Diazo reagents with small steric footprints for simultaneous arming/SAR studies of alcohol-containing natural products via O-H insertion

Article abstract of DOI:10.1021/cb2002686

Natural products are essential tools for basic cellular studies leading to the identification of medically relevant protein targets and the discovery of potential therapeutic leads. The development of methods that enable mild and selective derivatization of natural products continues to be of significant interest for mining their information-rich content. Herein, we describe novel diazo reagents for simultaneous arming and structure-activity relationship (SAR) studies of alcohol-containing natural products with a small steric footprint, namely, an α-trifluoroethyl (HTFB) substituted reagent. The Rh(II)-catalyzed O-H insertion reaction of several natural products, including the potent translation inhibitor lactimidomycin, was investigated, and useful reactivity and both chemo- and site (chemosite) selectivities were observed. Differential binding to the known protein targets of both FK506 and fumagillol was demonstrated, validating the advantage of the smaller steric footprint of α-trifluoroethyl derivatives. A p-azidophenyl diazo reagent is also described that will prove useful for photoaffinity labeling of low affinity small molecule protein receptors.

Full text of DOI:10.1021/cb2002686

LETTERS
pubs.acs.org/acschemicalbiology
Diazo Reagents with Small Steric Footprints for Simultaneous Arming/
SAR Studies of Alcohol-Containing Natural Products via OꢀH Insertion
Supakarn Chamni,† Qing-Li He,‡ Yongjun Dang,‡ Shridhar Bhat,‡ Jun O. Liu,‡ and Daniel Romo†,*
†Department of Chemistry, Texas A&M University, P.O. Box 30012, College Station, Texas 77842-3012, United States
‡Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, 725 North Wolfe St., Baltimore,
Maryland 21205, United States
S Supporting Information
b
ABSTRACT: Natural products are essential tools for basic
cellular studies leading to the identification of medically relevant
protein targets and the discovery of potential therapeutic leads.
The development of methods that enable mild and selective
derivatization of natural products continues to be of significant
interest for mining their information-rich content. Herein, we
describe novel diazo reagents for simultaneous arming and
structureꢀactivity relationship (SAR) studies of alcohol-con-
taining natural products with a small steric footprint, namely, an
α-trifluoroethyl (HTFB) substituted reagent. The Rh(II)-cata-
lyzed OꢀH insertion reaction of several natural products, including the potent translation inhibitor lactimidomycin, was
investigated, and useful reactivity and both chemo- and site (chemosite) selectivities were observed. Differential binding to the
known protein targets of both FK506 and fumagillol was demonstrated, validating the advantage of the smaller steric footprint of
α-trifluoroethyl derivatives. A p-azidophenyl diazo reagent is also described that will prove useful for photoaffinity labeling of low
affinity small molecule protein receptors.
ver the past 50 years, the discovery and study of novel
natural products from diverse terrestrial and marine organ-
and site nonselective OꢀH insertions with rhodium carbenoids
derived from hexynyl-α-p-bromophenyl diazo acetate (1, HBPA) as
a donor/acceptor carbenoid precursor.10 The arming process
leads to natural product derivatives that are equipped with
tethered alkynes for subsequent conjugation to various tags via
SharplessꢀH€uisgen cycloaddition13 to generate cellular probes.
The utility of this strategy was demonstrated with a panel of
natural products including FK506. An FK506-HBPA-biotin con-
jugate was employed to successfully pull-down the entire “immu-
nosuppressive complex” consisting of calcineurins A/B, calmodulin,
and FKBP12. However, the large steric size of the p-bromophe-
nyl group and its close proximity to the point of attachment to
the natural product was a potential liability for retaining bio-
activity and subsequent affinity chromatography experiments. We
thus sought to develop sterically smaller diazo reagents with
similar reactivity to the p-bromophenyl substituted reagent
(HBPA) that may improve retention of bioactivity. Herein, we
describe the development of an α-trifluoroethyl (HTFB) sub-
stituted carbenoid precursor that leads to greatly reduced steric
footprints yet provides similar reactivities, including chemosite
selectivities, as the previously described p-bromophenyl reagent 1.
Differences in IL-2 reporter assay activity and affinity chroma-
tography experiments with the derived FK506 conjugates highlight
O
isms has had a profound impact on human health.1,2 Indeed,
natural products are an immense source of chemical diversity.3
These complex molecules are capable of interacting with numer-
ous human cellular proteins and typically have intrinsic cell
permeability.4 As a result, many natural products or natural product-
inspired small molecules are currently in clinical use as anti-
biotics, antitumor agents, immunosuppressants, antiviral drugs,
and enzyme inhibitors.5 Furthermore, natural products have a
rich history as tools for basic cellular studies leading to the discovery
of potential cellular targets for intervention of human disease6
and in this way contain enormous information content that
should be exploited fully. Gaining an understanding of the detailed
mode of action of biologically active natural products, including
the identification of putative protein targets, continues to be a
highly useful strategy for the discovery of human therapeutics.7,8
These studies are greatly facilitated by detailed structureꢀactivity
relationship (SAR) studies and the synthesis of cellular probes
derived from a particular natural product of interest. These cellular
probes, which typically contain a functional group for in vitro or
in vivo attachment of reporter tags such as biotin or a fluorophore,
continue to be of great importance for such studies.9
We previously described mild and versatile strategies for
simultaneous arming (with a reactive functional group or array)
and SAR studies that facilitate mechanism of action studies of
natural products.10,11 One such method involves chemosite selective12
Received: February 2, 2011
Accepted: August 26, 2011
Published: September 06, 2011
r
2011 American Chemical Society
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Scheme 1. Strategy for Bioactive Polyol Natural Product Derivatization and Synthesis of Required Diazo Reagents with Varying
Steric Footprintsa
a (a) Simultaneous arming/SAR studies via OꢀH insertion of polyol natural products using diazo reagents with varied steric footprints and subsequent
tag conjugation. (b) General synthesis of new diazo alkyne reagents 3aꢀd. For full details of the synthesis of diazo reagents 3aꢀd, see Supporting
Information.
the profound changes in protein binding and capture. Impor-
tantly, the α-trifluoroethyl (HTFB) substituted conjugates may
offer greater utility for identifying cellular receptors of bioactive
natural products due to their smaller steric size as further demon-
strated by comparison of HBPA- versus HTFB-fumagillol deri-
vatives in the hMetAP2 inhibition assay and affinity chromatog-
raphy experiments.
derived natural product derivatives (cyano, 35.10 Å3; 1,1,1-
trifluoroethyl, 65.51 Å3; p-bromophenyl, 117.02 Å3). Molecular
volumes were calculated using DFT-B3LYP/6-31++G(2d, 2p)
level of theory (Spartan '08 v1.2.0). In addition, the hex-5-ynyl
2-(4-p-azidophenyl)-2-diazoacetate (3d)17 was prepared for its
potential as a trifunctional linker for photoaffinity labeling of low
affinity binding proteins,18 and the derived carbenoid was
expected to exhibit reactivity similar to that of the p-bromophe-
nyl reagent 1. To enable studies of their reactivity, these new
diazo reagents were successfully prepared on gram scale (∼5 g)
by esterification and subsequent base-promoted diazo transfer
(Scheme 1b, see Supporting Information for complete details).
We found that diazo reagents 3a, 3c, and 3d are stable at RT and
storable at ꢀ10 °C with no decomposition after 1 year. However,
the α-trifluoromethyl reagent 3b is highly unstable and decom-
posed at 23 °C.
The reactivity of diazo reagents 3a, 3c, and 3d were initially
studied with gibberellic acid methyl ester (4), which contains
several potentially reactive sites (Figure 1a, highlighted in red)
including an internal and a terminal disubstituted olefin, and two
sterically differentiated alcohols (secondary and tertiary). This
natural product derivative thus serves as an ideal substrate to
study chemosite selectivity. Under the standard OꢀH inser-
tion conditions with Rh2(OAc)4, diazo reagents 3a and 3d
exhibited stability and reactivity similar to that of the p-bromophenyl
We sought diazo reagents with substantially reduced steric
footprints (i.e., smaller α-substituents) compared to the pre-
viously described p-bromophenyl substituted reagent 1 due
to the proximity of these substituents to the native natural
product. We therefore set out to study the α-trifluoroethyl (3a),
α-trifluoromethyl (3b), and α-cyano (3c) diazo reagents that pos-
sess different α-substituents with varied steric sizes and electron-
withdrawing potential, which would also alter the reactivity of the
derived rhodium carbenoids (Scheme 1a). While the proposed
diazo reagents would lead to acceptor/acceptor carbenoids,14,15
the fluorinated hex-5-ynyl 2-diazo-4,4,4-trifluorobutanoate (3a,
HTFB) and hex-5-ynyl 2-diazo-3,3,3-trifluoropropanoate (3b)
have the added advantage of enabling 19F NMR analysis of
crude natural product derivatization reactions. In contrast, the
hex-5-ynyl 2-cyano-2-diazoacetate (3c) was expected to provide
a highly reactive metallocarbenoid species.16 Computational
studies supported the significant steric size difference based on
calculated molecular volumes that could be expected between the
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Figure 1. Rh(II)-catalyzed OꢀH insertion reactions using diazo reagents 3a, 3c-d with various natural products. (a) Comparison of reactivity and
chemosite selectivity of novel diazo reagents 3a, 3c-d with various Rh(II) catalysts (potential reactive sites highlighted in red). (b) Scope of Rh2(OAc)4-
catalyzed chemosite selective OꢀH insertion of HTFB (3a) with various natural products (reactive alcohols and amines highlighted in blue).
diazo 1 (Figure 1a, entry 1), providing monoethers 5a and 5d
derived from OꢀH insertion with the more accessible
secondary alcohol along with recovered ester 4 (Figure 1a,
entries 2, 3). However, the more reactive α-cyano reagent 3c
gave a complex mixture likely resulting from both OꢀH insertion
and cyclopropanation19 with no recovery of starting material
(not shown). Overall, the α-trifluoro diazo reagent 3a provided
the best results with regard to stability, reactivity, and selectivity
of derived rhodium carbenoid compared to the p-bromophe-
nyl diazo 1. With respect to catalysts, Rh2(esp)2 described by
DuBois20 gave optimal conversion to the monoether 5a
(66%) and higher diastereoselectivity (4:1); however, this
was accompanied by 25% of diether 6 (Figure 1a, entry 4).
Rh2(OAc)4 was the most chemosite selective catalyst, favor-
ing the less hindered and nucleophilic secondary alcohol at
C3 (Figure 1a, entry 2).
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Figure 2. Comparative affinity experiments with HTFB and HBPA-FK506-biotin conjugates. (a) Synthesis of FK506-HTFB-biotin (16) and FK506-
HBPA-biotin (17) conjugates. (b) IC50 values for IL-2 inhibition by FK506 derivatives of Jurkat T cells transiently transfected with an IL-2 promoter-
driven luciferase reporter. (c) Affinity chromatography experiments with biotin conjugates 16 and 17 (left panel: silver staining of all retained proteins;
right panel: Western blotting by indicated antibodies).
We next investigated various known chiral Rh(II) catalysts in
conjunction with the α-trifuoroethyl diazo reagent 3a as a means
to alter chemosite selectivity of the OꢀH insertion. Several
commercially available chiral rhodium catalysts including those
of Davies21,22 and Doyle23 were investigated. The use of chiral
catalysts enables a type of “double diastereoselectivity”24,25 that
could in principle alter chemosite selectivity. Of the four chiral
catalysts studied (Figure 1a, entries 6ꢀ9), Rh2(S-DOSP)4
provided the highest yield of monoetherification (75%) of ester
4 with good diastereoselectivity (dr, 3:1) and a high degree of
chemosite selectivity (Figure 1a, entry 9). However, Rh2(OCOCF3)4
and Rh2(MEOX)4, which are known to lead to more reactive
carbenoids compared to Rh2(OAc)4, gave lower chemosite
selectivity as expected. Thus, these catalysts are ideal for obtain-
ing the greatest number of derivatives for initial SAR studies of a
novel polyol natural product.
The scope of this OꢀH insertion with the new α-trifluoroethyl
diazo ester (3a) was assessed with several commercially available
natural products and derivatives (Figure 1b). In general, OꢀH
insertion with metallocarbenoids is governed by both steric and
electronic effects.26 Despite the smaller steric size of the α-trifluoro-
ethyl group, Rh2(OAc)4 catalyzed OꢀH insertions with diazo
reagent 3a with the natural products studied showed chemosite
selectivity similar to that of the p-bromophenyl reagent 1,10
suggesting that electronic effects play the predominant role for
chemosite selectivity. Brefeldin A (8a) presented two potentially
reactive alcohols; however, the more accessible secondary alco-
hol (C13) was selectively alkylated over the more electron-rich
allylic alcohol (C1). Paclitaxel (9a) has two electronically and
sterically distinct secondary alcohols and a tertiary alcohol. The
more accessible secondary alcohol (C20) was selectively deriva-
tized with no reaction detected at the other alcohols or the amide
NꢀH. To determine the reactivity of amines in comparison to
alcohols, ephedrine (10a), which bears a secondary alkyl amine
and a secondary alcohol was studied. As expected, the major
adduct isolated was that derived from NꢀH insertion due to
greater nucleophilicity; however, some bis-derivatization derived
from both NꢀH and OꢀH insertion was also detected. In the
case of FK506 (11a), OꢀH insertion led to chemosite selec-
tivity at the more accessible cyclohexyl secondary alcohol (C32)
in 66% yield and with good diastereoselectivity (dr, 7:1). No
OꢀH insertion at the macrocyclic secondary alcohol (C24) or
dietherification was observed. Previously, dietherification of FK506
was observed when HBPA (1) was added in one portion;10
therefore, slow addition of 6 equiv of HTFB (3a) over 2 h again
prevented dietherification. Overall, these results are consistent
with chemoselectivities previously observed for OꢀH insertion
with Rh(II)-carbenoids derived from HBPA, and while not all
OꢀH environments were explored, the same reactivity patterns
(primary ROH ≈ secondary alkyl NH > secondary alkyl OH >
secondary allylic OH g aryl NH > phenolic OH > tertiary alkyl
OH > indole NH and no reaction with amide NH or alkenes)
would be expected on the basis of observations to date.10
Lactimidomycin (12a) is a macrocyclic natural product with a
pendant cycloheximide that has gained much interest due to its
potent inhibitory effect on eukaryotic translation elongation
properties leading to dramatic antitumor activities.27,28 However,
lactimidomycin is highly acid- and base-sensitive, which limits
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Figure 3. Comparative affinity experiments with HTFB and HBPA-fumagillol derivatives in cellulo. (a) Synthesis of fumagillol derivatives. (b) Affinity
chromatography experiments were performed using azide 21 to afford biotinylated fumagillol dervatives, and the target of fumagillol was analyzed by a
blotting membrane using streptavidin horseradish peroxidase or an antibody against hMetAP2. (c) IC50 values of fumagillol derivatives against HUVEC
cells and EC50 values in the hMetAP2 assay.
derivatization strategies. Thus, this became an excellent substrate
for testing the mildness of this OꢀH insertion process. Use
of HTFB (3a) provided ∼15% yield of isolated, purified
(preparative HPLC) ether 12b on a reaction scale of 2 mg at
50% conversion with some loss of material attributed to degrada-
tion during purification. The successful derivatization of such a
sensitive natural product demonstrates the utility of the de-
scribed derivatization strategy as it proceeds under essentially
neutral conditions, works efficiently on small scale (∼1ꢀ2 mg),
and allows for the recovery of unreacted starting material.
We were next interested in studying the effect of having
different α-substituents (i.e., α-trifluoroethyl vs p-bromophenyl)
on ethers obtained from OꢀH insertion of natural products in a
cellular assay and also affinity chromatography experiments with
derived biotin conjugates. For these studies, the diastereomeric
FK506-derived ethers 11b/11b0 and 15/150 were separated by
preparative TLC. An FK506-HTFB-biotin probe (16) was
prepared by SharplessꢀH€uisgen cycloaddition of the major
diastereomeric HTFB ether 11b and biotin azide 14. The
FK506-HBPA-biotin conjugate 17 was also prepared in a similar
fashion (Figure 2a). In our previous study, the use of an FK506-
HBPA-biotin probe led to pull-down of the entire immunosup-
pressive complex including FKBP12, calcineurin A/B, and
calmodulin.10 In the IL-2 reporter assay, the FK506-HTFB
derivatives 11b and 11b0 showed diminished inhibition of IL-2
production (∼2 fold, IC50 15.9 ( 2.8 and 19.2 ( 14.5 nM
respectively) compared to the FK506-HBPA derivatives 15 and
150 (IC50 10.2 ( 3.8 and 7.3 ( 0.6 nM, respectively) (Figure 2b).
A similar trend was observed when the biotin conjugates were
assayed in the IL-2 reporter assay with the FK506-HTFB-biotin
probe 16 showing a >4-fold decrease in IC50 value compared to
the corresponding FK506-HBPA-biotin conjugate 17. In the
most dramatic demonstration of the impact of a smaller steric
footprint, side by side comparison of the two FK506-biotin
probes 16 and 17 showed that the smaller α-trifluoroethyl
conjugate led only to pull-down of FKBP12, whereas the p-
bromophenyl conjugate led once again to pull-down of the entire
ternary complex containing both FKBP12 and calcineurin
(Figure 2c). These results demonstrate the profound effect of a
smaller α-trifluorethyl versus a larger p-bromophenyl substituent
in pull-down experiments and is consistent with C32-aryl sub-
stituted FK506 derivatives, which were previously reported to
increase binding to calcineurin.29 This previous study also
reported increased IL-2 inhibition by C32-aryl versus C32-alkyl
substituted FK506 derivatives proposed to be due to favorable π-
π interactions with calcineurin A.
Fumagillol (13a, Figure 3b) is a natural product known to
inhibit angiogenesis through irreversible inhibition of human
type 2 methionine aminopeptidase (hMetAP2).30 Previous SAR
studies have shown that the structure of the C6-substituent can
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LETTERS
dramatically affect bioactivity.31 We thus set out to evaluate the
difference between α-trifluoroethyl and p-bromophenyl substi-
tuted fumagillol derivatives obtained from OꢀH insertion of the
C6-alcohol of fumagillol. Docking experiments with HTFB- and
HBPA-fumagillol using the X-ray structure of hMetAP2 sug-
gested that the p-bromophenyl derivative would suffer from
greater unfavorable interactions upon binding to hMetAP2
compared to the α-trifluoroethyl derivative (see Supporting
Information for details). The OꢀH insertions proceeded in
good yield to provide HTFB-fumagillol 13b/13b0 (63%) and
HBPA-fumagillol 18/180 (58%) without competing cyclopropa-
nation or epoxide degradation. The mixture of diastereomeric
fumagillol ethers 13b/13b0 and 18/180 were not readily sepa-
rated; therefore, they were used as a mixture of diastereomers for
in cellulo protein profiling (Figure 3a). Side by side assays with
fuma-alkyne (19) and TNP470 (20) were performed for com-
parison. As seen in Figure 3b, affinity experiments with fuma-
gillol-HTFB (13b/13b0) led to pull-down of hMetAP2 greater
than that of fumagillol-HBPA (18/180) (Figure 3b). Consistent
with these results, fumagillol-HTFB 13b/13b0 also showed
greater inhibitory activity in a HUVEC proliferation assay
compared to the fumagillol-HBPA derivatives 18/180 (∼5-fold
decrease, IC50 115.3 ( 35.4 and 568.2 ( 215.1 nM, respectively).
A similar trend was observed when 13b/13b0 and 18/180 were
assayed in the hMetAP2 enzymatic assay (Figure 3c). The
fumagillol-HTFB 13b/13b0 showed ∼4-fold increase in EC50
value compared to fumagillol-HBPA 18/180 (EC50 0.27 ( 0.03
and 0.95 ( 0.23 nM, respectively). Taken together, the compara-
tive results of both fumagillol and FK506-HBPA and HTFB
alcohol derivatives highlight the significance of the smaller steric
footprint and demonstrate the utility of the novel α-trifluoroethyl
diazo reagent 3a for natural product derivatization.
product (1.0 equiv) and rhodium catalyst (0.05 equiv) were placed into a
flame-dried, round-bottomed flask under a nitrogen atmosphere at
23 °C . Dry dichloromethane was added to make the final concentration
of natural product 0.01 mM, providing a slurry. A solution of HTFB (3a,
3.0 equiv) in dry dichloromethane (0.05 mM) was slowly added via
syringe pump over a 1 h period. Following complete addition, the
reaction mixture was stirred at 23 °C for an additional 1 h. The solvent
was evaporated under reduced pressure (rotary evaporator), and the
residue was purified by preparative thin layer chromatography to afford
the desired HTFB-ethers.
’ ASSOCIATED CONTENT
S
Supporting Information. Experimentals and full charac-
b
terization of all new compounds including H and 13C NMR
1
spectra. Experimental details for IL-2 reporter, hMetAP2, and
proliferation assays in addition to affinity chromatography and
modeling/docking experiments. This material is available free of
charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: romo@tamu.edu.
’ ACKNOWLEDGMENT
We thank the NIH (GM 086307 to D.R. and CA 78743 to J.O.L.)
for generous support of this work. We thank the Royal Thai
Government for a Graduate Fellowship to S.C. We also thank
Ms. Mingchien Li for performing preparative HPLC and Mr. Mikail
Abbasov for performing the molecular volume calculations.
In conclusion, we developed two new diazo reagents, a
α-trifluoroethyl diazo reagent 3a (HTFB) and a p-azidophenyl
diazo reagent 3d, for simultaneous arming and SAR studies of
bioactive natural products via OꢀH insertion. HTFB (3a)
possesses a reduced steric footprint compared to that of the
p-bromophenyl reagent (1) and enables the use of 19F NMR to
facilitate small-scale, crude derivatization reaction analysis.
Furthermore, this reagent showed comparable reactivity and
good chemosite selectivity compared to HBPA (1); secondary
(and likely primary) amines, if present, exhibit greater reactivity
over alcohols diminishing to an extent the degree of chemos-
electivity. The difference in steric footprint and binding affinity of
an FK506-HTFB derivative for FKBP12 was demonstrated by
measurement of IC50 values in the IL-2 reporter assay; in
addition, affinity chromatography experiments in side by side
comparisons of FK506-HTFB-biotin (16) and FK506-HBPA-
biotin (17) led to dramatic differences in proteins captured.
Furthermore, HTFB- and HBPA-fumagillol derivatives pre-
pared by these methods also demonstrated the advantage of the
α-trifluoroethyl substituent in terms of smaller steric footprint
leading to increased binding to hMetAP2. The p-azidophenyl
diazo reagent 3d should prove useful for photoaffinity experiments
with low affinity natural product receptors. Further applications of
these reagents to natural product derivatization and their subse-
quent use for receptor isolation are under active investigation.
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