Diazirine-containing photoactivatable isoprenoid: Synthesis and application in studies with isoprenylcysteine carboxyl methyltransferase

Article abstract of DOI:10.1021/jo402600b

Photoaffinity labeling is a useful technique employed to identify protein-ligand and protein-protein noncovalent interactions. Photolabeling experiments have been particularly informative for probing membrane-bound proteins where structural information is difficult to obtain. The most widely used classes of photoactive functionalities include aryl azides, diazocarbonyls, diazirines, and benzophenones. Diazirines are intrinsically smaller than benzophenones and generate carbenes upon photolysis that react with a broader range of amino acid side chains compared with the benzophenone-derived diradical; this makes diazirines potentially more general photoaffinity-labeling agents. In this article, we describe the development and application of a new isoprenoid analogue containing a diazirine moiety that was prepared in six steps and incorporated into an a-factor-derived peptide produced via solid-phase synthesis. In addition to the diazirine moiety, fluorescein and biotin groups were also incorporated into the peptide to aid in the detection and enrichment of photo-cross-linked products. This multifuctional diazirine-containing peptide was a substrate for Ste14p, the yeast homologue of the potential anticancer target Icmt, with K_m (6.6 μM) and V_max (947 pmol min^-1 mg^-1) values comparable or better than a-factor peptides functionalized with benzophenone-based isoprenoids. Photo-cross-linking experiments demonstrated that the diazirine probe photo-cross-linked to Ste14p with observably higher efficiency than benzophenone-containing a-factor peptides.

Full text of DOI:10.1021/jo402600b

Article
pubs.acs.org/joc
Diazirine-Containing Photoactivatable Isoprenoid: Synthesis and
Application in Studies with Isoprenylcysteine Carboxyl
Methyltransferase
Jeffrey S. Vervacke,† Amy L. Funk,‡ Yen-Chih Wang,† Mark Strom,† Christine A. Hrycyna,*,‡
and Mark D. Distefano*,†
†Departments of Chemistry and Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, United States
‡Department of Chemistry, Purdue University, West Lafayette, Indiana 47907−2084, United States
S
* Supporting Information
ABSTRACT: Photoaffinity labeling is a useful technique
employed to identify protein−ligand and protein−protein
noncovalent interactions. Photolabeling experiments have
been particularly informative for probing membrane-bound
proteins where structural information is difficult to obtain. The
most widely used classes of photoactive functionalities include
aryl azides, diazocarbonyls, diazirines, and benzophenones.
Diazirines are intrinsically smaller than benzophenones and
generate carbenes upon photolysis that react with a broader
range of amino acid side chains compared with the
benzophenone-derived diradical; this makes diazirines poten-
tially more general photoaffinity-labeling agents. In this article,
we describe the development and application of a new
isoprenoid analogue containing a diazirine moiety that was prepared in six steps and incorporated into an a-factor-derived
peptide produced via solid-phase synthesis. In addition to the diazirine moiety, fluorescein and biotin groups were also
incorporated into the peptide to aid in the detection and enrichment of photo-cross-linked products. This multifuctional
diazirine-containing peptide was a substrate for Ste14p, the yeast homologue of the potential anticancer target Icmt, with Km (6.6
μM) and Vmax (947 pmol min−1 mg−1) values comparable or better than a-factor peptides functionalized with benzophenone-
based isoprenoids. Photo-cross-linking experiments demonstrated that the diazirine probe photo-cross-linked to Ste14p with
observably higher efficiency than benzophenone-containing a-factor peptides.
carboxymethylation is catalyzed by Icmt, a SAM-dependent
enzyme.2 Attachment of the hydrophobic isoprenoid causes
membrane association3 and is essential for membrane-
associated functions involving protein−protein interactions,4
signal transduction,5,6 and cellular homeostasis regulation.7
Because of the fact that Ras prenylation is required for its
proper cellular localization and function,8 numerous human
clinical trials have been conducted to inhibit this critical signal
transduction event that is particularly relevant to cancer.9,10
Importantly, an area of growing interest is developing inhibitors
for the other enzymes in the processing pathway including
Icmt, which is localized to the membrane of the endoplasmic
reticulum.
INTRODUCTION
■
Photoaffinity labeling is a useful technique employed to identify
noncovalent interactions between molecules, including pro-
tein−ligand and protein−protein complexes. The utility of
photoreactive probes to study such interactions is highly
dependent upon three criteria, including how closely the
photolabeling moiety mimics the native structure, what type of
reactive intermediates are formed, and the spatial relationship
between the two interacting molecules. The most widely used
classes of photoactive functionality include aryl azides,
diazocarbonyls and diazirines, and benzophenones, which give
rise to nitrenes, carbenes, and diradicals, respectively, upon
photolysis.1
Prenylated proteins are a class of membrane-associated
polypeptides that employ a farnesyl or geranylgeranyl
isoprenoid (sometimes two in the latter case) to anchor the
proteins into membranes. A three-step process consisting of
prenylation, proteolysis, and carboxymethylation is frequently
required to produce the final mature active proteins.
Prenylation is performed by farnesyl- or geranylgeranyltrans-
ferases, and proteolysis is carried out by Rce1 or Ste24, whereas
Photoactive analogues of farnesyl diphosphate have been
used extensively to study the interactions between soluble
proteins and their ligands, including isoprenoids and prenyl-
transferases. This class of photoprobes includes molecules
containing aryl azides,11 diazoesters,12−15 and benzophe-
Received: November 22, 2013
Published: February 6, 2014
© 2014 American Chemical Society
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Figure 1. Photoactivatable isoprenoid analogues. Left: farnesol, diazirine 1, benzophenone 2, and DATFP 3. Right: space-filling models of the
isoprenoid analogues. Hydrogen atoms are omitted for clarity. Color scheme: carbon, green; oxygen, red; nitrogen, blue; and fluorine, white. Spheres
are shown at 0.6 van der Waals radii for clarity.
nones,16−20 with the latter being the most commonly
employed. Peptides containing these photoactive isoprenoids
have also been used to study the interactions between
prenylated proteins and their cognate receptors.21 Recently,
we used peptides functionalized with benzophenone-containing
isoprenoids in photo-cross-linking experiments with mem-
brane-associated proteins that also recognize isoprene moieties,
including the Ras-converting enzyme Rce1 and the isoprenyl-
cysteine carboxyl methyltransferase, Icmt, two enzymes
involved in the CaaX protein post-translational processing
pathway.22,23 Although we showed that those enzymes could be
successfully photolabeled, the yield of cross-linking was not
sufficient to generate quantities of material adequate for mass
spectrometric sequencing. In addition, the benzophenone-
containing peptides were generally poorer substrates than those
incorporating a natural farnesyl group, likely because of the
larger size of the benzophenone moiety. To address this size
question and potentially increase the yield of cross-linked
protein, we wanted to explore the use of smaller isoprene unit
surrogates. Although a number of diazoester-containing
isoprenoid analogues have been previously reported,12,13,17,19
Figure 2. a-Factor-based peptides for the in vitro study of Icmt.
their synthesis is complicated by the need to use phosgene gas
and trifluorodiazoethane.24 In contrast, diazirines are much
simpler to prepare from ketones. Because diazirines are
intrinsically smaller than benzophenones and more closely
approximate the size and shape of an isoprene unit, they should
be effective isoprenoid mimics; moreover, because they
generate carbenes upon photolysis, they also have the potential
to react with a wider range of amino acid side chains, thereby
increasing photo-cross-linking efficiency.
were necessary for detection of cross-linked protein, in-gel
fluorescence via a fluorophore incorporated into the peptide
was used here to detect the cross-linked purified enzyme
successfully. These results suggest that such multifunctional
diazirine-containing peptides should be useful for identifying
active-site residues in Icmts and potentially other enzymes
involved in the processing of prenylated proteins.
Herein, we describe the synthesis of a diazirine-containing
isoprenoid unit (1) (Figure 1) and its incorporation into a
biotinylated and fluorescently labeled a-factor peptide analogue
(16) (Figure 2); the dodecapeptide, a-factor, is a naturally
occurring farnesylated peptide found in yeast and known to be
generated via methylation by Icmt.25 The ability of this
multifunctional photoactive peptide to bind and serve as a
substrate for Icmt was then evaluated using Ste14p, the Icmt
from the yeast Saccharomyces cerevisiae, as a model enzyme. The
diazirine-containing a-factor analogue demonstrated an in-
creased Vmax and lower Km relative to benzophenone-containing
probes. Cross-linking studies showed that the efficiency of
photolabeling achieved with the diazirine-based probe was
greater than that of benzophenone-containing probes. In
contrast to previous work where antibody-based methods
RESULTS AND DISCUSSION
■
Design and Synthesis of Diazirine-Containing Iso-
prenoid. To prepare the desired multifunctional a-factor
peptide for in vitro kinetic and photo-cross-linking analyses of
Ste14p, the initial strategy was first to design an isoprenoid
moiety containing a diazirine group. Compound 1 was selected
as the target based on the overall similarity in size of the
analogue compared with farnesyl diphosphate (FPP). Compar-
ison of 1 with benzophenone- and DATFP-containing FPP
analogues (2 and 3) reveals that 1 is closest in size to FPP
(Figure 1). Although potentially less stable than ether-linked
analogues such as 2, the ester analogue 1 was chosen for ease of
synthesis. A convergent strategy was used to prepare 1 based on
previously published routes for the production of the
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Scheme 1. Synthesis of a Diazirine-Containing Analogue of a Farnesyl Group
isoprenoid building block, 9,17 and the diazirine-derivative
Scheme 2. Synthesis of Prenylated a-Factor Precursor
Peptide 16
pentanoic acid, 6 (Scheme 1).26 Coupling of 9 and 6 in the
presence of DIC and DMAP afforded the ester (10). Removal
of the THP protecting group was performed with PPTS in
EtOH followed by conversion of the free alcohol, 11, to the
corresponding bromide, 12, by reaction with CBr4 and PPh3.
Purification of the bromide was carried out using a small
reversed-phase cartridge to minimize decomposition of the
allylic bromide on silica gel. The reversed-phase purification
also appears to remove colored impurities that interfere with
the subsequent peptide alkylation; although this process does
not remove unreacted alcohol starting material, that compound
does not interfere with the subsequent alkylation chemistry.
Synthesis of Prenylated a-Factor. We incorporated
isoprenoid analogue 12 into a peptide based on the structure
of the farnesylated dodecapeptide mating pheromone a-factor,
an in vivo substrate for Ste14p that is produced via the same
three-step process as larger prenylated proteins (Scheme 2).25
This peptide (16) contains a free C-terminal carboxylate to
serve as the methyl acceptor in the enzymatic reaction.
Solid-phase peptide synthesis was used to construct peptide
13 utilizing standard Fmoc/HCTU coupling conditions. An
additional N-terminal lysine residue was added to the a-factor
sequence to provide a second handle (via the side chain) for
fluorophore attachment. After the peptide was elongated, its
free N-terminus was biotinylated to yield 14. Biotin-Peg4-NHS
was chosen for installation of the biotin label because of its
efficient reaction kinetics and inexpensive cost. After specific
on-resin cleavage of the Dde protecting group from the side-
chain ε-amino group of the N-terminal lysine with a 10%
hydrazine/DMF solution, the resulting free amine was acylated
with 5-carboxylfluorescein succinimidyl ester (5-Fam) in the
presence of DIEA to give the fluorescently labeled peptide on
resin. 5-Fam was chosen as the fluorescent reporter because of
its reactivity with primary amines via the succinimidyl ester and
its convenient emission wavelength that makes it easy to detect
using most commercially available fluorescence scanners.
Cleavage from the resin and acidic deprotection was carried
out simultaneously by treatment with Reagent K to afford an
orange peptide 15, with a C-terminal cysteine bearing a free
thiol. It is interesting to note that previous reports have noted
that some epimerization of C-terminal cysteine residues can
occur when Cys-functionalized Wang resin and Reagent K
cleavage conditions are used;27 however, after RP-HPLC
purification, no evidence for epimerization was observed.
MS/MS analysis (see Supporting Information Figure S5)
revealed a large ensemble of b-type and y-type fragments
consistent with the proposed structure for 15. Alkylation of the
free thiol was performed using bromide 12 (1.5 equiv) in the
presence of Zn(OAc)2 (0.1 equiv) in acidic DMF to yield
prenylated peptide 16, whose identity was determined via ESI-
MS and purity was confirmed by analytical RP-HPLC. As was
observed for 15, MS/MS analysis of 16 (see Supporting
Information Figure S6) showed the presence of a variety of b-
type and y-type fragments, consistent with the proposed
structure for 16. Fragmentation via loss of the farnesyl group
was also observed as has been previously reported for
prenylated peptides.23,28 Farnesylated control peptide 17 and
benzophenone-containing peptide 19 were prepared by a
similar procedure, whereas 18 was prepared as previously
described.22
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Table 1. In Vitro Reaction Kinetics for a-Factor Peptides Methylated by His-Ste14p
a
b
ab
,
compound
Vmax (pmol min−1 mg−1
)
Km(app) (μM)
Vmax/Km (pmol min−1 mg−1 μM−1
)
AFC22
870 15
15.9 0.9
6.6 0.2
12.2 0.1
14.6 0.3
54
16
947
4
143
157
52
17
1919 30
762 28
1822
19
217
3
27.8
1
7.0
a
b
Reported as pmol of methyl groups transferred to substrate. Because these experiments were performed at a single concentration of SAM and the
biphasic nature of the membrane suspension makes the concentration of the peptide substrate unknown (because of partitioning into the lipid
membranes), these experiments provide only apparent values for Km.
Diazirine-Modified a-Factor Is a Substrate for His-
Ste14p. To assess the ability of 16 to act as a substrate for
Icmt, we used a recombinant form of yeast Icmt (Ste14p) that
incorporates both a His10 tag for purification and a triply
iterated myc tag to facilitate detection by immunoblot analysis
(His-Ste14p).7 The ability of 16 to act as a substrate for His-
Ste14p was determined using an in vitro methyltransferase
vapor-diffusion assay.7 The Km and Vmax values (Table 1) for 16
were measured using crude membrane fractions prepared from
yeast expressing His-Ste14p using N-acetyl-S-farnesyl-L-cysteine
(AFC) as a positive control. Comparison of the kinetic
parameters obtained for diazirine 16 and the benzophenone-
containing analogues 1822 (previously reported) and 19 reveals
that of those three probes containing photoactive isoprenoids
the diazirine manifests the highest Vmax value and lowest Km
value, making it the most efficient substrate. Although the Vmax
for 16 was lower than that for a farnesylated analogue of a-
factor precursor, 17, 16 demonstrated a lower Km value, making
the catalytic efficiency of 16 (Vmax/Km = 143 pmol min−1 mg−1
μM−1) comparable to that of 17 (Vmax/Km = 157 pmol min−1
mg−1 μM−1). Both of these values are higher than those
Figure 3. Analysis of cross-linking reactions containing purified His-
Ste14p and different photoactive probes. (A) Fluorescent imaging, (B)
immunoblot analysis with NeutrAvidin HRP, and (C) immunoblot
analysis with α-Ste14 . Experiments were performed with 16, 18, and
19. For this experiment, purified His-Ste14p (0.25 μg) was incubated
with the probes indicated (50 μM) and irradiated on ice for 30 min
followed by fractionation via SDS-PAGE. The resulting gel was
visualized using (A) a fluorescence scanner or transferred to a
nitrocellulose membrane and visualized using (B) NeutrAvidin HRP
or (C) an α-Ste14 antibody. The data shown is from one of three
replicates of this experiment.
obtained for benzophenone-based probes 1822 and 19 (Vmax
/
Km = 52 and 7.0 pmol min−1 mg−1 μM−1, respectively)
suggesting that the smaller diazirine moiety is a superior mimic
of an isoprene unit.
His-Ste14p Cross-Linking with the Diazirine-Modified
a-Factor. The ability of His-Ste14p to be covalently modified
with the diazirine- and benzophenone-modified a-factor
peptides was assessed via photoaffinity-labeling studies (Figure
3). Purified His-Ste14p (0.25 μg) was incubated with 50 μM of
each peptide and irradiated with UV light (365 nm) for 30 min
on ice, and the cross-linked material was resolved by SDS-
PAGE. The identity of the Ste14p−a-factor conjugate was
determined by three different methods: detection of the biotin
moiety with NeutrAvidin HRP (Figure 3B), detection of His-
Ste14 using a α-Ste14p antibody (Figure 3C), and ultimately
through direct fluorescence detection of the 5-Fam fluorophore
(Figure 3A).
Following separation of the reaction mixture by SDS-PAGE,
immunoblot analysis with an α-Ste14p antibody demonstrated
that His-Ste14p was present in each lane at the expected
molecular weight (∼36 kDa) (Figure 3C). UV-dependent
photo-cross-linking of the probes to His-Ste14p was visualized
using NeutrAvidin HRP. Biotinylated His-Ste14p was only
present in samples subjected to UV light (Figure 3B, lanes 3, 5,
and 7). The strongest signal was obtained with diazirine-
containing peptide 16, suggesting that this compound photo-
labeled His-Ste14p with the highest efficiency. Additionally,
UV-dependent fluorescent labeling was also observed using 16
and 19 (Figure 3A, lanes 5 and 7). Probe 18 does not contain a
5-Fam moiety and hence could not be visualized in a similar
manner. Furthermore, the increased labeling efficiency with 16
observed with NeutrAvidin HRP detection was recapitulated
with the fluorescence data. This increased labeling, which can
be readily visualized via direct fluorescence scanning of the gel,
suggests that a larger number of enzyme molecules are cross-
linked. These results are in contrast to those previously
reported for Rce1 with benzophenone-based probes23,29 and
bode well for future experiments that will be aimed at
identifying the site(s) of cross-linking. Moreover, the
experimental simplicity of following the cross-linking reactions
via fluorescence scanning in lieu of more complicated western
blotting or NeutrAvidin HRP detection should greatly facilitate
optimization of the experimental conditions. Overall, the
presence of both a biotin for enrichment and a fluorescent
label for direct visualization should make these new probes
particularly useful. In addition to the direct analysis of cross-
linking reactions shown in Figure 3, pulldown experiments with
16 and 19 were also performed (see Supporting Information
Figure S7) and showed that the biotin handle could be used to
recover the cross-linked products from the reaction mixture.
Comparing the efficiency of cross-linking of His-Ste14p to
16, 18, or 19, it is clear that the diazirine probe is the more
efficient photoaffinity-labeling reagent. Because the cross-
linkling experiments were performed with a concentration of
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each probe that was ∼2−8 times greater than their individual
Km values, the increase in labeling is likely not attributable to
differences in affinities between the different probes. Rather, it
must be due to either the increased reactivity of the carbene
intermediate generated from diazirine 16 compared to the
diradical intermediate produced from the benzophenone
photophores of 18 and 19 or the greater proximity or more
favorable geometry of the peptide in the active site. Currently,
additional diazirine- and benzophenone-containing probes are
being prepared, and we will determine whether the former
types always yield higher levels of cross-linking.
to benzophenone-containing peptides. Lastly, the incorporation
and use of this diazirine-modified isoprenoid in a peptide probe
equipped with a fluorophore greatly simplified the detection of
cross-linked products. The greater yield of photo-cross-linked
His-Ste14p coupled with the ease of analysis obtained with this
new class of isoprenoid mimics should facilitate the
identification of active-site residues in His-Ste14p. Such
experiments are currently underway for this important
membrane protein target. Given their improved features
compared with other types of photoactive isoprenoid probes,
this new class of analogues should be useful for a variety of
studies of enzymes that act on terpene-derived molecules.
Lastly, in addition to confirming the formation of the cross-
linked protein, the specificity of the photolabeling of His-
Ste14p with 16 was examined in a competition experiment
(Figure 4). A biotinylated a-factor precursor peptide, 17, was
EXPERIMENTAL SECTION
■
General Information. All solvents and reagents used for the
synthesis of the diazirine isoprenoid analogue and solid-phase peptide
synthesis of the photoactivatable peptides were of analytical grade and
purchased from Peptides International (Louisville, KY), NovaBio-
Chem (Nohenbrunn, Germany), or Sigma-Aldrich (St. Louis, MO).
NHS-PEG4-Biotin was obtained from Thermo Scientific. The
benzophenone-containing isoprenoid bromides, C10-m-Bp-Br (2),
was prepared as previously described.15,24−27 HR-ESI-MS analysis
was performed using a Bio-TOF-II mass spectrometer
3-(3-Methyldiaziridin-3-yl)propanoic Acid (5). Levulinic acid 4
(1.61 g, 13.8 mmol, 1 equiv) was dissolved in 7 N NH3 in CH3OH
(13.5 mL, 91.0 mmol, 7 equiv). The resulting solution was stirred
under N2 on ice for 3 h. A solution of hydrolxylamine-O-sulfonic acid
(1.80 g, 15.9 mmol, 1.2 equiv) in CH3OH (12 mL) was added
dropwise at a rate of 1 s−1. The reaction mixture was stirred for 20 h
and allowed to warm to rt. N2 was bubbled through the solution for 1
h to remove NH3 gas. Vacuum filtration and concentration resulted in
a yellow oil that was used in the next step without purification.
3-(3-Methyl-3H-diazirin-3-yl)propanoic Acid (6). Diaziridine 5
was redissolved in CH3OH (10 mL) and stirred on ice for 5 min in a
tin foil-covered flask. Triethylamine (3.00 mL, 21.5 mmol) was added
and allowed to stir for 5 min. Slowly, chips of I2 were added until the
solution remained a brown−red color for longer than 5 min after the
last addition. The reaction solution was diluted with EtOAc and
washed with 1 M HCl and aqueous 10% sodium thiosulfate until the
organic layer was colorless. The aqueous layer was further extracted
with EtOAc (2 × 20 mL). The organic layers were combined, dried
over MgSO4, and concentrated to afford diazirine acid 5 as a brown
residue (0.785 g, 45%). 1H NMR (300 MHz, CDCl3): δ 1.05 (s, 3H),
1.73 (t, 2H, J = 6.8 Hz), 2.23 (t, 2H, J = 6.8 Hz). 13C NMR (75.0
MHz, CDCl3): δ 20.4, 29.2, 30.0, 72.3, 179.1. HR-ESI-MS: calcd for
C5H8N2O2 [M−H]−, 127.0513; found, 127.0493.
Figure 4. Competition of photolabeling of His-Ste14p by 16 using a
biotinylated a-factor precursor peptide 17. For this experiment, His-
Ste14p crude membrane protein (100 μg) was mixed with 16 and 17
at the concentrations indicated and irradiated on ice for 30 min
followed by enrichment with neutravidin-agarose beads. The samples
were then eluted and resolved via SDS-PAGE. The resulting gel was
blotted to a nitrocellulose membrane and visualized using an α-Ste14
antibody. The data shown is from one of two replicates of this
experiment.
chosen as a competitor, as it was a substrate for His-Ste14p
(Table 1) and closely mimicked the structure of 16 (Figure 2).
Crude membranes prepared from a Δste14 deletion strain
overexpressing His-Ste14p were incubated with 16 in the
presence or absence of increasing concentrations of the
competitor 17 in the presence of UV light (Figure 4).
Following incubation, the samples were enriched for biotiny-
lated proteins via neutravidin-agarose beads, and the proteins
were separated by SDS-PAGE. Immunoblot analysis with an α-
Ste14p antibody revealed that the amount of cross-linked His-
Ste14p decreased as the concentration of the competitor, 17,
was increased from 5 (Figure 4, lane 3) to 200 μM (Figure 4,
lane 8). These data suggest that 16 is labeling the substrate
binding site of His-Ste14p.
(E)-2-((3,7-Dimethylocta-2,6-dien-1-yl)oxy)tetrahydro-2H-
pyran (8). This compound was prepared via a modification of a
previously described procedure.17 To a solution of geraniol 7 (3.11 g,
20.2 mmol, 1 equiv) in CH2Cl2 (3.5 mL) were added DHP (2.54 g,
30.3 mmol, 1.5 equiv) and PPTS (0.502 g, 2.0 mmol. 0.1 equiv), and
the resulting solution was stirred overnight at rt. The reaction mixture
was quenched with saturated aqueous NaHCO3 and extracted with
CH2Cl2. The organic layer was dried over MgSO4 and concentrated to
1
afford 8 as a clear oil (4.79 g, 99%). H NMR (300 MHz, CDCl3): δ
1.58 (s, 3H), 1.65 (s, 6H), 1.48−1.85 (m, 6H), 2.04−2.22 (m, 4H),
3.47−3.54 (m, 1H), 3.85−3.92 (m, 1H), 3.98−4.05 (dd, 1H, J = 7.2,
12 Hz), 4.21−4.27 (dd, 1H, J = 6.3, 12 Hz), 4.62 (t, 1H, J = 2.7 Hz),
5.16 (t, 1H, J = 6.3 Hz), 5.39 (t, 1H, J = 6.2 Hz). HR-ESI-MS: calcd
for C15H26O2 [M + H]+, 239.1919; found, 239.1932.
(2E,6E)-2,6-Dimethyl-8-((tetrahydro-2H-pyran-2-yl)oxy)octa-
2,6-dien-1-ol (9). This compound was prepared via a modification of
a previously described procedure.17 Protected geraniol 8 was dissolved
in CH2Cl2 (28 mL). In turn, 70% t-Bu-OOH in H2O (8.5 mL, 60.9
mmol, 3 equiv), salicylic acid (0.279 g, 2.02 mmol, 0.1 equiv), and
SeO2 (0.205 g, 2.03 mmol, 0.1 equiv) were added, and the resulting
solution was stirred overnight at rt. The reaction mixture was
quenched with saturated aqueous NaHCO3, extracted with CH2Cl2,
CONCLUSIONS
■
Herein, we have described the development and application of
a new class of isoprenoid analogue containing a photoexcitable
diazirine moiety. The photoactive farnesyl mimic was prepared
in six steps and incorporated into a multifunctional peptide
produced via solid-phase synthesis. Kinetic analyses with His-
Ste14p showed that the diazirine-containing peptide was an
efficient substrate for the enzyme compared to similar peptides
functionalized with benzophenone-based isoprenoids. The
diazirine-based probe cross-linked to His-Ste14p upon UV
irradiation with an observable increase in efficiency compared
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and dried over MgSO4. After concentration, the residue was purified
by flash chromatography (hexanes/Et2O, 3:2, v/v) on silica gel to
obtain 2.36 g (46%) of alcohol 9 as a clear oil. H NMR (300 MHz,
was washed with CH2Cl2, dried in vacuo overnight, weighed, and
divided into three portions for further synthesis on a reduced scale.
Using 83.0 μmol of peptide, the free amino terminus was biotinylated
in DMF (5 mL) with NHS-PEG4-Biotin (0.49 mg, 83.0 μmol, 1 equiv)
catalyzed by DIEA (14.4 μL, 8.3 μmol, 0.1 equiv) for 16 h. After
acylation, the resin-bound peptide was washed thoroughly with
CH2Cl2 and dried in vacuo for 4 h. The peptide was then reacted with
5% hydrazine in DMF (5 mL, v/v) to orthogonally remove they Dde-
protected side chain. After verifying the deprotection was complete by
ninhydrin analysis, the peptide was washed with CH2Cl2, dried, and
then reacted 5-FAM SE (45 mg, 86.0 μmol, 1 equiv) catalyzed by
DIEA (14.4 μL, 8.3 μmol, 0.1 equiv) overnight. The peptide was
cleaved from the resin along with simultaneous side-chain depro-
tection by treatment with Reagent K containing TFA (10 mL),
crystalline phenol (0.5 g), 1,2-ethanedithiol (0.25 mL), thioanisole
(0.5 mL), and H2O (0.5 mL) for 2 h at rt. The released peptide was
collected and combined with TFA washes of the resin before
precipitation of the peptide in chilled Et2O (100 mL). The crude solid
peptide was collected by centrifugation, the supernatant was removed,
and the resulting pellet was washed two times with cold Et2O (50 mL),
repeating the centrifugation and supernatant-removal steps each time.
The crude peptide was purified using a semipreparative C18 RP-HPLC
column with detection at 280 nm and eluted with a gradient of solvent
A (H20/0.1% TFA, v/v) and solvent B (CH3CN/0.1% TFA, v/v). The
crude peptide (150 mg) was dissolved in a DMF/H2O solution (1:5 v/
v, 25 mL), applied to the column equilibrated in solvent A, and washed
with 15% solvent B for 15 min. The peptide was eluted using a linear
gradient of (15−65% solvent B over 1.5 h at a flow-rate of 5 mL/min).
Fractions were analyzed using an analytical C18 RP-HPLC column
employing a linear gradient (0−100% solvent B over 60 min at a flow
rate of 1 mL/min) and detected at 214 nm. Fractions containing
peptide product of at least 90% purity were pooled and concentrated
by lyophilization to yield 55 mg (37% yield) of a yellow solid. A small
amount (<1 mg) of the resulting purified peptide was dissolved in 10
μL of 0.1% TFA/CH3CN and diluted 1:50 in a mixture of CH3CN/
H2O (1:1 v/v) prior to MS analysis. MS was performed using a 50 μL
injection and collecting 3000 scans. ESI-MS: calcd for
C117H155N19O30S2 [M + 2H]+2, 1186.0334; found, 1186.0453.
Synthesis of Biotin-Peg4-K(5-Fam)YIIKGVFWDPAC(C10-Dia-
zirine)-OH (16). Peptide 15 (20 mg, 5.3 μmol, 1 equiv) was dissolved
in DMF/n-butanol/H2O (0.10% TFA) (3:1:1, v/v/v, 6 mL).
Isoprenoid bromide 12 (15 mg, 54.5 μmol, 10 equiv) was dissolved
in 0.50 mL of DMF and loaded onto a C18 Sep-Pak column that had
been equilibrated with 5% CH3CN in aqueous 0.10% TFA. The
column was washed with 5% CH3CN (5 mL) followed by 30%
CH3CN (10 mL). The purified bromide was then eluted from the
column with 3.0 mL of DMF directly into the reaction flask that
contained the dissolved peptide. Zn(OAc)2·2H2O (5.4 mg, 25 μmol, 5
equiv) was then added to initiate the alkylation reaction. After 4 h, the
reaction was analyzed by analytical RP-HPLC, purified by semi-
preparative C18 RP-HPLC, and identified via ESI-TOF MS. This
reaction yielded 5.1 mg (21%) of desired alkylated peptide 16. Purity
by HPLC: 96.1%. ESI-MS: calcd for C132H177N21O32S2 [M + 2H]+2,
1317.1223; found, 1317.1172.
1
CDCl3): δ 1.65 (s, 6H), 1.48−1.85 (m, 6H), 2.04−2.22 (m, 4H),
3.47−3.54 (m, 1H), 3.85−3.92 (m, 1H), 3.91 (s, 2H), 3.98−4.05 (dd,
1H, J = 7.2, 12 Hz), 4.21−4.27 (dd, 1H, J = 6.3, 12 Hz), 4.62 (t, 1H, J
= 2.7 Hz), 5.16 (t, 1H, J = 6.3 Hz), 5.39 (t, 1H, J = 6.2 Hz). HR-ESI-
MS: calcd for C15H26O3Na [M + Na]+, 277.1790; found, 277.1763.
(2E,6E)-2,6-Dimethyl-8-((tetrahydro-2H-pyran-2-yl)oxy)octa-
2,6-dien-1-yl 3-(3-Methyl-3H-diazirin-3-yl)propanoate (10). To
a solution of protected alcohol 9 (1.94 g, 7.60 mmol, 1 equiv) and
DMAP (92.8 mg, 0.76 mmol, 0.1 equiv) in CH2Cl2 (7 mL) was added
a solution of the diazirine acid 6 (0.97 g, 7.60 mmol, 1 equiv) in
CH2Cl2 (10 mL). DIC (0.959 g, 7.60 mmol, 1 equiv) was added, and
the resulting solution was stirred at rt for 16 h. The reaction mixture
was filtered and concentrated. Purification by flash chromatography
(hexanes/EtOAc, 5:1) afforded 1.07 g (57%) of diazirine 6 as a clear
1
oil. H NMR (300 MHz, CDCl3): δ 1.03 (s, 3H), 1.48−1.85 (m,
14H), 2.04−2.22 (m, 6H), 3.47−3.54 (m, 1H), 3.85−3.92 (m, 1H),
3.98−4.05 (dd, 1H, J = 7.2, 12 Hz), 4.21−4.27 (dd, 1H, J = 6.3, 12
Hz), 4.46 (s, 2H), 4.62 (t, 1H, J = 2.7 Hz), 5.36 (t, 1H, J = 6.3 Hz),
5.44 (t, 1H, J = 6.2 Hz). 13C NMR (75.0 MHz, CDCl3): δ 13.16, 16.4,
20.5, 23.7, 25.1, 26.6, 30.6, 32.9, 39.7, 44.2, 63.3, 65.6, 69.3, 72.3,
105.5, 118.5, 130.4, 130.7, 141.8, 173.1. IR (NaCl, cm−1): 2940 (s),
2870 (m), 1737 (s), 1669 (w), 1587 (w), 1447 (m), 1385 (m), 1175
(m), 1117 (m), 1023 (m). HR-ESI-MS: calcd for C20H32N2O4Na [M
+ Na]+, 387.2260; found, 387.2234.
(2E,6E)-8-Hydroxy-2,6-dimethylocta-2,6-dien-1-yl 3-(3-
Methyl-3H-diazirin-3-yl)propanoate (11). To a solution of 10
(99 mg, 0.271 mmol, 1 equiv) in EtOH (1.60 mL) was added PPTS
(6.81 mg, 271 μmol, 0.1 equiv). The reaction flask was fitted with a
septum and stirred at 60 °C for 4 h. The reaction mixture was
concentrated in vacuo and purified by flash chromatography (hexanes/
EtOAc, 2:1) to afford 75 mg (99%) of diazirine alcohol 11 as a clear
oil. 1H NMR (300 MHz, CDCl3): δ 1.03 (s, 3H), 1.58−1.75 (m, 8H),
2.05−2.22 (m, 6H), 4.15 (t, 2H, J = 6.0 Hz), 4.47 (s, 2H), 5.38−5.45
(m, 2H). 13C NMR (75.0 MHz, CDCl3): δ 13.6, 16.4, 23.5, 26.4, 32.9,
39.7, 44.2, 58.9, 69.3, 72.1, 124.6, 130.4, 130.7, 141.8, 173.1. IR (NaCl,
cm−1): 3395 (br), 2925 (s), 2870 (m), 1735 (s), 1669 (w), 1587 (w),
1447 (m), 1385 (m), 1176 (m), 1000 (m). HR-ESI-MS: calcd for
C15H24N2O3Na [M + Na]+, 303.1685; found, 303.1659.
(2E,6E)-8-Bromo-2,6-dimethylocta-2,6-dien-1-yl 3-(3-Meth-
yl-3H-diazirin-3-yl)propanoate (12). Diazirine alcohol 11 (69.5
mg, 0.247 mmol, 1 equiv) was converted to the corresponding
bromide in the presence of resin-bound PPh3 (250 mg, 1.08 mmol, 4
equiv) and CBr4 (350 mg, 1.08 mmol, 4 equiv) dissolved in CHCl3 (6
mL). The resulting solution was allowed to stir for 2 h at rt. After the
reaction was complete, excess CBr4 and resin-bound PPh3 were
removed from the mixture by passing the reaction through a C18 Sep-
Pak column. Removal of the solvent afforded 68.1 mg (80%) of
1
diazirine bromide 12 as a clear oil. H NMR (300 MHz, CDCl3): δ
1.03 (s, 3H), 1.58−1.75 (m, 8H), 2.05−2.22 (m, 6H), 3.99 (d, 2H, J =
8.4 Hz), 4.47 (s, 2H), 5.39 (t, H, J = 6 Hz) 5.51 (t, 2H, J = 6.1 Hz).
13C NMR (75.0 MHz, CDCl3): δ 13.6, 15.4, 23.5, 26.4, 30.8, 32.9,
38.3, 44.2, 69.3, 72.1, 123.3, 130.4, 130.7, 143.2, 173.1. IR (NaCl,
cm−1): 2925 (m), 2870 (m), 1736 (s), 1656 (w), 1586 (w), 1446 (m),
1385 (m), 1173 (m). HR-ESI-MS: calcd for C15H23BrN2O2Na [M +
Na]+, 365.0840 (79Br); found, 365.0862; 367.0819, (81Br); found,
367.0831.
Synthesis of Biotin-Peg4-K(5-Fam)YIIKGVFWDPAC-OH (15).
Peptide synthesis was carried out using an automated solid-phase
peptide synthesizer (PS3, Protein Technologies Inc., Memphis, TN)
employing standard Fmoc/HCTU-based chemistry. Synthesis began
on preloaded Fmoc-Cys(Trt)-Wang resin (0.25 mmol), and the
peptide chain was elongated using HCTU/N-methylmorpholine-
catalyzed, single coupling steps with 4 equiv of both protected
amino acids and HTCU for 30 min. Following complete chain
elongation, the peptide’s N-terminus was deprotected with 10%
piperidine in DMF (v/v), and the presence of the resulting free amine
was confirmed by ninhydrin analysis. The resin containing the peptide
Synthesis of Biotin-Peg4-YIIKGVFWDPAC(Fr)-OH (17). Solid-
phase peptide synthesis and purification were carried out in the same
fashion as described above. Following complete chain elongation, the
peptide’s N-terminus was deprotected with 10% piperidine in DMF
(v/v), and the presence of the resulting free amine was confirmed by
ninhydrin analysis. Using 83.0 μmol of peptide, the free amino
terminus was biotinylated in DMF (5 mL) with NHS-PEG4-Biotin
(0.49 mg, 83.0 μmol, 1 equiv) catalyzed by DIEA (14.4 μL, 8.3 μmol,
0.1 equiv) for 16 h. After Reagent K cleavage and HPLC purification,
the free thiol-containing peptide (20 mg, 13 μmol, 1 equiv) was
prenylated with farnesyl bromide (10 mg, 65 μmol, 5 equiv) using the
same conditions as 16. Purity by HPLC: 92.4%. ESI-MS: calcd for
C105H157N17O23S2 [M + 2H]+2, 1045.0551; found, 1045.0499
Synthesis of Biotin-Peg4-K(5-Fam)YIIKGVFWDPAC(C5-m-
BP)-OH (19). Starting with 15, as previously described, the peptide
was prenylated using the same conditions and quantities as used for 16
1976
dx.doi.org/10.1021/jo402600b | J. Org. Chem. 2014, 79, 1971−1978

The Journal of Organic Chemistry
Article
with replacement of the isoprenoid analogue 12 with C5-m-Bp-Br that
was prepared as previously described.19,21 Purity by HPLC: 94.2%.
ESI-MS: calcd for C136H173N19O32S2 [M + 2H]+2, 1325.0951; found,
1325.0994.
three times with PBST, and the protein bands were visualized using
ECL.
ASSOCIATED CONTENT
* Supporting Information
■
Protein Isolation and in Vitro Methyltransferase Vapor-
Diffusion Assays. For experiments using crude protein, membrane
preparations from the yeast strain CH2704 overexpressing His-Ste14p
were prepared as previously described, with minor modifications.7,30
Following centrifugation at 100 000g for 1 h, the membrane pellet was
resuspended in 10 mM Tris-HCl, pH 7.5, aliquoted, flash-frozen in
liquid N2, and stored at −80 °C. In vitro assays for methyltransferase
activity were performed using crude membranes as previously
described.22 In brief, reactions contained crude membrane prepara-
tions, 200 μM AFC, 20 μM S-adenosyl-L-[methyl-14C]methionine
([14C]SAM) (50−60 mCi/mmol), and 100 mM Tris-HCl, pH 7.5, in
60 μL. The reactions were incubated at 30 °C for 30 min and
terminated by the addition of 50 μL of 1 M NaOH and 1% SDS (v/v).
Each reaction (100 μL) was then spotted onto a filter paper that was
wedged into the neck of a vial containing 10 mL of scintillation fluid
and allowed to diffuse at rt for 2.5 h. The filters were discarded, and
the base-labile [14C]methyl groups transferred were measured via
liquid scintillation counting. For experiments performed with purified
His-Ste14p, His10myc3N-Ste14p (His-Ste14p) was expressed in a
Δste14 deletion strain30 and purified as previously described.7
Photo-Cross-Linking and Neutravidin-Agarose Pulldown
Assays in Crude Membranes. Photo-cross-linking assays were
performed as described previously, with minor modifications.20,22
One-hundred micrograms of crude membrane preparation expressing
His-Ste14p in 10 mM Tris-HCl, pH 7.5, was preincubated with
increasing concentrations of the competition peptide before addition
of the photolabeling reagent and incubated at 4 °C for 5 min. The
samples were irradiated at 365 nm in 96-well plates for 30 min on ice.
The resulting protein samples were solubilized in 800 μL of
radioimmunoprecipitation assay (RIPA) buffer (25 mM Tris-HCl,
pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate,
and 0.1% sodium dodecylsulfate)/10% SDS and incubated with 50 μL
of a 50% neutravidin/RIPA bead slurry for 2 h at 4 °C. The beads were
centrifuged at 13 000g for 1 min and washed three times with RIPA/
10% SDS. The cross-linked protein was eluted from the neutravidin
beads by the addition of 50 μL of 2× SDS sample buffer (0.5 M Tris-
HCl, pH 6.8, 30% sucrose (w/v), 10% sodium dodecylsulfate (w/v),
3.5 M 2-mercaptoethanol, and 0.1% bromophenol blue (w/v)).
Samples were heated for 30 min at 65 °C and subjected to 12% SDS-
PAGE and immunoblot analysis.
S
1H NMR, 13C HMR, and IR for all new compounds; ESI-MS
and analytical HPLC chromatograms for reported peptides; and
analysis of cross-linking reactions containing purified His−
Ste14p and photoactive probes 16 and 19 after pulldown with
streptavidin. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*E-mail: Hrycyna@purdue.edu (C.A.H.).
*E-mail: Diste001@umn.edu (M.D.D.).
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This research was supported by funds from the NIH (grants
R01 GM084152 to M.D.D. and R01 GM106082 to C.A.H.).
■
REFERENCES
■
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