Evaluation of α-pyrones and pyrimidones as photoaffinity probes for affinity-based protein profiling

Article abstract of DOI:10.1021/jo201281c

α-Pyrones and pyrimidones are common structural motifs in natural products and bioactive compounds. They also display photochemistry that generates high-energy intermediates that may be capable of protein reactivity. A library of pyrones and pyrimidones was synthesized, and their potential to act as photoaffinity probes for nondirected affinity-based protein profiling in several crude cell lysates was evaluated. Further "proof-of-principle" experiments demonstrate that a pyrimidone tag on an appropriate scaffold is equally capable of proteome labeling as a benzophenone.

Full text of DOI:10.1021/jo201281c

ARTICLE
pubs.acs.org/joc
Evaluation of r-Pyrones and Pyrimidones as Photoaffinity Probes for
Affinity-Based Protein Profiling
Oliver A. Battenberg, Matthew B. Nodwell, and Stephan A. Sieber*
Department Chemie, Center for Integrated Protein Science CIPSM, Institute of Advanced Studies, Technische Universit€at M€unchen,
Lichtenbergstrasse 4, 85747 Garching, Germany
S Supporting Information
b
ABSTRACT: R-Pyrones and pyrimidones are common struc-
tural motifs in natural products and bioactive compounds. They
also display photochemistry that generates high-energy inter-
mediates that may be capable of protein reactivity. A library of
pyrones and pyrimidones was synthesized, and their potential to
act as photoaffinity probes for nondirected affinity-based pro-
tein profiling in several crude cell lysates was evaluated. Further
“proof-of-principle” experiments demonstrate that a pyrimi-
done tag on an appropriate scaffold is equally capable of
proteome labeling as a benzophenone.
’ INTRODUCTION
intrinsic photoreactivity include steroid enones,10,11 some aryl
chlorides,12 and some thioethers.13,14
Many disease states arise from aberrant protein function,
and it is thus a major goal to determine the molecular, cellular,
and physiological functions for the multitude of proteins en-
coded by eukaryotic and prokaryotic genomes, a goal that cannot
be accomplished by analysis of genome sequences alone. Because
proteins do not function in isolation but rather as parts of
complex regulatory networks, a method to examine proteins in
their natural state has been developed. Termed activity-based
protein profiling (ABPP), this chemical-proteomic approach
involves the interrogation of an entire native proteome with
carefully designed probes.1ꢀ4 These probes typically contain an
active-site-directed group for binding and covalent modification
of the target protein, a linker, and a tag that allows for visualiza-
tion or enrichment of the labeled proteins.
Typically, ABPP probes achieve covalent protein modification
of enzyme active sites either by electrophilic labeling of com-
plementary protein nucleophilic sites or by photo-cross-linking,
which is the strategy of choice for probes that lack an electrophilic
function. Photo-cross-linking does not necessarily represent a
mechanism-based labeling of target proteins and therefore can
also be referred to as “affinity-based protein profiling”.
R-Pyrones and pyrimidones are structural motifs found in
many natural products and bioactive molecules. Examples in-
clude citreoviridin,15 bufadienolides,16 aureothins,17 fusapyrone,18
pyrimidone-thiazolidinediones,19 and Zebularine20 (Figure 1).
In addition to their presence in bioactive compounds, pyrones
and pyrimidones also display well-documented photoreactivities.
R-Pyrones (1 or 4), upon irradiation with UV light, undergo an
isomerization to either ketene 2 or a bycyclic β-lactone 5,
depending on the nature of the substituents on the pyrone
ring.21ꢀ23 These isomerized forms can then undergo reactions
with nucleophiles. In the case of 4-alkyl-substituted pyrone 1,
simple attack of the nucleophile, in this example, methanol, on
ketene 2 leads to methyl β-acetonylcrotonate 3. In the case of
4-alkoxy substituted pyrone, which initially forms bicyclic lactone
5 upon irradiation, the reaction pathway involves zwitterionic
intermediate 6.24 MeOH addition to 6 followed by thermal
conrotatory ring opening of 7 forms intermediate 8, which can
undergo ring closure to orthoester product 9. Hydrolysis of 9 in
aqueous media results in formation of β-carbomethoxymethyl-
crotonic acid 10 (Scheme 1).
Pyrimidones (11) upon irradiation undergo a Norrish type I
reaction (11 to 12) to form either an isocyanate 13 or bicyclic
intermediate 14, which can then react with nucleophiles to form
the tautomeric products 15 and 15a as shown in Scheme 2.25,26
Common examples of photo-cross-linking groups5,6 are aryl
azides,7 diazirines,8 and benzophenones.9 Each group has its
disadvantages and advantages; however, each involves the in-
corporation of a non-natural moiety into a probe structure that
can drastically perturb probeꢀprotein interaction. This limita-
tion can be circumvented by the application of intrinsically
photoreactive probes, i.e., probes whose natural structures gen-
erate highly reactive intermediates upon irradiation. Examples of
Received: April 5, 2011
Published: July 05, 2011
r
2011 American Chemical Society
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Figure 1. Pyrone and pyrimidone-containing bioactive compounds.
Scheme 123
Scheme 226
We found this photoreactivity, coupled with the occurrence of
R-pyrones and pyrimidones in various natural product and
bioactive compounds to be an intriguing approach to intrinsic
photoreactivity. Natural products and other bioactive com-
pounds with R-pyrones or pyrimidones in their structures could
be photoactivated to a reactive species as illustrated in Schemes 1
and 2. This reactive species could then react with a nearby amino
acid to covalently modify the binding protein (Scheme 3).
In this report, we outline syntheses of R-pyrone- and pyrimi-
done-containing compounds and attempts to use these as
nondirected photoactivatible affinity probes.
Kinetics of photoisomerization and reaction of the inter-
mediates with nucleophiles has been the subject of several
studies.27ꢀ31 Whereas the above photoisomerizations to active
species are fast, the reaction of these species with nucleophiles is
relatively slow. However, given that the formation of the active
species is largely reversible and in a proteinꢀsubstrate complex,
the nucleophile concentration is high, and a protein-labeling
event is likely to occur.
’ RESULTS AND DISCUSSION
The design of the probes required careful consideration
for their structural elements. We sought to have a wide variety of
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Scheme 3
Figure 3. Zebularine and probe 16.
Scheme 4
Figure 4. Trimethoprim and probe 17.
probes can be broadly divided in two classes: those that are
inspired by compounds with known bioactivity (biology-inspired
probes) and those that are not (diversity-oriented probes).
Probe 16 was designed as a mimic of the DNA methyla-
tion inhibitor Zebularine20 (Figure 3).
Formation of the glycosidic bond was accomplished by
utilizing a one-pot procedure as outlined by Vorbr€uggen et al.35,36
between pyrimidine 24 and peracetylated ribofuranose 25 to give
26 in 12% yield. Deprotection of the acetyl groups by ammonia in
MeOH gives nucleoside 27 in 81% yield.37 At this point, the
alkyne tag was installed by coupling of 5-hexynoic acid to
nucleoside 27 in DMF. Probe 16 was isolated from a mixture
of isomers, and its regiochemistry was confirmed by HMBC
correlations between the 50 ribofuranose methylene and the ester
carbonyl (Scheme 4).
Figure 2. Biology- and diversity-inspired probes.
probe structures in order to maximize the opportunities for
specific biological interactivity, in this case with bacterial pro-
teomes. In addition to the R-pyrone or pyrimidone, within each
structure is a sterically benign alkyne tag suitable for the Cu(I)-
catalyzed Huisgen [3 + 2]-azideꢀalkyne cycloaddition,32ꢀ34
which would allow the attachment of a fluorescent or a biotin
tag for visualization and enrichment, respectively. Figure 2 shows
the probes used in our proteome labeling experiments. These
Probe 17 was designed to resemble the dihydrofolate reduc-
tase inhibitor trimethoprim (Figure 4).38
Therefore, vanillin was alkylated by tosylate 29 to yield
aldehyde 30.39 Functional group interchanges40,41 yielded bro-
mide 31, which was then substituted with pyrimidone 24 to yield
probe 17 (Scheme 5).
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Scheme 5
Scheme 6
Standard deprotection (TBAF/THF) then yields probe 20
(Scheme 8).
Scheme 7
Probe 21 was based on a naphthalene core and was synthe-
sized in a manner analogous to probe 17 starting with 2-hydroxy-
1-napthaldehyde (40) (Scheme 9).
Probe 22 was based on an amino acid scaffold, in this case,
serine. Protection of the C and N terminals of serine as a methyl
ester and tert-butyl carbamate, respectively, gives 44.46
Bromination47 of the primary alcohol to yield 45 followed by
substitution with pyrimidone 24 gives 46, albeit with racemiza-
tion of the chiral center. Deprotection of the Boc group under
standard conditions, followed by amide formation with 5-hex-
ynoic acid, yields probe 22 (Scheme 10).
The final pyrone probe, 23, was synthesized via deprotonation
of bromide 33 at C7, followed by quenching of the anion with
biphenyl-4-carboxaldehyde to yield 47.48,49 Sonogashira reaction
with trimethylsilylacetylene and 47, followed by standard depro-
tection, yields probe 23 (Scheme 11).
R-Pyrone probe 18 has been reported to possess broad-
spectrum antibacterial activity42 and was prepared via bromina-
tion and subsequent Sonogashira reaction with trimethylsilyla-
cetylene of the commercially available triacetic acid lactone 32
(Scheme 6).43
Probe 19 is based upon 17R-ethynylestradiol (35), a common
estrogenic component in oral contraceptives that conveniently
has a terminal alkyne in its structure. While 17R-ethynylestradiol
will in all probability display no appreciable biological activity
toward bacterial proteins, the core steroid structure is a common
one and may impart specific protein binding in the bacterial
proteome. Reaction of 17R-ethynylestradiol with bromide 33
under basic conditions results in a Michael additionꢀβ-elimina-
tion to yield probe 19 (Scheme 7).44
The diversity-oriented class of probes were not based upon
bioactive scaffolds but were chosen to present a variety of
structural elements to the proteome. Probe 20 was synthesized
by iodination of pyrimidone 24 with N-iodosuccinimide,45 fol-
lowed by installation of a benzyl group to yield 36. Sonogashira
reaction between iodide 36 and trimethylsilylacetylene yields 37.
With probes 16ꢀ23 in hand, we wanted to verify their light-
induced reactivity with nucleophiles. We picked two probes, 18
and 20, as test compounds.
Irradiation of a methanolic solution of 18 at 350 nm resulted in
the loss of starting material and the formation of a peak that
corresponds to the expected ester photoproduct 49 (Figure 5).
As LCꢀMS was problematic for this compound, it was isolated
1
and the H and 13C NMR spectra of 49 were recorded. This
reaction was complete after less than 10 min.
Irradiation of 20 in MeOH at 350 nm results in almost
complete conversion after 30 min, accompanied by the formation
of a peak that corresponds to the proposed photoproduct 50 as
determined by LCꢀMS (Figure 6).
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Scheme 8
Scheme 9
Scheme 10
Scheme 11
Once we were satisfied that our probes were reacting suffi-
ciently with weak nucleophiles, we set labeling parameters for
optimal probe reaction with crude cell lysate preparations. Using
either E. coli or S. aureus Mu50 cytosol preparations as our
standard proteome preparations, we screened representative
probes for time and concentration dependence with respect to
photolabeling. In a typical experiment, a crude cell lysate
(proteome) is diluted with PBS to give 43 μL of 1 mg/mL
protein concentration. One microliter of the desired probe is
then added in DMSO to give the final probe concentration.
Irradiation is carried out over ice cooling in a 96-well plate. The
irradiated solutions are then transferred back to a reaction vessel,
and [3 + 2]-azideꢀalkyne “click chemistry” is performed on the
solution with rhodamine azide for 1 h at rt. Figure 7 shows that
for probe 19, 100 μM and 1 h yields saturated protein labeling.
These conditions also result in saturated labeling for 17;
however, for 20, while 100 μM remains an optimal concentra-
tion, only 20 min is necessary for saturated labeling (Figures S1
and S2, Supporting Information). Increases in irradiation time
only increase background signal, and probe concentrations over
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Figure 5. UV-dependent conversion of 18 to 49.
Figure 6. UV-dependent conversion of 20 to 50.
100 μM do not result in significantly increased signal-to-
noise ratios. Nonirradiated controls demonstrate a clear light-
dependent labeling for some protein bands (Figure 7, lanes
5ꢀ8), emphasizing that photoinduced reactivity leads to protein
labeling.
With the key parameters set, we then compared all probes
for labeling patterns in both L. welshimerii and E. coli crude
cell lysates using the optimized labeling parameters as descri-
bed above. A variety of structures as shown in Figure 2 should,
by virtue of their differing noncovalent interactions, show a
wide variety of labeling patterns as well. However, Figure 8
demonstrates that while a variety of structural types are displayed
to the bacterial proteomes, the labeling patterns show only a few
differences (see also Figure S3, Supporting Information for E. coli
labeling).
The similarity in labeling patterns may be due to the relatively
long lifespan of the photogenerated intermediates of the R-
pyrones or pyrimidones (Schemes 1 and 2) that allows for
mobility from the probes original binding site. This mobile
intermediate can then equilibrate among the reaction medium
and then react with available protein nucleophiles, and hence the
probes designed herein reflect a preference for type of reactant
instead of type of binding site. However, a striking feature of cell
lysate labeling with probes 16ꢀ23 is the moderate reactivity of
the photogenerated species with respect to the native proteomes
emphasizing that the photoinduced electrophiles display a
certain selectivity for available protein nucleophiles. Concerned
with this apparent lack of selectivity that probes 16ꢀ23
demonstrate toward bacterial lysates, we sought a more com-
parative approach toward demonstrating the feasibility of our
hypothesis. Realizing that biology-based probes 16ꢀ19 display
significant structural deviations from their original inspirations
that may drastically reduce their target binding50ꢀ52 and that
diversity-based probes 20ꢀ23 may not possess enough structural
elements to prevent diffusion after photoactivation, we chose to
modify a substrate whereby the additional elements (photolabel
and alkyne tag) would constitute a minimal structural change. A
recent account from our group details the synthesis of vanco-
mycin-based photoaffinity probes that selectively label autolysin
(ATLam) and an ABC transporter protein (pABC) in S. aureus
and E. faecalis, respectively (Figure 9).53
We reasoned that by substituting the benzophenone in probe
51 for a pyrone or pyrimidone, we could directly compare the
labeling efficacy of a well-established photo-cross-linking moiety
with a novel cross-linking group. For synthetic ease, we chose to
examine a pyrimidone in this position. Compound 55 was
prepared according to Scheme 12 by a coupling of primary
amine 53 with modified vancomycin 54.
With probe 55 in hand, we then compared the labeling
patterns of 51 and 55 in BL21 E. coli recombinant clone cell
lysates overexpressing both ATLam and pABP. To our gratifica-
tion, the labeling patterns for both 51 and 55 were identical at 1
and 10 μM, respectively, while nonirradiated controls demon-
strated little to no labeling at 100 μM 55. Furthermore, attempts
to photolabel the overexpressed E. coli cell lysates with 100 μM
22 gave no appreciable labeling, showing patterns similar to
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Figure 7. Time- and concentration-dependent labeling of E. coli. crude cellular lysate with 19.
This result represents an important validation of the hypothesis
that pyrimidones can be used as photo-cross-linkers. The selec-
tivity of probe 55 relative to probes 16ꢀ23 can be explained by
the much greater molecular complexity of vancomycin, which
results in greater noncovalent protein binding, allowing the
photoactivated species to remain target-bound while the iso-
cyanate reacts the protein. Given the results displayed in
Figure 10, we are confident that a high affinity natural product
or bioactive compound containing a native pyrone or pyrimi-
done, appropriately modified with an alkyne tag, will display
selective photolabeling of complex proteomes. Work is currently
underway to adapt this approach to affinity labeling of proteomes
with natural structures.
’ CONCLUSION
Using intrinsic photoreactivity represents a powerful approach
to photoaffinity labeling that precludes the necessity of additional
photoreactive groups, which can perturb native substrateꢀtarget
interaction. We have attempted to use nondirected R-pyrone and
pyrimidone probes to demonstrate the feasibility of these groups
as intrinsic photolabels. We synthesized a series of biology- and
diversity-inspired probes 16ꢀ23 and demonstrated that repre-
sentative samples of these probes displayed light-induced reac-
tion with MeOH and also displayed light-dependent labeling of
crude bacterial lysates. However, these probes displayed little to
no selectivity in bacterial proteomes, prompting us to attempt a
secondary “proof of principle” strategy. In this strategy, vanco-
mycin-based probe 55 displays selective labeling of ATLam
and pABC proteins in E. coli in a manner identical to that for
the similar benzophenone-containing vancomycin probe 51.
Figure 8. Labeling of L. welshimerii crude cell lysate with probes 16ꢀ23.
Figure 8, demonstrating the key role of the vancomycin scaffold
in the proteome labeling of 55 (Figure 10).
While 51 appears to label more effectively than 55, both
probes show identical selectivity toward the bacterial proteome.
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Figure 9. Full and shorthand illustrations of probe 51.
a colorless solid. 1H NMR (400 MHz, CDCl3) δ 1.91 (s, 3H), 1.96 (s,
6H), 4.23 (d, J = 3.4 Hz, 2H), 4.26ꢀ4.32 (m, 1H), 5.12ꢀ5.18 (m, 1H),
5.32 (dd, J = 3.4, 5.5 Hz, 1H), 5.88 (d, J = 3.3 Hz, 1H), 6.29 (dd, J = 4.1,
6.8 Hz, 1H), 7.94 (dd, J = 2.8, 6.8 Hz, 1H), 8.45 (dd, J = 2.9, 4.0 Hz, 1H);
13C NMR (100 MHz, CDCl3) δ 20.3, 20.3, 20.6, 62.4, 69.2, 73.6, 79.6,
90.1, 104.3, 143.4, 155.0, 166.6, 169.3, 169.3, 170.0. HRMS (ESI): [M +
Na]+ C15H18O8N2Na calcd 377.0955, found 377.0954. Rf (10%
MeOH/CH2Cl2) = 0.6.
Scheme 12
Zebularine (27). Compound 27 was prepared by the removal of the
acetyl groups in 26 with NH3/MeOH to yield 27 (603 mg, 2.6 mmol,
81%) as a colorless solid. The spectral data matched with those reported
in the literature.20 1H NMR (400 MHz, CD3CD) δ 3.81 (dd, J = 2.4,
12.5 Hz, 1H), 3.99 (dd, J = 2.1, 12.5 Hz, 1H), 4.09ꢀ4.19 (m, 3H), 5.87
(s, 1H), 6.59 (dd, J = 4.3, 6.7 Hz, 1H), 8.58 (dd, J = 2.7, 4.2 Hz, 1H), 8.79
(dd, J = 2.7, 6.7 Hz, 1H); 13C NMR (100 MHz, CD3OD) δ 59.5, 68.2,
75.2, 84.4, 92.2, 104.6, 145.3, 156.1, 165.9. HRMS (ESI): [2 M + Na]+
C18H24N4NaO10 calcd 479.1385, found 479.1382.
50-O-Hex-5-ynoyl-Zebularine (16). HOBt (432 mg, 3.2 mmol)
and 5-hexynoic acid (353 μL, 3.2 mmol) were dissolved in DMF (5 mL),
and N,N0-diisopropylcarbodiimide (496 μL, 3.2 mmol) was added. The
reaction mixture was stirred for 30 min at rt. A solution of 27 (1.1 g,
3.2 mmol) in DMF (5 mL) was added. The mixture was stirred for 16 h
at 50 °C. The reaction mixture was concentrated to dryness and purified
by by column chromatography (CH2Cl2/MeOH) to yield 16 (121 mg,
0.4 mmol, 13%) as a colorless solid. 1H NMR (500 MHz, CD3OD) δ
1.79ꢀ1.88 (m, 2H), 2.24ꢀ2.30 (m, 3H), 2.54 (t, J = 7.4 Hz, 2H), 4.05
(dd, J = 5.0, 7.9 Hz, 1H), 4.21 (dd, J = 1.5, 4.9 Hz, 1H), 4.29ꢀ4.34
(m, 1H), 4.40ꢀ4.51 (m, 2H), 5.83 (d, J = 1.2 Hz, 1H), 6.64 (dd, J = 4.2,
6.7 Hz, 1H), 8.40 (dd, J = 2.7, 6.7 Hz, 1H), 8.61 (dd, J = 2.9, 4.1 Hz, 1H);
13C NMR (90 MHz, CD3OD) δ 17.0, 23.4, 32.2, 62.6, 69.0, 69.1,
74.6, 81.4, 82.6, 93.0, 104.7, 144.4, 156.0, 166.2, 172.8. HRMS (ESI):
[M + H]+ C15H18N2O6 calcd 323.1238, found 323.1220. Rf (10%
MeOH/CH2Cl2) = 0.5.
This result validates our hypothesis and encourages us to attempt
this approach with natural product structures already containing
a pyrone or pyrimidone.
’ EXPERIMENTAL SECTION
General Methods. All nonaqueous reactions were carried out in
flame-dried glassware unless otherwise noted. Air- and moisture-sensi-
tive liquid reagents were manipulated via a dry syringe. Anhydrous
tetrahydrofuran (THF) was obtained from distillation over sodium. All
other solvents and reagents were used as obtained from commercial
sources without further purification. Flash chromatography was per-
formed using Merck Silica Gel 60 (40ꢀ63 μm). HPLC analysis was
accomplished with a C18 column 5 μm (4.6 ꢁ 100 mm) and a PDA
detector. Mobile phase (HPLC grade): A, 0.1% (v/v) TFA in H2O; B,
0.1% TFA in acetonitrile. Flow: 0.5 mL/min.
20,30,50-Tri-O-acetyl-Zebularine (26). Pyrimidine 24 (8.9 g,
67 mmol) and a catalytic amount of (NH4)2SO4 were refluxed for 4 h
in HMDS (25 mL) under an inert atmosphere. Excessive HMDS was
removed under reduced pressure, and the residue was dissolved in dry
CH2Cl2 (10 mL). A solution of 25 (7.2 g, 23 mmol) and SnCl4 (4 mL,
34 mmol) was then combined in 5 mL of dry CH2Cl2, and the solution
was stirred at rt for 16 h. The mixture was washed with saturated
NaHCO3 solution, and the resulting emulsion was filtered over silica.
The aqueous layer was extracted into CH2Cl2. The organic layer was
dried over MgSO4, filtered, and concentrated. The crude product was
purified by column chromatography to yield 26 (2.9 g, 8 mmol, 12%) as
But-3-yn-1-yl 4-Methylbenzenesulfonate (29)39. 4-Methyl-
benzenesulfonyl chloride (20.4 g, 110 mmol) was added to a solution of
28 (5.4 mL, 70 mmol) and pyridine (11.5 mL, 140 mmol) in CH2Cl2 at
0 °C, warmed to rt, and stirred for 16 h. The reaction mixture was treated
with water and extracted with CH2Cl2. The combined organic layers
were washed with 1 M HCl, saturated NaHCO3, H2O, and brine. The
solution was dried over MgSO4 and concentrated to dryness. The crude
product was purified by column chromatography to yield 29 (13.6 g, 61
mmol, 87%) as a colorless oil. 1H NMR (360 MHz, CDCl3) δ 1.98 (t, J =
2.7 Hz, 1H), 2.46 (s, 3H), 2.56 (td, J = 2.7, 7.0 Hz, 1H), 4.11 (t, J = 7.0
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Figure 10. Labeling of E. coli overexpressing ATLam and pABC with 51 and 55.
Hz, 2H), 7.36 (d, J = 8.0 Hz, 2H), 7.81 (d, J = 8.3 Hz, 2H); 13C NMR
(90 MHz, CDCl3) δ 19.4, 21.6, 67.5, 70.8, 78.4, 127.9, 129.9, 132.8,
145.0. HRMS (EI): [M]+ C11H12O332S calcd 224.0502, found
224.0504. Rf (hexanes) = 0.7.
NMR (90 MHz, CDCl3) δ 19.4, 34.2, 56.0, 67.2, 70.2, 80.1, 112.9, 113.6,
121.6, 131.1, 148.0, 149.7. HRMS (EI): [M]+ C12H1379BrO2 calcd
268.0093, found 268.0085. Rf (50% EtOAc/hexanes) = 0.4.
1-(4-(But-3-yn-1-yloxy)-3-methoxybenzyl)pyrimidin-2(1H)-
one (17). Bromide 31 (675 mg, 2.5 mmol) was added to a suspension
of K2CO3 (1.0 g, 7.5 mmol) and pyrimidine 24 (398 mg, 3.0 mmol) in
DMF (25 mL). The reaction mixture was stirred for 16 h at 70 °C and
concentrated to dryness. The crude product was purified by column
chromatography (CH2Cl2/MeOH) to yield 17 (336 mg, 1.2 mmol,
48%) as a yellow solid. 1H NMR (500 MHz, CD3OD) δ 2.33 (t, J = 2.7
Hz, 1H), 2.66 (td, J = 2.7, 6.9 Hz, 2H), 3.86 (s, 3H), 4.11 (t, J = 6.9 Hz,
2H), 5.15 (s, 2H), 6.64ꢀ6.71 (m, 1H), 6.99 (d, J = 0.9 Hz, 2H), 7.10 (s,
1H), 8.45ꢀ8.50 (m, 1H), 8.63ꢀ8.68 (m, 1H); 13C NMR (125 MHz,
CD3OD) δ 18.7, 54.2, 55.2, 67.2, 69.6, 79.9, 104.6, 112.9, 114.0, 121.4,
127.6, 148.5, 150.0, 153.2, 153.6, 163.3. HRMS (ESI): [M + H]+
C16H17N2O3 calcd 285.1234, found 285.1228. Rf (10% MeOH/
CH2Cl2) = 0.4
4-Ethynyl-6-methyl-2H-pyran-2-one (18). This compound
was prepared according to Fairlamb et al.43 The characterization data
for this compound matched those reported in the literature.
3-(6-Methyl-2H-pyran-2-one)-17R-ethynylestradiol (19).
17R-Ethynylestradiol 35 (45.9 mg, 0.16 mmol), bromide 33 (30 mg,
0.16 mmol), and K2CO3 (21.4 mg, 0.16 mmol) were stirred in 1 mL of
DMF at rt for 24 h. EtOAc was added, and the resulting suspension was
extracted with 3 ꢁ H2O. The organic phase was dried over Na2SO4,
filtered, and concentrated to dryness. The crude product was purified by
column chromatography to yield 19 (16.8 mg, 0.04 mmol, 25%) as a
colorless solid. 1H NMR (360 MHz, CDCl3) δ 0.93 (s, 3H), 1.35ꢀ1.62
(m, 4H), 1.63ꢀ1.86 (m, 4H), 1.87ꢀ1.98 (m, 1H), 2.00ꢀ2.14 (m, 2H),
2,27 (s, 3H), 2.34ꢀ2.45 (m, 2H), 2.62 (s, 1H), 2.88 (m, 2H), 5.24
(s, 1H), 5.99 (s, 1H), 6.79 (s, 1H), 6.84, (dd, J = 2.2, 8.1 Hz, 1H), 7.34
4-(But-3-yn-1-yloxy)-3-methoxybenzaldehyde (30)39. A
suspension of vanillin (5.1 g, 34 mmol), 29 (7.5 g, 33 mmol), K2CO3
(13.9, 101 mmol), and NaI (510 mg, 3.4 mmol) in CH3CN was
refluxed for 16 h. The solution was concentrated and treated with
EtOAc/H2O, and the aqueous layer was extracted with EtOAc. The
combined organic layers were washed with H2O and brine, dried over
MgSO4, and concentrated to dryness to yield 30 (4.2 g, 21 mmol,
63%) as a colorless solid. 1H NMR (500 MHz, CDCl3) δ 2.07 (t, J =
2.6 Hz, 1H), 2.76 (td, J = 2.6, 7.3 Hz, 2H), 3.90 (s, 3H), 4.21 (t, J = 7.3
Hz, 2H), 6.98 (d, J = 8.2 Hz, 1H), 7.37ꢀ7.45 (m, 2H), 9.83 (s, 1H);
13C NMR (90 MHz, CDCl3) δ 19.3, 56.0, 66.9, 70.5, 79.6, 109.6,
111.9, 126.5, 130.5, 149.9, 153.3, 190.8. HRMS (ESI): [M + H]+
C12H13O3 calcd 205.0856, found 205.0854. Rf (50% EtOAc/
hexanes) = 0.3.
4-(Bromomethyl)-1-(but-3-yn-1-yloxy)-2-methoxybenzene
(31). At 0 °C NaBH4 (445 mg, 12 mmol) was slowly added to a cooled
solution of 30 (1.2 g, 6 mmol) in dry THF. After 30 min the reaction
mixture was treated with H2O and extracted with EtOAc. The combined
organic layers were washed with H2O and brine, dried over MgSO4, and
concentrated. The crude product was used without further purification.
The alcohol was added to a solution of CBr4 (2.8 g, 8.5 mmol) and PPh3
(2.2 g, 8.5 mmol) in THF and stirred for 16 h. The precipitate was
removed by filtration, and the filtrate was concentrated to dryness. The
crude product was purified by column chromatography to yield 31 (1.4
g, 5.2 mmol, 87%) as a yellow solid. 1H NMR (360 MHz, CDCl3) δ 2.06
(t, J = 2.7 Hz, 1H), 2.74 (td, J = 2.7, 7.4 Hz, 2H), 3.89 (s, J = 2.0 Hz, 3H),
4.16 (t, J = 7.4 Hz, 2H), 4.50 (s, J = 5.5 Hz, 2H), 6.81ꢀ6.98 (m, 3H); 13C
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(d, J = 8.1 Hz, 1H); 13C NMR (90 MHz, CDCl3) δ 12.7, 20.0, 22.8, 26.3,
26.9, 29.6, 32.7, 38.9, 39.0, 43.7, 47.1, 49.5, 74.1, 79.8, 87.5, 90.9, 100.4,
118.0, 120.1, 127.1, 138.7, 139.2, 150.1, 163.1, 164.9, 171.1. HRMS (EI):
[M]+ C26H28O4 calcd 404.1982, found 404.1971. Rf (40% EtOAc/
hexanes) = 0.5.
the aqueous layer was extracted with EtOAc. The combined organic
layers were washed with H2O and brine, dried over MgSO4, and
concentrated to dryness. The crude product was purified by column
chromatography (hexane/EtOAc) to yield 41 (1.49 g, 6 mmol, 75%) as a
1
slightly yellow solid. H NMR (500 MHz, CDCl3) δ 1.75ꢀ1.82 (m,
2H), 1.99ꢀ2.07 (m, 3H), 2.29ꢀ2.36 (m, 2H), 4.24 (t, J = 6.2 Hz, 2H),
7.25 (d, J = 9.1 Hz, 1H), 7.43 (ddd, J = 1.1, 6.9, 8.0 Hz, 1H), 7.63 (ddd,
J = 1.4, 6.9, 8.5 Hz, 1H), 7.77 (d, J = 8.1 Hz, 1H), 8.03 (d, J = 9.1 Hz, 1H),
9.30 (d, J = 8.7 Hz, 1H), 10.93 (s, 1H); 13C NMR (125 MHz, CDCl3) δ
18.1, 25.0, 28.3, 68.8, 69.0, 83.8, 113.4, 116.6, 124.7, 124.9, 128.2, 128.4,
129.9, 131.5, 137.6, 163.5, 192.0. HRMS (EI): [M]+ C17H16O2 calcd
252.1145, found 252.1131. Rf (25% EtOAc/hexanes) = 0.5.
1-Benzyl-5-iodopyrimidin-2(1H)-one (36). Pyrimidine 24
(304 mg, 2.3 mmol) and N-iodosuccinimide (541 mg, 2.4 mmol) in
dry DMF (5 mL) were stirred at rt for 48 h. The reaction mixture was
treated with Et2O, and the resulting precipitate was collected by
filtration. The crude product was washed with MeOH and used
without further purification. The crude iodide, K2CO3 (934 mg,
6.7 mmol), and benzyl bromide (321 μL, 2.7 mmol) were dissolved in
DMF (10 mL). The reaction mixture was stirred for 16 h at rt
and concentrated to dryness. The crude product was purified by
column chromatography (CH2Cl2/MeOH) to yield 36 (647 mg,
2.1 mmol, 92%, 2 steps) as a yellow solid. 1H NMR (360 MHz,
CDCl3) δ 5.06 (s, 2H), 7.31ꢀ7.43 (m, 5H), 7.75 (d, J = 3.1 Hz, 1H),
8.59 (d, J = 3.1 Hz, 1H); 13C NMR (90 MHz, CDCl3) δ 54.3, 63.9,
128.8, 129.0, 129.3, 134.2, 151.2, 154.3, 170.3. HRMS (ESI): [M +
H]+ C11H10ON2I calcd 312.9832, found 312.9825. Rf (5% MeOH/
CH2Cl2) = 0.5.
1-Benzyl-5-((trimethylsilyl)ethynyl)pyrimidin-2(1H)-one
(37). A solution of 36 (298 mg, 1.0 mmol), ethynyltrimethylsilane
(675 μL, 4.8 mmol), 10% Pd/C (2 mol % based on Pd), PPh3 (2.5 mol
%), and CuI (4 mol %) in 1:1 Et3N/CH3CN (10 mL) was refluxed for
4 h under an inert atmosphere. Solids were removed by centrifugation,
and the remaining mixture was concentrated to dryness. The crude
product was purified by column chromatography (CH2Cl2/MeOH) to
yield 37 (134 mg, 0.5 mmol, 51%) as a yellow solid. 1H NMR (500 MHz,
CDCl3) δ 0.23 (s, 9H), 5.10 (s, 2H), 7.34ꢀ7.44 (m, 5H), 7.78 (d, J =
3.1 Hz, 1H), 8.62 (s, 1H); 13C NMR (125 MHz, CDCl3) δ ꢀ0.3, 54.3,
96.4, 98.8, 102.2, 128.8, 128.9, 129.3, 134.2, 149.7, 154.6, 167.7. HRMS
(ESI): [M + H]+ C16H19N2OSi calcd 283.1261, found 283.1256. Rf
(50% EtOAc/hexanes) = 0.5.
1-(Bromomethyl)-2-(hex-5-yn-1-yloxy)naphthalene (42).
At 0 °C NaBH4 (151 mg, 4.0 mmol) was slowly added to a cooled
solution of 41 (504 mg, 2.0 mmol) in dry MeOH (5 mL). After 30 min
the reaction mixture was treated with H2O and extracted with EtOAc.
The combined organic layers were washed with H2O and brine, dried
over MgSO4, and concentrated. The crude product was used without
further purification. The alcohol was added to a solution of CBr4
(975 mg, 2.9 mmol) and PPh3 (772 mg, 2.9 mmol) in THF (20 mL)
and stirred for 16 h. The precipitate was removed by filtration, and the
filtrate was concentrated to dryness. The crude product was purified by
column chromatography (hexane/EtOAc) to yield the bromide 42
1
(515 mg, 1.6 mmol, 81%) as a yellow solid. H NMR (500 MHz,
CDCl3) δ 1.83ꢀ1.88 (m, 2H), 2.03ꢀ2.07 (m, 3H), 2.34ꢀ2.40 (m, 2H),
4.22 (t, J = 6.2 Hz, 2H), 5.13 (s, 2H), 7.23 (d, J = 9.1 Hz, 1H), 7.36 (d, J =
8.1 Hz, 1H), 7.42 (t, J = 7.5 Hz, 1H), 7.62 (t, J = 7.5 Hz, 1H), 7.81ꢀ7.87
(m, 1H), 8.06 (d, J = 8.6 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 18.3,
25.1, 25.5, 28.5, 68.6, 69.0, 84.2, 114.0, 118.3, 122.7, 123.9, 127.3, 128.7,
129.1, 129.9, 132.3, 154.6. HRMS (EI): [M]+ C17H17O79Br calcd
316.0457, found 316.0447. Rf (25% EtOAc/hexanes) = 0.6.
1-((2-(Hex-5-yn-1-yloxy)naphthalen-1-yl)methyl)pyrimidin-
2(1H)-one (21). Bromide 46 (201 mg, 0.63 mmol) was added to a
suspension of K2CO3 (249 mg, 1.80 mmol) and pyrimidine 24 (100 mg,
0.75 mmol) in DMF (5 mL). The reaction mixture was stirred for 16 h at
70 °C and concentrated to dryness. The crude product was purified by
column chromatography (CH2Cl2/MeOH) to yield 21 (52 mg, 0.17
mmol, 26%) as a yellow solid. 1H NMR (500 MHz, CDCl3) δ
1.67ꢀ1.76 (m, 2H), 1.94ꢀ2.03 (m, 3H), 2.25ꢀ2.32 (m, 2H), 4.24 (t,
J = 6.3 Hz, 2H), 5.64 (s, 2H), 6.12 (dd, J = 4.1, 6.5 Hz, 1H), 7.35 (d, J =
9.1 Hz, 1H), 7.38ꢀ7.43 (m, 2H), 7.53 (t, J = 7.3 Hz, 1H), 7.84 (d, J = 8.1
Hz, 1H), 7.90ꢀ7.97 (m, 2H), 8.52 (s, 1H); 13C NMR (125 MHz,
CDCl3) δ 18.1, 24.9, 28.4, 43.1, 68.7, 69.0, 83.7, 104.2, 113.5, 114.3,
122.7, 124.3, 128.2, 128.7, 129.1, 131.8, 133.0, 146.4, 155.8, 156.8, 165.1.
HRMS (ESI): [M + H]+ C21H21N2O2 calcd 333.1598, found 333.1594.
1-Benzyl-5-ethynylpyrimidin-2(1H)-one (20). 37 (124 mg,
0.4 mmol) was added to a solution of TBAF 3 H2O (274 mg,
0.8 mmol) and AcOH (48 μL, 0.8 mmol) in THF (5 mL). The
reaction mixture was stirred for 1 h at rt, concentrated to dryness, and
purified by column chromatography (CH2Cl2/MeOH) to yield 20 (68
1
mg, 0.3 mmol, 81%) as a slightly yellow solid. H NMR (500 MHz,
CDCl3) δ 3.16 (s, 1H), 5.11 (s, 2H), 7.34ꢀ7.47 (m, 5H), 7.79 (d, J =
3.0 Hz, 1H), 8.65 (s, 1H); 13C NMR (125 MHz, CDCl3) δ 54.3, 75.7,
81.2, 101.0, 128.9, 129.1, 129.4, 134.0, 150.1, 154.6, 167.6. HRMS
(ESI): [M + H]+ C13H11ON2 calcd 211.0866, found 211.0862. Rf
(10% MeOH/CH2Cl2) = 0.5.
Hex-5-yn-1-yl 4-Methylbenzenesulfonate (39).39 4-Methyl-
benzenesulfonyl chloride (20.0 g, 105 mmol) was added to a solution of
38 (7.7 mL, 70 mmol) and pyridine (11.3 mL, 140 mmol) in CH2Cl2
(150 mL) at 0 °C, warmed to rt, and stirred for 16 h. The reaction
mixture was treated with water and extracted with CH2Cl2.The com-
bined organic layers were washed with 1 M HCl, saturated NaHCO3,
H2O, and brine. The solution was dried over MgSO4 and concentrated
to dryness. The crude product was purified by column chromatography
(hexane/EtOAc) to yield 39 (14.0 g, 55 mmol, 79%) as a colorless oil.
1H NMR (360 MHz, CDCl3) δ 1.53ꢀ1.63 (m, 2H), 1.75ꢀ1.86 (m,
2H), 1.94 (t, J = 2.7 Hz, 1H), 2.19 (td, J = 2.7, 6.9 Hz, 2H), 2.47 (s, 3H),
4.08 (t, J = 6.3 Hz, 2H), 7.34ꢀ7.40 (m, 2H), 7.78ꢀ7.84 (m, 2H); 13C
NMR (90 MHz, CDCl3) δ 17.7, 21.6, 24.2, 27.8, 69.0, 69.9, 83.4, 127.9,
129.8, 133.1, 144.7. HRMS (EI): [M]+ C13H16O332S calcd 252.0815,
found 252.0808. Rf (20% EtOAc/hexanes) = 0.4
2
Rf (10% MeOH/CH Cl2) = 0.5
(S)-Methyl 2-((tert-Butoxycarbonyl)amino)-3-hydroxy-
propanoate (44). Compound 44 was prepared according to the
method of Chamberlain et al.46 to yield 44 (7.2 g, 33 mmol, 73%) as a
colorless oil. The spectral data matched with those reported in the
literature. 1H NMR (360 MHz, DMSO) δ 1.39 (s, 9H), 3.56ꢀ3.71 (m,
4H), 4.01ꢀ4.14 (m, 1H), 4.88 (t, J = 6.1 Hz, 1H), 6.92 (d, J = 8.0 Hz,
1H); 13C NMR (90 MHz, DMSO) δ 28.6, 52.2, 56.7, 61.8, 78.8, 155.7,
171.9. HRMS (ESI): [M + H]+ C9H18NO5 calcd 220.1180, found
220.1170.
(R)-Methyl 3-Bromo-2-((tert-butoxycarbonyl)amino)propano-
ate (45). Alcohol 44 (4.0 g, 18.3 mmol), PPh3 (7.2 g, 27.5 mmol), and
CBr4 (9.1 g, 27.5 mmol) were dissolved in CH2Cl2 (100 mL). The
reaction mixture was stirred for 48 h at rt. Et2O was added, and the
resulting precipitate was removed by filtration. The organic layer was
washed with saturated NaHCO3 and brine, dried over MgSO4, and
concentrated to dryness. The crude product was purified by column
chromatography to yield 45 (3.3 g, 12 mmol, 65%) as a yellow solid. 1H
NMR (250 MHz, CDCl3) δ 1.40 (s, 9H), 3.59ꢀ3.87 (m, 4H),
2-(Hex-5-yn-1-yloxy)-1-naphthaldehyde (41). A suspension
of aldehyde 40 (1.4 g, 8 mmol), 39 (3.0 g, 12 mmol), K2CO3 (3.3,
24 mmol), and NaI (120 mg, 0.8 mmol) in CH3CN was refluxed for
16 h. The solution was concentrated and treated with EtOAc/H2O, and
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4.62ꢀ4.80 (m, 1H), 5.36ꢀ5.54 (m, 1H); 13C NMR (63 MHz, CDCl3)
δ 28.2, 34.0, 52.9, 53.9, 80.4, 154.9, 169.6. HRMS (ESI): [M + H]+
C9H17BrNO4 calcd 282.0336, found 282.0336. Rf (30% EtOAc/
hexanes) = 0.5.
were combined in 1.5 mL of dry CH3CN/2.2 mL of dry Et3N.
Trimethylsilylacetylene (64.5 mL, 0.46 mmol) was then added, and
the solution was heated to 80 °C for 16 h. The reaction mixture was then
cooled to rt, and the solids were filtered off and washed with EtOAc. The
resulting organic solution was then washed with 2 ꢁ 1 M HCl and 1 ꢁ
satd NaHCO3. The organic phase was dried over Na2SO4, filtered, and
concentrated to dryness. The crude product was then passed through a
silica plug and was used in the next step without further purification.
Crude 48 was dissolved in 2 mL of THF and cooled to ꢀ78 °C. A
solution of TBAF (300 μL, 1.0 M in THF, 300 mmol) was slowly added,
and the solution was stirred for 1.5 h at ꢀ78 °C. The cold reaction was
quenched by the addition of water, and the resulting solution was
allowed to warm to rt. EtOAc was added, and the organic phase was
washed with 1 ꢁ satd NaHCO3. The organic phase was then dried over
Na2SO4, filtered, and concentrated to dryness. The crude product was
purified by column chromatography to yield 23 (31 mg, 0.097 mmol,
36%, 2 steps) as a yellow solid. 1H NMR (360 MHz, CDCl3) δ 2.57 (s,
br, 1H), 2.81ꢀ2.98 (m, 2H), 3.50 (s, 1H), 5.20 (m, 1H), 6.11 (s, 1H),
6.33 (s, 1H), 7.32ꢀ7.56 (m, 5H), 7.55ꢀ7.66 (m, 4H); 13C NMR (90
MHz, CDCl3) δ 43.5, 71.1, 79.3, 86.6, 106.9, 116.9, 126.0, 127.0, 127.5,
128.9, 137.8, 140.6, 141.1, 141.9, 161.9, 162.1. HRMS (EI): [M]+
C21H16O3 calcd 316.1094, found 316.1087. Rf (20% EtOAc/hexanes) =
0.3.
(()-Methyl 2-((tert-Butoxycarbonyl)amino)-3-(2-oxopyri-
midin-1(2H)-yl)propanoate (46). A suspension of bromide 45
(2.3 g, 8.3 mmol), pyrimidine 24 (1.3 g, 10 mmol), and K2CO3
(3.5 g, 25 mmol) in DMF (50 mL) was stirred for 16 h at rt. The
reaction mixture was concentrated to dryness and the crude product was
purified by column chromatography (CH2Cl2/MeOH) to yield 46
(0.8 g, 2.8 mmol, 33%) as a slightly brown solid. Optical rotation
measurements demonstrated that racemization of the chiral center had
occurred during the reaction. 1H NMR (360 MHz, CDCl3) δ 1.35 (s,
9H), 3.74 (s, 3H), 4.15ꢀ4.29 (m, 1H), 4.38ꢀ4.51 (m, 1H), 4.56ꢀ4.68
(m, 1H), 5.89 (d, J = 6.8 Hz, 1H), 6.29 (dd, J = 4.2, 6.4 Hz, 1H),
7.69ꢀ7.80 (m, 1H), 8.53ꢀ8.58 (m, 1H); 13C NMR (90 MHz, CDCl3)
δ 28.2, 52.1, 52.8, 80.3, 104.1, 149.2, 155.4, 156.5, 162.5, 166.4, 170.2.
HRMS (ESI): [M + H]+ C13H20N3O5 calcd 298.1398, found 298.1393.
2
Rf (10% MeOH/CH Cl2) = 0.6.
(()-Methyl 2-(Hex-5-ynamido)-3-(2-oxopyrimidin-1(2H)-
yl)propanoate (22). A solution of pyrimidone 46 (298 mg,
1.0 mmol) in 1:1 TFA/CH2Cl2 (2 mL) was stirred for 5 h (0 °Cꢀrt).
The reaction mixture was treated with saturated NaHCO3, and the
aqueous phase was extracted with CH2Cl2. The organic phase was dried
over MgSO4 and concentrated. The crude product was used without
further purification. HOBt (270 mg, 2.0 mmol) and 5-hexynoic acid
(220 μL, 2.0 mmol) were dissolved in DMF (1 mL), and DIC (309 μL,
2.0 mmol) was added. The reaction mixture was stirred for 30 min at rt.
A solution of the amine in DMF (1 mL) was added. The mixture was
stirred for 2 h at rt. The reaction mixture was concentrated to dryness
and purified by by column chromatography (CH2Cl2/MeOH) to yield
(E)-Methyl 3-Ethynyl-5-oxohex-2-enoate (49). Compound
18 (16.2 mg, 0.12 mmol) was dissolved in 6 mL of MeOH and irradiated
at 350 nm at a distance of 1 cm for 30 min. The solvent was then
removed, and the resulting residue was purified by flash chromatogra-
phy. The major product was 49 (9.9 mg, 0.060 mmol, 50%). 1H NMR
(360 MHz, CDCl3) δ 1.62 (s, 3H), 2.71 (m, 2H), 3.39 (s, 3H), 3.53 (s,
1H), 6.26 (s, 1H); 13C NMR (90 MHz, CDCl3) δ 22.9, 39.3, 50.1, 80.4,
87.7, 103.7, 124.2, 135.4, 162.5. MS (EI): [M]+ C9H10O3 calcd 166.06,
found 166.1. Rf (20% EtOAc/hexanes) = 0.35.
1
22 (175 mg, 0.6 mmol, 60%) as a yellow solid. H NMR (500 MHz,
tert-Butyl (2-(2-Oxopyrimidin-1(2H)-yl)ethyl)carbamate (52).
A suspension of 2-(Boc-amino)ethylbromide (200 mg, 0.9 mmol), 24
(108 mg, 0.8 mmol) and K2CO3 (340 mg, 2.5 mmol) in DMF (5 mL)
was stirred for 48 h at rt. The reaction mixture was concentrated to
dryness, and the crude product was purified by column chromatography
(CH2Cl2 /MeOH) to yield 52 (186 mg, 0.7 mmol, 88%) as a colorless
solid. 1H NMR (360 MHz, CDCl3) δ 1.33 (s, 9H), 3.46 (t, J = 5.5 Hz,
2H), 4.01 (t, J = 5.5 Hz, 2H), 6.25 (dd, J = 4.2, 6.4 Hz, 1H), 7.65ꢀ7.75
(m, J = 3.9 Hz, 1H), 8.45ꢀ8.55 (m, 1H). 13C NMR (90 MHz, CDCl3) δ
28.3, 38.9, 51.2, 79.5, 104.0, 149.0, 156.2, 156.5, 166.0. HRMS (ESI):
[M + H]+ C11H18O3N3 calcd 240.1343, found 240.1344. Rf (10%
MeOH/CH2Cl2) = 0.3.
1-(2-Aminoethyl)pyrimidin-2(1H)-one (53). A solution of
pyrimidone 52 (97 mg, 0.4 mmol) in 1:1 TFA/CH2Cl2 (2 mL) was
stirred for 5 h (0 °Cꢀrt). The reaction mixture was poured into ice cold
Et2O. The precipitate was collected by centrifugation and dried under
reduced pressure. The crude product 53 (101 mg, 0.4 mmol, quant)
was used without further purification. 1H NMR (500 MHz, CD3OD) δ
3.48 (s, br, 2H), 4.40 (s, br, 2H), 6.74 (s, br, 1H), 8.50 (s, br, 1H), 8.74
(s, br, 1H). 13C NMR (125 MHz, CD3OD) δ 38.2, 49.5, 105.3, 153.3,
155.1, 164.9. HRMS (ESI): [M + H]+ C6H10ON3 calcd 140.0818,
found 140.0819.
N0-(Hex-5-ynamide)-vancomycin (54). This compound was
prepared according to Eirich et al. The characterization data for this
compound matched with those reported in the literature.53
N0-(Hex-5-ynamide)-C-[1-(2-aminoethyl)pyrimidin-2(1H)-
one]-vancomycin (55). N0-(Hex-5-ynamide)-vancomycin (54)
(30 mg, 19 μmol), pyrimidone 53 (10 mg, 39 μmol), and DIPEA
(16 μL, 95 μmol) were dissolved in DMF. PyBOP (11 mg, 21 μmol) and
HOBt (4 mg, 21 μmol) were added, and the reaction mixture was stirred
for 16 h at rt. The solution was poured into cold Et2O, and the
precipitate was collected by centrifugation and dried under reduced
CD3OD) δ 1.72ꢀ1.80 (m, 2H), 2.17ꢀ2.22 (m, 2H), 2.28 (t, J = 2.7 Hz,
1H), 2.32ꢀ2.36 (m, 2H), 3.78 (s, 3H), 4.13 (dd, J = 8.8, 13.3 Hz, 1H),
4.60 (dd, J = 5.2, 13.3 Hz, 1H), 4.93 (dd, J = 5.2, 8.8 Hz, 1H), 6.54 (dd,
J = 4.3, 6.5 Hz, 1H), 8.02 (dd, J = 2.8, 6.5 Hz, 1H), 8.61 (dd, J = 2.8, 4.3
Hz, 1H); 13C NMR (90 MHz, CD3OD) δ 18.5, 25.6, 35.2, 51.6, 53.2,
53.2, 70.4, 84.0, 106.1, 151.9, 158.3, 168.0, 171.1, 175.6. HRMS (ESI):
[M + H]+ C14H18O4N3 calcd 292.1292, found 292.1287. Rf (10%
MeOH/CH2Cl2) = 0.5
6-(2-([1,10-bBiphenyl]-4-yl)-2-hydroxyethyl)-4-bromo-2H-
pyran-2-one (47). Diisopropylamine (90.4 μL, 0.64 mmol) was
n
dissolved under Ar in 5 mL of dry THF. BuLi (0.27 mL, 2.4 M in
hexanes, 0.64 mmol) was then added, and the solution was stirred at rt
for 15 min. The reaction mixture was then cooled to ꢀ78 °C, and 100 μL
HMPA was added. After stirring at ꢀ78 °C for 30 min, a solution of
bromide 33 (100 mg, 0.53 mmol) in 1 mL of dry THF was slowly added.
The reaction mixture was stirred cold for an additional 40 min, and then
a solution of biphenyl-4-carboxaldehyde (116 mg, 0.64 mmol) in 1 mL
of dry THF was slowly added. The reaction mixture was stirred
at ꢀ78 °C for 1 h, then water was added to the cold solution, and the
mixture was allowed to warm to rt. EtOAc was added, and the organic
phase was washed with 2 ꢁ 1 M HCl and 1 ꢁ satd NaHCO3. The
organic phase was dried over Na2SO4, filtered, and concentrated to
dryness. The crude product was purified by flash chromatography to
yield 47 (100 mg, 0.27 mmol, 50%) as a white solid. 1H NMR (360 MHz,
CDCl3) δ 2.79ꢀ2.99 (m, 3H), 5.20 (m, 1H), 6.32 (s, 1H), 6.48 (s, 1H),
7.32ꢀ7.52 (m, 5H), 7.55ꢀ7.66 (m, 4H); 13C NMR (90 MHz, CDCl3)
δ 43.5, 71.0, 110.0, 115.4, 126.0, 127.1, 127.5, 128.9, 140.5, 141.1, 141.4,
141.9, 160.8, 162.0. HRMS (EI): [M]+ C19H15O379Br1 calcd 370.0199,
found 370.0181. Rf (20% EtOAc/hexanes) = 0.2.
6-(2-([1,10-Biphenyl]-4-yl)-2-hydroxyethyl)-4-ethynyl-2H-
pyran-2-one (23). Bromide 47 (100 mg, 0.27 mmol), 10% Pd/C
(5.73 mg), PPh3 (17.5, 0.067 mmol), and CuI (2.1 mg, 0.011 mmol)
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pressure. The crude product was purified by HPLC to yield 55 (22 mg,
13 μmol, 68%) as a colorless solid. HRMS (ESI): [M + H]+ C78H89-
O25N12Cl37Cl calcd 1665.5404, found 1665.5488.
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’ ASSOCIATED CONTENT
S
Supporting Information. Copies of NMR spectra and
b
SDS-PAGE gels for time and concentration proteome photo-
labeling with 17 and 20, as well as labeling E. coli cytosolic
proteins with probes 16ꢀ23. This material is available free of
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’ AUTHOR INFORMATION
Corresponding Author
*E-mail: stephan.sieber@tum.de.
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’ ACKNOWLEDGMENT
We thank Mona Wolff for excellent scientific support and
J€urgen Eirich for E. coli clones and a sample of probe 51. S.A.S.
was supported by the Deutsche Forschungsgemeinschaft (Emmy
Noether), SFB749, FOR1406, an ERC starting grant and the
Center for Integrated Protein Science Munich CIPSM. M.B.N.
thanks the Alexander von Humboldt foundation for financial
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