2N
N
O
H
H
F
F
NH3
F
F
accompanying supplemental information section, see Supplemen-
tal Figure 1). While other NOS substrate analogues have previously
been found to undergo alternate transformations when acted upon
by NOS,18,28,29 the difluoro analogue remains ‘on path’ in the en-
zyme reaction, an important consideration in establishing the
validity of such compounds as representative mechanistic probes.
AcOH
NHCbz
10
1
Scheme 1. Synthesis of 4,4-difluoro-L-arginine 1. Reagents and conditions:
(a) 1—CbzCl, NaHCO3, EtOAc/H2O; 2—2,2-dimethoxypropane, acetone, BF3ꢀEt2O;
3—DIBAL Et2O, ꢁ78 °C, 74%, 3 steps; (b) 1—ethyl bromodifluoroacetate, Zn(s), THF;
2—thiocarbonyldiimidazole, THF, 40 °C, 48 h, 64%, 2 steps; (c) Et3SiH, benzoyl
peroxide, dioxane, 110 °C, 82%; (d) NH4OH, MeOH, quantitative; (e) 1—Red-Al,
toluene, 0 °C; 2—N,N0-di-Cbz-N00-triflylguanidine, NEt3, CH2Cl2 35 °C, 16 h, 81%, 2
steps; (f) 1,2-ethanedithiol, BF3ꢀEt2O, CH2Cl2 82%; (g) CrO3, H5IO6, MeCN/H2O, 96%;
(h) H2, Pd/C, MeOH/H2O (1% AcOH), 93%.
Once 4,4-difluoro-
tically representative analogue of
intermediate analogue, 4,4-difluoro-NG-hydroxy-
L
-arginine had been confirmed as a mechanis-
-arginine, the NG-hydroxylated
-arginine 2, was
L
L
pursued so as to permit independent study of the two discrete
NOS reaction steps. The synthesis of the NG-hydroxylated difluoro
analogue, however, was significantly more challenging than that of
the non-hydroxylated species and required the development of a
new methodology for incorporation of the NG-hydroxyguanidine
moiety.30 In the manner shown in Scheme 2, 4,4-difluoro-NG-hy-
droxy-L-arginine was ultimately prepared in 16 linear steps with
an overall yield of 23%.
With 1 and 2 in hand, the NOS activity for both fluorinated ana-
logues was assessed using an indirect spectral assay making use of
the NO-induced oxidation of oxyhemoglobin to methemoglobin
observable at 401 nm. Both 4,4-difluoro-
fluoro-NG-hydroxy-
-arginine 2 were found to be NO-forming sub-
strates for iNOS. 4,4-Difluoro- -arginine 1 was found to have a Km
value of 814 24 M and a kcat value approximately 10% of that
measured for -argi-
-arginine (Fig. 4A). 4,4-Difluoro-NG-hydroxy-
nine 2 was determined to be a slightly better substrate for iNOS
with a Km value of 328 16 M and a kcat value approximately
30% of that measured for NG-hydroxy-
-arginine (Fig. 4B). The Km
L-arginine 1 and 4,4-di-
L
L
l
L
L
l
HO
L
NH2
O
N
O
values for both fluorinated substrate analogues were found to be
significantly higher than those measured for the native substrates
H2N
N
H
O
H2N
N
O
H
F
F
F
F
NH3
NH3
L
-arginine
(Km = 52
(Km = 8.6 0.5
lM).
l
M)
and
NG-hydroxy-
L-arginine
1
2
3
The binding of both difluorinated analogues 1 and 2 to the iNOS
active site was next investigated. Using a previously described
Figure 3. NOS substrate analogues 4,4-difluoro-
hydroxy- -arginine 2.
L
-arginine 1 and 4,4-difluoro-NG-
L

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N. I. Martin et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1758–1762
O
OH
O
O
a
b
OEt
O
F
HO
OCH3
OCH3
O
F
F
NH3Cl
NBoc
NBoc
3
11
12
O
NHCbz
OEt
c
d
e
O
O
F
F
F
NBoc
NBoc
13
14
O
f
g
tBuO
NHCbz
HO
NHCbz
F
F
F
F
NHBoc
NHBoc
15
16
HO
OTHP
N
O
O
N
h
H2N
TFA
N
O
tBuO
N
H
NHCbz
H
F
F
NH3
F
F
NHBoc
17
2
Scheme 2. Synthesis of 4,4-difluoro-NG-hydroxy-
L-arginine 2. Reagents and condi-
tions: (a) 1—Boc2O, NEt3, MeCN; 2—2,2-dimethoxypropane, acetone, BF3ꢀEt2O, 81%,
2 steps; (b) 1—DIBAL, Et2O, ꢁ78 °C; 2—ethyl bromodifluoroacetate, Zn(s), THF, 69%,
2
steps; (c) 1—thiocarbonyldiimidazole, THF, 40 °C, 40 h; 2—Et3SiH, benzoyl
peroxide, dioxane, 110 °C, 2 steps, 60%; (d) 1—NH4OH, MeOH; 2—Red-Al, toluene,
0 °C; 3—CbzCl, NaHCO3, H2O/dioxane, 90%, 3 steps; (e) pTsOH, MeOH, 50 °C, 85%; (f)
1—NaClO2/NaOCl, TEMPO, MeCN/NaHPO4 buffer; 2—O-tert-butyl-dicyclohexylisou-
rea, CH2Cl2, 35 °C 16 h, 2 steps, 93%; (g) 1—H2, Pd/C, EtOAc; 2—CbzNCS; 3—H2 N-
OTHP, EDCI, NEt3, CH2Cl2, 96%, 3 steps30; (h) TFA/thioanisole, 5 h, quantitative.
spectral binding assay, the affinity of both 1 and 2 for iNOSheme was
measured (iNOSheme binds heme, H4B and substrate(s) in the same
fashion as the full-length enzyme).1 As with the native substrates
L
-arginine and NG-hydroxy-
L-arginine, binding of 4,4-difluoro-L-
arginine 1 to H4B-bound iNOSheme induces a low- to high-spin state
conversion at the heme center resulting from loss of water (FeIII-
aqua ? FeIII-unligated). This spin-state transition is accompanied
by an observable decrease in absorbance at ꢂ420 nm and an in-
crease at ꢂ396 nm in the UV–visible spectrum (Fig. 5A illustrates
the spectral transition observed when iNOSheme is titrated with
Figure 4. Km determination for: (A) 4,4-difluoro-
hydroxy- -arginine 2 (results of triplicate analysis).
L
-arginine 1 and (B) 4-difluoro-NG-
4,4-difluoro-L-arginine 1). As depicted in Figure 5B, the binding
L
constant (spectral Kd) of 1 for iNOSheme is determined by applying
the saturation binding equation (DDAbs = (DDAbsmax ꢃ [com-
pound])/(Kd + [1])). Like the iNOS activity-based Km value, the
spectral Kd measured for 4,4-difluoro-
is much higher than that for the non-fluorinated native substrate
-arginine (7.0 0.7 M). The results of the spectral binding analy-
sis with NG-hydroxy-4,4-difluoro-
-arginine 2, however, were quite
L
-arginine 1 (3.1 0.2 mM)
added to iNOSheme but only in the absence of H4B. Based on further
EPR and resonance Raman findings, they proposed a model where-
by NG-hydroxyguanidines are able to directly ligate the iNOS FeIII
heme center via the NG-hydroxy oxygen atom. The resultant com-
plexes are described as low-spin, 6-coordinate species with UV–
visible absorbance maxima at 430 and 368 nm. They suggest that
this mode of binding of NG-hydroxyguanidines to iNOSheme is
dependent upon the relative acidity of the –OH group in the NG-hy-
droxy moiety.32 Consistent with this proposal, difluorination at the
L
l
L
unexpected. Upon addition of 2 to iNOSheme, a dramatic change in
the UV–visible spectrum was observed leading to a split Soret
absorbance with maxima at 431 and 368 nm (Fig. 6A). This spectral
shift is much different than that seen with the corresponding
non-fluorinated native substrate. Addition NG-hydroxy-
L
-arginine
to iNOSheme (measured spectral Kd = 5.1 0.4
to high-spin state change similar to that observed for both
nine and 4,4-difluoro-
in the absorbance spectrum, a spectral Kd value of 116
l
M) causes a low-
C4 position of NG-hydroxy-
L-arginine likely increases the acidity of
L
-argi-
M for
the NG-hydroxy moiety, weakening the O–H bond (possibly leading
to deprotonation) allowing for oxygen ligation of the heme iron
(Fig. 7).
L
-arginine 1. Despite the unexpected change
4
l
NG-hydroxy-4,4-difluoro- -arginine 2 was determined by plotting
The ability of certain N-hydroxyguanidines to ligate heme ob-
served by Mansuy and Stuehr was reported to be also governed
by steric effects, requiring H4B to be absent from the enzyme active
site.31 If, as our spectral data suggests, addition of analogue 2 to
H4B-bound iNOSheme leads to direct heme ligation, the observation
would appear to be the first of its kind. In the case of difluorinated
analogue 2, H4B does not pose a steric impediment towards a liga-
tion-based mode of binding. The proposed mode of NOS binding by
analogue 2 is further supported by the work of Hoffman and
DDAbs (DDAbs =
D
Abs431L ꢁ
DAbs395) as a function of the concen-
tration of analogue added (Fig. 6B).
Interpretation of the spectral shift data obtained upon addition
of NG-hydroxy-4,4-difluoro-
L-arginine 2 to iNOSheme is aided by
previous reports describing comparable effects observed in the
addition of non-amino acid NG-hydroxyguanindines to NOS and
microperoxidase 8 (MP8).31,32 Mansuy and Stuehr found that cer-
tain NG-hydroxyguanidines induce similar spectral shifts when

N. I. Martin et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1758–1762
1761
Figure 6. Spectral Kd determination for NG-hydroxy-4,4-difluoro-
Spectral transition effected by addition of NG-hydroxy-4,4-difluoro-
L
-arginine. (A)
L
-arginine 2 to
iNOSheme. (B) Titration data and fit used in determination of the spectral Kd for NG-
hydroxy-4,4-difluoro- -arginine 2 (results of triplicate analysis).
Figure 5. Spectral Kd determination for 4,4-difluoro-
spin state transition effected by addition of 4,4-difluoro-
(B) Titration data used in determination of the spectral Kd for 4,4-difluoro-
1 (results of triplicate analysis).
L
-arginine 1. (A) Low- to high-
-arginine 1 to iNOSheme.
L
L
L-arginine
co-workers who described intermediates observed during the
cryoreduction of a FeII-O2- -arginine NOS complex.33 When the
hydroxylation of -arginine by NOS was carried out at low temper-
L
L
ature (77 K), formation of a spectrally similar low-spin FeIII species
was observed, proposed to be the oxygen-ligated complex.33 This
complex was stable up to 165 K and at higher temperatures re-
verted to the typically observed, high-spin, five-coordinate FeIII
species. In contrast to the native substrate NG-hydroxy-
L-arginine,
Figure 7. Proposed mode of binding of NG-hydroxy-4,4-difluoro-
active site H4B-bound iNOSheme
L-arginine 2 to the
our observations would suggest that difluorinated analogue 2 is
capable of stably coordinating the FeIII heme center at room tem-
perature. Furthermore, if binding by 2 does involve ligation of
the FeIII heme center, it is likely dynamic, as both analogues 1
and 2 support NO formation with full-length iNOS (i.e., the enzyme
is still capable of binding and reducing O2 in the presence of the
fluorinated analogues). The unique NOS binding properties of diflu-
orinated analogue 2 may provide for a useful tool in future mech-
anistic investigations.
.
conversion of L-arginine to NO and L-citrulline. Difluorinated ana-
logues 1 and 2 may also serve as useful molecular probes for future
investigations into the NOS reaction mechanism using NMR and
EPR spectroscopic methods.
Acknowledgment
In summary, we have successfully prepared and studied the C4
L
-arginine and NG-
This work was supported by the Aldo DeBenedictis Fund, the
Natural Sciences and Engineering Council of Canada, and the Alber-
ta Heritage Foundation for Medical Research (postdoctoral fellow-
ships to N.I.M.).
difluorinated analogues of the NOS substrates
hydroxy- -arginine. While both analogues exhibit
L
a reduced
binding affinity for the enzyme, they were each shown to be NO-
forming substrates and appear to follow the native reaction path-
way. 4,4-difluoro-NG-hydroxy-
L-arginine also displays a mode of
binding to iNOS previously unobserved with amino acid substrates
at room temperature. This binding likely involves a direct ligation
of the FeIII heme center in the enzyme and provides additional in-
sight into reaction pathway followed during the NOS mediated
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.01.076.

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N. I. Martin et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1758–1762
18. Luzzi, S. D.; Marletta, M. A. Bioorg. Med. Chem. Lett. 2005, 15, 3934.
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