N-Activated β-lactams as versatile reagents for acyl carrier protein labeling

Article abstract of DOI:10.1039/c2ob06846j

Acyl carrier proteins are critical components of fatty acid and polyketide biosynthesis. Their primary function is to shuttle intermediates between active sites via a covalently bound phosphopantetheine arm. Small molecules capable of acylating this prosthetic group will provide a simple and reversible means of introducing novel functionality onto carrier protein domains. A series of N-activated β-lactams are prepared to examine site-specific acylation of the phosphopantetheine-thiol. In general, β-lactams are found to be significantly more reactive than our previously studied β-lactones. Selectivity for the holo over apo-form of acyl carrier proteins is demonstrated indicating that only the phosphopantetheine-thiol is modified. Incorporation of an N-propargyloxycarbonyl group provides an alkyne handle for conjugation to fluorophores and affinity labels. The utility of these groups for mechanistic interrogation of a critical step in polyketide biosynthesis is examined through comparison to traditional probes. In all, we expect the probes described in this study to serve as valuable and versatile tools for mechanistic interrogation.

Full text of DOI:10.1039/c2ob06846j

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PAPER
N-Activated β-lactams as versatile reagents for acyl carrier protein labeling†
Gitanjeli Prasad, Jon W. Amoroso, Lawrence S. Borketey and Nathan A. Schnarr*
Received 2nd November 2011, Accepted 21st December 2011
DOI: 10.1039/c2ob06846j
Acyl carrier proteins are critical components of fatty acid and polyketide biosynthesis. Their primary
function is to shuttle intermediates between active sites via a covalently bound phosphopantetheine arm.
Small molecules capable of acylating this prosthetic group will provide a simple and reversible means of
introducing novel functionality onto carrier protein domains. A series of N-activated β-lactams are
prepared to examine site-specific acylation of the phosphopantetheine-thiol. In general, β-lactams are
found to be significantly more reactive than our previously studied β-lactones. Selectivity for the holo
over apo-form of acyl carrier proteins is demonstrated indicating that only the phosphopantetheine-thiol is
modified. Incorporation of an N-propargyloxycarbonyl group provides an alkyne handle for conjugation
to fluorophores and affinity labels. The utility of these groups for mechanistic interrogation of a critical
step in polyketide biosynthesis is examined through comparison to traditional probes. In all, we expect the
probes described in this study to serve as valuable and versatile tools for mechanistic interrogation.
Nearly all biosynthetic steps associated with modular PKS
systems rely on acyl-ACPs. Therefore, functional analysis of
Introduction
Polyketide natural products are an enormous class of secondary
metabolites with broad-spectrum applications as antibiotic, anti-
tumor, and immunosuppressive agents.1 The looming menace of
accelerating drug resistance demands the continued production
of new and diversified polyketides to retain this therapeutic
efficacy. The genetic and chemical manipulation of existing
polyketide systems provides a promising route to such next-
generation products, but significant technical challenges remain.
Simplified mechanistic and diagnostic tools are thus a major
current objective in metabolic engineering.
Polyketides are biosynthesized by repeated decarboxylative
condensation of malonate-derived extender units via collabora-
tive enzyme clusters termed polyketide synthases (PKSs).2–6
Each condensation step requires an extender unit-primed acyl
carrier protein (ACP) and a ketosynthase (KS)-bound polyketide
intermediate (Fig. 1). In both cases, the small molecule com-
ponent is attached to the corresponding protein through a thioe-
ster linkage. While the KS active site harbors a typical cysteine
residue for this purpose, the apo form of ACP is posttranslation-
each enzymatic event requires a robust method for both generat-
ing these entities and monitoring their fate. Traditionally, this has
been accomplished through enzymatic incorporation of radio-
labeled acyl-CoA substrates onto ACP domains. Unfortunately,
this strategy is often fraught with difficult syntheses and very
costly reagents. As genomics-based technologies continue to
provide an abundance of new PKS systems, the need for comp-
lementary methods utilizing simple, reliable, and structurally
flexible ACP-acylating agents has never been greater.
Our work in this area began with a simple idea of using elec-
trophilic β-lactones as acylating agents for ACPs.7 The resulting
ACP-bound β-hydroxythioester represents a common structural
motif in modular polyketide biosynthesis. Selectivity for the
Ppant-thiol over competing nucleophiles, including the KS
active site, was examined for a panel of substituted lactones.
Although all structures investigated showed some level of Ppant-
acylation, monosubstituted β-lactones were generally both more
reactive and selective. However, even the best acylating agents
required a very large excess to obtain complete loading of the
ACPs. Therefore, we were compelled to consider alternate elec-
trophiles for future experimentation.
ally modified with
a thiol-terminated phosphopantetheine
(Ppant) group to generate the mature holo-ACP. These two sites
serve as the primary points of covalent attachment throughout
polyketide biogenesis.
We were initially worried that increased reactivity may result
in decreased selectivity for the Ppant group when competing
nucleophiles, most prominently the KS-active site, were present.
However, recent work by Khosla and coworkers has helped to
change the landscape of possibilities surrounding PKS
interrogation.8–11 Using limited proteolysis of intact modules,
they were able to identify conserved regions that served as effec-
tive boundary points for each domain, thus affording “broken
Department of Chemistry, University of Massachusetts, 710 N. Pleasant
Street, Amherst, Massachusetts 01003. E-mail: scharr@chem.umass.edu
†Electronic supplementary information (ESI) available. See DOI:
10.1039/c2ob06846j
1992 | Org. Biomol. Chem., 2012, 10, 1992–2002
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Fig. 1 Schematic diagram for polyketide formation in modular PKSs. (A) KS-mediated decarboxylation of ACP-bound malonate forms an ACP-
bound enolate (B) Claisen-like condensation between the KS-bound chain and extender unit produces an ACP-bound β-ketothioester. (C) The ketore-
ductase domain reduces the β-ketothioester to a β-hydroxythioester. (D) The dehydratase domain dehydrates the β-hydroxythioester to an enoyl-thioe-
ster. (E) The enoyl reductase domain reduces the enoyl group to form a saturated acyl-chain. See text for abbreviations.
modules” where KS and ACP domains could be cloned and
expressed separately. Most importantly, the separated domains
retain their function when reintroduced in vitro. As a result, the
challenge of designing Ppant-acylating agents is reduced to one
of selectivity for holo-ACP over apo-ACP alone since the KS
domain can be introduced after ACP-loading has occurred
without limiting the scope of its utility.
Confident that all downstream applications of our probes
would proceed smoothly using separated domains, we began to
explore β-lactams as thiol-reactive acylating agents for isolated
ACP domains. Relative to β-lactones, the amide nitrogen of
β-lactams offers an additional site for ring substitution that is
electronically coupled to the carbonyl, thus providing a reliable
means of tuning electrophilicity. Herein, we describe our efforts
to establish N-activated β-lactams as efficient and versatile acy-
lating agents for ACP domains. The fluorescence and affinity
labeling applications described set the stage for downstream
studies aimed at increasing our understanding of KS-ACP recog-
nition and intermodular polyketide transfer between these
components.
Results and discussion
We expected that β-lactams could have a reactive advantage rela-
tive to β-lactones through appropriate activation of the ring nitro-
gen.12 A variety of common nitrogen-protecting groups were
introduced onto the parent azetidinone (compound 1) via estab-
lished methods to produce compounds 2–5 (Fig. 2).13–16 Satur-
ation curves were prepared by varying the number of equivalents
of each lactam relative to isolated holo-ACP2 (module 2) and
holo-ACP3 (module 3) from 6-deoxyerythronolide B synthase
(DEBS) in analogous fashion to our previous work with β-lac-
tones.7 Briefly, lactam was added to a buffered solution of ACP
and allowed to react for 1 h at room temperature. The mixture
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Fig. 2 Structures of N-activated β-lactams.
was subjected to trypsin digestion and subsequently quenched
with formic acid. The resulting array of peptide fragments was
analyzed by LC-MS. The extent of loading was estimated from
the ratio of acylated Ppant-containing peptide to total Ppant-
containing peptide, assuming similar ionization potentials.
To our satisfaction, compounds 2–5 exhibited excellent acyla-
tion efficiencies with both holo-ACPs. All but the Boc-activated
lactam (compound 5) showed around 80% loading at a mere 10
equivalents (Fig. 3). In addition, the thioester-ACP generated
from compound 6 underwent a mere 10% hydrolysis after 24 h
at room temperature, suggesting that the acyl-ACPs are suitably
stable for all foreseeable applications (see supporting infor-
mation). A direct competition between compound 6 and the
most promising β-lactone from our previous study, 4-methyloxe-
tan-2-one, reveals the substantial reactive advantage of β-lactams
for acylation of holo-ACP (Fig. 4). Again, the Ppant-thiol is the
only competent nucleophile present as these ACPs do not
contain solvent-exposed cysteine residues. As expected,
however, the general thiol-reactivity of these warheads renders
them unsuitable for selective ACP-acylation in full modules due
to competitive acylation of the KS-active site (see supporting
information†).
To further examine selectivity for holo- over apo-ACP, we
wished to employ a gel-based fluorescent assay. Based on work
by Sieber and coworkers,17 a [3 + 2] cycloaddition (click reac-
tion) was planned for coupling acylated ACPs to a rhodamine-
based fluorophore.18–20 ACP2 and ACP3 (both holo and apo)
were mixed with 1, 5, and 10 equivalents of 6 and allowed to
react at room temperature for 1 h. Rhodamine azide was then
added along with all necessary click reaction components and
the resulting protein products were analyzed via SDS-PAGE.
Bright bands were observed for the holo-ACPs while apo-ACPs
provided only background fluorescence (Fig. 5). It should be
noted that fluorescence can be readily observed at an impressive
5 equivalents of lactam for both ACPs.
Fig. 3 Saturation curves as determined by tandem proteolysis-mass
spectrometry for ACP2 (Top) and ACP3 (Bottom) with compounds
2–6. Equivalents of lactam are per protein molecule. Lines are added for
clarity. Error bars represent one standard deviation (n = 3).
6 with the biotin azide followed by 1 h reaction with the ACP
resulted in low yields of the acylated protein. Competition from
other proteinaceous nucleophiles was observed for longer reac-
tion times which forced us to consider an alternative order of
addition.
To our satisfaction, [3 + 2] cycloaddition of the biotin azide
with pre-acylated holo-ACP resulted in 30% recovery following
streptavidin–sepharose binding and subsequent treatment of the
bound material with hydrazine, which restores the Ppant-thiol
via nucleophilic attack on the thioester carbonyl. To remove
excess lactam and biotin following the initial acylation and sub-
sequent [3 + 2] cycloaddition, respectively, the samples were
filtered twice prior to treatment with the streptavidin beads.
Analysis of protein concentration at each stage revealed that the
primary source of ACP loss in the process was these filtration
steps. In fact, more than 60% of the protein subjected to the
streptavidin was recovered upon treatment with hydrazine while
almost half of the initial mixture is lost to the filtration mem-
brane before this step (see supporting information†). We expect
that upon scale-up, where small losses of protein to filtration are
To further explore Ppant selectivity and utility of the alkyne
handle, we turned our focus to mixtures of apo and holo ACP2.
In particular, we wanted to ask the question of whether holo-
ACP2 could be isolated from a mixture of apo and holo by an
affinity label conjugated to compound 6. We chose a biotin-
streptavidin interaction as the basis for ACP purification as this
system (1) has been used extensively for protein separations21–24
and (2) relies on a small molecule that we envisioned could
readily be conjugated to the lactam warhead. As proof of prin-
ciple and to assess the efficiency of each step, we began with
samples of pure holo-ACP. Attempts to pre-conjugate compound
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Fig. 4 β-lactam vs. β-lactone ACP-acylation. ACP2 was treated simultaneously with 10 equivalents of compound 6 and 4-methyloxetan-2-one for
1 h. The acylated-ACP was trypsinized and the resulting peptide fragments were analyzed by LC-MS. All labeled peaks arise from m/z = +2.
However, as expression levels increase, the transferase struggles
to keep up and mixtures of holo- and apo-ACP are often
observed. To examine the viability of our approach for separating
holo- from apo-ACP we prepared an 80 : 20 (holo : apo) mixture
of ACP2, a typical ratio observed from overexpression of ACP in
BAP1, and subjected it to the same process described above.
Analysis of the final solution obtained from hydrazine treatment
of the bound material by LC-MS revealed only holo-ACP2
(Fig. 6). Taken together with the fluorescence assay, these exper-
iments nicely illustrate the remarkable Ppant-selectivity and ver-
satility of compound 6.
To confirm that the purification process did not alter the
protein structure, the ability of eluted holo-ACP2 to accept a
methylmalonyl-CoA unit from an acyltransferase was examined.
To a buffered solution of DEBS module 6 ketosynthase-acyl-
transferase didomain11,25–27 ([KS6AT6]) was added methylmalo-
nyl-CoA and purified holo-ACP2. After 30 min of reaction time,
the mixture was subjected to trypsinolysis and the resulting
peptide fragments were separated and analyzed via LC-MS
(Fig. 7). The prominent peak at 809.1 mass units suggests that
the purified ACP remains competent for AT-ACP methylmalo-
nate transfer as seen previously.27
Fig. 5 Fluorescence SDS-PAGE analysis of DEBS ACP2 (apo and
It was now clear that carrier proteins could be efficiently acy-
holo), and ACP3 (apo and holo) reaction with compound 6 and sub-
lated with β-lactams. However, to demonstrate the utility of these
sequent click reaction with rhodamine-azide. Lanes are marked above
reagents for PKS examination, a direct comparison to traditional
with the corresponding protein component and lactam equivalents (per
small molecule probes was necessary. In particular, we were
interested in ACP to KS transfer of the β-aminothioester product
(Fig. 8). As a primary determinant for proper flow of intermedi-
protein molecule). Markers to the left indicate expected bands for the
indicated species.
ates during polyketide biosynthesis, intermediate transfer
less significant, percent recovery will be drastically improved. As
proof of principle, however, we were confident that these recov-
ery levels would suffice for further experimentation.
Overexpression of ACP in BAP1, an engineered strain of
E. coli with a phosphopantetheinyl transferase embedded in
the genome, results in primarily the holo (Ppantylated)-form.
between ACP and KS plays a critical role in product generation
for all PKS systems. The most common method for direct acyla-
tion of KS active sites involves incubation of the KS with an
N-acetylcysteamine thioester (SNAc) that mimics the terminal
portion of the Ppant group.28–31 To qualitatively determine the
kinetic competency of our lactam-derived acyl-ACPs, relative to
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Fig. 6 Whole protein mass spectra of (top) an initial 80 : 20 (holo : apo) mixture of ACP2 and (bottom) the result of β-lactam based purification of
holo-ACP2. The lack of apo-ACP2 in the latter indicates that the β-lactam (compound 6) is highly selective for holo-ACP2. Peaks shown span m/z
values from +21 through +24 for both species. See supporting information for full spectra.†
propionyl-SNAc and the potent KS-inhibitor, cerulenin,32 a gel-
based fluorescence assay was employed.
Acylation of holo-ACP2 was executed as before using 10
equivalents of compound 6 followed by Cu-mediated [3 + 2]
cycloaddition with rhodamine azide. The resulting acyl-ACP
was incubated with [KS6AT6] for 1 h at room temperature with
or without competing KS-acylating/alkylating agents to explore
ACP to KS transfer (Fig. 9). From these data, it is clear that flu-
orescence transfers from the ACP to KS in the absence and pres-
ence of both cerulenin and propionyl-SNAc, indicating that the
lactam-derived acyl-ACPs are, at a minimum, competitive with
traditional small molecule probes (Lanes 2, 4, and 6). The lack
of KS-fluorescence observed when [KS6AT6] is pre-treated with
each competitor (Lanes 3 and 5) confirms that transfer is
1996 | Org. Biomol. Chem., 2012, 10, 1992–2002
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Fig. 7 Methylmalonyl-CoA transfer. Mass spectrum of purified holo-ACP2 digest following treatment with [KS6AT6] and methylmalonyl-CoA (see
text). The prominent peak at 809.1 mass units confirms that the ACP functions properly after being subjected to β-lactam-based affinity purification.
Both MS peaks correspond to m/z = +2.
Fig. 8 Schematic diagram of ACP to KS acyl transfer. Fluorescently
labeled ACP is mixed with a [KSAT] didomain in the presence or
absence of competing KS probes and transfer of fluorescence from ACP
to KS is monitored. The red star represents a rhodamine fluorophore.
occurring between the Ppant arm and KS active site. Finally, the
absence of a fluorescent band when [KS6AT6] is treated directly
Fig. 9 Fluorescent competition assay for ACP to KS acyl transfer. Flu-
with the compound 6-rhodamine click product implies that the
orescent ACP2 (ACP*) is prepared via click coupling of rhodamine
ACP is required for transfer to occur (Lane 7). In all, these
azide with ACP2, preacylated with compound 6. ACP2* is then incu-
results suggest that β-lactam-derived, acyl-ACPs behave in ana-
bated with [KS6AT6] under various conditions. Lanes are marked with
logous fashion to natural polyketide intermediates and are kineti-
cally competitive with traditional PKS probes and inhibitors.
Further experimentation to quantitatively determine the transfer
rate is currently ongoing in the lab but, at this point, it is clear
that activated β-lactams offer a greatly simplified and readily
adaptable set of carrier protein modifiers.
1
the corresponding components for each reaction. [KS6AT6] was mixed
with competitor (cerulenin or propionyl-SNAc) for 1 h prior to addition
of ACP* (Lanes 3 and 5). 2ACP* and competitor were introduced to
3
[KS6AT6] simultaneously (Lanes 4 and 6). [KS6AT6] was mixed with
10 eq of the click product in the absence of ACP2 (Lane 7). SNAc =
N-acetylcysteimine.
are significantly more reactive than the β-lactones that we have
previously reported and all are available in a single synthetic
step from commercially available starting materials. Both carba-
mate and sulfamate moieties effectively activate the lactam ring
Conclusion
In summary, we have demonstrated the utility of N-activated
β-lactams for acylation of holo-ACPs. The structures examined
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for ACP-acylation. The resulting acylation products maintain the
thioester linkage common to all polyketide biosynthetic inter-
mediates and the reaction can be readily reversed by treatment
with hydrazine.
We are confident that the applications of β-lactams covered in
this manuscript just begin to scratch the surface of their capabili-
ties. The N-propargyloxycarbonyl group (compound 6) provides
a versatile chemical handle for nearly limitless elaboration of
structure. Although we have focused exclusively on proteins
involved in polyketide biosynthesis, we expect the general thiol-
reactivity of these simple β-lactams to translate well to other
systems bearing natural or engineered thiols.
stirred for 1 h. Benzylchloroformate (256 mg, 1.5 mmol) was
added in portions and stirred for 2 h at −78 °C. The reaction
mixture was allowed to warm to ambient temperature and stirred
for an additional 4 h. The reaction mixture was diluted with
water and extracted with DCM (50 mL × 3). Combined organic
phases were washed with brine and dried over Na2SO4. Solvent
was removed by rotary evaporation. Purification of crude product
by flash chromatography with 5 : 1 hexane : ethyl acetate yields
(285 mg, 1.39 mmol) 93% of product as an oil.
1H NMR (400 MHz, CDCl3) δ: 7.28–7.16 (m, 5H), 5.1 (s,
2H), 3.44–3.4 (t, J = 5.3, 2H), 2.85–2.81(t, J = 5.3, 2H).13C
NMR (400 MHz, CDCl3) δ 23.7, 34.7, 36.1, 65.9, 126.1, 126.8,
147.1, 155.0, 167.8.
IR: ν 3040, 2985, 1811, 1724, 1388, 1330, 1214, 1177, 1120,
1044, 763, 699 cm−1
,
Methods
MS [M + H]+ = 206
General information
All moisture sensitive reactions were performed under argon in
flame-dried glassware with Teflon-coated stir bars. Reagents and
solvents for organic synthesis were used as received unless other-
wise noted. n-Butyl lithium was used as a 2.5 M solution in
hexanes. Dichloromethane was distilled from calcium hydride
and stored over activated molecular sieves. Acetonitrile was
dried over activated molecular sieves for at least 24 h prior to
use. Thin-layer chromatography (TLC) was conducted on glass-
backed silica plates visualized with either UV illumination or
basic permanganate stain. Flash chromatography was conducted
on 60 Å silica gel.
(9H-Fluoren-9-yl)methyl 2-oxoazetidin-1-carboxylate (4).
2-Azetidinone (72 mg, 1 mmol) was dissolved in THF (5 mL),
and LiHDMS (167 mg, 1 mmol) dissolved in THF (2 mL) was
added dropwise at −78 °C over a 15 min period and stirred for
an additional 30 min. 9-Fluorenylmethyl chloroformate (259 mg,
1 mmol) dissolved in THF (2 mL) was added dropwise at
−78 °C. The resulting mixture was stirred for 1 h at −78 °C,
then allowed to warm to R.T, and stirred for 1 h further at which
point the starting material had been completely consumed (moni-
tored by TLC). The reaction mixture was poured into water and
extracted with DCM (3 × 25 mL). The combined extracts were
washed with brine and dried over Na2SO4. After removal of the
solvent by rotary evaporation, the crude reaction mixture was
purified by flash chromatography with DCM : ethyl acetate,
95 : 5, producing the product as an off-white solid (62 mg,
0.62 mmol) in 86% yield.
Synthesis
1-[(4-Methylphenyl)sulfonyl]azetidin-2-one.33 (2). 2-Azetidi-
none (72 mg, 1 mmol) was dissolved in THF (5 mL) at −78 °C.
NaHDMS (367 mg, 2 mmol) dissolved in THF (1 mL) was
added in portions. 4-Toluenesulfonyl chloride (763 mg, 4 mmol)
dissolved in THF (5 mL) was added to the reaction mixture in
portions slowly over 15 min. The reaction mixture was stirred
until all the starting material was consumed (monitored by
TLC), approximately 8 h. The reaction was washed with sodium
bicarbonate (10 ml ×3) and extracted with DCM (25 mL ×3).
The combined organic layers were dried over Na2SO4, and the
solvent was removed by rotary evaporation. The crude material
was purified by flash chromatography with 1 : 1 hexane : ethyl
acetate. Yield: (178 mg, 0.79 mmol) 79% of final product as col-
orless solid.
1H NMR (400 MHz, CDCl3) δ 3.13 (t, 2H), 3.68 (t, 2H), 4.32
(t, 1H), 4.47 (d, 2H), 7.26–7.80 (m, 8H).
13C NMR (400 MHz, CDCl3) δ 162.1, 148.3, 141.3, 139.4,
126.4, 125.5, 123.5, 118.1, 66.8, 44.3, 34.8, 36.2
IR: ν 3015, 2922,1770, 1722, 1449, 1388, 1313,1119, 1044,
964, 738 cm−1
HRMS (EI+) Calcd for C18H15NO3, 293.1052; Found,
293.1046
tert-Butyl 2-oxoazetidine-1-carboxylate.34 (5). 2-Azetidinone
(72 mg, 1 mmol) and DMAP (12.2 mg, 0.1 mmol) were dis-
solved in acetonitrile (5 mL) at 0 °C. Di-tert-butyl dicarbonate
(218 mg, 1.1 mmol) was added to the reaction mixture in por-
tions. The reaction mixture was stirred at 0 °C for 2 h, allowed to
warm to ambient temperature and stirred overnight. The reaction
mixture was diluted with ethyl acetate and washed with 1 N aq.
HCl and brine. The aqueous layer was extracted with ethyl
acetate (25 mL ×3). Combined organic layers were dried over
Na2SO4 and the solvent was removed by rotary evaporation. The
crude product was purified by flash chromatography using 2 : 1,
hexanes : ethyl acetate (154 mg, 0.9 mmol) 90% of product as an
oil.
1H NMR (400 MHz, CDCl3) δ: 7.9–7.87 (d, J = 8.29, 2H),
7.32–7.41 (d, J = 8.48, 2H), 3.67–3.64 (t, J = 5.2, 2H),
3.06–3.02 (t, J = 5.1, 2H), 2.46 (s, 3H).
13C NMR (400 MHz, CDCl3) δ: 161.9, 143.4, 128.3, 125.7,
38.0, 34.9, 19.9.
IR: ν 2973, 1776, 1362, 1154, 682, 98 cm−1
,
MS [M + H]+ = 227
Benzyl 2-oxoazetidine-1-carboxylate.14 (3). HDMS (250 mg,
1.5 mmol) was dissolved in THF (8 mL) and cooled to −78 °C.
nBuLi (96 mg, 1.5 mmol) was added dropwise and stirred for
30 min. In a separate RBF, 2-azetidinone (106.6 mg, 1.5 mmol)
was dissolved in 5 mL of THF at −78 °C. The contents of the
first flask were transferred via cannula to the second flask and
1H NMR (400 MHz, CDCl3) δ: 3.56–3.52 (t, J = 5.1, 2H),
3.03–2.87 (t, J = 5.2, 2H), 1.5 (s, 9H).
13C NMR (400 MHz, CDCl3) δ: 205.4, 162.1, 146.5, 81.2,
36.8, 35.0, 29.8, 26.2.
1998 | Org. Biomol. Chem., 2012, 10, 1992–2002
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IR: ν 2984, 1795, 1719, 1331, 1157, 1043 cm−1
,
(5 × 50 mL portions). Solvent was removed by rotary evapor-
ation and the material was used without further purification. The
material from the ester coupling was dissolved in 5 mL of DMF
and NaN3 (40 mg, 0.600 mmol) and a catalytic amount of KI
were added. A reflux condenser was attached and the reaction
mixture was heated to 80 °C for 16 h. The material was diluted
with 100 mL of ethyl acetate and washed with brine. The solvent
was removed by rotary evaporation and the crude material
purified by flash chromatography (5% MeOH in DCM) to yield
67 mg, 0.182 mmol of product (61%) as a waxy pale yellow
solid.
MS [M + H]+ = 172
Prop-2-yn-1-yl 2-oxoazetidine-1-carboxylate (6). 2-Azetidi-
none (107 mg, 1.5 mmol) was dissolved in THF (7 mL) at
−78 °C. LiHDMS (330 mg, 2 mmol) dissolved in THF (2 mL)
was added in portions over 10 min and the reaction mixture was
stirred for 30 min. Propargyl chloroformate (181 mg, 1.5 mmol)
dissolved in THF (2 mL) was added in portions over 10 min and
the reaction mixture was stirred at −78 °C for 2 h then allowed
to warm to ambient temperature. The reaction mixture was
diluted with water and extracted with DCM (50 mL ×3). The
combined organic layers were washed with brine and dried over
Na2SO4. Solvent was removed by rotary evaporation. Purifi-
cation of crude product by flash chromatography with 95 : 5
DCM : methanol yields (106 mg, 0.69 mmol) 46% of product as
a pale yellow solid.
1H NMR (400 MHz, CDCl3) δ (ppm) 1.32–1.52 (m, 6 H)
1.56–1.80 (m, 8 H) 2.33 (t, J = 7.45, 2 H) 2.74 (d, J = 12.76, 1
H) 2.91 (dd, J = 12.82, 4.99, 1 H) 3.16 (ddd, J = 8.34, 6.38,
4.74, 1 H) 3.28 (t, J = 6.88, 2 H) 4.06 (t, J = 6.63, 2 H) 4.31
(ddd, J = 7.67, 4.71, 1.14, 1 H) 4.51 (dd, J = 7.71, 5.05, 1 H)
5.67 (s, 1 H) 6.03 (s, 1 H).
1H NMR (400 MHz, CDCl3) δ 4.82–4.81(d, J = 23.3, 1H),
3.68–3.65 (t, J = 5.3, 2H), 3.10–3.07 (t, J = 5.3, 2H), 2.55–2.53
(t, J = 2.4, 1H).
13C NMR (400 MHz, CDCl3) δ (ppm) 173.7, 163.7, 65.6,
61.2, 60.8, 56.1, 51.3, 40.5, 33.9, 29.9, 28.7, 28.4, 27.2, 26.3,
25.5, 24.8
13C NMR (400 MHz, CDCl3) δ 34.9, 36.1, 51.8, 74.0, 146.3,
162.2.
IR: ν 3230, 2934, 2101, 1733,1700, 1264, 1175 cm−1
HRMS (EI+) Calcd for C16H27N5O3S, 369.1848; Found,
369.1814
IR: ν 3251, 2925, 2134, 1782, 1722, 1313, 1187, 1114, 1045,
615 cm−1
HRMS (EI+) Calcd for C7H7NO3, 153.0426; Found,
153.0402
Propionyl-N-acetylcysteamine (Propionyl-SNAc)
Sulforhodamine-B azide (S1).7 3-Azido-1-aminopropane35
(22 mg, 0.22 mmol) and triethylamine (51 mg, 0.5 mmol) were
dissolved in 1 mL of 5 : 1 DCM : DMF and cooled to 0 °C. Sul-
forhodamine B, sulfonyl chloride [mixture of ortho- and para-
sulfonyl chloride isomers] (115 mg, 0.20 mmol) was added
portion-wise over ca. 30 min and stirred overnight at room temp-
erature. Solvent was removed in vacuo, and the product was
purified by flash chromatography over silica gel with 90 : 5 : 5
DCM : acetonitrile : methanol mobile phase. The ortho isomer
was distinguished by its reversible color change in pH 9.0
buffer.36 para Isomer: (22 mg 0.034 mmol) 17%, ortho isomer
(18 mg, 0.028 mmol) 14%. Both obtained as a red solid with a
metallic green luster. The ortho isomer was found to perform
best in the click reaction and was utilized for labeling
experiments.
To a solution of triethylamine (2.80 mmol) in dichlromethane
(5 mL) was added propionic acid (1.40 mmol), (3-dimethylami-
nopropyl)-3-ethylcarbodiimide hydrochloride (1.40 mmol),
1-hydroxybenzotriazole (HOBt) (1.40 mmol) and N-acetyl
cysteamine (SNAc)(1.35 mmol). The reaction mixture was
stirred overnight. The organic layer was washed with saturated
NaHCO3 solution, 0.1 N HCl solution and brine. It was then
dried over anhydrous sodium sulfate, concentrated under
vacuum, and purified by flash column chromatography (1 :
1 hexane : ethyl acetate) to provide compound (140 mg, 74%) as
a pale yellow oil.
1H NMR (400 MHz, CDCl3) δ (ppm) 0.86 (t, J = 7.58 Hz,
3H) 1.69 (s, 3H) 2.28 (q, J = 7.4 Hz, 2H) 2.72 (t, J = 6.8 Hz,
2H) 3.07 (q, J = 6.6 Hz, 2H) 7.39 (br s, 1H)
13C NMR (400 MHz, CDCl3) δ (ppm) 199.4, 170.6, 39.0,
36.8, 28.0, 22.6, 9.4
NMR spectra are of poor quality due to long relaxation times
(see supporting information†). Product is pure by LC-MS.
IR: ν 2975, 2100, 2590, 1339, 1180 cm−1
IR: ν 3282,2979,1690,1650,1546,1373,1288,1090,935 cm−1
MS [M + H]+ = 176
HRMS (EI+) Calcd for C30H37N6O6S2, 641.2210. Found,
641.2216.
Protein expression and purification
Biotin azide (S2). D(+)-biotin (73 mg, 0.300 mmol) was dis-
solved in 3 mL of DMF by briefly heating the mixture with a
heat gun. The mixture was cooled to 0 °C in an ice-water
bath and 6-chloro-1-hexanol (120 mg, 0.900 mmol), and
N,N-dimethylamino pyridine (73 mg, 0.600 mmol) were added.
N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride
(115 mg, 0.600 mmol) was added in portions over ca. 60 min
and the reaction mixture was allowed to come to room tempera-
ture. Stirring was continued for 24 h. When biotin had been con-
sumed (monitored by TLC), the reaction mixture was diluted
with ethyl acetate (100 mL). The organic phase was washed with
concentrated NaHCO3 (3 × 50 mL), and pH 2 saturated Na2SO4
ACP2 was expressed from pNW06,37 ACP3 was expressed from
pVYA05.11 [KS6AT6] was expressed from pAYC11.26 apo-
ACPs were harvested from E. coli BL-21 and holo-ACPs were
harvested from E. coli. BAP-138 pNW6 and pVYA05 contain
kanamycin resistant vectors.
General procedure for protein expression and isolation
Bacteria were grown in 1 L shake cultures of LB-antibiotic
media at 37 °C in an incubating shaker until the OD600 was
between 0.6 and 0.8. Over expression was induced with 200 μL
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Org. Biomol. Chem., 2012, 10, 1992–2002 | 1999

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of 1 M IPTG (per liter of culture) and carried out at 18 °C for
18 h. After this point all work was carried out at 4 °C. Cells
were pelleted by spinning at 3000 RPM for 10 min and resus-
pended in 50 mL of lysis buffer (20 mM Tris-HCl, 150 mM
NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM
sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM
sodium Na3VO4, 1 μg mL−1 leupeptin, pH 7.5). Cells were lysed
using an ultrasonic converter. The lysate was pelleted at 10,000
rpm for 60 min. The supernatant was equilibrated with 3 mL of
Ni-NTA slurry (per liter of culture) for 60 min by stirring with a
PTFE-coated stir bar at minimal speed. The mixture was then
poured into a fritted column and the supernatant eluted. The
resin bed was washed with two 15 mL portions of wash buffer
(50 mM phosphate, 300 mM NaCl, 20 mM imidazole, pH 8.0),
and eluted with 3 mL (per liter of culture) of elution buffer
(50 mM phosphate, 300 mM NaCl, 250 mM imidazole, pH 8.0).
The purified protein was loaded into a 3 kDa NMW centrifugal
concentrator and diluted to 15 mL with storage buffer (100 mM
Tris, 1 mM EDTA, 1 mM dithioerythritol, 10% glycerol pH 8)
and spun until the final volume was ≤500 μL. Dilution and fil-
tration was repeated a total of three times. Protein concentration
was determined by Bradford assay with a BSA standard curve,
average final concentration was approximately 3 mM. Proteins
were flash frozen and stored at −80 °C until use.
proteins were imaged on a UV-transilluminator. Total protein
was stained using Coomassie stain. For the ACP to KS transfer
experiments (Fig. 8), proteins were separated using a 4–20% gra-
dient HEPES-PAGE gel (100V, 50mA, 90 min).
Proteolysis
Sequencing grade modified trypsin was added to prepared
samples so that the final trypsin : ACP ratio was 1 : 50 (w/w).
The mixture was incubated at 30 °C for 18 h. Digestion was
quenched by addition of an equal volume of 10% formic acid.
Digests were flash frozen in liquid nitrogen and stored at −20 °C
until analysis.
Affinity purification of holo-ACPs
Lactam modification of ACPs
Unless otherwise stated, phosphate buffer refers to 100 mM,
pH 7.0 phosphate. 1 μL of an 80 : 20 mixture of holo : apo-
ACP2 (25 μM total protein) was equilibrated at ambient tempera-
ture for 15 min in phosphate buffer containing 2.5 mM TCEP. 6
(25× with respect to total ACP concentration) was added and the
mixture was equilibrated at ambient temperature for 1 h. To
remove excess 6, the mixture was loaded into a 3kDa NMW con-
centrator and the volume reduced to 100 μL by spinning in a
centrifuge cooled to 4 °C. The mixture was diluted to 500 μL
with phosphate buffer, and then concentrated to 100 μL again.
This process was repeated a total of 3 times. Protein was
removed from the concentrator by inverting it and spinning. The
concentrator was then washed several times with phosphate
buffer which was added to the protein sample. The filtered
protein was reconstituted to 900 μL.
Proteins acylation with β-lactams
Labeling reactions for gel assays were performed at 20 μL total
volume, reactions for LC-MS were performed at 50 μL total
volume. Final concentrations reported in procedure. Phosphate
buffer (100 mM, pH 7), tris(2-carboxyethyl)phosphine (TCEP)
(2.5 mM), and protein (0.025 mM) were reacted at ambient
temperature for 15 min. β-Lactam was added to achieve the
appropriate final concentration (0.5, 1, 5, 10, 20, 50x) with
respect to protein. The mixture was equilibrated at ambient temp-
erature for 60 min.
[3 + 2] Cycloaddition
In a separate microfuge tube, DMSO (100 μL), S2 (50× with
respect to protein), THPTA39(1.1 mM), NaAsc (10 mM), and
CuSO4 (1 mM) were combined. Best results are obtained if these
reagents are first combined in a separate container and then
added to the protein. The combined click reagents are added to
the protein from the previous step and allowed to react at
ambient temperature for 6 h. Excess biotin reagent was removed
using the same procedure for the removal of excess lactam. The
modified protein was reconstituted to 1000 μL in phosphate
buffer.
β-Lactam/β-lactone competition loading of ACP
10 equivalents of compound 6 and 4-methyloxetan-2-one were
mixed and introduced to buffered holo-ACP2 for 1 h. The
sample was trypsinized analyzed by LC-MS as described
previously.7
Immobilization of modified ACPs
Streptavidin Sepharose High Performance (10× with respect
to protein) was washed 3× with 1000 μL of phosphate buffer and
the supernatant was removed. The modified protein from the pre-
vious step was added to the beads, and the mixture equilibrated
at ambient temperature for 60 min with agitation on an orbit
shaker. The supernatant was removed and the beads were washed
5× with 1000 μL of phosphate buffer, 5× with 1000 μL of TRIS
buffer (100 mM, pH 9.0), and once with 1000 μL of 100 mM
TRIS, +10 mM N2H4, pH 9.0.
Chromophore attachment
The reaction was carried out at 25 μL, final concentrations
reported. S1, (2× with respect to 6), sodium ascorbate (1 mM),
and copper(II) sulfate (1 mM) were added to samples which had
been labeled with 6. The reaction was performed at ambient
temperature for 60 min.
Elution of immobilized ACPs
Gel assay
The washed beads from the previous step were resuspended in
1000 μL of 100 mM ammonium formate containing 100 mM
N2H4 (100 mM pH 9.0) and equilibrated overnight at 4 °C with
agitation on an orbit shaker. Eluted protein was concentrated in a
3kDa NMW concentrator and subjected to buffer exchange for
Labeled samples were diluted to 35 μL with gel-loading buffer.
ACP 2 was separated by 12% and ACP 3 with 15% SDS-PAGE,
each with a 5% stacking gel run at 100 V, 50 mA, 135 min. Gels
were developed in 10% acetic acid to visualize S1. Labeled
2000 | Org. Biomol. Chem., 2012, 10, 1992–2002
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8 F. T. Wong, A. Y. Chen, D. E. Cane and C. Khosla, Protein-Protein Rec-
ognition between Acyltransferases and Acyl Carrier Proteins in Multi-
modular Polyketide Synthases, Biochemistry, 2009, 49, 95–102.
9 V. Y. Alekseyev, C. W. Liu, D. E. Cane, J. D. Puglisi and C. Khosla, Sol-
ution Structure and Proposed domain-domain Recognition Interface of an
Acyl Carrier Protein Domain from a Modular Polyketide Synthase,
Protein Sci., 2007, 16, 2093–2107.
10 Y. Tang, A. Y. Chen, C. Y. Kim, D. E. Cane and C. Khosla, Structural
and Mechanistic Analysis of Protein Interactions in Module 3 of the 6-
Deoxyerythronolide B Synthase, Chem. Biol., 2007, 14, 931–943.
11 C. Y. Kim, V. Y. Alekseyev, A. Y. Chen, Y. Tang, D. E. Cane and
C. Khosla, Reconstituting Modular Activity from Separated Domains
of 6-Deoxyerythronolide B Synthase, Biochemistry, 2004, 43, 13892–
13898.
12 I. Staub and S. A. Sieber, β-Lactam Probes as Selective Chemical-
Proteomic Tools for the Identification and Functional Characterization of
Resistance Associated Enzymes in MRSA, J. Am. Chem. Soc., 2009,
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13 M. Ito, L. W. Koo, A. Himizu, C. Kobayashi, A. Sakaguchi and
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further experiments, or combined with TCEP for direct infusion
LC-MS experiments.
Fluorescent assay for ACP to KS transfer
Holo-ACP2 (25 μM) was labeled with 10 equivalents of 6 fol-
lowed by [3 + 2] cycloaddition with S2 as described above. For
transfer of the fluorescent product, [KS6AT6] (25 μ M) was
introduced to acyl-ACP2 (25 μM) and incubated for 1 h. For
lanes 3 and 5 (Fig. 9), [KS6AT6] was pretreated with cerulenin
(5 mM) or propionyl SNAc (10 mM) for 1 h before introducing
it to acyl-ACP2. For lanes 4 and 6, acyl-ACP2 (25 μM) was
mixed with cerulenin (5 mM) or propionyl-SNAc (10 mM) fol-
lowed by 1 h incubation with [KS6AT6] (25 μM). The
[KS6AT6] control (lane 7) was executed under the same acyla-
tion and click conditions as in other samples but without the
ACP or small molecule competitors.
14 S. Gérard, G. Dive, B. Clamot, R. Touillaux and J. Marchand-Brynaert,
Synthesis, Hydrolysis, Biochemical and Theoretical Evaluation of 1, 4-
Bis (Alkoxycarbonyl) Azetidin-2-Ones as Potential Elastase Inhibitors,
Tetrahedron, 2002, 58, 2423–2433.
LC-MS
15 S. Gérard, G. Nollet, J. Vande Put and J. Marchand-Brynaert, 1-Alkoxy-
carbonyl-3-Halogenoazetidin-2-Ones as Elastase (PPE) Inhibitors,
Bioorg. Med. Chem., 2002, 10, 3955–3964.
16 R. M. Adlington, J. E. Baldwin, G. W. Becker, B. Chen, L. Cheng, S.
L. Cooper, R. B. Hermann, T. J. Howe, W. McCoull and A. M. McNulty,
Design, Synthesis, and Proposed Active Site Binding Analysis of Mono-
cyclic 2-Azetidinone Inhibitors of Prostate Specific Antigen, J. Med.
Chem., 2001, 44, 1491–1508.
17 T. Böttcher and S. A. Sieber, β-Lactones as Privileged Structures for the
Active-Site Labeling of Versatile Bacterial Enzyme Classes, Angew.
Chem., Int. Ed., 2008, 47, 4600–4603.
18 H. C. Kolb, M. G. Finn and K. B. Sharpless, Click Chemistry: Diverse
Chemical Function from a Few Good Reactions, Angew. Chem., Int. Ed.,
2001, 40, 2004–2021.
Separation was performed with a Waters 1525 system. The gradi-
ent employed was A = water + 0.1% formic acid, B = aceto-
nitrile + 0.1% formic acid, 5–95% B over 60 min with a Waters
XBridge C18 5u column (4.6 × 150 mm). Mass spectra were
acquired with a Waters Micromass ZQ mass detector in EI+
mode: Capillary voltage = 3.50 kV, cone voltage = 30 V, extrac-
tor = 3 V, RF lens = 0.0 V, source T = 100 °C, desolvation
T = 200 °C, desolvation gas = 300 L h−1, desolvation gas =
0.0 L h−1 The system was operated by and spectra were pro-
cessed using the Waters Empower software suite.
19 W. G. Lewis, L. G. Green, F. Grynszpan, Z. Radić, P. R. Carlier,
P. Taylor, M. G. Finn and K. B. Sharpless, Click Chemistry in Situ:
Acetylcholinesterase as a Reaction Vessel for the Selective Assembly of a
Femtomolar Inhibitor from an Array of Building Blocks, Angew. Chem.,
Int. Ed., 2002, 41, 1053–1057.
20 V. V. Rostovtsev, L. G. Green, V. V. Fokin and K. B. Sharpless, A Step-
wise Huisgen Cycloaddition Process: Copper (I)-catalyzed Regioselective
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2002, 41, 2596–2599.
Acknowledgements
This work was supported by start-up funding from the University
of Massachusetts, Amherst (to N.A.S). The authors would like
to thank Professor Chaitan Khosla for the generous gifts of
BAP1, pVYA05, pNW06, and pAYC11. We also thank Pro-
fessors Alan Kennan and James Chambers for helpful
discussions.
21 N. M. Green, Avidin and Streptavidin, Methods Enzymol., 1990, 184,
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to Affinity-Based Separations, J. Chromatogr., A, 1990, 510, 3–11.
23 M. Wilchek and E. A. Bayer, The Avidin-Biotin Complex in Bioanalyti-
cal Applications, Anal. Biochem., 1988, 171, 1–32.
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