Bioavailable affinity label for collagen prolyl 4-hydroxylase

Article abstract of DOI:10.1016/j.bmc.2013.04.057

Collagen is the most abundant protein in animals. Its prevalent 4-hydroxyproline residues contribute greatly to its conformational stability. The hydroxyl groups arise from a post-translational modification catalyzed by the nonheme iron-dependent enzyme, collagen prolyl 4-hydroxylase (P4H). Here, we report that 4-oxo-5,6-epoxyhexanoate, a mimic of the α-ketoglutarate co-substrate, inactivates human P4H. The inactivation installs a ketone functionality in P4H, providing a handle for proteomic experiments. Caenorhabditis elegans exposed to the esterified epoxy ketone displays the phenotype of a worm lacking P4H. Thus, this affinity label can be used to mediate collagen stability in an animal, as is desirable in the treatment of a variety of fibrotic diseases.

Full text of DOI:10.1016/j.bmc.2013.04.057

Bioorganic & Medicinal Chemistry 21 (2013) 3597–3601
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Bioavailable affinity label for collagen prolyl 4-hydroxylase
James D. Vasta a, Joshua J. Higgin b, Elizabeth A. Kersteen a, Ronald T. Raines a,b,
⇑
a Department of Biochemistry, University of Wisconsin–Madison, 433 Babcock Drive, Madison, WI 53706-1544, USA
b Department of Chemistry, University of Wisconsin–Madison, 1101 University Avenue, Madison, WI 53706-1322, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 30 April 2013
Collagen is the most abundant protein in animals. Its prevalent 4-hydroxyproline residues contribute
greatly to its conformational stability. The hydroxyl groups arise from a post-translational modification
catalyzed by the nonheme iron-dependent enzyme, collagen prolyl 4-hydroxylase (P4H). Here, we report
Keywords:
Affinity label
that 4-oxo-5,6-epoxyhexanoate, a mimic of the a-ketoglutarate co-substrate, inactivates human P4H. The
inactivation installs a ketone functionality in P4H, providing a handle for proteomic experiments. Caeno-
rhabditis elegans exposed to the esterified epoxy ketone displays the phenotype of a worm lacking P4H.
Thus, this affinity label can be used to mediate collagen stability in an animal, as is desirable in the treat-
ment of a variety of fibrotic diseases.
a-Ketoglutarate
Caenorhabditis elegans
Collagen
Prolyl 4-hydroxylase
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
analogs to inhibit catalysis by human P4H.11 Epoxy ketone 1, which
also resembles 5-aminolevulinic acid, is a known competitive
Collagen is the most abundant protein in animals, and main-
tains the structural integrity of various connective tissues.1 The
overproduction of collagen is associated with fibrotic diseases,
including myocardial infarction, stroke, peripheral vascular dis-
ease, diabetes, and severe anemias.2 The stability of collagen relies
on catalysis by collagen prolyl 4-hydroxylase (P4H).3 This enzyme
is located in the lumen of the endoplasmic reticulum, where it cat-
alyzes a remarkable post-translational modification—the conver-
sion of (2S)-proline residues (Pro) to (2S,4R)-4-hydroxyproline
residues (Hyp) in protocollagen strands. The human enzyme con-
inhibitor of 5-aminolevulinic acid dehydratase but does not
alkylate that enzyme.13
sists of an a2b2 tetramer in which each a subunit contains an active
site. P4H is essential for animals,4–6 as the conformational stability
of mature collagen relies on its hydroxylation.7–9 Moreover, P4H is
a validated target for the treatment of fibrotic diseases.2
P4H is a nonheme iron(II) dioxygenase that uses O2 and a-keto-
glutarate as co-substrates (Fig. 1A). The three-dimensional struc-
ture of mammalian P4H is unknown. We reasoned that an
electrophilic analog of
a-ketoglutarate could serve as an irrevers-
ible inhibitor of the enzyme and, hence, a useful probe for active-
site residues. Inspired by natural products such as trapoxin and
epolactaene as well as artificial affinity labels,10 we designed 4-
oxo-5,6-epoxyhexanoate (1) for this purpose. Our thought was that
the epoxide oxygen would chelate to the active-site iron and there-
by be activated for nucleophilic attack (Fig. 1B). We also noted that
a-ketoglutarate and epoxy ketone 1 have the same number and a
similar arrangement of nonhydrogen atoms. This attribute is
important because of the inability of larger
a-ketoglutarate
Figure 1. (A) Reaction catalyzed by prolyl 4-hydroxylase. (B) Notional image of a-
ketoglutarate (left) or epoxy ketone 1 (right) bound in the active site of P4H and
interacting with an enzymic base, B. Alkylation is more likely to occur by
nucleophilic attack at C6 than C5.12
⇑
Corresponding author. Tel.: +1 608 262 8588.
E-mail address: rtraines@wisc.edu (R.T. Raines).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.04.057

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J. D. Vasta et al. / Bioorg. Med. Chem. 21 (2013) 3597–3601
effect of this putative affinity label on the ability of P4H to catalyze
2. Results and discussion
the hydroxylation of a fluorescent peptidic substrate, dansyl-
Gly–Phe–Pro–Gly-OEt.14–17 The data revealed that epoxy ketone
1 elicits irreversible inactivation of P4H, and does so in a concen-
2.1. Effect of epoxy ketone 1 on catalysis by P4H
tration-dependent manner (Fig. 2A).
P4H against inactivation (Fig. 2B), suggesting that epoxy ketone 1
a-Ketoglutarate protected
We produced human P4H by heterologous expression in Esche-
richia coli,14–18 and we synthesized racemic 1. We assayed the
competes with this co-substrate for binding to the active site.
2.2. Utility of epoxy ketone 1 as a proteomic probe
Noting that a ketone has chemical reactivity orthogonal to that
in natural amino acids,20 we examined the utility of epoxy ketone 1
in a model proteomic experiment. Epoxy ketone 1 was incubated
with P4H in the presence of iron(II). The mixture was treated with
biocytin hydrazide and then NaBH4 to reduce the incipient N-acyl
hydrazone (which is unstable21). Small molecules were removed
by gel-filtration chromatography. The P4H was added to wells of
a microplate containing immobilized streptavidin. The wells were
washed and exposed to a monoclonal antibody directed against
P4H. Visualization with a secondary antibody conjugated to horse-
radish peroxidase revealed that P4H is associated with streptavidin
only in the presence of epoxy ketone 1 (Fig. 2C). The conjugation
was confirmed by SDS–PAGE (data not shown). These data indicate
that epoxy ketone 1 could serve as a chemo-selective probe for
P4H. Thus, epoxy ketone 1 complements a (2S,4S)-4-fluoroproline
substrate that spawns a ketone upon P4H catalysis.16
2.3. Effect of epoxy ketone 1 on an animal
Finally, we determined the effect of epoxy ketone 1 on a live
animal. Like all animals (including dinosaurs22), Caenorhabditis ele-
gans has an abundance of collagen. In wild-type worms, P4H exists
largely as a tetramer of two
a subunits (DPY-18 and PHY-2, which
each contain an active site) and two b subunits.23 The dpy-18 gene
is essential for body morphology, whereas the function of the phy-2
gene is not apparent.4,5 The deletion of both genes results in em-
bryos that grow to the twofold stage, but are unable to maintain
their shape and consequently explode.5 These two forms of the
a
subunit have 56.5% amino-acid sequence identity with each other
and ꢀ50% identity with their human homologues. -Ketoglutarate
a
Figure 3. In vivo assay of epoxy ketone 1. Dpy-18 C. elegans worms (four in stage
L4) were exposed to known concentrations of epoxy ketone 1 in the growth
medium. Lethality refers to eggs that do not hatch. Insets: Image of lethality
observed in the embryos of either a dpy-18/phy-2 animal or a dpy-18 animal
exposed to epoxy ketone 1 (top); image of an adult dpy-18 animal not exposed to
epoxy ketone 1 (bottom).
Figure 2. In vitro assays of epoxy ketone 1. (A) Kitz–Wilson plot19 showing the
effect of different concentrations of epoxy ketone 1 on the catalytic activity of P4H.
a-ketoglutarate concentration
on the inactivation of P4H by epoxy ketone 1. (C) Immobilization of P4H after its
kinact/Ki = 0.09 MÀ1 sÀ1. Inset: raw data. (B) Effect of
reaction with epoxy ketone 1. Wells 1 and 2, A490 = 0.14; wells 3 and 4, A490 = 0.98.

J. D. Vasta et al. / Bioorg. Med. Chem. 21 (2013) 3597–3601
3599
analogs can eliminate the remaining P4H activity in dpy-18 C. ele-
gans at concentrations that do not affect the wild-type worm.5 This
synthetic lethality provided the basis for our assay.
Dpy-18 worms were placed onto solid medium containing
known concentrations of esterified epoxy ketone 1. The ester
group, which can be hydrolyzed by cellular esterases, served to
mask the anionic carboxylate and thus enhance uptake.5,24 Con-
centration-dependent embryonic lethality of dpy-18 C. elegans
was observed with LD50 = 0.11 mM (Fig. 3). These data are consis-
tent with lethality being due to P4H inactivation.
3. Conclusions
The need for small-molecule probes and inhibitors of P4H is evi-
dent from the lack of structural information about this essential
enzyme as well as the list of fibrotic diseases associated with col-
lagen overproduction.2 The newly discovered role of P4Hs in hy-
poxia25–27 and cancer,28 and the cardioprotection conferred by
P4H inhibitors29–31 increase the imperative.32 Our identification
of 4-oxo-5,6-epoxyhexanoate as an affinity label for P4H that is
bioavailable upon esterification provides a means to address this
need. On-going efforts in our laboratory are directed at identifying
the alkylated enzymic residue, improving the potency of the epoxy
ketone as an affinity label, and evaluating its selectivity towards
other P4Hs.
4. Experimental procedures
4.1. General
Scheme 1. Route for the synthesis of epoxy ketone 1.
in benzyl alcohol (31.6 mL, 33 g, 300 mmol), and the resulting solu-
tion was heated at reflux for 4 h. The reaction mixture was dis-
solved in ether (100 mL), and the insoluble succinic acid was
removed by filtration. The filtrate was extracted with saturated
aqueous Na2CO3 (3 Â 100 mL), and the combined aqueous extracts
were acidified with 2 M HCl (2.0 L). The precipitate was collected
by filtration and dried under vacuum to give 2 (30 g, 48%). 1H
NMR (CDCl3, 300 MHz) d 7.35 (m, 5H), 5.15 (s, 2H), 2.70 (m, 4H).
13C NMR (CDCl3, 75 MHz) d 177.7, 172.1, 135.7, 128.8, 128.5,
128.4, 66.9, 29.1, 29.0.
BakerDry™ solvents in cyclotainers™ (DMF, 620 ppm water;
CH2Cl2, 630 ppm water; THF, 610 ppm water) were from Baker
(Phillipsburg, NJ). Unless noted otherwise, all other chemicals were
from Aldrich Chemical (Milwaukee, WI), Fisher Scientific (Pitts-
burgh, PA), or NovaBiochem (San Diego, CA), and were used with-
out further purification.
Human collagen prolyl 4-hydroxylase (P4H) was produced in
E. coli and purified as described previously.14–18 Dansyl-Gly–Phe–
Pro–Gly-OEt, a fluorescent P4H substrate, was synthesized as de-
scribed previously.14–17
In the synthetic procedures, the term ‘concentrated under re-
duced pressure’ refers to the removal of solvents and other volatile
materials using a rotary evaporator at water aspirator pressure
(<20 torr) while maintaining the water-bath temperature below
50 °C.
4.3.2. Benzyl 4-chlorooxobutyrate (3)
Benzyl 4-chlorooxobutyrate was synthesized by following a
known procedure.34 Here, benzyl succinic acid (4.14 g, 20 mmol)
was dissolved in CH2Cl2 (150 mL), and the resulting solution was
cooled to 0 °C. Oxalyl chloride (13.8 g, 109 mmol) was added
slowly, and the reaction mixture was stirred at 0 °C for 5 min.
The reaction mixture was allowed to warm to 15 °C and stirred
for an additional 30 min. The reaction mixture was then concen-
trated under reduced pressure, and the residue was dissolved in
benzene. The residue was concentrated under reduced pressure
to give 3 (4.5 g, 99%) as a clear viscous liquid. 1H NMR (CDCl3,
300 MHz) d 7.34 (m, 5H), 5.13 (s, 2H), 3.19 (t, J = 7 Hz, 2H), 2.69
(t, J = 7 Hz, 2H). 13C NMR (CDCl3, 75 MHz) d 173.2, 170.9, 135.6,
128.8, 128.6, 128.4, 67.1, 29.5, 28.5.
4.2. Instrumentation
NMR spectra were obtained with a Bruker AC+ 300 MHz, Bruker
DMX 400 WB, or Bruker Avance III 500i spectrometer. Mass spectra
were obtained with a Micromass LCTÒ electrospray ionization,
time-of-flight analyzer from Waters (Milford, MA). HPLC analyses
were preformed with an instrument from Waters controlled with
the Millenium (version 3.20) software package and equipped with
two 515 pumps, a 717 plus autosampler, and a 996 photodiode ar-
ray detector.
4.3.3. Benzyl 4-oxo-5-hexenoate (4)
Benzyl 4-oxo-5-hexenoate (4) was synthesized in 82% yield
from benzyl 4-chlorooxobutyrate in the same manner as was ethyl
4-oxo-5-hexenoate (6) from ethyl 4-chlorooxobutyrate, vide infra.
1H NMR (CDCl3, 300 MHz) d 7.35 (m, 5H), 6.38 (dd, J = 10 and
18 Hz, 1H), 6.26 (dd, J = 1 and 18 Hz, 1H), 5.86 (dd, J = 1 and
10 Hz, 1H), 5.13 (s, 2H), 2.94 (t, J = 7 Hz, 2H), 2.70 (t, J = 7 Hz, 2H).
4.3. Synthesis of 4-oxo-5,6-epoxyhexanoate (1)
Epoxy ketone 1 was synthesized in five steps with an overall
yield of 38% by the route shown in Scheme 1 and described below.
4.3.1. Benzyl succinic acid (2)
4.3.4. Benzyl 4-oxo-5,6-epoxyhexanoate (5)
Benzyl 4-oxo-5,6-epoxyhexanoate (5) was synthesized in 98%
yield from benzyl 4-oxo-5-hexenoate in the same manner as was
Benzyl succinic acid was synthesized by following a known pro-
cedure.33 Here, succinic anhydride (30 g, 300 mmol) was dissolved

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J. D. Vasta et al. / Bioorg. Med. Chem. 21 (2013) 3597–3601
ethyl 4-oxo-5,6-epoxyhexanoate (6) from ethyl 4-oxo-5-hexenoate
(7), vide infra. 1H NMR (CDCl3, 300 MHz) d 7.34 (m, 5H), 5.10 (s,
2H), 3.46 (dd, J = 2 and 5 Hz, 1H), 2.99 (dd, J = 5 and 6 Hz, 1H)
2.94 (dd, J = 2 and 6 Hz, 1H), 2.64 (m, 4H). 13C NMR (CDCl3,
75 MHz) d 206.1, 172.4, 135.9, 128.7, 128.5, 128.4, 66.6, 53.7,
46.3, 31.3, 27.5.
dried over MgSO4(s) and concentrated under reduced pressure to
ꢀ20 mL. A half-saturated solution of KF in MeOH (25 mL) was
added to precipitate the SnBu3F, which was removed by filtration
through CeliteÒ. The filtrate was concentrated under reduced pres-
sure and purified by chromatography on silica gel, eluting with
ethyl acetate (10% v/v) in CH2Cl2 to give 7 (1.944 g, 82%). ESI MS
m/z: [M+Na]+ 179.0, calcd 179.1. 1H NMR (CDCl3, 400 MHz) d
6.35 (dd, J = 10 and 18 Hz, 1H), 6.23 (dd, J = 1 and 18 Hz, 1H),
5.84 (dd, J = 1 and 10 Hz, 1H), 4.10 (q, J = 7 Hz, 2H), 2.89 (t,
J = 7 Hz, 2H), 2.59 (t, J = 7 Hz, 2H), 1.21 (t, J = 7, 3H). 13C NMR
(CDCl3, 100 MHz) d 198.5, 172.7, 136.2, 128.4, 60.6, 34.0, 27.9, 14.1.
4.3.5. 4-Oxo-5,6-epoxyhexanoic acid (1, free acid)
Benzyl 4-oxo-5,6-epoxyhexanoate (340 mg, 1.45 mmol) was
dissolved in EtOAc (20 mL). Pd/C (10% w/w, 40 mg) was added,
and the reaction mixture was stirred under H2(g) for 4 h. The reac-
tion mixture was filtered through CeliteÒ, and concentrated under
reduced pressure to give the free acid of epoxy ketone 1 (183 mg,
99%). ESI MS EMM m/z: [MÀH]À 143.0340, calcd 143.0344. 1H
NMR (CDCl3, 300 MHz) d 10.74 (br s, 1H), 3.48 (dd, J = 2 and
5 Hz, 1H). 3.03 (dd, J = 5 and 5 Hz, 1H), 2.96 (dd, J = 2 and 5 Hz,
1H), 2.64 (m, 4H). 13C NMR (CDCl3 75 MHz) d 206.1, 178.3, 53.7,
46.4, 31.1, 27.2.
4.4.2. Ethyl 4-oxo-5,6-epoxy-hexanoate
Ethyl 4-oxo-5,6-epoxyhexanoate was synthesized by following
a known procedure.13 Here, ethyl 4-oxo-5-hexenoate (1.107 g,
7.09 mmol) and H2O2 (30% v/v, 1.75 mL) were dissolved in tBuOH
(11.81 mL) and H2O (5.9 mL) at 0 °C. Saturated aqueous K2CO3
(606 lL) at 0 °C was added slowly. The reaction mixture was stir-
red for 1 h at 0 °C, allowed to warm to room temperature, and then
stirred for an additional 3 h. The reaction mixture was diluted with
H2O (100 mL) and extracted with diethyl ether (3 Â 30 mL). The
combined organic extracts were dried over MgSO4(s) and concen-
trated under reduced pressure. The product was purified by chro-
matography on silica gel, eluting with ethyl acetate (10% v/v) in
CH2Cl2 to give 6 (1.04 g, 85%). ESI MS m/z: [M+Na]+ 195.0, calcd
195.1. 1H NMR (CDCl3, 300 MHz) d 4.11 (q, J = 7 Hz, 2H), 3.48 (dd,
J = 3 and 4 Hz, 1H), 3.03 (dd, J = 4 and 6 Hz, 1H), 3.01 (dd, J = 3
and 6 Hz, 1H), 2.66 (m, 4H), 1.25 (t, J = 7 Hz, 3H). 13C NMR (CDCl3,
75 MHz) d 206.3, 172.6, 60.9, 53.7, 46.3, 31.3, 27.5, 14.3.
4.3.6. Sodium 4-oxo-5,6-epoxyhexanoate (1, sodium salt)
4-Oxo-5,6-epoxyhexanoic acid (206 mg, 1.4 mmol) was dis-
solved in aqueous NaHCO3 (3.6 mL, 400 mM), and the resulting
solution was stirred for 1 h. The reaction mixture was concentrated
to dryness by lyophilization to give the water-soluble sodium salt
of epoxy ketone 1 (237 mg, quant), which was used for assays
in vitro. 1H NMR (D2O, 500 MHz) d 3.69 (dd, J = 3 and 5 Hz, 1H),
3.00 (t, J = 5 and 6 Hz, 1H), 2.80 (dd, J = 3 and 6 Hz, 1H), 2.70 (m,
2H), 2.34 (t, J = 7 Hz, 2H). 13C NMR (D2O, 125 MHz) d 211.7,
182.1, 54.2, 48.7, 36.1, 31.5.
4.4. Synthesis of ethyl 4-oxo-5,6-epoxy-hexanoate (6, esterified
1)
4.5. In vitro assay of P4H activity
The enzymatic activity of P4H was assayed as described previ-
ously.14 Briefly, substrate (dansyl-Gly–Phe–Pro–Gly-OEt) was
added to 1 mL of 50 mM Tris–HCl buffer, pH 7.8, containing P4H
Esterified 1 was synthesized in two steps with an overall yield
of 70% by the route shown in Scheme 2 and described below.
(0.43
ascorbate (2 mM), DTT (0.1 mM), and
Aliquots (30 L) were removed at 60, 180, and 300 s, quenched
by boiling for 30 s, and injected onto a Microsorb-MV C18 re-
versed-phase HPLC column (4.6 Â 250 mm, 100 Å pore size) from
Varian (Palo Alto, CA). The column was eluted at 1 mL/min with
aqueous acetonitrile (50% v/v), and the absorbance of the eluent
was monitored at 337.5 nm. All assays were preformed in
triplicate.
l
M), FeSO4 (0.1 mM), BSA (1 mg/mL), catalase (0.1 mg/mL),
4.4.1. Ethyl 4-oxo-5-hexenoate (7)
a-ketoglutarate (0.5 mM).
Ethyl 4-oxo-5-hexenoate was synthesized by following
a
l
known procedure.35 Here, ethyl 4-chlorooxobutyrate (2.5 g,
15.2 mmol) was dissolved in HMPA (10 mL). Tributylvinyltin
(5.0 g, 15.2 mmol) and benzylchlorotriphenylphosphine palla-
dium(II) (52 mg) were added, and the reaction mixture was heated
open to air to 65 °C for 5 min. At that time, the reaction mixture
turned black. The reaction mixture was allowed to cool to room
temperature. H2O (50 mL) and NaCl (1.0 g) were added, and the
resulting solution was stirred for 5 min. The solution was extracted
with diethyl ether (3 Â 60 mL). The combined organic extracts
were washed with H2O (4 Â 60 mL), and the organic layer was
4.6. In vitro assay for irreversible inhibition of P4H
P4H (43 lM) was incubated for 2.5, 5, 10, or 15 min with FeSO4
(2 mM) and epoxy ketone 1 in 50 mM Tris–HCl buffer, pH 7.8. BSA
(1 mg/mL), catalase (0.1 mg/mL), ascorbate (2 mM), DTT (0.1 mM),
and a-ketoglutarate (0.5 mM) were added to this reaction mixture.
Substrate (dansyl-Gly–Phe–Pro–Gly-OEt) was added to initiate the
reaction, and assays for P4H activity were performed as described
above. All assays were preformed in triplicate. Data are reported as
activity relative to control reaction mixtures that lacked epoxy ke-
tone 1. Half-times for inactivation at various inhibitor concentra-
tions are determined from linear least-squares regression
analysis of the log of the relative rates. Half-times for inactivation
were plotted against the reciprocal of inhibitor concentration in a
Kitz–Wilson plot19 to determine the value of kinact/Ki.
4.7. Protection of P4H by a-ketoglutarate
Epoxy ketone 1 (356 mM), P4H (43
were incubated for 5 min with -ketoglutarate (0, 15, 30, or
50 mM) in 50 mM Tris–HCl buffer, pH 7.8. BSA (1 mg/mL), catalase
lM), and FeSO4 (2 mM)
a
Scheme 2. Route for the synthesis of 6 (esterified 1).

J. D. Vasta et al. / Bioorg. Med. Chem. 21 (2013) 3597–3601
3601
(0.1 mg/mL), ascorbate (2 mM), DTT (0.1 mM), and substrate (dan-
syl-Gly–Phe–Pro–Gly-OEt) (2.5 mM) were added to initiate the
reaction. Aliquots were removed at 15, 60, 120, 180, 240, and
300 s, quenched by boiling for 30 s, and injected onto the C18 re-
versed-phase HPLC column described above. The column was
eluted at 1 mL/min with aqueous acetonitrile (50% v/v), and the
absorbance of the eluent was monitored at 337.5 nm. Reaction
rates were compared to the reactions without inhibitor.
H. Jensen and Lisa Friedman for guidance with chemical synthesis
and in vivo assays, respectively. This work was supported by Grant
R01 AR044276 (NIH). J.D.V. was supported by Molecular Biosci-
ences Training Grant T32 GM007215 (NIH). E.A.K. was supported
by Biotechnology Training Grant T32 GM083149 (NIH). The Micro-
mass LCTÒ Mass Spectrometer was purchased with funds from
Grant CHE-9974839 (NSF). This study made use of the National
Magnetic Resonance Facility at Madison, which was supported by
Grants P41 RR002301 and P41 GM066326 (NIH). Additional NMR
equipment was purchased with funds from the University of Wis-
consin, the NIH (P41 RR002781, P41 RR008438), the NSF (DMB-
8415048, OIA-9977486, BIR-9214394), and the USDA.
4.8. Immobilization of P4H
P4H (50 lg) was incubated for 30 min at 37 °C with FeSO4
(0.1 mM), ascorbate (5 mM), and epoxy ketone 1 (13 mM) in
1 mL of PBS. Biocytin hydrazide was added (to 11 mM), and the
reaction mixture was incubated for an additional 30 min at 37 °C.
NaBH4 (30 mg/mL in 10 mM NaOH) was added to the reaction mix-
ture to reduce the acylhydrazone. The P4H complex was desalted
using a NICK™ column from Amersham Pharmacia (Uppsala, Swe-
den) and incubated on Reacti-Bind™ streptavidin coated microtiter
plates from Pierce Chemical (Rockford, IL) for 1 h at 37 °C. The
wells were washed (4Â) with blocking buffer (0.1% w/v BSA and
0.05% v/v Tween 20Ò in PBS). A mouse monoclonal antibody spe-
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This Letter is dedicated to Professor Ashraf Brik on the occasion
of his winning the 2013 Tetrahedron Young Investigator Award in
Bioorganic & Medicinal Chemistry. We are grateful to Drs. Katrina