Studies of 1-Amino-2,2-difluorocyclopropane-1-carboxylic Acid: Mechanism of Decomposition and Inhibition of 1-Aminocyclopropane-1-carboxylic Acid Deaminase

Article abstract of DOI:10.1021/acs.orglett.5b01570

1-Amino-2,2-difluorocyclopropane-1-carboxylic acid (DFACC) is of interest in the study of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase due to the increased reactivity of its cyclopropyl functionality. It is shown that DFACC is unstable under near

Full text of DOI:10.1021/acs.orglett.5b01570

Letter
pubs.acs.org/OrgLett
Studies of 1‑Amino-2,2-difluorocyclopropane-1-carboxylic Acid:
Mechanism of Decomposition and Inhibition of
‑Aminocyclopropane-1-carboxylic Acid Deaminase
1
Cheng-Hao Liu, Shao-An Wang, Mark W. Ruszczycky, Huawei Chen, Keqiang Li,§
†
,⊥
†,⊥
‡
§,∥
§
,†,‡,§
Kazuo Murakami, and Hung-wen Liu*
†
‡
Department of Chemistry and Division of Medicinal Chemistry, College of Pharmacy, University of Texas at Austin, Austin, Texas
7
8712, United States
§
Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, United States
*
S Supporting Information
ABSTRACT: 1-Amino-2,2-difluorocyclopropane-1-carboxylic acid (DFACC) is
of interest in the study of 1-aminocyclopropane-1-carboxylic acid (ACC)
deaminase due to the increased reactivity of its cyclopropyl functionality. It is
shown that DFACC is unstable under near-physiological conditions where it
primarily decomposes via specific-base catalysis to 3-fluoro-2-oxobut-3-enoic acid
−1
with a rate constant of 0.18 ± 0.01 min . Upon incubation with ACC
deaminase, DFACC is found to be a slow-dissociating inhibitor of ACC
deaminase with submicromolar affinity.
1
-Aminocyclopropane-1-carboxylic acid (ACC) deaminase is a
along with the role played by PLP in this process remains an
pyridoxal-5′-phosphate (PLP)-dependent enzyme that catalyzes
the decomposition of ACC (1) to α-ketobutyrate (2) and
ammonia (see Scheme 1). It has been identified in a wide
unresolved and intriguing question for which a number of
7
possibilities have been proposed.
1
It is well documented that species containing a gem-
difluorocyclopropane are susceptible to nucleophilic attack at
8
−11
Scheme 1. Reaction Catalyzed by ACC Deaminase (1 → 2)
and the Structure of DFACC (3)
both the CH and CF centers of the cyclopropane ring.
2 2
Likewise, alkyl-substituted gem-difluorocyclopropanes have also
been proposed to undergo 1,4-elimination reactions whereby
1
2−14
fluoride is eliminated concomitant with ring opening.
Hence, the gem-difluoro analog of ACC, 1-amino-2,2-difluoro-
cyclopropane-1-carboxylic acid (DFACC, 3), has been
recognized for its potential to provide additional insight into
the mechanism of ACC deaminase on account of the greatly
enhanced reactivity of the gem-difluorocyclopropane moi-
14−16
1
ety.
range of bacteria and fungi. The substrate of ACC deaminase
Little is known, however, about the impact of introducing a
basic amino functionality at the α-carbon of gem-difluorocyclo-
propyl amino acids. Such a modification may promote ring
opening in aqueous systems at near-neutral pH. This would
have consequences for the use of DFACC in both mechanistic
is also the immediate biosynthetic precursor to ethylene, which
is produced in plants via oxidation of ACC.
is an essential plant hormone that regulates fruit ripening, seed
germination, and leaf senescence, it can also be detrimental
causing senescence, chlorosis, and abscission when produced in
excessive quantities under conditions of stress. The potential
use of ACC deaminase to mitigate the deleterious effects of
ethylene production has thus made it a focus of enzymological
research to better understand its atypical mechanism of
2
−4
While ethylene
4
,5
15
4
−6
studies of ACC deaminase and as a potential ligand for
biological receptors. Herein we report the synthesis of racemic
DFACC and show that it undergoes a pH-dependent ring-
opening reaction that may involve a carbanionic intermediate.
Interestingly, despite its instability, DFACC is still effective at
6b,7
action.
reversibly inhibiting ACC deaminase with a K in the
i
The opening of the cyclopropane ring of ACC catalyzed by
ACC deaminase is essentially a redox-neutral deamination
reaction. The chemistry of ACC deaminase thus stands in
contrast to that of typical PLP-dependent transaminases which
instead couple the oxidative deamination of amino acids to the
reductive amination of β-keto acids. The mechanism by which
ACC is both deaminated and linearized by ACC deaminase
submicromolar range.
The chemical synthesis of rac-DFACC began with silylation
of 2-(4′-methoxyphenyl)-2-propen-1-ol (4) and subsequent
Received: May 28, 2015
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.5b01570
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
cyclization using difluorocarbene to generate the difluoro-
substituted cyclopropane 6 (see Scheme 2). The silyl protecting
decomposition products were also consistent with additional
measurements made using various two-dimensional NMR
techniques ( H COSY, H− C HSQC, and H− C
HMBC), which are provided in the SI.
In addition to 11 and its hydrate (12), a new compound
which makes up roughly 10% of the total product distribution
1
1
13
1
13
Scheme 2. Chemical Synthesis of rac-DFACC (3)
(
see SI) was also found when the reaction was run in H O. This
2
new product was identified as 3,3-difluoro-2-oxobutyric acid
15) by spectroscopic analysis (see Figure S3.5). Notably, 15
(
was not produced at a detectable level in the reactions run in
D O, and there was no evidence for deuterium incorporation at
2
C4 of either 11 or its hydrate in D O.
2
Based on these observations, four mechanisms describing the
decomposition of DFACC in aqueous solution were considered
(
see Scheme 3). The first of these (A), which can account for
Scheme 3. Hypotheses for the Decomposition of DFACC
3)
(
group of 6 was removed, and the resulting alcohol (7) was
oxidized to afford the carboxylic acid 8. A Curtius rearrange-
ment of 8 was then performed to produce the Boc-protected
amine 9. Next, the 4′-methoxyphenyl functionality of 9 was
oxidatively cleaved using ruthenium tetroxide generated in situ
to yield 10. After deprotection of the Boc group of 10, the crude
product was purified using DOWEX 50WX8 cation exchange
resin to afford DFACC (3) as the HCl salt.
Despite its stability as a HCl salt, when DFACC is incubated
in D O at neutral pD, it undergoes decomposition with a half-
2
life of ∼4 min (see below). This results in the production of
1
two new species along with release of a free fluoride ion. H
NMR of the major (ca. 75%) product demonstrated an ABX
spin system (X = F) with the AB protons appearing in the
the formation of 15 in buffered H O, involves ring opening of
2
5
.65−5.85 ppm region and a J coupling constant of 4.8 Hz
the conjugate base of DFACC (13) whereby a solvent-derived
proton is transferred to the C3-carbon (13 → 14). The
resulting intermediate would then undergo hydrolysis to
produce the stable species 15. Mechanism B in Scheme 3
involves formation of a halohydrin intermediate (16). This
mechanism begins with nucleophilic attack by hydroxide at C3
and is reminiscent of the reaction of 1-acetyl-2,2-difluoro-3-
AB
1
9
(
see Figure S3.3). The F NMR likewise exhibited a doublet of
doublets splitting pattern with JAF = 15.0 Hz and JBF = 46.0 Hz
see Figure S3.3). The magnitudes of JAF and JBF are most
(
consistent with three-bond couplings between the fluorine and
both the A and B protons rather than two-bond couplings,
17−19
13
which can exceed 80 Hz.
The C NMR of the major
8
,9
product also revealed the presence of two carbonyl carbons in
phenylcyclopropane with phenylthiolate and the addition of
the 165−195 ppm region and thus implicated an α-keto acid
bromide to (2,2-difluorocyclopropyl)-phenylketone in N-
10
(
see Supporting Information (SI)). Moreover, electrospray
pentylpyridinium bromide. While the α-amino group of
DFACC is not directly involved in the ring-opening step of
mechanism B (i.e., 3 → 16), it is important for the conversion
of 17 to 18/11.
ionization mass spectrometry (ESI-MS) of the product mixture
exhibited a signal at 117.00 m/z (negative ion mode) consistent
−
with a composition of C H FO (calculated mass: 116.9993
4
2
3
Da). The major decomposition product was thus assigned as 3-
fluoro-2-oxobut-3-enoic acid (11; see Scheme 2).
Alternatively, the amino functionality could play a direct role
in the DFACC ring opening as shown in mechanisms C and D
of Scheme 3. Both of these mechanisms, like A, also involve an
initial rapid acid−base equilibrium; however, in the case of C,
the proposed ring opening results in a transient carbanion
intermediate (19) that can undergo either protonation (19→
14) or elimination (19 → 18). In contrast, mechanism D
involves a one-step (i.e., (intra-D )D ) elimination to produce
The NMR spectra of the minor decomposition product (ca.
5%) showed features very similar to those of 11. Again an
2
ABX system was observed (X = F); however, the two AB
quartets are found shifted upfield to the 4.70−4.95 ppm region.
1
3
Likewise, the C2 C resonance, which in the case of 11 is
observed as a doublet at 189.4 ppm and split by the fluorine at
N
N
1
3
C3, is replaced by a doublet C resonance at 91.4 ppm (see
SI). This implies that the C2 carbonyl seen in 11 is absent and
led to the assignment of the second product as the
corresponding hydrate (12). The assignments for the two
18 directly from 13. Neither mechanism B nor D, however, can
explain the coproduction of 15 in H O−solvent systems and
2
would thus require the participation of a second, competing
process such as 13 → 14 in mechanism A.
B
DOI: 10.1021/acs.orglett.5b01570
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Organic Letters
Letter
To examine these hypotheses, the role of the α-amino group
in the decomposition of DFACC was investigated more closely.
N-Acetyl rac-DFACC was prepared from 9 (see SI) and found
to be stable at all pH values considered. This result
demonstrates the importance of a basic α-amino group for
the observed decomposition of DFACC. The pH-dependence
of the DFACC decomposition rate was then determined by
following the disappearance of DFACC at different pH’s using
In order to evaluate the potential of DFACC as a mechanistic
probe of ACC deaminase, we first checked to see whether it
would interact with the enzyme. The activity of ACC
deaminase in the presence of DFACC (3) was thus assayed
by following the deamination of ACC to α-ketobutyrate (2) at
1
pH 7.0 using H NMR spectroscopy as shown in Figure 1. Each
1
9
F NMR. Triplicate reactions were run in 1.0 M succinate or
phosphate (H O) buffers at pH values ranging from 4.5 to 8.0
2
and a constant ionic strength of 3 M. DFACC was observed to
decay via a first-order process with a pH-dependent rate
constant (see Section S4 in the SI). This plot demonstrates that
the decomposition of DFACC is slowed at low pH and reaches
a plateau under alkaline conditions indicating base catalysis.
In order to obtain a measure of the apparent pK and
a
maximum first-order rate constant, k, for decomposition of
DFACC, the data in Figure S4.3 were fit using eq 1,
−
pKa
−pKa
+ 10−pH)] + log k
log k = log [10 /(10
app
1
0
10
10
(1)
Figure 1. Inhibition of ACC deaminase with rac-DFACC (3).
Reactions were run at rt with 0.5 μM enzyme in 500 μL of 100
mM NaPi buffer (pH 7.0) with 20 mM ACC and 0 (black), 5 (white),
or 50 μM (gray) rac-DFACC. Reactions were followed by H NMR
(see SI). The uninhibited time course was fit to a straight line to
This provided values of 5.88 ± 0.06 for the pK and 0.18 ±
a
1
0
.01 min− for k. The observed pK is too low to correspond to
a
1
the second ionization constant of phosphoric acid, which is 6.8
20
at an ionic strength of 3 M. However, this pK is consistent
a
with those for the α-amino group of β,β-difluoroalanine and
obtain the rate v (broken line), while the inhibition time courses were
fit simultaneously using eqs 2 and 3. Fits exclude points where product
is undetectable except at t = 0 min.
0
β,β-difluorophenylalanine, which have pK values roughly 2
a
21
units lower than their nonfluorinated counterparts. Therefore,
22
given the pK of 8.15 for ACC, the corresponding pK for
a
a
DFACC is expected be ca. 6.0. This result suggests that
decomposition of DFACC involves specific-base catalysis where
the conjugate base of DFACC (i.e., 13) is the reactive species
that formally undergoes ring opening. This conclusion argues
against formation of a halohydrin intermediate as shown in
mechanism B. The above result along with the observation that
decomposition of 3/13 is unaffected by the addition of a weak
acid such as phenol (see SI) also casts doubt on the signifcance
of pathway A, which involves general-acid catalysis.
time trace exhibits a lag period during which the concentration
of 2 increases to detectable levels. In the absence of rac-
DFACC, this period is less than 1 h. In the presence of 5 and
50 μM DFACC, however, this period is extended to at least 2
and 3 h, respectively. Following the lag, each reaction reaches a
roughly constant rate (ca. 16 μM/min with no DFACC) for the
remainder of the observation period. This is consistent with a
<10% drop in enzyme saturation assuming a Michaelis constant
7g
of 4 mM and indicated an enzyme specific activity of ∼1
−1
−1
Both mechanisms C and D in Scheme 3, however, are
consistent with these results. The (intra-D )D ring opening of
μmol·min mg . Furthermore, when fresh ACC was added to
each reaction after 48 h, by which time all of the original ACC
had been consumed, equivalent rates of turnover were observed
in each of the three reactions. These observations revealed that
DFACC is a reversible instead of an irreversible inhibitor for
ACC deaminase. Furthermore, no evidence of inhibition was
observed when ACC deaminase was incubated in the presence
of DFACC following its decomposition (see Figure S7.3 in the
SI). This indicates that the decomposition product 11 does not
react with the enzyme despite being a potential Michael
acceptor and that DFACC itself is responsible for the
inhibition.
N
N
mechanism D is analogous to an E1cB version of the 1,4-
elimination reactions that have been proposed for the
dehydrohalogenation of substituted gem-difluorocyclo-
1
2−14
propanes
and is consistent with the propensity of
cyclopropanes to behave more as conjugated π-systems rather
3
23
than sp -hybridized carbocycles. However, mechanism D
would require a competing process such as A in order to
explain 15 and the product distributions in H O versus D O. In
2
2
contrast, mechanism C can account for the formation of all
observed products. More importantly, it possesses a product-
determining step (i.e., 19 → 18/14) that would be susceptible
to a normal solvent deuterium kinetic isotope effect and thus
explain the formation of 15 in H O as opposed to D O buffers.
The rapid decomposition of DFACC in comparison to the
lag times suggested a simplified kinetic model from which
estimates of the DFACC dissociation constant K and
i
2
2
dissociation rate constant k could be made using the progress
While mechanism C involves the intermediacy of a β-
d
−
curves in Figure 1. This model assumes that a binding
equilibrium between DFACC and enzyme is rapidly established
fluorocarbanion (i.e., RCF CH ), this species could be
2
2
24
stabilized through negative hyperconjugation. Though a
competing α-fluorinated carbanion (i.e., RCH CF ) could
−
such that the initial fraction f of enzyme being inhibited is
0
2
2
given by
likewise be stabilized inductively, its stability may be offset by
orbital repulsion between the lone pairs on the α-carbanion and
f = x K /[K (K + s ) + x K ]
25−27
0
0
M
i
M
0
0
M
(2)
the adjacent fluorines,
resulting in preferential formation
of the β-fluorocarbanion 19. Taken together, the available
evidence seems to be most consistent with mechanism C.
where x and s are the initial concentrations of DFACC and
0 0
ACC, respectively, and K is the Michaelis constant (4 mM). If
M
C
DOI: 10.1021/acs.orglett.5b01570
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
dissociation of the enzyme−inhibitor complex is slow versus
DFACC decomposition, then the observed time course of
product formation will be given by
Zhou, Q.; Tao, Z.; Zhang, H.; Liu, H.-w. Angew. Chem., Int. Ed. 2004,
4
3, 3425. (i) Karthikeyan, S.; Zhou, Q.; Zhao, Z.; Kao, C.-L.; Tao, Z.;
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0
0
d
d
(3)
0
where v is ∼16 μM/min (see SI for derivation). When eqs 3/2
(9) Kobayashi, Y.; Taguchi, T.; Morikawa, T.; Takase, T.; Takanashi,
H. J. Org. Chem. 1982, 47, 3232.
0
were fit simultaneously to both inhibition time courses in
−1
(
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i
d
3
(
were obtained. It should be emphasized that the true value of Ki
may be significantly smaller if there is no rapid equilibration
between the enzyme and inhibitor. The kinetic parameter
estimates are nevertheless consistent with the conclusion that
rac-DFACC is a reversible, slow-dissociating inhibitor of ACC
deaminase that binds with submicromolar affinity.
(
(
5
(
ASSOCIATED CONTENT
Supporting Information
■
(
*
S
Details regarding the synthetic procedures, enzyme assays,
kinetic analyses, and spectroscopic characterization of all
compounds discussed. The Supporting Information is available
free of charge on the ACS Publications website at DOI:
(
John Wiley & Sons: NJ, 2009; pp 61−74.
(18) Baklouti, A.; Chaabouni, M. M. J. Fluorine Chem. 1981, 19, 181.
(19) Nguyen, T.; Wakselman, C. J. Org. Chem. 1989, 54, 5640.
(20) Ellis, K. J.; Morrison, J. F. Method. Enzymol. 1982, 87, 405.
1
0.1021/acs.orglett.5b01570.
(
21) Schlosser, M.; Brugger, N.; Schmidt, W.; Amrhein, N.
Tetrahedron 2004, 60, 7731.
22) Shoukry, M. M.; Hassan, S. S. Spectrochim. Acta A: Mol. Biomol.
AUTHOR INFORMATION
Corresponding Author
E-mail: h.w.liu@mail.utexas.edu.
■
*
(
Spectrosc. 2014, 118, 146.
(23) (a) Lowry, T. H.; Richardson, K. S. Mechanism and Theory in
Organic Chemistry, 3rd ed.; HarperCollins Publishers: New York, 1989;
pp 31−33. (b) Smith, M. B.; March, J. March’s Advanced Organic
Chemistry, 5th ed.; John Wiley & Sons: New York, 2001; pp 181−182.
Present Address
∥Oncology iMed, AstraZeneca, Waltham, MA.
Author Contributions
⊥C.-H.L. and S.-A.W. contributed equally.
Notes
(
24) Dixon, D. A.; Fukunaga, T.; Smart, B. E. J. Am. Chem. Soc. 1986,
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1
(
(
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported in part by grants from the National
Institutes of Health (GM040541) and the Welch Foundation
■
(F-1511).
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DOI: 10.1021/acs.orglett.5b01570
Org. Lett. XXXX, XXX, XXX−XXX