Ipomoeassin F Binds Sec61α to Inhibit Protein Translocation
Transcript of Ipomoeassin F Binds Sec61α to Inhibit Protein Translocation
doi.org/10.26434/chemrxiv.7581764.v1
Ipomoeassin F Binds Sec61α to Inhibit Protein TranslocationWei Shi, Guanghui Zong, Zhijian Hu, Sarah O’Keefe, Dale Tranter, Ludivine Baron, Michael Iannotti, BelindaHall, Katherine Corfield, Anja Paatero, Mark Henderson, Peristera Roboti, Jianhong Zhou, Xianwei Sun,Mugunthan Govindarajan, Jason M. Rohde, Nicolas Blanchard, Rachel Simmonds, James Inglese, YuchunDu, Caroline Demangel, Stephen High, Ville Paavilainen
Submitted date: 13/01/2019 • Posted date: 14/01/2019Licence: CC BY-NC-ND 4.0Citation information: Shi, Wei; Zong, Guanghui; Hu, Zhijian; O’Keefe, Sarah; Tranter, Dale; Baron, Ludivine; etal. (2019): Ipomoeassin F Binds Sec61α to Inhibit Protein Translocation. ChemRxiv. Preprint.
The ipomoeassin family of natural resin glycosides is underexplored chemical space with potent antitumoractivity revealed in the NCI-60 cell lines screen; however, its mode of action has so far remained unexplored.In this manuscript, we report our chemical proteomics and subsequent biology studies that transform ourcollective knowledge of the ipomoeassin glycolipids from Organic Synthesis and Medicinal Chemistry tobiological mechanism and provide a step change in our understanding of its action at a cellular level. Hence,we created an ipomoeassin F-based biotin affinity probe and used it in live cells to isolate the ER membraneprotein Sec61α as its presumptive molecular target. A direct interaction between Sec61α and ipomoeassin Fwas confirmed by cell imaging, pulldown from purified ER membranes and competition studies using aphoto-crosslinking analogue of the cyclodepsipeptide cotransin, a known Sec61α inhibitor. Crucially, we thenshowed that ipomoeassin F binding has a profound effect on Sec61 function, using both in vitro and in vivoassays for protein translocation and protein secretion respectively. Although structurally quite distinct, thepotency of ipomoeassin F is comparable to that of mycolactone, a recently identified and intensely studiedinhibitor of Sec61. The ~1,000 fold increase in the ipomoeassin F resistance of two cell lines expressingmutant forms of Sec61α strongly supports our conclusion that the effect of the compound on Sec61α is theprimary basis for its potent cytotoxicity. However, we also provide evidence that ipomoeassin F ismechanistically distinct from known Sec61α inhibitors, suggesting that it is a novel structural class that mayoffer new opportunities to explore the Sec61 protein translocation complex as a therapeutic target for drugdiscovery.
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Ipomoeassin F Binds Sec61α to Inhibit Protein
Translocation
Guanghui Zong,a,b,† Zhijian Hu,a,† Sarah O’Keefe,c,# Dale Tranter, d,e,# Michael J. Iannotti,f,# Ludivine
Baron,g,h,# Belinda Hall,i,# Katherine Corfield,i, # Anja O. Paatero,d,e,# Mark J. Henderson,f,# Peristera
Roboti,c,# Jianhong Zhou,j Xianwei Sun,a,k Mugunthan Govindarajan,a,l Jason Rohde,f Nicolas Blanchard,m
Rachel Simmonds,i,* James Inglese,f,* Yuchun Du,j,* Caroline Demangel,g,h,* Stephen High,c,* Ville O.
Paavilainen,d,e,* and Wei Q. Shia,*
a Department of Chemistry and Biochemistry, and j Department of Biological Sciences, J. William Fulbright
College of Arts & Science, University of Arkansas, Fayetteville, Arkansas, 72701, USA
b Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742,
United States
c School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester,
Manchester, M13 9PT, United Kingdom.
d University of Helsinki, HiLIFE, and e Institute of Biotechnology, Helsinki, Finland
f National Center for Advancing Translational Sciences, National Institutes of Health, Rockville,
Maryland 20850, USA.
g Institut Pasteur, Immunobiology of Infection Unit, 75015 and h INSERM, U1221, 75005 Paris, France
i Department of Microbial Sciences, School of Biosciences and Medicine, University of Surrey, Guildford,
Surrey GU2 7XH, United Kingdom
k Department of Radiology, Baylor College of Medicine, Houston, TX, 77030, USA
l Emory Institute for Drug Development, Emory University, 954 Gatewood Road, Atlanta, Georgia, 30329,
USA
m Université de Haute-Alsace, Université de Strasbourg, CNRS, LIMA, UMR 7042, 68000 Mulhouse,
France
† Equal contribution as first-author
# Equal contribution as second-author
* Corresponding authors
ABSTRACT
Ipomoeassin F is a potent natural cytotoxin that inhibits growth of many tumor cell lines with single-digit
nanomolar potency by an undefined mechanism. However, its biological and pharmacological properties
have remained largely unexplored. Building upon our earlier achievements in total synthesis and medicinal
chemistry, we used chemical proteomics to identify Sec61α (protein transport protein Sec61 subunit
alpha isoform 1), the pore-forming subunit of the Sec61 protein translocon, as a direct binding partner of
ipomoeassin F in living cells. The interaction is specific and strong enough to survive lysis conditions,
enabling a biotin-analogue of ipomoeassin F to pull down Sec61α from live cells, yet it is also reversible
as judged by fluorescent streptavidin staining. Sec61α provides the central subunit of the ER protein
translocation complex, and the binding of ipomoeassin F results in a substantial, yet selective, inhibition of
protein translocation in vitro and a broad ranging inhibition of protein secretion in live cells. Lastly, the
unique resistance profile demonstrated by mutant cells provides compelling evidence that Sec61α is the
primary molecular target of ipomoeassin F and strongly suggests that the binding of this natural product to
Sec61α is distinctive. Therefore, ipomoeassin F represents the first plant-derived, carbohydrate-based
member of a novel structural class that offers new opportunities to explore Sec61α function and to further
investigate its potential as a therapeutic target for drug discovery.
INTRODUCTION
Historically, natural products have significantly contributed to the development of drugs for human
disorders,1 most notably as anticancer chemotherapeutics.2 Structurally and functionally unique natural
products also provide a spectrum of valuable chemical tools for examining biological systems in
translational biomedical research.3-4 To continue our battle against various unsolved problems of human
health, including those arising from drug resistance, it is crucial to systematically investigate underexplored
areas of existing chemical space such as those offered by bioactive natural products with unique structures
and/or mechanisms.5
Resin glycosides, also called glycoresins, are a large collection of amphiphilic glycolipids isolated from
the morning glory family of plants,6 and these compounds are considered active ingredients of many
morning glory-based traditional medicines that are used worldwide. Resin glycosides consist of a
differently-acylated oligosaccharide glycosylated with a mono- or dihydroxy C14 or C16 fatty acid, with
more than 300 family members discovered to date. Through an ester bond, the fatty acid chain is usually
folded back to form a macrolactone ring of various sizes spanning one or more carbohydrate units. Because
of their unique macrocyclic architecture with embedded carbohydrates, and their broad spectrum of
biological activities exhibited in phenotypic screens, resin glycosides have attracted considerable attention
from the synthetic chemistry community, but not much beyond.7-9
In 2005, a new family of glycoresins, ipomoeassins A–E, was isolated from the leaves of Ipomoea
squamosa found in the Suriname rainforest and shown to inhibit the proliferation of A2780 human ovarian
cancer cells.10 Among these compounds, ipomoeassin D (Figure 1) displayed the greatest potency with an
IC50 value of 35 nM. Two years later, a new member of the family, ipomoeassin F (Figure 1), was isolated
and showed a cytotoxicity comparable to ipomoeassin D.11 Because of their promising antiproliferative
activity and unique molecular skeleton with carbohydrates being part of the macrocycle, these newly
discovered natural glycoconjugates quickly inspired synthetic chemists, including ourselves, to tackle their
total syntheses.12-16 Subsequently, ipomoeassin F was confirmed to be the most cytotoxic resin glycoside
discovered to date with single-digit nanomolar IC50 values against several cancer-derived cell lines.13
Intriguingly, ipomoeassins A and F (see Figure 1) have distinct cytotoxicity profiles, as revealed in the NCI
60-cell lines screen, suggesting that the ipomoeassins may possess an unusual mode of action.16-17 On this
basis, ipomoeassin F is a very promising candidate for molecular probe and chemotherapeutic development;
however, the absence of knowledge about the cellular targets of ipomoeassin F has significantly impeded
such efforts. To overcome this challenge, after improving our understanding of the structure–activity
relationship (SAR) of ipomoeassin F through medicinal chemistry studies,18-20 we employed a chemical
proteomics approach to identify its binding partner(s) in human cells. Here we describe the evolution of
our chemoproteomic studies that enabled the discovery of Sec61α (protein transport protein Sec61 subunit
alpha isoform 1) as a primary molecular target of ipomoeassin F, and our subsequent mechanistic
investigations which validate this component as a key target. To this end, we compared the activity of
ipomoeassin F to mycolactone, a recently discovered broad-spectrum inhibitor of Sec61-dependent
translocation.21-22 This polyketide lactone is the pathogenic exotoxin of Buruli ulcer disease
(Mycobacterium ulcerans infection) that has long-established immunosuppressive and cytotoxic
activities.23-24 We define the inhibitory effects of ipomoeassin F on Sec61-mediated protein translocation
into and across the membrane of the endoplasmic reticulum (ER), corroborating our contemporaneous
observations of the potent inhibition of cellular protein secretion by ipomoeassin F.
Figure 1. Structures of ipomoeassins A, D, and F.
RESULTS AND DISCUSSION
Design, Synthesis and Proteomic Evaluation of Chemical Probes derived from Ipomoeassin F.
Michael acceptor systems are present in many electrophilic natural products, allowing the formation of
covalent adducts with bio-macromolecules, which are often responsible for their biological activities.25
Because of significant activity loss (up to 160-fold) after reducing one of the two double bonds in the α,β-
unsaturated esters to a single bond,18 we hypothesized that the cinnamate and/or the tiglate (Figure 1) may
enable irreversible binding between ipomoeassin F and its biological target(s). Therefore, we initially
designed and synthesized a potential activity-based probe26-28 3 (Scheme 1) by introducing a small
propargyloxy group to the para-position of the benzene ring.
Probe 3 was synthesized in two steps from key intermediate 1 obtained during the total synthesis of
ipomoeassin F.15 The alkyne-functionalized cinnamic acid 2 was synthesized as previously described.29
DCC-mediated Steglich esterification was applied to couple 1 with 2 in the presence of a catalytic amount
of DMAP to give the TBS-protected intermediate in 90% yield. Subsequently, exposure of the intermediate
to TBAF led to the removal of the TBS silyl protecting groups and delivered the final product 3 in good
yield. As expected, the small alkyne bioorthogonal tag did not affect cytotoxicity and 3 was equally potent
as compared to the original natural product (Table 1).
Scheme 1. Synthesis of Ipomoeassin F Probes 3, 5 and 7
a Reagents and conditions: a) 4-propargyloxycinnamic acid (2), DCC, DMAP, CH2Cl2, 0 oC→RT,
overnight, 90%; b) TBAF, THF, –10 °C, 6 h, 80%; c) CuSO4, sodium ascorbate, CH2Cl2/t-BuOH 3/2.
Table 1. Cytotoxicity (IC50, nM) of Ipomoeassin F, Cotransin (CT8) and Probes 3, 5 and 7–15a
Cell Line: MDA-MB-231b MCF7 MCF-10A HCT116 b
Ipomoeassin F 6.3 36.4 5.3 8
CT8 ‒ c ‒ ‒ 243
3 6.3 22.2 9.6 ‒
5 29.5 187.4 21.7 ‒
7 7.0 19.8 5.1 ‒
8 > 25,000 > 25,000 ‒ 2832
9 > 25,000 > 25,000 ‒ ‒
10 8.9 172.2 11.3 ‒
11 2.8 21.1 2.0 ‒
12 253.4 172.2 133.8 ‒
14 98018 256018 ‒ 1874
15 42918 47718 ‒ 480
a The data were obtained from at least two independent experiments, and the standard errors are within
20%; b See Figure S1 for IC50 curves; c “‒” indicates “not determined”.
With probe 3 in hand, we conducted activity-based protein profiling experiments for target
identification both in the soluble fraction of cell lysates made from MDA-MB-231 breast cancer cells and
live MDA-MB-231 cells. Unfortunately, after in situ click chemistry with a fluorescent rhodamine azide
S2730 (see Supporting Information), we detected no differences in labeled proteins between negative control
(lane 1), probe 3 (lane 2), and probe 3 plus ipomoeassin F (competition; lane 3) (Figure S2A), while an
unrelated probe used as a positive control for pulldown gave clear signals (Figure S2A, lanes 4 and 5). We
considered two possible explanations for this negative result: firstly, ipomoeassin F is not a covalent protein
modifier; secondly, target proteins of ipomoeassin F are in low abundance. Due to the dramatic loss in
activity with alkene-reduced compounds, we favored the second possibility.
In order to enrich for relatively low abundance cellular proteins, we next synthesized a biotin-labeled
analogue 5 of ipomoeassin F by reacting the alkyne probe 3 with biotin azide 431 under chemo-selective
copper-catalyzed alkyne-azide coupling (CuAAC) conditions (Scheme 1).32 The synthesis was successful
in producing biotin probe 5 that remained cytotoxic (Table 1), showing only ~5-fold decrease. After
incubating 5 with either detergent solubilized cell lysate or intact cells, we used streptavidin beads to
recover the biotin probe 5-bound proteins followed by electrophoresis and silver staining of the captured
material. However, no proteins were selectively enriched by probe 5 (Figures S3A and S3C, lanes 1 to 3),
nor was any enrichment apparent when these samples were probed using a fluorophore-labeled streptavidin
(Figure S3B, lanes 1 to 3).
We hypothesized that if the target protein(s) of ipomoeassin F is of relatively low abundance, then
endogenously biotinylated proteins could outcompete, and thereby mask, any positive signal. To minimize
interference from endogenously biotinylated proteins, we synthesized a new biotin probe 7 (Scheme 1)
containing a photo-cleavable nitrobenzene moiety33 by coupling the alkyne analogue 3 with the biotin azide
fragment 6 containing an o-nitrobenzyl photolabile linker, the latter synthesized by modifying previously
reported procedures31-33 (see Supporting Information). Notably, the capacity of 7 to inhibit cancer cell
proliferation was close to that of parental ipomoeassin F (Table 1), despite the large size of the appendage
at the para-position of the cinnamate benzene ring. We then used probe 7 to label target proteins in live
MDA-MB-231 cells, performed a streptavidin pulldown on the resulting cell lysate and selectively released
bound proteins by UV photocleavage. When the resulting products were analyzed by SDS-PAGE, probe 7
strongly enriched a ~40 kDa protein and, to a lesser extent, a second protein of ~100 kDa (Figure 2A, lane
2, red arrows), both of which were absent from the negative control (Figure 2A, lane 1). In support of their
bona fide interaction with 7, these two proteins were lost when a 100-fold excess of unmodified
ipomoeassin F was included during the incubation (Figure 2A, lane 3). Because no proteins were selectively
recovered with probe 7 when it was incubated with detergent-solubilized (1% NP40 and 0.25% sodium
deoxycholate) extracts of MDA-MB-231 cells (Figure S3C, lanes 1, 4 and 5), we believe that the interaction
between ipomoeassin F and its target protein(s) may require a particular subcellular environment that is
detergent sensitive.
We next investigated whether photocleavage is essential for the selective recovery of the two putative
target protein bands using probe 7. Strikingly, the ~40 kDa protein was also recovered without
photocleavage. It was substantially enriched by 7 when total streptavidin-bound products were analyzed,
and was lost in the presence of excess ipomoeassin F (Figure 2B, lanes 1–3). In contrast, an enrichment of
the ~100 kDa component under these conditions was not apparent (Figures 2A and 2B). Since
photocleavage was not essential for probe 7 to selectively recover the ~40 kDa protein, it is very likely that
the increased linker length between the biotin moiety and the ipomoeassin F region, but not its capacity for
selective cleavage, significantly improved the performance of probe 7 over probe 5. Our data also suggest
that, once formed, the complex between probe 7 and the ~40 kDa protein is tight and can survive detergent-
containing cell lysis. This is further supported by pulldown of the ~40 kDa target protein with low
concentrations of probe 7 (10 nM) that was out-competed by a 5-fold excess of ipomoeassin F (50 nM)
(Figure S4A, lanes 1–3). Fluorescent streptavidin staining confirmed that a stable covalent adduct between
the ~40 kDa protein and probe 7 was not formed under our experimental conditions (Figure S3B, lanes 4
and 5). We next submitted both the ~40 kDa protein(s) (Figure 2B, lane 2) and the corresponding regions
of the gel for the negative control (Figure 2B, lane 1) and the ipomoeassin F competition sample (Figure
2B, lane 3) for mass spectrometry analysis. When spectral counts were compared, Sec61α (protein transport
protein Sec61 subunit alpha isoform 1) stood out as the only protein that was both substantially enriched in
the pulldown with probe 7 and showed a corresponding reduction when excess ipomoeassin F was present
(Table S1). The selective recovery of Sec61α by probe 7 was further confirmed by Western blotting (Figure
2C, cf. signals between input and AP).
Figure 2. Affinity pulldown using probe 7 with (A) or without (B) photo cleavage. Red arrows indicate
specifically pulled down protein species; (C) Target validation by western blot with a Sec61α antibody.
Ipom-F, ipomoeassin F; AP, affinity pulldown.
To further support Sec61α as a target protein responsible for the biological activity of ipomoeassin F,
we synthesized two inactive reference compounds 8 and 9 (Figure 3 and Table 1) by removing both double
bonds in cinnamate and tiglate (see Supporting Information). As predicted, pulldown experiments with
inactive biotin probe 9 revealed a loss of the ~40 kDa protein, even with 500 nM of 9 (Figure S4B, lanes 1
and 4). Additionally, inactive analogue 8 was ineffective at competing with probe 7 binding, even when 8
was present in 50-fold excess (Figure S4B, cf. lanes 1 to 3). Taken together, these findings further support
the specificity of Sec61α as a primary cellular target of ipomoeassin F.
Figure 3. Structures of two inactive derivatives of ipomoeassin F.
Cell Imaging Studies. The Sec61 protein translocon plays an essential role in translocating newly
synthesized membrane and secretory polypeptides into and across the membrane of the ER.34-35 Sec61α
forms the membrane conduit that nascent secretory polypeptides pass through. To examine the subcellular
localization of ipomoeassin F, we performed live cell imaging studies using three fluorescent analogues
10–12 (Figure 4) prepared from alkyne probe 3 using CuAAC (see Supporting Information). Both
coumarin-coupled probe 10 and NBD-coupled probe 11 retained substantial cytotoxicity with 11 being even
more potent than ipomoeassin F (see Table 1). Unfortunately, 11 underwent photobleaching very easily
and could not be used for imaging studies. In contrast, 10 gave a rather weak fluorescent signal, presumably
due to an internal photo-induced electron transfer effect combined with its shorter excitation maximum
(365 nm). To overcome these technical limitations, rhodamine analogue 12 was synthesized from the
rhodamine azide precursor S27. Although 12 is between 5- and 40-fold less potent than ipomoeassin F
depending on the cell type analyzed, it retains substantial cytotoxicity when compared to inactive analogues
8 and 9 (see Table 1), and we therefore used it for our imaging studies.
Figure 4. Structures of fluorescent derivatives of ipomoeassin F.
Subsequently, we confirmed that 12 could penetrate the cell membrane in a concentration- and time-
dependent manner and gave a strong fluorescent signal. Hence, 12 stained cells within 30 mins when
present at 2 μM, but took >3 h to give clear images when used at 20 nM. When cells labelled with 12 were
co-stained with markers for either the ER or the nucleus, it was apparent that 12 strongly labeled the ER
(Figure 5), but not the nucleus (Figure S5A). To confirm the specificity of ER labelling by 12, competition
experiments were performed using ipomoeassin F or inactive compound 8. The fluorescence signal of 12
was almost completely abolished when the cells were preincubated for 30 minutes with a 100-fold excess
of free ipomoeassin F (Figure S5B), yet preincubation with compound 8 had little effect (Figure S5C). The
apparent ER localization of the cellular interacting partner(s) of ipomoeassin F strongly supports the notion
that its target is Sec61α.
12, 0.2 μM ER Tracker Merge
Figure 5. Cell imaging studies with fluorescent probe 12 in MDA-MB-231 cells. Rhodamine-conjugated
ipomoeassin F analogue 12 (0.2 μM) was added to cells and after 1 h, cells were imaged to analyze
localization of 12 relative to ER staining.
Pulldown from ER Microsomes. As evidenced by the negative results from our pulldown experiments
using cell lysates prepared with detergent, we propose that the membrane environment of the ER may be
required to maintain Sec61α in a conformation that can bind to ipomoeassin F effectively. In this regard,
it would be unlikely that purified Sec61α alone could recapitulate a direct interaction with the natural
product. However, since the biological function of Sec61α is maintained in isolated ER microsomes,22 and
once pre-bound to probe 7 in intact cells the resulting complex appears stable to detergent treatment (Figures
2A and 2B), we attempted to pulldown Sec61α from purified ER-derived microsomes. After incubation
with probe 7 and detergent solubilization (1% n-dodecyl-ß-D-maltoside) to release biotin-bound proteins
from the phospholipid bilayer, only a single protein was visible after pulldown (Figure S6A). The identity
of this product was confirmed as Sec61α by western blotting (Figure S6B). This finding strongly supports
our previous conclusion made with live cells, namely that Sec61α directly interacts with ipomoeassin F.
Competition of Ipomoeassin F with Cotransin for Binding to Sec61α. To test whether ipomoeassin
F competes for binding to Sec61α with known Sec61 ligands, such as cotransin and mycolactone,36-38 we
exploited CT7,39 a cotransin photoaffinity probe that can covalently label Sec61α. As reported earlier,36
mycolactone efficiently competes with CT7 and prevents its photo-crosslinking to Sec61α (Figure 6). A
similarly potent competition was observed with ipomoeassin F, suggesting it binds directly to Sec61α,
potentially at the same or a partially overlapping interaction site as that used by cotransin. Ipomoeassin F
competes with CT7 photo-crosslinking with similar potency as mycolactone, consistent with its ability to
inhibit protein ER translocation with high potency.
Figure 6. Sheep rough microsomes were preincubated with ipomoeassin F (Ipom-F) or mycolactone (Myco)
at 10 μM. Subsequently 100 nM photocotransin CT7 was added and samples photolyzed. The covalent
CT7/Sec61α adduct was detected using click chemistry to install a rhodamine-azide reporter and in-gel
fluorescent scanning.
In Vitro Inhibition of Protein Translocation by Ipomoeassin F. In order to understand the
consequences of ipomoeassin F binding for Sec61 function, we used a well-established in vitro system to
study the Sec61-dependent translocation of radiolabeled precursor proteins into ER microsomes derived
from canine pancreas.22-23 In the presence of 1 µM ipomoeassin F, the translocation of two model secretory
proteins, bovine preprolactin and yeast prepro-alpha-factor, was almost completely prevented. The
blockade of protein translocation by ipomoeassin F was comparable to that achieved using 1 µM
mycolactone, and resulted in a loss of signal sequence cleavage and protein N-glycosylation (see Figures
7A and 7B; cf. lanes 1, 3 and 4), both events specific to the ER lumen. The unusual ability of tail-anchored
membrane proteins to insert into the ER membrane independently of the Sec61 complex provides a useful
control for validating the specificity of ER translocation inhibitors.40 The in vitro integration of two model
tail-anchored proteins bearing C-terminal N-glycosylation reporters was unaffected by ipomoeassin F
treatment (Figures 7C and 7D, cf. lanes 1 and 3), mirroring the specificity of the Sec61-selective inhibitor
mycolactone21-22 (see also Figures 7C and 7D, lane 4). Furthermore, despite the presence of carbohydrate
moieties in ipomoeassin F (see Figure 1), it has no effect on the N-glycosylation of our model tail-anchored
proteins, and we conclude that protein N-glycosylation of model substrates provides a robust reporter for
the ipomoeassin F-mediated inhibition of Sec61-dependent protein translocation.
Figure 7. Ipomoeassin F inhibits Sec61-mediated protein translocation into and across the ER in a substrate
selective manner. Phosphorimages of membrane associated in vitro products resolved by SDS-PAGE
together with outline structures of the protein substrates generated in the presence or absence of
ipomoeassin F (Ipom-F) or mycolactone (Myco): secretory proteins (A) bovine preprolactin, PPL and (B)
yeast prepro-alpha-factor ppF; tail-anchored proteins (C) C-terminally tagged Sec61 subunit,
Sec61OPG2 and (D) C-terminally tagged cytochrome b5, Cytb5OPG2; type I single pass transmembrane
protein (TMP) (E) vascular cell adhesion molecule 1, VCAM1; type II single pass TMP (F) isoform 2
(residues 17 to 232) of the short form of HLA class II histocompatibility antigen gamma chain, Ii; type III
single pass TMP (G) glycophorin C, GypC; short secretory protein (H) C-terminally tagged Hyalophora
cecropia preprocecropin A, ppcecAOPG2. Samples were treated with endoglycosidase H (Endo H) where
indicated to distinguish N-glycosylated (XGly) from non-glycosylated (0Gly) products. nc, signal sequence
not cleaved; sc, signal sequence cleaved; TM1, transmembrane domain 1. Proteins are of human origin
unless otherwise stated.
Known small molecule inhibitors of Sec61-mediated protein translocation show varying degrees of
substrate selectivity,23,41 and we therefore studied the effects of ipomoeassin F on the Sec61-mediated
translocation of four other classes of precursor proteins (see Figure S7) into and across the ER membrane
using the in vitro system outlined above.22,42 We find that ipomoeassin F potently inhibits the membrane
insertion of representative type I and type II integral membrane proteins that have a single transmembrane
spanning domain (Figures 7E and 7F). In contrast, the compound has no effect on the membrane insertion
of the single-spanning type III integral membrane protein glycophorin C and only a marginal impact on the
translocation of the short secretory protein prepro-cecropin A (Figures 7G and 7H). Interestingly, it has
recently been shown that type III membrane proteins have the unusual ability to exploit an alternative,
Sec61-independent, mechanism for ER insertion, which relies on the ER membrane protein complex acting
as an integrase.43 On this basis, we conclude that the integration of glycophorin C is completely refractive
to ipomoeassin F (this study) and mycolactone (See Ref. 42) because it can insert into the ER via a Sec61-
independent mechanism. Based on the model membrane and secretory proteins that we have studied here,
the substrate selectivity for the ipomoeassin F-mediated inhibition of ER protein translocation appears
remarkably similar to that of mycolactone.42
To further investigate ipomoeassin F specificity, we used the ER insertion of the type II membrane
protein Ii (HLA class II histocompatibility antigen gamma chain; cf. Figure 7F) to compare the
effectiveness of a subset of structural variants. The differences we observe in this in vitro assay (see Figure
S8) are strikingly similar to the variations in cytotoxicity that the same compounds display in cell-based
assays (Table 1). Hence, ipomoeassin F and the open-chain analogue 1318 (Figure 8) strongly inhibit
membrane insertion, whilst 1418 (Figure 8) is a far less effective inhibitor and analogue 8 (Figure 3) has no
measurable inhibitory activity in vitro (see Figures S8A and S8B). Ii membrane insertion was also used to
estimate IC50 values for ipomoeassin F (~50 nM) and 13 (~120 nM) (see Figures S8C and S8D). The fact
that we observe IC50 values in the nM range using in vitro translocation inhibition and cell-based
cytotoxicity assays, combined with the similarity in the structure-activity relationship observed in both
systems, leads us to conclude that the inhibitory effect of ipomoeassin F on Sec61-mediated protein
translocation is most likely the principal molecular basis for its cytotoxic activity. Furthermore, since the
ER acts as the entry point into the eukaryotic secretory pathway, our in vitro findings predict that if cells
are exposed to ipomoeassin F their capacity to produce and deploy membrane and/or secretory proteins is
likely compromised.
Figure 8. Structures of ipomoeassin F analogues 13–15.
Inhibition of Protein Secretion by Ipomoeassin F in Live Cells. Through a partnership with the
National Center for Advancing Translational Sciences (NCATS), ipomoeassin F was included within a
collection of natural products for screening against a panel of assays to interrogate a variety of biological
pathways and assess cell type-dependent toxicity.44 Ipomoeassin F was identified as an inhibitor of protein
secretion in two distinct quantitative high-throughput screening (qHTS) assays using different cell lines and
secretory reporters.45 In U2-OS human osteosarcoma cells, a protein reporter consisting of secreted
NanoLuc (secNLuc) fused to the Z mutant of alpha-1 antitrypsin (secNLuc-ATZ) was designed to monitor
the extracellular accumulation of this protein.46 Ipomoeassin F demonstrated acute, highly potent secretion
inhibition (EC50 = ~5 nM) in U2-OS cells prior to the onset of cytotoxicity as assessed by decreasing cellular
ATP after 24 h (Figure 9A). A second reporter of protein secretion was also examined, using SH-SY5Y
human neuroblastoma cells expressing a secreted Gaussia luciferase (GLuc). Ipomoeassin F inhibited
secretion of the reporter protein with a lower potency (EC50= ~120 nM) than in U2-OS cells (Figure 9B)
reflecting a potential cell type-dependent functional effect. The secretion phenotype in cells treated with
ipomoeassin F is strongly consistent with Sec61α as its inhibitory protein target, since most secretory
proteins must pass through the Sec61 translocon into the ER prior to traversing the secretory pathway for
extracellular secretion. The difference in potency between different cell lines is, again, in line with previous
observations with mycolactone.47-48
Figure 9. Differential effect of acute ipomoeassin F treatment on protein secretion and cell viability.
Bioluminescence outputs from secretion sensor proteins (•)and cellular viability based on total well ATP
content (). (A) secNLuc-ATZ secretion and cell viability measured in U2-OS cells after 24 h. (B) GLuc
secretion and cell viability measured in SH-SY5Y cells after 48 h. Error bars represent the SEM for n=2
or 3 replicates.
Selective Prevention of Biogenesis of Secreted Proteins by Ipomoeassin F. To assess ipomoeassin
F effects on cellular protein production, we employed a metabolic labeling strategy. Cells were pretreated
with ipomoeassin F or its analogue 8 (Figure 3), 14 or 15 (Figure 8), for 30 minutes before addition of 35S-
labelled methionine/cysteine for further 90 minutes. The lack of inhibition of nascent protein biogenesis
observed in total cellular lysates and cytosolic fractions (Figures 10A and 10B) demonstrates that
ipomoeassin F does not influence global cellular processes such as transcription or translation, and is in line
with previous observations using mycolactone.21,49 In contrast, strong inhibition was observed for the
production of secreted proteins for cells treated with 100 nM ipomoeassin F (Figure 10C). Importantly,
ipomoeassin F inhibits the production of most newly synthesized secretory proteins, suggesting its
mechanism of action leads to an inhibition of the biogenesis of a broad range of Sec61 substrate proteins
that is comparable to known Sec61 inhibitors such as mycolactone21,36 and apratoxin A38,50. Ipomoeassin F
analogues 8, 14, and 15 all inhibit the production of secreted proteins less potently when tested at 1 µM
concentration. Furthermore, the rank order of potencies with which ipomoeassin F and its analogues inhibit
protein secretion (Ipom-F > 15 > 14 > 8) correlates with their cytotoxicity against HCT-116 cells (cf. Table
1 and Figure S1C), suggesting a link between cytotoxicity and inhibition of cellular protein secretion, as
previously suggested for other Sec61 inhibitors.36,38,51-52
Figure 10. HCT-116 cells were washed twice with PBS and incubated in the presence of indicated
concentrations of compounds under media lacking methionine and cysteine for 30 minutes. 35S-labelled
methionine and cysteine were then introduced to the media, and cells incubated for a further 90 minutes.
Cells were harvested by scraping into ice cold PBS, homogenized and analyzed by SDS-PAGE and
autoradiography. (A). The collected cells were subsequently homogenized and analyzed by SDS-PAGE
and autoradiography; (B) As for (A) but the samples are the cytosolic contents of harvested cells following
partial permeabilization with 0.15% Digitonin; (C) As for (A) but the samples are from TCA-precipitated
culture medium from the same experiment. Ipom-F denotes ipomoeassin F. AprA denotes control samples
treated with Apratoxin A to block protein translocation into the ER. CHX denotes samples treated with
cycloheximide and chloramphenicol to inhibit total cellular protein synthesis. Cotransin CT8 is a substrate-
selective inhibitor of protein translocation into the ER.
HepG2 cells secrete a range of endogenous proteins, but these products are almost completely absent
following treatment with the ER translocation inhibitor eeyarestatin I or the vesicular transport inhibitor
brefeldin A.40 To extend our studies with HCT116 cells (Figure 10), we therefore treated HepG2 cells with
ipomoeassin F or mycolactone overnight and then followed the fate of newly synthesized proteins by pulse
chase analysis. When the cell media was analyzed for secretory proteins, a substantial reduction was
observed for cells treated with ipomoeassin F or mycolactone when compared to control cells treated with
DMSO or compound 8 (see Figure S9, media, lanes 1 to 4). Whilst the effect of these Sec61 inhibitors was
akin that of brefeldin A, treatment with the proteasome inhibitor bortezomib had no effect (see Figure S9,
media, lanes 3 to 6). When the levels of total radiolabeled proteins recovered from the same cells were
analyzed, they were directly comparable across all treatments confirming that ipomoeassin F did not inhibit
global protein synthesis during the treatment used (Figure S9, whole cell extract, cf. lanes 1 to 4). In fact,
overnight exposure to ipomoeassin F increases at least one prominent cellular component (Figure S9, whole
cell extract, lane 3, 80 kDa), perhaps reflecting the early stages of a cellular stress response.51,53
Immunomodulatory Effects of Ipomoeassin F. While ultimately cytotoxic in most cell types,
mycolactone has immediate immunosuppressive effects.23 Recent studies have indicated that these effects
are a direct consequence of mycolactone’s inhibitory activity on Sec6121,36,53-54, and we find the effects of
ipomoeassin F and mycolactone are equivalent in cell-free assays of Sec61-dependent protein translocation
(Figure 7). By blocking Sec61, mycolactone prevents the production of both secreted proteins and
membrane receptors that are key to the generation of immune responses21, as is clearly illustrated by the
biogenesis of CD62L, a cell surface receptor mediating naive lymphocyte homing to secondary lymphoid
organs, which is highly susceptible to mycolactone.36 In the human Jurkat T cell line, we find that
ipomoeassin F and mycolactone downregulated CD62L expression with comparable potency (Figure 11A).
Likewise, they were also equivalent in capacity to prevent Interleukin (IL)-2 production by activated T cells
(Figure 11B). Together, these data suggest that, like mycolactone,55 ipomoeassin F may display systemic
immunosuppressive effects in vivo.
A. B.
Figure 11. Compared effects of Ipomoeassin F and synthetic mycolactone (A/B form) in assays of CD62L
expression (A) and activation-induced production of IL-2 (B) by Jurkat T cells. Data are mean +/- SEM of
biological duplicates and are representative of two independent experiments.
Resistance of Sec61α Mutants to Ipomoeassin F Cytotoxicity. Having established that binding of
ipomoeassin F to Sec61α potently impairs protein translocation at the ER both in vitro and in vivo, we
sought further evidence that this interaction also underlies the cytotoxic effects of ipomoeassin F (cf. Table
1).
Chemogenetic screening has identified mutations in the SEC61A1 gene that confer resistance to known
inhibitors of Sec61α-dependent protein translocation,56 and established that these mutations can confer
resistance when engineered into a naïve cell background.37-38 We therefore compared the effects of
ipomoeassin F and mycolactone on the growth of HEK293 and HCT116 cells expressing either wild type
Sec61α, or a previously characterized point mutant of Sec61α that is resistant to mycolactone.36,41 As
previously observed using mycolactone in HEK293 cells engineered to express mutant alleles of
Sec61α,36,41 point mutations R66I and S82P confer significant and dominant desensitization to ipomoeassin
F in a viability assay of HEK293 cells, yet T86M confers resistance to mycolactone but not to ipomoeassin
F (see Figures 12A and 12B). Furthermore, whilst isolated drug-resistant HCT116 cells carrying mutations
to Sec61α of R66K and D60G were strongly resistant to mycolactone (Figure 12D) as previously reported51,
any decrease in sensitivity to ipomoeassin F was comparatively modest (Figure 12C). Taken together, these
results strongly suggest that ipomoeassin F and mycolactone bind to a similar region as other known
inhibitors of Sec61, but indicate that the interaction of ipomoeassin F may be partially dependent upon the
basic side chain of residue R66 but does not require conservation of D60 or T86.
A. B.
C. D.
Figure 12. Human HEK293 cells or cells engineered to express Sec61α alleles bearing indicated point
mutations were treated with increasing concentrations of ipomoeassin F (A) or mycolactone (B), and cell
viability assayed at 72h with the Alamar Blue assay. (Mean ± SD; n = 4 technical quadruplicates).
Parental or HCT116 cells containing mutations in Sec61A1 were seeded at 3 x 104 /well in 96-well plates
in triplicate. ipomoeassin F (C) or mycolactone (D) were added at various concentrations with the carrier
DMSO used as a control. Cell viability was measured at 96h with the Alamar Blue assay (Mean ± SEM of
n = 3 independent experiments performed in triplicate).
CONCLUSIONS
Here, we report comprehensive chemical biology studies of ipomoeassin F to understand the molecular
basis for its potent cytotoxicity. By designing and evolving our chemical probes on an empirical basis,
Sec61α was ultimately identified and validated as a direct and physiologically relevant target of
ipomoeassin F. The absence of fluorescent signals for the potential activity-based probe 3 or for the active
biotinylated analogue 5 or 7 upon Western blotting with fluorescent streptavidin indicates ipomoeassin F
is unlikely to form a stable covalent complex with Sec61α, despite the presence of two Michael acceptor
systems in its structure (cinnamate and tiglate, Figure 1). We therefore conclude that the pulldown of
Sec61α with probe 7 represents a successful biotin affinity enrichment of a non-covalent ligand-membrane
protein complex formed in live cells, most likely reflecting a slow disassociation rate of ipomoeassin F
from Sec61α. The binding of ipomoeassin F to cellular Sec61α is supported by the ER staining seen with a
fluorescent version of the compound, the highly selective pulldown of Sec61α from ER-derived
microsomes and the ability of ipomoeassin F to compete with cotransin for Sec61α binding.
The impact of ipomoeassin F on Sec61 function was first investigated in vitro, revealing a strong
inhibition of protein translocation into the ER that affected secretory, type I and type II membrane proteins.
The in vitro effect of ipomoeassin F translated into potent secretion inhibition in two cellular models
utilizing independent luciferase secretory reporters. Likewise, ipomoeassin F treatment downregulated the
release of a broad range of endogenous secretory proteins from cultured cells, acting at a stage after their
translation. At this point in our studies, it was striking that, although structurally quite distinct, the molecular
mechanisms of ipomoeassin F, and the functional consequences of its application, closely resemble those
seen with the immunosuppressive macrolide mycolactone that is released by the human pathogen
Mycobacterium ulcerans.23 We therefore studied the effects of ipomoeassin F on immune cells and found
that its inhibition of membrane receptor expression and cytokine production is comparable to that achieved
with mycolactone, Previous studies have illustrated the translational potential of mycolactone against
inflammatory disorders55, but further exploitation of mycolactone-inspired molecules has been limited by
their intrinsic structural complexity that required extensive synthetic efforts.57-62 Given that ipomoeassin
variants are easier to generate by synthetic chemistry and equally inhibitory in cellular assays of
inflammation, future studies of their pharmacokinetics and therapeutic efficacy against inflammatory
diseases and inflammatory pain are an obvious area to pursue in future.
Lastly, we could establish an excellent correlation between the functional Sec61 inhibition and the
cytotoxic potency of ipomoeassin F using cell lines expressing inhibitor-resistant mutants of Sec61α mutant.
The loss of cytotoxicity we observed in HEK293 cells expressing R66I and S82P mutants is consistent with
the notion that ipomoeassin F binds to a location that overlaps with the interaction site for other small
molecule inhibitors of Sec61α, most likely at or near its so-called lateral gate.41 However, differences
between ipomoeassin F and mycolactone in response to other mutant cell lines strongly suggest that the
mode of ipomoeassin F binding to Sec61α is distinctive from other known Sec61α inhibitors41. The
sustained sensitivity of the mycolactone resistant T86M (in HEK293) and D60G (in HCT116) mutant cell
lines to ipomoeassin F that we report is particularly intriguing, highlighting that ER protein translocation
can be complex63 and our current understanding is far from complete.43
In short, we provide compelling evidence that the core α subunit of the Sec61 translocation complex is
the major target for ipomoeassin F in a cellular context, thereby opening up the way for the exploitation of
ipomoeassin F for target-based drug discovery. More broadly, given its comparatively advantageous
synthesis, we anticipate that ipomoeassin F and its analogues/probes will provide accessible new molecular
tools that will help our efforts to fully define the molecular basis for Sec61-mediated protein translocation
at the ER membrane.
ASSOCIATED CONTENT
Supporting Information. Experimental procedures and analytical data for all the new compounds and
biological assays. This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author: * E-mail: [email protected]; [email protected];
[email protected]; [email protected]; [email protected]; [email protected];
[email protected], [email protected].
Author Contributions: G.Z. and J.Z. contributed equally as first-author. S.O’K, D.T., M.J.I., L.B., B.H.,
K.C., A.O.P., M.J.H. and P.R. contributed equally as second-author.
Notes: The authors report no conflicts of interest.
ACKNOWLEDGMENTS
We thank the Arkansas Nano & Bio Materials Characterization Facility at the Institute for Nano Sciences
& Engineering for our imaging studies, and Prof Yoshito Kishi (Harvard University) for the kind gift of
synthetic mycolactone A/B used by S.H. and R.S. W.S. is supported by Grant Number R15GM116032 from
the National Institute of General Medical Sciences of the National Institutes of Health (NIH) and startup
funds from the University of Arkansas. This work was also supported in part by Grant Number P30
GM103450 from the National Institute of General Medical Sciences of the National Institutes of Health
(NIH) and by seed money from the Arkansas Biosciences Institute (ABI). S.O’K. is the recipient of a
Biotechnology and Biological Sciences Research Council (BBSRC) Doctoral Training Programme Award
BB/J014478/1 and S.H. holds a Welcome Trust Investigator Award in Science 204957/Z/16/Z. The alpha-
1 antitrypsin work was supported by the Alpha-1 Foundation (J.I. and M.J.I.). J.I. and M.J.H were supported
by the intramural program of NCATS, National Institutes of Health, projects 1ZIATR000048-03 (JI) and
ZIATR000063-04 (MJH). RS holds Welcome Trust Investigator Award in Science 202843/Z/16/Z. CD
received funding from the Institut Pasteur, the Institut National de la Santé et de la Recherche Médicale and
the Fondation Raoul Follereau. NB’s synthesis and chemical biology studies of mycolactone were
supported by CNRS, Université de Strasbourg, Fondations Potier et Follereau and the Investissement
d’Avenir (Idex Unistra). V.O.P is supported by the Academy of Finland (grants 289737 and 314672), and
the Sigrid Juselius Foundation.
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Table of Content Graphic
download fileview on ChemRxivManuscript.pdf (1.19 MiB)
S1
Supporting Information for:
Ipomoeassin F Binds Sec61α to Inhibit Protein Translocation
Guanghui Zong,a,b,† Zhijian Hu,a,† Sarah O’Keefe,c,# Dale Tranter, d,e,# Michael J. Iannotti,f,# Ludivine
Baron,g,h,# Belinda Hall,i,# Katherine Corfield,i, # Anja O. Paatero,d,e,# Mark J. Henderson,f,# Peristera
Roboti,c,# Jianhong Zhou,j Xianwei Sun,a,k Mugunthan Govindarajan,a,l Jason Rohde,f Nicolas
Blanchard,m Rachel Simmonds,i,* James Inglese,f,* Yuchun Du,j,* Caroline Demangel,g,h,* Stephen
High,c,* Ville O. Paavilainen,d,e,* and Wei Q. Shia,*
a Department of Chemistry and Biochemistry, and j Department of Biological Sciences, J. William Fulbright College
of Arts & Science, University of Arkansas, Fayetteville, Arkansas, 72701, USA
b Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, United
States
c School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester,
M13 9PT, United Kingdom.
d University of Helsinki, HiLIFE, and e Institute of Biotechnology, Helsinki, Finland
f National Center for Advancing Translational Sciences, National Institutes of Health, Rockville,
Maryland 20850, USA.
g Institut Pasteur, Immunobiology of Infection Unit, 75015 and h INSERM, U1221, 75005 Paris, France
i Department of Microbial Sciences, School of Biosciences and Medicine, University of Surrey, Guildford, Surrey
GU2 7XH, United Kingdom
k Department of Radiology, Baylor College of Medicine, Houston, TX, 77030, USA
l Emory Institute for Drug Development, Emory University, 954 Gatewood Road, Atlanta, Georgia, 30329, USA
m Université de Haute-Alsace, Université de Strasbourg, CNRS, LIMA, UMR 7042, 68000 Mulhouse,
France
† Equal contribution as first-author
# Equal contribution as second-author
* Corresponding authors
S2
Table of Contents
CHEMISTRY
General Methods S3
Synthetic Procedures and Analytical Data S3 – S16
BIOLOGY
General Methods S17
Experimental Procedures of Biological Assays S17 – S27
Figures S1–S9 S28 – S37
Table S1. Spectral Counts for Proteins Identified in ~40 kDa Bands S38 – S44
REFERENCES S45
S3
CHEMISTRY
General Methods
Reactions were carried out in oven-dried glassware. All reagents were purchased from commercial
sources and were used without further purification unless noted. Unless stated otherwise, all reactions
were carried out under a nitrogen atmosphere and monitored by thin layer chromatography (TLC) using
Silica Gel GF254 plates (Agela) with detection by charring with 5% (v/v) H2SO4 in EtOH or by visualizing
in UV light (254 nm or 365 nm). Column chromatography was performed on silica gel (230–450 mesh,
Sorbent). The ratio between silica gel and crude product ranged from 100 to 50:1 (w/w). NMR data were
collected on a Bruker 400 MHz NMR spectrometer and a Bruker 400 MHz system. 1H NMR spectra were
obtained in deuteriochloroform (CDCl3) with chloroform (CHCl3, δ = 7.27 for 1H) as an internal reference.
13C NMR spectra were proton decoupled and were in CDCl3 with CHCl3 (δ = 77.0 for 13C) as an internal
reference. Chemical shifts are reported in ppm (δ). Data are presented in the form: chemical shift
(multiplicity, coupling constants, and integration). 1H data are reported as though they were first order.
The errors between the coupling constants for two coupled protons were less than 0.5 Hz, and the average
number was reported. Proton assignments, when made, were done so with the aid of COSY NMR spectra.
For some compounds, HSQC and HMBC NMR were also applied to assign the proton signals. Optical
rotations were measured on an Autopol III Automatic Polarimeter at 25 ± 1 oC for solutions in a 1.0 dm
cell. High resolution mass spectrum (HRMS) and were acquired in the ESI mode.
Synthetic Procedures and Analytical Data
Compound 3.
4-Propargyloxy cinnamic acid (17.4 mg, 0.086 mmol) was added in one portion to a solution of 1 (40
mg, 0.043 mmol), DCC (17.8 mg, 0.086 mmol) and 4-dimethylaminopyridine (2.6 mg, 0.022 mmol) in
CH2Cl2 (2 mL). The reaction was stirred at room temperature for 12 h. At this point, TLC (silica, 1:3
EtOAc–hexanes) showed the reaction was complete. The reaction mixture was diluted with ether (2 mL)
and hexanes (1 mL), stirred for 20 min then filtered thru a pad of celite using ether (3 mL) as the eluent
and the filtrate concentrated in vacuo. The residue was purified by flash column chromatography (silica,
EtOAc–hexanes, 1:3→ 1:1) gave the protected intermediate (43 mg, 90%) as a colorless syrup. To a
solution of the intermediate (41 mg, 0.037 mmol) in THF (2 mL) was added TBAF (1M solution in THF,
S4
0.22 mL, 0.22 mmol, 6 equiv) at –10 oC. The reaction mixture was stirred at the same temperature for 6
h at which point TLC (silica, 2:1 EtOAc–hexanes) showed it was complete. The reaction mixture was
diluted with Et2O (20 mL), washed with 1M HCl (10 mL), saturated aqueous NaHCO3 (10 mL), brine (10
mL). The aqueous layer was extracted with Et2O (20 mL). The combined organic layer was dried over
Na2SO4 and concentrated under reduced pressure. The residue was purified by column chromatography
(silica, EtOAc–hexanes, 1:1→ 2:1) gave analogue 3 (26.0 mg, 80%) as a white solid. [α]D25 –55.3° (c 1
CHCl3). 1H NMR (400 MHz, CDCl3) δ 7.58 (d, J = 16.0 Hz, 1H, Ph-CH=CH-), 7.50 – 7.43 (m, 2H, 2 ×
ArH), 7.01 – 6.95 (m, 2H, 2 × ArH), 6.92 – 6.85 (m, 1H, Me-CH-C(Me)-C=O), 6.22 (d, J = 16.0 Hz, 1H,
Ph-CH=CH-), 5.30 (t, J = 10.0 Hz, 1H, H-4-Glup), 5.18 – 5.10 (m, 2H, H-3-Glup, H-4-Fucp), 4.73 (d, J
= 2.4 Hz, 2H, HC≡C-CH2), 4.63 – 4.58 (m, 2H, H-1-Glup, OH), 4.45 (dd, J = 12.4, 3.2 Hz, 1H, H-6-
Glup), 4.40 (d, J = 7.6 Hz, 1H, H-1-Fucp), 4.14 (dd, J = 12.4, 2.4 Hz, 1H, H-6-Glup), 4.04 (br, 1H, OH),
3.91 (dd, J = 9.6, 3.2 Hz, 1H, H-3-Fucp), 3.78 – 3.72 (m, 1H, H-5-Glup), 3.71 – 3.59 (m, 4H, H-2-Glup,
H-2-Fucp, H-5-Fucp, -CH2-CH-CH2-), 2.83 – 2.39 (m, 7H), 2.18 (s, 3H, CH3-C=O), 1.79 – 1.72 (m, 6H,
CH3-CH-C(CH3)-C=O), 1.70 – 1.20 (m, 18H), 1.18 (d, J = 6.4 Hz, 3H, H-6-Fucp), 0.89 (t, J = 6.8 Hz,
3H). 13C NMR (100 MHz, CDCl3) δ 210.04, 171.76, 171.70, 168.82, 165.61, 159.43, 145.55, 139.74,
129.89(2), 127.56, 127.55, 115.21(2), 114.62, 105.66, 100.19, 82.74, 79.73, 77.91, 75.98, 75.85, 73.95,
72.68, 72.59, 72.42, 68.79, 67.32, 61.78, 55.79, 41.82, 37.56, 34.32, 33.08, 31.89, 29.10, 29.02, 28.28,
24.66, 24.48, 23.45, 22.63, 20.92, 16.32, 14.57, 14.08, 11.95. HRMS (ESI) m/z calcd for C47H64NaO16
[M+Na]+ 907.4092. Found: 907.4089.
Compound 5.
Biotin linker 4 was prepared from D-biotin and 1-amino11-azido-3,6,9-trioxaundecane (S1) following
the literature reported procedure1 with a yield of 90%. Compound 3 (24.8 mg, 0.028 mmol) and biotin
linker 4 (6.23 mg, 0.014 mmol) were dissolved in a mixture of CH2Cl2/t-BuOH (3:2, w/w, 1 mL). To the
mixture were added an aqueous solution of CuSO4 (0.3 M, 1 eq, 93 μL) and a aqueous solution of sodium
S5
ascorbate (1 M, 2 eq, 56 μL) and the mixture was vigorously stirred without light for 24 h. TLC analysis
(silica, 1:10 MeOH–CH2Cl2) showed the reaction was done. The reaction mixture was concentrated under
reduced pressure. The residue was purified by column chromatography (silica, MeOH–CH2Cl2, 1:30 →
1:20 → 1:10) gave compound 5 (17.4 mg, 94%) as a white solid. 1H NMR (400 MHz, CDCl3) δ 7.86 (s,
1H, triazole-H), 7.57 (d, J = 16.0 Hz, 1H, Ph-CH=C-), 7.45 (d, J = 8.4 Hz, 2H, ArH), 7.00 (d, J = 8.4 Hz,
2H, ArH), 6.93 – 6.83 (m, 1H, Me-CH-C(Me)-C=O), 6.61 (br, 1H, NH), 6.31 – 6.18 (m, 2H, Ph-CH=CH-
, NH), 5.35 – 5.21 (m, 3H, H-4-Glup, , triazole-CH2O-), 5.18 – 5.11 (m, 2H, H-3-Glup, H-4-Fucp), 4.63
(d, J = 8.0 Hz, 1H, H-1-Glup), 4.57 (t, J = 5.2 Hz, 2H, PEG-CH2), 4.54 – 4.44 (m, 2H, biotin NCH, H-6-
Glup), 4.40 (d, J = 7.6 Hz, 1H, H-1-Fucp), 4.36 – 4.29 (m, 1H, biotin NCH), 4.16 – 4.08 (m, 1H, H-6-
Glup), 3.96 – 3.87 (m, 3H, H-3-Fucp, PEG CH2), 3.78 – 3.35 (m, 20H, H-2-Glup, H-5-Glup, H-2-Fucp,
H-5-Fucp, -CH2-CH-CH2-, PEG CH2), 3.20 – 3.08 (m, 2H), 2.95 – 2.38 (m, 8H), 2.28 – 2.10 (m, 5H),
1.79 – 1.20 (m, 28H), 1.17 (d, J = 6.4 Hz, 3H, H-6-Fucp), 0.89 (t, J = 6.8 Hz, 3H). 13C NMR (75 MHz,
CDCl3) δ 210.1, 173.3, 171.7, 168.7, 165.6, 163.6, 160.2, 145.5, 143.3, 139.5, 130.0(×2), 127.6, 127.3,
124.3, 115.1(×2), 114.6, 105. 7, 100.4, 82.7, 79.9, 77.4, 75.8, 73.9, 72.8, 72.6, 72.4, 70.7, 70.5, 70.4, 70.1,
69.8, 69.4, 68.8, 67.4, 62.0, 60.3, 55.4, 50.7, 50.4, 41.8, 40.4, 39.2, 37.6, 35.7, 34.4, 33.2, 31.9, 29.1, 29.1,
28.3, 28.1, 28.0, 25.4, 24.7, 24.5, 23.5, 22.6, 21.0, 16.4, 14.6, 14.1, 12.0. HRMS (ESI) m/z calcd for
C65H96N6NaO21S [M+Na]+ 1351.6247, found: 1351.6238.
Biotin Linker with photo cleavage moiety
S6
Compound S3.
BnBr (1.54 g, 9.0 mmol) was added in one portion to a solution of compound S2 (1.0 g, 6.0 mmol),
K2CO3 (1.66 g, 12.0 mmol) and KI (200 mg, 1.2 mmol) in DMF (20 mL). The reaction mixture was stirred
at 90 oC overnight. At this point, TLC (silica, 1:3 EtOAc–hexanes) showed the reaction was complete.
The reaction mixture was cooled to room temperature, and then filtered through a pad of Celite using
Ethyl Acetate (50 mL) as the eluent and the filtrate concentrated. The residue was purified by column
chromatography (silica, EtOAc–hexanes, 1: 4) gave desired compound (1.4 g, 91%) as a white solid. To
a solution of this (1.3 g, 5.1 mmol) in AcOH (20 mL) was added HNO3 (5 mL) at 0 oC. The reaction
mixture was stirred at the same temperature for 12 h at which point TLC (silica, 1:3 EtOAc–hexanes)
showed the reaction was complete. The reaction mixture was diluted with Ice-Water (60 mL), and then
filtered, washed with Ice-Water (20 mL). The precipitated product was recrystallized from ethanol to yield
product (1.1g, 75%) as a yellow solid. The yellow solid (1.1 g, 4.3 mol) was slowly added to TFA (15
mL) at 0 oC for 1 h, and then the result mixture was stirred at room temperature overnight at which point
TLC (silica, 1:3 EtOAc–hexanes) showed the reaction was complete. The reaction mixture was diluted
with Ice-Water (60 mL), and then filtered, washed with Ice-Water (20 mL). The precipitated product was
purified by column chromatography (silica, EtOAc–hexanes, 1:4 → 1:1) gave compound S3 (590 mg,
69%) as a white solid. 1H NMR (400 MHz, CDCl3) δ 7.67 (s, 1H), 6.81 (s, 1H), 4.02 (s, 3H), 2.49 (s, 3H).
13C NMR (100 MHz, CDCl3) δ 199.83, 150.98, 146.61, 131.86, 110.70, 108.58, 56.74, 30.27.
Compound S5.
Compound S4 (425 mg, 1.8 mmol) was added in one portion to a solution of compound S3 (250 mg,
1.2 mmol), K2CO3 (327 mg, 2.4 mmol) and KI (39 mg, 0.24 mmol) in DMF (10 mL). The reaction mixture
was stirred at 90 oC overnight. At this point, TLC (silica, 1:3 EtOAc–hexanes) showed the reaction was
complete. The reaction mixture was cooled to room temperature, and then filtered through a pad of Celite
using Ethyl Acetate (50 mL) as the eluent and the filtrate concentrated. The residue was purified by
column chromatography (silica, EtOAc–hexanes, 1: 4) gave desired compound S5 (390 mg, 89%) as a
yellow oil. 1H NMR (400 MHz, CDCl3) δ 7.69 (s, 1H), 6.75 (s, 1H), 4.20 (t, J = 5.2 Hz, 2H), 4.03 (t, J =
5.2 Hz, 2H), 3.94 (s, 3H), 2.49 (s, 3H), 0.89 (s, 9H), 0.09 (s, 6H).
Compound S8.
NaBH4 (54 mg, 1.4 mmol) was slowly added to a solution of compound S5 (500 mg, 1.4 mmol) in
THF/MeOH (16mL, 1:1). The reaction mixture was stirred at room temperature 0.5 h. At this point, TLC
(silica, 1:2 EtOAc–hexanes) showed the reaction was complete. The reaction mixture was evaporated,
and then diluted with Et2O (20 mL), washed with 1M HCl (10 mL), saturated NaHCO3 (10 mL), and brine
(10 mL). The aqueous layer was extracted with Et2O (20 mL). The combined organic layer was dried over
Na2SO4 and concentrated under reduced pressure. The residue was purified by column chromatography
S7
(silica, EtOAc–hexanes, 1:4 → 1:1) gave compound S6 (450 mg, 91%) as a yellow oil. A solution of
compound S6 (400 mg, 1.1 mmol), Et3N (326 mg, 3.2 mmol) and DMAP (13 mg, 0.1 mmol) in THF (20
mL) was slowly added to Compound S7 (325 mg, 1.6 mmol) in THF. The reaction mixture was stirred at
0 oC 3h. At this point, TLC (silica, 1:3 EtOAc–hexanes) showed the reaction was complete. The reaction
mixture was quenched with 5% HCl (20 mL), and the aqueous layer was extracted with Ethyl Acetate (20
mL), washed with saturated NaHCO3 (10 mL), and brine (10 mL). The combined organic layer was dried
over Na2SO4 and concentrated under reduced pressure. The residue was purified by column
chromatography (silica, EtOAc–hexanes, 1: 4) gave desired compound S8 (510 mg, 88%) as a yellow oil.
1H NMR (400 MHz, CDCl3) δ 8.26 (d, J = 9.2 Hz, 2H), 7.68 (s, 1H), 7.34 (d, J = 9.2 Hz, 2H), 6.75 (s,
1H), 6.53 (q, J = 6.4 Hz, 1H), 4.12 (t, J = 5.2 Hz, 2H), 4.03 (t, J = 5.2 Hz, 2H), 3.99 (s, 3H), 1.77 (d, J =
5.2 Hz, 3H), 0.89 (s, 9H), 0.09 (s, 6H).
Compound S10.
Compound S9 (107 mg, 0.5 mmol) was slowly added to a solution of compound S8 (200 mg, 0.37
mmol) in THF (6mL). The reaction mixture was stirred at room temperature 0.5 h. At this point, TLC
(silica, 1:1 EtOAc–hexanes) showed the reaction was complete. The reaction mixture was quenched with
5% HCl (5 mL), and the aqueous layer was extracted with Ethyl Acetate (20 mL), washed with saturated
NaHCO3 (10 mL), and brine (10 mL). The combined organic layer was dried over Na2SO4 and
concentrated under reduced pressure. The residue was purified by column chromatography (silica,
EtOAc–hexanes, 2: 1) gave desired compound S10 (220 mg, 96%) as a yellow oil. 1H NMR (400 MHz,
CDCl3) δ 7.66 (s, 1H), 7.07 (s, 1H), 6.40 (q, J = 6.4 Hz, 1H), 4.30-4.24 (m, 2H), 4.22 (t, J = 5.2 Hz, 2H),
4.16 (t, J = 5.2 Hz, 2H), 4.02 (s, 3H). 3.72-3.65 (m, 12H), 3.40-3.36 (m, 2H). 1.66 (d, J = 5.2 Hz, 3H),
0.89 (s, 9H), 0.09 (s, 6H).
Compound S11.
2M HCl (3 mL) was slowly added to a solution of compound S10 (200 mg, 0.33 mmol) in THF (6mL).
The reaction mixture was stirred at room temperature 0.5 h. At this point, TLC (silica, 3:1 EtOAc–
hexanes) showed the reaction was complete. The reaction mixture was quenched with saturated NaHCO3
(10 mL), and the aqueous layer was extracted with Ethyl Acetate (20 mL), washed with brine (10 mL).
The combined organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue
was purified by column chromatography (silica, EtOAc) gave desired compound S11 (152 mg, 92%) as
a yellow oil. 1H NMR (400 MHz, CDCl3) δ 7.63 (s, 1H), 7.09 (s, 1H), 6.38 (q, J = 6.4 Hz, 1H), 4.29-4.21
(m, 2H), 4.18 (t, J = 5.2 Hz, 2H), 4.01-3.99 (m, 2H), 3.97 (s, 3H). 3.71-3.64 (m, 12H), 3.39 (t, J = 5.2 Hz,
2H). 2.23 (t, J = 6.0 Hz,1H), 1.66 (d, J = 6.4 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 154.28, 154.04,
147.19, 139.67, 133.29, 109.72, 108.20, 72.09, 71.06, 70.65, 70.63(2), 70.51, 70.02, 68.85, 67.05, 60.96,
56,44, 50.66, 22.08
S8
Compound S12.
A solution of compound S11 (140 mg, 0.28 mmol), Et3N (85 mg, 0.84 mmol) and DMAP (4 mg, 0.03
mmol) in THF (10 mL) was slowly added to Compound S7 (85 mg, 0.42 mmol) in THF (5 mL). The
reaction mixture was stirred at 0 oC 3 h. At this point, TLC (silica, 2:1 EtOAc–hexanes) showed the
reaction was complete. The reaction mixture was quenched with 5% HCl (10 mL), and the aqueous layer
was extracted with Ethyl Acetate (20 mL), washed with saturated NaHCO3 (10 mL), and brine (10 mL).
The combined organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue
was purified by column chromatography (silica, EtOAc–hexanes, 1: 1) gave desired compound S12 (150
mg, 81%) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 8.27 (d, J = 9.2 Hz, 2H), 7.66 (s, 1H), 7.41 (d, J
= 9.2 Hz, 2H), 7.12 (s, 1H), 6.40 (q, J = 6.4 Hz, 1H), 4.70 (t, J = 4.4 Hz, 2H), 4.41-4.4.39 (m, 2H), 4.33-
4.21 (m, 2H), 3.99 (s, 3H). 3.71 (t, J = 4.8 Hz, 2H), 3.70-3.65 (m, 10H), 3.39 (t, J = 5.2 Hz, 2H). 1.67 (d,
J = 6.4 Hz, 3H).
Compound 6.
Compound S13 (75 mg, 0.18 mmol) was slowly added to a solution of compound S12 (100 mg, 0.15
mmol) in THF (6 mL). The reaction mixture was stirred at room temperature 0.5 h. At this point, TLC
(silica, 5:1 EtOAc–MeOH) showed the reaction was complete. The reaction mixture was quenched with
5% HCl (5 mL), and the aqueous layer was extracted with Ethyl Acetate (30 mL), washed with saturated
NaHCO3 (10 mL), and brine (10 mL). The combined organic layer was dried over Na2SO4 and
concentrated under reduced pressure. The residue was purified by column chromatography (silica,
EtOAc–MeOH, 10: 1) gave desired compound 6 (58 mg, 41%) as a yellow oil. 1H NMR (400 MHz,
MeOD) δ 8.02 (brs, 1H, NH), 7.70 (s, 1H), 7.20 (s, 1H), 6.30 (q, J = 6.4 Hz, 1H), 4.53 (dd, J1 = 4.4 Hz,
J2 = 7.6 Hz, 1H). 4.45 (brs, 2H), 4.36-4.33 (m, 3H), 4.30-4.26 (m, 2H), 4.00 (s, 3H). 3.74-3.65 (m, 22H),
3.59-3.56 (m, 4H), 3.26-3.22 (m, 1H), 2.97 (dd, J1 = 5.2 Hz, J2 = 12.8 Hz, 1H). 2.74 (d, J = 12.4 Hz, 1H),
2.26 (t, J = 6.4 Hz, 2H), 1.82-1.58 (m, 4H), 1.66 (d, J = 6.4 Hz, 3H), 1.52-1.43 (m, 2H). 13C NMR (100
MHz, MeOD) δ 176.29, 166.16, 158.88, 155.92, 155.86, 148.88, 141.43, 134.18, 110.94, 109.87, 73. 32,
71.73, 71.67, 71.60, 71.59, 71.33, 71.30, 71.21, 70.87, 70.14, 69.44, 68.41, 64.37, 63.48, 61.75, 57.22,
57.12, 51.93,41.88, 41.02, 40.43, 36.88, 29.89, 29.65, 26.98, 22.25.
S9
Photo Cleavable Biotin Probe 7
Compound 3 (28.0 mg, 0.0316 mmol) and the azide 6 (15.0 mg, 0.0158 mmol) were dissolved in a
mixture of CH2Cl2/t-BuOH (3:2, w/w, 2 mL). To the mixture were added an aqueous solution of CuSO4
(1 M, 1 eq, 32 μL) and an aqueous solution of sodium ascorbate (1 M, 2 eq, 63 μL) and the mixture was
vigorously stirred without light for 24 h. TLC analysis (silica, 1:20 MeOH–CH2Cl2) showed the reaction
was done. The reaction mixture was concentrated under reduced pressure. The residue was purified by
column chromatography (silica, MeOH–CH2Cl2, 1:30 → 1:20) gave compound 7 (20.9 mg, 72%) as a
white foam. [α]D25 –13.3° (c 1 CHCl3).
1H NMR (400 MHz, CDCl3) δ 7.84 (s, 1H, triazole-H), 7.63 (s,
1H, NO2-Ar-H), 7.56 (d, J = 16.0 Hz, 1H, Ph-CH=C-), 7.44 (d, J = 8.8 Hz, 2H, ArH), 7.06 (s, 1H, NO2-
Ar-H), 6.99 (d, J = 8.8 Hz, 2H, ArH), 6.93 – 6.80 (m, 1H, Me-CH-C(Me)-C=O), 6.49 (br, 1H, NH), 6.36
(q, 1H, J = 6.4 Hz, NO2-Ar-CH(CH3)-O-), 6.20 (d, J = 16.0 Hz, 1H, Ph-CH=CH-), 5.94 (br, 1H, NH),
5.58 (br, 1H, NH), 5.28 (t, J = 9.6 Hz, 1H, H-4-Glup), 5.23 (s, 3H, triazole-CH2O-, NH), 5.18 – 5.10 (m,
2H, H-3-Glup, H-4-Fucp), 4.83 (br, 1H, OH), 4.62 (d, J = 8.0 Hz, 1H, H-1-Glup), 4.56 (t, J = 5.2 Hz, 2H,
PEG-CH2), 4.53 – 4.42 (m, 5H, biotin-CH, PEG-CH2, OH), 4.40 (d, J = 7.6 Hz, 1H, H-1-Fucp), 4.35 –
4.10 (m, 6H, H-6-Glup, PEG-CH2), 3.94 (s, 3H, CH3OAr), 3.92 – 3.84 (m, 3H, H-3-Fucp, PEG-CH2),
3.78 – 3.52 (m, 26H, H-2-Glup, H-5-Glup, H-2-Fucp, H-5-Fucp, -CH2-CH-CH2-, PEG-CH2), 3.48 – 3.32
(m, 4H, PEG-CH2), 3.18 – 3.10 (m, 1H), 2.95 – 2.86 (m, 1H), 2.83 – 2.39 (m, 6H), 2.26 – 2.15 (m, 5H),
1.88 (br, 1H), 1.78 – 1.59 (m, 16H), 1.58 – 1.20 (m, 18H), 1.17 (d, J = 6.4 Hz, 3H, H-6-Fucp), 0.89 (t, J
= 6.8 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 210.0, 173.1, 171.7, 171.6, 168.6, 165.6, 163.5, 160.3,
156.2, 154.3, 154.1, 147.1, 145.5, 143.3, 139.7, 139.4, 133.1, 130.0(×2), 127.6, 127.2, 124.2, 115.1(×2),
114.5, 109.7, 108.4, 105.7, 100.4, 82.7, 79.8, 75.7, 74.0, 72.8, 72.6, 72.4, 72.1, 70.5, 70.4, 70.2, 70.1,
69.9, 69.8, 69.4, 68.8, 68.8, 68.1, 67.5, 67.0, 62.9, 62.0, 61.9, 61.8, 60.1, 56.5, 55.4, 50.3, 41.8, 40.5, 39.1,
37.6, 35.8, 34.4, 33.2, 31.9, 29.1, 29.0, 28.3, 28.1, 28.0, 25.4, 24.7, 24.5, 23.5, 22.6, 22.0, 20.9, 16.3, 14.5,
14.1, 12.0. HRMS (ESI) m/z calcd for C86H126N8NaO33S [M+Na]+ 1853.8046, found: 1853.8023.
S10
Syntheses of Negative Controls 8 and 9
Compound S18.
Compound S16 was prepared from 3-(4-hydroxyphenyl)acrylate following the literature reported
procedure2 with a yield of 93% over two steps. Propargyl bromide (0.28 mL, 2.55 mmol, 80wt.% solution
in toluene) was added to a mixture of S16 (247.7 g, 1.275 mmol), K2CO3 (352.5 mg, 2.55 mmol) and KI
(21.2 mg, 0.128 mmol) in THF (10 mL). The reaction mixture was stirred at reflux overnight. TLC (silica,
1:4 EtOAc–hexanes) showed it was complete. The reaction mixture was concentrated in vacuo and the
residue was diluted with CH2Cl2 (20 mL), washed with water (30 mL). The aqueous layer was extracted
with CH2Cl2 (3 × 20 mL). The combined organic layer was dried over Na2SO4 and concentrated under
reduced pressure. The residue was purified by column chromatography (silica, EtOAc–hexanes, 1:5
→1:4) gave compound S17 (231.4 mg, 78%) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 7.14 (d, J
= 8.4 Hz, 2H, ArH), 6.91 (d, J = 8.4 Hz, 2H, ArH), 4.67 (d, J = 2.4 Hz, 2H, alkyne CH2), 4.13 (q, J = 7.2
Hz, 2H, CH2CH3), 2.91, 2.59 (2t, J = 7.6 Hz, 4H, Ar-CH2CH2-), 2.52 (t, J = 2.4 Hz, 1H, alkyne CH), 1.24
(t, J = 7.2 Hz, 3H, CH2CH3). 13C NMR (100 MHz, CDCl3) δ 172.9, 156.0, 133.6, 129.2(×2), 114.9(×2),
78.7, 75.4, 60.3, 55.8, 36.1, 30.0, 14.2.
To a solution of ethyl ester S17 (204.3 mg, 0.880 mmol) in MeOH (5 mL) was added NaOH (2 M
solution in H2O, 2.2 mL, 4.40 mmol) at 0 oC. The mixture was slowly warmed to RT and stirred overnight.
TLC analysis (silica, 1:2 EtOAc–hexanes) showed it was complete. MeOH was evaporated and the
resulting mixture was acidified with 10% w/v aq. HCl until pH = 4. White precipitate was formed.
Filtration following washing with water (5 mL) and then dry in high vacuum gave S18 (155.4 mg, 87%)
as a white solid. 1H NMR (400 MHz, CDCl3) δ 11.45 (br, 1H, COOH), 7.16 (d, J = 8.4 Hz, 2H, ArH),
6.92 (d, J = 8.4 Hz, 2H, ArH), 4.68 (d, J = 2.4 Hz, 2H, alkyne CH2), 2.92, 2.67 (2t, J = 7.6 Hz, 4H, Ar-
CH2CH2-), 2.53 (t, J = 2.4 Hz, 1H, alkyne CH). 13C NMR (100 MHz, CDCl3) δ 179.0, 156.1, 133.2,
129.2(×2), 115.0(×2), 78.7, 75.4, 55.8, 35.7, 29.7. The 1H NMR data was in accordance with the
literature.3
S11
Compound 8.
2-Chloro-1-methylpyridinium iodide (CMPI, 64.1 mg, 0.251 mmol), N,N-dimethylaminopyridine
(DMAP, 10.2 mg, 0.0837 mmol) and Et3N (117 μL, 0.837 mmol) were added to a solution of S194 (155.9
mg, 0.167 mmol) and acid S18 (51.3 mg, 0.251 mmol) in dry CH2Cl2 (4 mL) at 0 oC. The reaction was
warmed to ambient temperature and stirred for 12 h. At this point, TLC (silica, 1:2 EtOAc–hexanes)
showed the reaction was complete. Evaporation of the solvent followed by purification of the residue by
flash column chromatography (silica, EtOAc−hexanes, 1:5 → 1:3) to give protected compound (161.7
mg, 89%) as a colorless syrup. To a solution of the protected compound (135.5 mg, 0.148 mmol) in THF
(5 mL) was added TBAF (1M solution in THF, 740 μL, 740 μmol, 5 equiv) at –10 oC. The reaction
mixture was stirred at the same temperature for 3 h at which point TLC (silica, 1:1 EtOAc–hexanes)
showed it was complete. The reaction mixture was diluted with Et2O (40 mL), washed with 1M HCl (20
mL), saturated NaHCO3 (20 mL), brine (20 mL). The aqueous layer was extracted with Et2O (40 mL).
The combined organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue
was purified by column chromatography (silica, EtOAc–hexanes, 1:2 → 1:1) gave compound 8 (79.6 mg,
71%) as a white solid. 1H NMR (400 MHz, CDCl3) δ 7.15 – 7.07 (m, 2H, ArH), 6.91 – 6.84 (m, 2H, ArH),
5.14 – 5.05 (m, 3H, H-3-Glup, H-4-Glup, H-4-Fucp), 4.66 (d, J = 2.4 Hz, 2H, alkyne CH2), 4.52 (d, J =
8.0 Hz, 1H, H-1-Glup), 4.48 – 4.35 (m, 2H, H-6-Glup, H-1-Fucp), 4.03 – 3.95 (m, 1H, H-6-Glup), 3.90
(dd, J = 9.6, 3.6 Hz, 1H, H-3-Fucp), 3.73 – 3.54 (m, 5H, H-2-Glup, H-5-Glup, H-2-Fucp, H-5-Fucp, -
CH2-CH-CH2-), 2.90 – 2.31 (m, 12H), 2.17 (s, 3H, CH3-C=O), 1.80 – 1.13 (m, 23H), 1.09, 1.06 (2d, J =
7.2, 6.8 Hz, 3H), 0.96 – 0.82 (m, 6H). 13C NMR (100 MHz, CDCl3) δ 210.0, 177.0, 177.0, 172.0, 172.0,
171.7, 171.7, 171.5, 171.4, 156.1, 133.2, 133.2, 129.2, 129.2, 114.9, 114.9, 105.7, 100.0, 82.6, 79.5, 78.7,
75.4, 74.7, 73.8, 73.7, 72.9, 72.7, 72.7, 72.5, 68.8, 67.2, 67.1, 61.3, 55.8, 41.8, 41.2, 41.0, 37.6, 35.6, 35.6,
34.4, 33.1, 31.9, 29.6, 29.6, 29.1, 29.0, 28.3, 26.5, 26.4, 24.7, 24.5, 23.5, 22.6, 20.9, 16.5, 16.4, 16.3, 14.1,
11.5, 11.5. HRMS (ESI) m/z calcd for C47H68NaO16 [M+Na]+ 911.4405, found: 911.4409.
S12
Compound S21.
Compound S1 (107 mg, 0.5 mmol) was slowly added to a solution of compound S8 (200 mg, 0.37
mmol) in THF (6mL). The reaction mixture was stirred at room temperature 0.5 h. At this point, TLC
(silica, 1:1 EtOAc–hexanes) showed the reaction was complete. The reaction mixture was quenched with
5% HCl (5 mL), and the aqueous layer was extracted with Ethyl Acetate (20 mL), washed with saturated
NaHCO3 (10 mL), and brine (10 mL). The combined organic layer was dried over Na2SO4 and
concentrated under reduced pressure. The residue was purified by column chromatography (silica,
EtOAc–hexanes, 2:1) gave desired compound S20 (220 mg, 96%) as a yellow oil. 2M HCl (3 mL) was
slowly added to a solution of compound S20 (200 mg, 0.33 mmol) in THF (6mL). The reaction mixture
was stirred at room temperature 0.5 h. At this point, TLC (silica, 3:1 EtOAc–hexanes) showed the reaction
was complete. The reaction mixture was quenched with saturated NaHCO3 (10 mL), and the aqueous
layer was extracted with Ethyl Acetate (20 mL), washed with brine (10 mL). The combined organic layer
was dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by column
chromatography (silica, EtOAc) gave desired compound S21 (152 mg, 92%) as a yellow oil. 1H NMR
(400 MHz, CDCl3) δ 7.63 (s, 1H), 7.04 (s, 1H), 6.38 (q, J = 6.4 Hz, 1H), 5.34 (brs, 1H, NH), 4.18 (t, J =
5.2 Hz, 2H), 4.00 (t, J = 5.2 Hz, 2H), 3.97 (s, 3H). 3.70-3.62 (m, 10H), 3.54-3.53 (m, 2H), 3.41-3.35 (m,
4H), 1.61 (d, J = 6.4 Hz, 3H).
Compound S22.
A solution of compound S21 (140 mg, 0.28 mmol), Et3N (85 mg, 0.84 mmol) and DMAP (4 mg, 0.03
mmol) in THF (10 mL) was slowly added to Compound S7 (85 mg, 0.42 mmol) in THF (5 mL). The
reaction mixture was stirred at 0 oC 3 h. At this point, TLC (silica, 2:1 EtOAc–hexanes) showed the
S13
reaction was complete. The reaction mixture was quenched with 5% HCl (10 mL), and the aqueous layer
was extracted with Ethyl Acetate (20 mL), washed with saturated NaHCO3 (10 mL), and brine (10 mL).
The combined organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue
was purified by column chromatography (silica, EtOAc–hexanes, 1: 1) gave desired compound S22 (150
mg, 81%) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 8.26 (d, J = 9.2 Hz, 2H), 7.63 (s, 1H), 7.39 (d, J
= 9.2 Hz, 2H), 7.05 (s, 1H), 6.35 (q, J = 6.4 Hz, 1H), 5.37 (t, J = 4.8 Hz 1H, NH), 4.67 (t, J = 4.4 Hz, 2H),
4.36 (t, J = 4.4 Hz, 2H), 3.96 (s, 3H). 3.69-3.61 (m, 10H), 3.52 (brs, 2H), 3.39-3.29 (m, 4H), 1.59 (d, J =
6.4 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 155.33, 154.18, 152.28, 146.33, 145.37, 139.40, 135.26,
125.94, 125.23, 121.68, 115.47, 109.95, 108.44, 70.58, 70.52, 70.48, 70.15, 69.96, 69.86, 68.74, 66.95,
66.86, 56.34, 50.56, 40.72, 22.10.
Compound S24.
Compound S23 (75 mg, 0.18 mmol) was slowly added to a solution of compound S22 (100 mg, 0.15
mmol) in THF (6 mL). The reaction mixture was stirred at room temperature 0.5 h. At this point, TLC
(silica, 5:1 EtOAc–MeOH) showed the reaction was complete. The reaction mixture was quenched with
5% HCl (5 mL), and the aqueous layer was extracted with Ethyl Acetate (30 mL), washed with saturated
NaHCO3 (10 mL), and brine (10 mL). The combined organic layer was dried over Na2SO4 and
concentrated under reduced pressure. The residue was purified by column chromatography (silica,
EtOAc–MeOH, 10: 1) gave desired compound S24 (58 mg, 41%) as a yellow oil. 1H NMR (400 MHz,
CDCl3) δ 7.66 (s, 1H), 7.20 (s, 1H), 6.28 (q, J = 6.4 Hz, 1H), 4.48 (dd, J1 = 4.4 Hz, J2 = 7.6 Hz, 1H). 4.41-
4.39 (m, 2H), 4.31-4.28 (m, 3H), 3.96 (s, 3H). 3.66-3.51 (m, 22H), 3.49-3.25 (m, 4H), 3.26-3.22 (m, 1H),
2.93 (dd, J1 = 5.2 Hz, J2 = 12.8 Hz, 1H). 2.70 (d, J = 12.8 Hz, 1H), 2.20 (t, J = 7.2 Hz, 2H), 1.74-1.60 (m,
4H), 1.60 (d, J = 6.4 Hz, 3H), 1.482-1.41 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 176.10, 166.05, 158.67,
157.87, 155.79, 148.41, 140.95, 134.89, 110.91, 109.81, 71.60, 71.58, 71.54, 71.52, 71.46, 71.21, 71.18,
71.08, 70.96, 70.61, 69.75, 69.29, 64.25, 63.34, 61.61, 57.02, 56.98, 51.77 ,41.75, 41.64, 41.04, 40.32,
36.73, 29.75, 29.49, 26.83, 22.46.
Compound 9.
S14
Compound 8 (21.5 mg, 0.0242 mmol) and azide S22 (13.7 mg, 0.0145 mmol) were dissolved in a
mixture of CH2Cl2/t-BuOH (3:2, w/w, 2 mL). To the mixture were added an aqueous solution of CuSO4
(1 M, 1 eq, 24 μL), an aqueous solution of sodium ascorbate (1 M, 2 eq, 48 μL) and ligand THPTA (2.1
mg, 0.2 eq) and the mixture was vigorously stirred in dark for 24 h. The reaction mixture was concentrated
under reduced pressure. The residue was purified by column chromatography (silica, MeOH–CH2Cl2,
1:20 → 1:10) gave compound 9 (20.5 mg, 77%) as a white film. 1H NMR (400 MHz, CDCl3) δ 7.82 (s,
1H, triazole-H), 7.63 (s, 1H, NO2-Ar-H), 7.08 (d, J = 8.4 Hz, 2H, ArH), 7.03 (s, 1H, NO2-Ar-H), 6.89 (d,
J = 8.4 Hz, 2H, ArH), 6.45 (br, 1H, NH), 6.35 (q, 1H, J = 6.0 Hz, NO2-Ar-CH(CH3)-O-), 5.79 (br, 1H,
NH), 5.59 (br, 1H, NH), 5.45 (br, 1H, NH), 5.22 – 5.14 (m, 3H, triazole-CH2O-, NH), 5.13 – 5.06 (m, 3H,
H-3-Glup, H-4-Glup, H-4-Fucp), 4.63 – 4.42 (m, 8H, H-1-Glup, H-6-Glup, biotin-CH, PEG-CH2, OH),
4.38 (d, J = 7.6 Hz, 1H, H-1-Fucp), 4.35 – 4.20 (m, 4H), 4.01 – 3.85 (m, 7H, H-6-Glup, H-3-Fucp, PEG-
CH2, CH3OAr), 3.70 – 3.22 (m, 32H, H-2-Glup, H-5-Glup, H-2-Fucp, H-5-Fucp, -CH2-CH-CH2-, PEG-
CH2), 3.18 – 3.11 (m, 1H), 2.95 – 2.64 (m, 6H), 2.62 – 2.30 (m, 7H), 2.24 – 2.13 (m, 5H), 1.82 –1.21 (m,
30H), 1.17 (d, J = 6.4 Hz, 3H, H-6-Fucp), 1.09, 1.05 (2d, J = 6.8, 7.2 Hz, 3H), 0.90 – 0.82 (m, 6H). 13C
NMR (100 MHz, CDCl3) δ 210.0, 176.8, 176.7, 173.1, 171.9, 171.7, 171.6, 171.5, 171.4, 156.8, 156.2,
155.4, 154.1, 146.8, 139.5, 134.7, 132.9, 129.3, 114.8, 109.9, 108.5, 105.7, 100.3, 82.5, 79.6, 74.6, 73.7,
73.6, 73.0, 72.7, 72.7, 72.4, 70.5, 70.5, 70.4, 70.4, 70.2, 70.0, 70.0, 69.8, 69.4, 68.7, 68.1, 67.4, 67.3, 63.0,
62.1, 61.8, 61.4, 60.2, 56.4, 55.4, 50.4, 41.8, 41.2, 41.0, 40.8, 40.7, 40.5, 39.1, 37.6, 35.8, 35.7, 34.4, 33.2,
31.9, 29.6, 29.6, 29.2, 29.0, 28.3, 28.1, 26.5, 26.4, 25.4, 24.7, 24.5, 23.6, 22.6, 22.2, 21.0, 16.5, 16.4, 16.3,
14.1, 11.5, 11.5. HRMS (ESI) m/z calcd for C86H132N9O32S [M+H]+ 1834.8699, found: 1834.8657.
Syntheses of Fluorescent Probes 10, 11 and 12.
S15
Compound 10.
Compound 3 (23.2 mg, 0.0262 mmol) and the azide S255 (5.3 mg, 0.0262 mmol) were dissolved in a
mixture of CH2Cl2/t-BuOH (3:2, w/w, 2 mL). To the mixture were added an aqueous solution of CuSO4
(1 M, 1 eq, 26 μL) and an aqueous solution of sodium ascorbate (1 M, 2 eq, 52 μL) and the mixture was
vigorously stirred without light for 24 h. TLC analysis (silica, 1:20 MeOH–CH2Cl2) showed the reaction
was done. The reaction mixture was concentrated under reduced pressure. The residue was purified by
column chromatography (silica, MeOH–CH2Cl2, 1:30 → 1:20) gave compound 10 (18.5 mg, 65%) as a
colorless film. [α]D25 –15.1° (c 0.3 CHCl3).
1H NMR (400 MHz, CDCl3) δ 8.73 (s, 1H, ArH), 8.43 (s, 1H,
ArH), 7.63 – 7.55 (m, 1H, ArH), 7.52 (d, J = 15.6 Hz, 1H, Ph-CH=C-), 7.43 (d, J = 8.8 Hz, 1H, ArH),
7.37 – 7.31 (m, 2H, ArH), 7.01 – 6.85 (m, 3H, ArH, Me-CH-C(Me)-C=O), 6.82 (d, J = 2.0 Hz, 1H, ArH),
6.15 (d, J = 15.6 Hz, 1H, Ph-CH=CH), 5.28 (t, J = 10.0 Hz, 1H, H-4-Glup), 5.24 (s, 2H, triazole-CH2O-
), 5.17 – 5.10 (m, 2H, H-3-Glup, H-4-Fucp), 4.68 – 4.62 (m, 3H, H-1-Glup, H-6-Glup, OH), 4.41 (d, J =
7.6 Hz, 1H, H-1-Fucp), 4.17 – 4.05 (m, 2H, H-6-Glup, OH), 3.98 – 3.90 (m, 1H, H-3-Fucp), 3.84 – 3.75
(m, 1H, H-5-Glup), 3.74 – 3.59 (m, 4H, H-2-Glup, H-2-Fucp, H-5-Fucp, -CH2-CH-CH2-), 2.93 – 2.38 (m,
6H), 2.19 (s, 3H, CH3-C=O), 1.79 – 1.71 (m, 6H, CH3-CH-C(CH3)-C=O), 1.71 – 1.60 (m, 2H), 1.55 –
1.22 (m, 16H), 1.20 (d, J = 6.4 Hz, 3H, H-6-Fucp), 0.89 (t, J = 6.8 Hz, 3H). 13C NMR (100 MHz, CDCl3)
δ 210.2, 172.7, 171.9, 169.1, 165.7, 161.3, 160.2, 156.2, 154.6, 145.7, 140.2, 134.2, 130.2, 129.9(×2),
127.5, 127.1, 119.7, 114.9(×2), 114.6, 114.2, 111.0, 105.9, 103.2, 100.3, 83.2, 79.9, 77.7, 76.2, 74.0, 72.7,
72.6, 68.8, 66.9, 61.8, 61.7, 41.8, 37.7, 34.4, 33.3, 31.9, 29.7, 29.2, 29.1, 28.4, 24.8, 24.6, 23.6, 22.6, 20.9,
16.3, 14.6, 14.1, 12.0. HRMS (ESI) m/z calcd for C56H69N3NaO19 [M+Na]+ 1110.4423, found: 1110.4420.
Compound 11.
Compound 3 (40.0 mg, 0.0452 mmol) and the azide S266 (15.5 mg, 0.0407 mmol) were dissolved in a
mixture of CH2Cl2/t-BuOH (3:2, w/w, 2 mL). To the mixture were added an aqueous solution of CuSO4
(1 M, 1 eq, 45 μL) and an aqueous solution of sodium ascorbate (1 M, 2 eq, 90 μL) and the mixture was
vigorously stirred without light for 24 h. TLC analysis (silica, 1:10 MeOH–EtOAc) showed the reaction
was done. The reaction mixture was concentrated under reduced pressure. The residue was purified by
column chromatography (Ialtra beads, MeOH–EtOAc, 1:20 → 1:10) gave compound 11 (44.8 mg, 87%)
as colorless syrup. 1H NMR (400 MHz, CDCl3) δ 8.42 (d, J = 8.8 Hz, 1H, NBD-H), 7.82 (s, 1H, Triazole-
H), 7.52 (d, J = 16.0 Hz, 1H, Ph-CH=C-), 7.39 (d, J = 8.8 Hz, 2H, ArH), 7.21 (br, 1H, NH), 6.94 (d, J =
8.8 Hz, 1H, ArH), 6.92 – 6.80 (m, 1H, Me-CH-C(Me)-C=O), 6.18 (d, J = 16.0 Hz, 1H, Ph-CH=CH-),
6.18 (d, J = 8.8 Hz, 1H, NBD-H), 5.26 (t, J = 9.6 Hz, 1H, H-4-Glup), 5.24 (s, 2H, triazole-CH2O-), 5.18
– 5.11 (m, 2H, H-3-Glup, H-4-Fucp), 4.71 (br, 1H, OH), 4.60 (d, J = 7.6 Hz, 1H, H-1-Glup), 4.56 (t, J =
4.8 Hz, 2H, PEG-CH2), 4.47 (dd, J = 12.8, 3.2 Hz, 1H, H-6-Glup), 4.38 (d, J = 7.6 Hz, 1H, H-1-Fucp),
S16
4.20 (br, 1H, OH), 4.15 – 4.04 (m, 2H, H-6-Glup, PEG-CH), 3.90 (t, J = 4.8 Hz, 2H, PEG-CH2), 3.85 –
3.70 (m, 3H, H-5-Glup, H-3-Fucp, PEG-CH), 3.69 – 3.55 (m, 14H, H-2-Glup, H-2-Fucp, H-5-Fucp, -
CH2-CH-CH2-, PEG-CH2), 2.82 – 2.35 (m, 6H), 2.15 (s, 3H, CH3-C=O), 1.79 – 1.70 (m, 6H, CH3-CH-
C(CH3)-C=O), 1.69 – 1.41 (m, 6H), 1.40 – 1.20 (m, 12H), 1.16 (d, J = 6.4 Hz, 3H, H-6-Fucp), 0.87 (t, J
= 6.8 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 210.1, 171.7, 171.6, 171.09, 168.6, 165.5, 160.1, 145.4,
144.2, 144.1, 143.9, 139.5, 136.5, 129.9(×2), 127.6, 127.2, 124.0, 123.6, 115.0(×2), 114.5, 105.7, 100.2,
98.8, 82.7, 79.7, 75.7, 73.9, 72.7, 72.6, 72.3, 70.5, 70.4, 70.4, 70.4, 69.3, 68.7, 68.1, 67.3, 61.9, 61.7, 60.3,
50.3, 43.6, 41.8, 37.6, 34.3, 33.1, 31.8, 29.1, 29.0, 28.2, 24.6, 24.4, 23.5, 22.6, 21.0, 20.9, 16.3, 14.5, 14.1,
14.0, 11.9. HRMS (ESI) m/z calcd for C61H84N7O22 [M+H]+ 1266.5669, found: 1266.5660.
Compound 12.
Compound 3 (50.1 mg, 0.0566 mmol) and azide S277 (30.0 mg, 0.0396 mmol) were dissolved in a
mixture of CH2Cl2/t-BuOH (3:2, w/w, 3 mL). To the mixture were added an aqueous solution of CuSO4
(1 M, 1 eq, 57 μL), sodium ascorbate (1 M, 2 eq, 113 μL) and ligand THPTA (5 mg). The mixture was
vigorously stirred without light for 24 h. TLC analysis (silica, 1:20 MeOH–CH2Cl2) showed the reaction
was done. The reaction mixture was concentrated under reduced pressure. The residue was purified by
column chromatography (silica, MeOH–CH2Cl2, 1:30 → 1:20) gave compound 12 (52.7 mg, 81%) as a
purple solid and starting material 3 (10.8 mg) was recovered. 1H NMR (400 MHz, CDCl3) δ 8.85 (d, J =
2.0 Hz, 1H, ArH), 8.03 (s, 1H, ArH), 7.98 (dd, J = 7.6, 1.6 Hz, 1H, ArH), 7.57 (d, J = 16.0 Hz, 1H, Ph-
CH=C-), 7.43 (d, J = 8.8 Hz, 2H, ArH), 7.27 – 7.23 (m, 2H, ArH), 7.20 (d, J = 8.0 Hz, 1H, ArH), 6.99 (d,
J = 8.4 Hz, 2H, ArH), 6.93 – 6.83 (m, 1H, Me-CH-C(Me)-C=O), 6.82 – 6.75 (m, 2H, ArH), 6.66 (d, J =
2.4 Hz, 2H, ArH), 6.25 – 6.15 (m, 2H, Ph-CH=CH-, SO2NH), 5.29 (t, J = 9.6 Hz, 1H, H-4-Glup), 5.21 (s,
2H, triazole-CH2O-), 5.18 – 5.10 (m, 2H, H-3-Glup, H-4-Fucp), 4.69 (br, 1H, OH), 4.65 (d, J = 7.6 Hz,
1H, H-1-Glup), 4.60 (t, J = 5.2 Hz, 2H, PEG-CH2), 4.45 (dd, J = 12.4, 3.6 Hz, 1H, H-6-Glup), 4.39 (d, J
= 7.6 Hz, 1H, H-1-Fucp), 4.23 – 4.07 (m, 2H, H-6-Glup, OH), 3.95 (t, J = 4.8 Hz, 2H, CH2), 3.90 (dd, J
= 9.6, 3.6 Hz, 1H, H-3-Fucp), 3.78 – 3.72 (m, 1H, H-5-Glup), 3.72 – 3.45 (m, 22H, H-2-Glup, H-2-Fucp,
H-5-Fucp, -CH2-CH-CH2-, PEG-CH2, N(CH2CH3)2), 3.28 – 3.21 (m, 2H, SO2NHCH2CH2O), 2.86 – 2.36
(m, 6H), 2.15 (s, 3H, CH3-C=O), 1.93 – 1.20 (m, 36H), 1.17 (d, J = 6.4 Hz, 3H, H-6-Fucp), 0.89 (t, J =
6.8 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 210.1, 171.7, 171.6, 168.9, 165.7, 160.5, 159.0, 157.9(×2),
155.5(×2), 148.4, 145.8, 143.0, 141.9, 139.8, 133.6, 133.4(×2), 129.9(×2), 129.6, 127.6, 127.2, 126.9,
124.8, 115.3, 114.3, 114.2, 113.5, 105.6, 100.4, 95.6, 82.6, 79.9, 76.0, 73.9, 72.8, 72.5, 72.4, 70.4, 70.2,
70.2, 69.4, 69.3, 68.8, 67.4, 61.9, 50.3, 45.8, 43.2, 41.8, 37.6, 34.4, 33.2, 31.9, 29.1, 29.0, 28.3, 24.7, 24.5,
23.5, 22.6, 20.9, 16.4, 14.6, 14.1, 12.5, 11.9. HRMS (ESI) m/z calcd for C82H111N6O25S2 [M+H]+
1643.7040, found: 1643.7048.
S17
BIOLOGY.
General Methods
All reagents were purchased from commercial sources listed below and were used without further
purification unless otherwise noted. DMEM high glucose culture medium was purchased from GE
Healthcare Life Sciences (SH30243.02). Protease inhibitor (0993C286), Resazurin sodium (0695),
Deoxycholic acid sodium salt (0613) and Glycerol(M152) were from Amresco. Tris(2-carboxyethyl)
phosphine (TCEP) was from Combi-blocks (OR-5119). High capacity streptavidin agarose beads and
Alamar Blue cell viability reagent (DAL1025) were from Thermo scientific (20361). Thiazoyl blue
tetrazolium bromide (00697), N-dodecyl-ß-D-maltoside (00127) and Sodium Dodecyl Sulfate (00270)
were from Chem Implex Intl Inc. Hepes (HB0264), Sodium orthovanadate (SB0869) and Micro BCA
protein assay kit (SK3061) were from Bio Basic. Tergitol NP40 was from Spectrum (T1279). Tris[(1-
benzyl-1H-1,2,3-triazol-4-yl) methyl] amine (TBTA) was from TCI Chemicals (T2993). Copper (II)
Sulfate Pentahydrate was from Alpha Aesar (14178). Sodium fluoride was from Avantor (3688-01). β-
Glycerophosphoric acid was from Acros (410991000). Fibronectin (Bovine) was from Akron Biotech
(AK8350-0001). Mountain medium was from Biotium (23001). Coverslip was from Electron Microscopy
Sciences (72296-08). ER-Tracker™ Blue-White DPX was from Invitrogen (E12353). 4',6-Diamidino-2-
Phenylindole, Dihydrochloride (DAPI) was from MP Biomedicals (157574). Expre35s35s protein
labelling mix (NEG072002MC) was from Perkin Elmer. Concanavalin A aragrose beads were from
Sigma-Aldrich (C7555). Antibody against Sec61α was from Abcam (ab15575). Anti-rabbit IgG-HRP
(C2818), anti-goat IgG-HRP (sc-2020) and antibody against actin (I-19) (sc-1616) were from Santa Cruz.
IRDye® 800CW StreptavidinSynergy™ (92632230) and Odyssey® Blocking Buffer (92640000) was
from Licor. Synergy™ H1 plate reader was from BioTek Instruments. Typhoon™ FLA 9500 was from
GE Healthcare Life Sciences. Leica TCS SP5 was from Leica Microsystems. Odyssey infrared imager
was from Licor. Synthetic mycolactone used in the pulse‐chase analysis of protein secretion in HepG2
cells was a gift from Yoshito Kishi, Harvard University, Cambridge, MA.
Experimental Procedures
Cytotoxicity Assay
Cell culture
Two human breast cancer cell lines (MDA-MB-231 and MCF7) and one human breast non-
tumorigenic epithelial cell line (MCF-10A) were maintained in a DMEM high glucose culture medium
S18
supplemented with 10% fetal bovine serum (FBS) and 2 mM L-glutamine. The cells were grown in a
humidified atmosphere of 5% CO2 and 95% air at 37 ºC. The culture medium was changed every 2–4
days depending on cell density. Cell cultures were passaged once a week using 0.125% trypsin-EDTA to
detach the cells from culture flasks/dishes.
The human colorectal cancer cell line HCT116 was maintained in McCoy’s 5A (modified) medium
supplemented with 10% fetal bovine serum (FBS). The human embryonic kidney (HEK293T and
HEK293 FRT TRex) cell line was maintained in DMEM high glucose culture medium supplemented with
10% fetal bovine serum (FBS).
HepG2 cells were cultured in DMEM supplemented with 10% fetal bovine serum and 2 mM L-
glutamine at 37°C under 5% CO2.
MTT Cytotoxicity Assay
Viable cells were counted with a hemocytometer immediately before each experiment. Experiments
were done in triplicate. First, 100 μL of MCF7 cells at the density of 50,000 cells/mL were seeded in a
96-well plate (5,000 cells/well), which was incubated at 37 ºC and 5% CO2 atmosphere for 24 h. The
compounds were dissolved in DMSO (dimethyl sulfoxide) to make drug stocks (10 mM). The stock
solutions were diluted with the complete DMEM high glucose culture medium to make a series of gradient
fresh working solutions right before each test. Subsequently, the cells were treated with 100 μL of the
freshly made gradient working solution in a total volume of 200 μL/well for 72 h. After that, the media
were discarded and 200 μL of the fresh complete medium containing 10% of MTT stock solution (5
mg/mL) was added to each well. The plate was then incubated at 37 ºC and 5% CO2 for 3 h. Next, 180
μL of the medium was discarded from each well, and 180 μL of DMSO was added to each well to dissolve
formazan crystals. Absorbance of formazan was detected by a microplate reader (BioTek Synergy H1)
at 570 nm with 650 nm as the reference wavelength. The percentage of viability compared to the negative
control (DMSO-treated cells) was determined. GraphPad Prism 6 software was used to make a plot of %
viability versus sample concentration and to calculate the concentration at which a compound exhibited
50% cytotoxicity (IC50).
AlamarBlue Cytotoxicity Assay
MDA-MB-231, MCF-10A and HCT116. Viable cells were counted with a hemocytometer
immediately before each experiment. Experiments were done in triplicate. First, 100 μL of MDA-MB-
231 cells or MCF-10A cells at the density of 50,000 cells/mL was seeded in a 96-well plate (5,000
cells/well) and then incubated at 37 ºC and 5% CO2 for 24 h. The compounds were dissolved in DMSO
(dimethyl sulfoxide) to make drug stocks (10 mM). The stock solutions were diluted with the complete
S19
DMEM high glucose culture medium to make a series of gradient fresh working solutions right before
each test. Subsequently, the cells were treated with 100 μL of the freshly made gradient working solution
in a total volume of 200 μL/well for 72 h. After that, the media were discarded and 200 μL of the fresh
complete medium containing 10% of AlamarBlue (resazurin) stock solution (3 mg/27.15mL) (5 mg/mL)
was added to each well. The plate was then incubated at 37 ºC and 5% CO2 for another 3 h. Emission of
each well at 620 nm was detected by a microplate reader (BioTek Synergy H1) at excitation 580 nm. For
HCT116 WT and mutant lines, cells were seeded at 1000 cells/ well in complete McCoys medium.
Compounds were diluted from a DMSO stock into culture medium as described above. Cells were treated
for 96 h, then viability measured with AlamarBlue, detected using the conditions described but with a
BMG Fluostar Optima Microplate Reader. The percentage of viability compared to the negative control
(DMSO-treated cells) was determined. GraphPad Prism 6 software was used to make a plot of % viability
versus sample concentration and to calculate the concentration at which a compound exhibited 50%
cytotoxicity (IC50).
HCT116 and HEK293T. Viable cells were counted using an automated cell counter (BioRad TC20)
before each experiment. Experiments were performed either in quadruplicate or duplicate sets. First 100µl
of HCT116 or HEK293T cells at a density of 25,000 cells/ml was seeded in black wall, clear bottom, 96-
well plates (2500 cells/well) (Viewplate-96 Perkin Elmer). HEK293 FRT Trex cells were seeded in
complete medium supplemented to 1ng/ml Doxycycline to induce expression of Sec61a constructs. Cells
were then incubated at 37 ºC and 5% CO2 for 24 h. The compounds were dissolved in DMSO to make
drug stocks (10 mM). The stock solutions were diluted with the complete DMEM or McCoy’s 5A culture
media supplemented with DMSO to make a series of gradient fresh working solutions at equal DMSO
percentage immediately prior to each test. Subsequently, the cells were treated with 25 μl of the freshly
made gradient in a total volume of 125 μl/well for 72 h. After that, the media were supplemented with
10% of AlamarBlue (resazurin) stock solution. The plate was then incubated at 37 ºC and 5% CO2 for
another 4 h. Emission of each well at 620 nm was detected by a microplate reader (Perkin Elmer EnSpire)
at excitation 580 nm. The percentage of viability compared to the negative control (DMSO-treated cells)
was determined. GraphPad Prism 7 software was used to make a plot of % viability versus sample
concentration and to calculate the concentration at which a compound exhibited 50% cytotoxicity (IC50).
Activity Based Protein Profiling
MDA-MB-231 cells were seeded in five groups of 6 cm petri dishes with 3 ml DMEM high glucose
culture and incubated at 37 ºC and 5% CO2 until 90% confluency. Each group was treated with fresh
medium containing 20 µM competitor or an equal volume of DMSO vehicle for 30 min at 37 ºC and 5%
S20
CO2, followed by addition of 0.2 µM probe or an equal volume of DMSO. After 1 h incubation at 37 ºC
and 5% CO2, the cells were harvested and washed with cold PBS for 3 times. The cells were then lysed
in 100 µl of lysis buffer 1 (50 mM Hepes, pH 9.0, 100 mM NaCl, 10% glycerol, 5 mM MgCl2, 1 mM
CaCl2, 0.2% NP40, 1x protease inhibitor) using ultrasound sonication. The supernatant of each group was
collected after centrifugation at 21,100 g and 4 ºC for 10 min, and the protein concentration was quantified
with a BCA kit. 10% SDS was then added into protein solution to make a final SDS concentration of
0.4%, and the solution was incubated at 95 ºC for 5 min. 1 µl TECP (100 mM stock solution in H2O) was
mixed with 1 µl TBTA (10 mM stock solution in a 4:1 ratio of t-butanol and DMSO), 1 µl CuSO4 (100
mM stock solution in H2O), and 1 µl rhodamine-azide (1 mM stock solution in DMSO). The mixed
solution was added into the previously prepared protein solution containing 0.25 mg protein. The total
volume was adjusted to 100 µl with the lysis buffer 1 to make a final protein concentration of 2.5 mg/ml,
and the click reaction was performed at room temperature for 1.5 h. Proteins in 15 µl of the reactant were
fractionated by an SDS-PAGE gel. The protein gel was scanned with a typhoon scanner (Typhoon™ FLA
9500) at 532 nm excitation with 700 mV gain setting and then stained with Coomassie blue.
Biotin Affinity Pulldown
Live Cell-based Pulldown
According to the experimental needs, MDA-MB-231 cells were seeded into 3-7 groups of 15-cm tissue-
culture dishes and incubated at 37 ºC and 5% CO2 until 90% confluency. Each group was treated with
fresh medium containing a competitor or an equal volume of DMSO vehicle for 30 min at 37 ºC and 5%
CO2, followed by addition of a probe or an equal volume of DMSO. After 1 h incubation at 37 ºC and 5%
CO2, the cells were harvested and washed with cold PBS for 3 times and lysed with the lysis buffer 2 (50
mM Tris, pH 7.4, 150 mM NaCl, 1% NP40, 0.25% sodium deoxycholate, 10 mM NaF, 10 mM β-
glycerophosphate, and 1 mM Na3VO4). The supernatant of each group was collected after centrifugation
at 4 ºC and 21,100 g for 10 min, and the protein concentration was quantified with the BCA kit. After
adjusting protein concentration with lysis buffer 2, each protein solution with equal amount of total protein
in the same volume was incubated with 20 µl of washed streptavidin bead at 4 ºC for 2 h with rotation.
After incubation, the beads were washed with washing buffer (50 mM Tris, pH 7.4, 150mM NaCl, 1%
NP40, 0.25% sodium deoxycholate, 10 mM NaF, 10 mM β-glycerophosphate, and 1 mM Na3VO4) for 3
times and boiled at 95ºC for 5 min in 2x sample buffer (125 mM Tris-HCl, pH 6.8, 20% glycerol, 2%
SDS, 2% β-mercaptoethanol and 0.02% bromophenol blue). After centrifugation at 21,100 g and 4 ºC for
1 min, the cleared eluted proteins were divided into two parts. One part was fractionated by a 12% SDS-
S21
PAGE and visualized by silver staining. The other part was analyzed by Western blotting, in which
proteins on the nitrocellulose membrane were visualized by either fluorescently labeled streptavidin or an
anti-Sec61α antibody.
Cell Lysate-based Pulldown
MDA-MB-231 cells from seven 15-cm tissue culture dishes were harvested and washed with PBS for 3
times and lysed in buffer 2 using ultrasound sonication. The supernatant of each group was collected after
centrifugation at 21,100 g and 4 ºC for 10 min, and the protein concentration was quantified with the BCA
kit and adjusted to be between 3.5–4 mg/mL with lysis buffer 2. Next, the cell lysate was equally divided
into 7 fractions, 1 ml/each. Each fraction was treated with either a competitor or an equal volume of
DMSO vehicle for 30 min at 4 ºC, followed by the addition of a probe or an equal volume of DMSO. All
the samples were then incubated at 4 ºC for 1 h. After incubation, 20 µl of streptavidin washed bead was
added to each sample followed by 2 h incubation at 4 ºC with rotation. The remaining procedures were
the same as the live cell-based pulldown (see above).
ER Microsome-based Pulldown
1 ul of Ipomoeassin F (2 mM stock concentration) or DMSO vehicle was added to 198.6 ul ER microsome
suspension in one of the three Eppendorf tubes to produce a final ipomoeassin F concentration of 10 µM
and two blank tubes. The resulting mixtures were incubated at 4 ºC for 30 min, followed by addition of
0.2 µM probe (final concentration) to two tubes or an equal volume of DMSO to a blank tube. After 1 h
incubation at 4 ºC, 10% DDM (n-dodecyl-ß-D-maltoside) was added to each tube to give the final
concentration of 1%, and the mixtures were further incubated at 4 ºC for another 1 h. The supernatant of
each group was collected after centrifugation at 4 ºC and 21,100 g for 10 min. 20 µl of washed streptavidin
beads was added to each sample followed by 2 h incubation at 4 ºC with rotation. After washing with
washing buffer (50 mM Tris, pH 7.4, and 150 mM NaCl) for 3 times, the bound proteins were eluted by
were boiling the beads in 20 µl of 2x sample buffer at 95 ºC for 5 min. The eluted proteins were analyzed
by SDS-PAGE or Western blotting as described above.
Mass Spectrometry (MS) Analysis and MS Data Processing
In the pulldown, the bound proteins were eluted via boiling the high capacity streptavidin agarose beads
in 1x sample buffer. The eluted proteins were fractionated with a 12% SDS-PAGE and the fractionated
S22
proteins were visualized by blue silver staining.8 The ~40 kDa protein band from the probe 7-enriched
sample and the corresponding regions of the gel for the negative control and the ipomoeassin F
competition sample were cut, in-gel digested, the resulting peptides were analyzed by an Orbitrap Fusion
mass spectrometer (Thermo Fisher Scientific), and the MS/MS spectra were searched against a composite
target-decoy UniProtkB human protein database (2018_02, 20317 entries) using Mascot (Matrix Science,
London, UK; version 2.5.1) as described previously.9-10 The parameters for database searching were as
follows: MS tolerance of 3.0 ppm and MS/MS tolerance of 0.5 Da; tryptic enzyme specificity with a
maximum of 2 missed cleavages; fixed modification, carbamidomethyl of cysteine; and variable
modifications, oxidation of methionine and acetylation of the N-terminus. Search results were further
processed by Scaffold software (version 4.8.6; Proteome Software, Portland, OR) for viewing protein and
peptide identification information. In the Scaffold analysis, peptide identifications were accepted if they
could achieve a false discovery rate (FDR) of <1.0% by the Scaffold Local FDR algorithm. Protein
identifications were accepted if they could achieve an FDR of <1.0% and contained at least 2 identified
peptides. Total spectral counts were exported from Scaffold and used to represent relative protein
abundance from different samples based on the positive, linear correlation between spectral count and the
abundance of a protein in complex samples.11-13 Contaminant proteins such as keratins were discarded.
For reliable representation of protein abundance, proteins identified with <4 spectra in the probe 7-
enriched sample were discarded.12
Cell Imaging
MDA-MB-231 cells were seeded on fibronectin-coated coverslips at 70% confluency in a 48-well plate
and cultured at 37 ºC and 5% CO2. After 24 h, culture medium was replaced with fresh medium containing
a 20 µM competitor or an equal volume of DMSO vehicle for 30 min at 37 ºC and 5% CO2, followed by
addition of 0.2 µM (final concentration) fluorescent probe 12. After 1 h incubation, the coverslips were
mounted onto a glass slide with a mounting medium with or without 0.5 µg/ml DAPI. The fluorescent
images were acquired with a confocal microscope (Leica TCS SP5) using 405 nm excitation and 562 nm
excitation. For the ER co-localization studies, 1 uM ER-Tracker™ Blue-White DPX was added to the
medium after 1 h incubation with probe 12. After 5 min incubation, the coverslips were mounted onto a
glass slide and the images were acquired as described above.
S23
Photo-Affinity Labeling.
CT7 photo-affinity labeling and click chemistry were done as previously described.14 Briefly, SRMs
(sheep rough microsomes, isolated as described in reference15) containing 100 nM Sec61 were treated
with 10 µM ipomoeassin F/mycolactone or 1% DMSO for 30 min at 0°C, followed by incubation with
100 nM CT7 for 10 min RT. Samples were then photolyzed for 10 min and crosslinked proteins were
detected by click chemistry, SDS-PAGE, and in-gel fluorescent scanning (Typhoon Trio, GE Healthcare).
In Vitro Membrane Translocation Assays.
DNA constructs
Unless otherwise stated, all cDNAs are of human origin and as previously described: CytB5OPG2,16
GypC,17 ppF (Saccharomyces cerevisiae),16 ppcecAOPG2 (Hyalophora cecropia),16 PPL (Bos
Taurus),16 Sec61OPG216 and VCAM1.17 Ii encodes residues 17 to 232 of the short form of human HLA
class II histocompatibility antigen gamma chain (Ii; P04232, isoform 2). Linear DNA templates were
generated by PCR and transcribed into RNA using T7 or SP6 polymerase (Promega).
In vitro translation and translocation into ER microsomes
Translation reactions (20 μL) were performed in nuclease-treated rabbit reticulocyte lysate (Promega) in
the presence of EasyTag EXPRESS 35S Protein Labelling Mix containing [35S] methionine (Perkin Elmer)
(0.533 MBq; 30.15 TBq/mmol). Amino acids minus methionine (Promega) were added to 25 M each,
5% (v/v) compound (concentration as indicated in figures, from stock solutions in DMSO), or an
equivalent volume of DMSO, and nuclease-treated ER microsomes (from stock with OD280 = 44/mL)
were added to 6.5% (v/v). Lastly, 10% volume of in vitro transcribed mRNA (from 200-1000 ng/μL
stocks) encoding the relevant precursor protein was added and translation reactions performed for 20 min
at 30°C. Following incubation with 0.1 mM puromycin for 5 min at 30°C to ensure translational
termination and ribosome release, membrane-associated fractions were isolated by centrifugation through
an 80 μL high-salt cushion (0.75 M sucrose, 0.5 M KOAc, 5 mM Mg(OAc)2, 50 mM Hepes-KOH, pH
7.9) at 100,000 x g for 10 min at 4°C in a TLA100 rotor (Beckmann). Membrane pellets were suspended
directly in SDS (sodium dodecyl sulphate) sample buffer (0.02% bromophenol blue, 62.5 mM, 4% (w/v)
SDS, 10% (v/v) glycerol, pH 7.6, 1 M dithiothreitol) and, where indicated, treated with 1,000 U of
Endoglycosidase H (New England Biolabs) for 30 min at 37°C prior to analysis. All samples were
denatured for 10 min at 70°C prior to resolution by SDS-PAGE (12% or 16% polyacrylamide gels, 120
S24
V, 120 min), gels were fixed (20% MeOH, 10% AcOH) for 5 min, dried (BioRad gel dryer) for 2 h at
65°C and, following exposure to a phosphorimaging plate for 24-72 h, radiolabelled products were
visualised using a Typhoon FLA-7000 (GE Healthcare).
Statistical Analysis
Quantitative analysis of Ii membrane insertion was performed using AIDA v.5.0 (Raytest
Isotopenmeβgeräte) whereby the ratio of the signal intensity for the 2Gly/0Gly forms was used as a read-
out to estimate efficiency of integration into the ER membrane. This value was then expressed relative to
the matched DMSO control in order to derive the mean relative ER insertion from three independent
experiments (n=3). Statistical significance of the effectiveness of Ipom-F analogues (RM one-way
ANOVA) was determined using Tukey’s multiple comparisons test. IC50 value estimates were determined
using non-linear regression to fit data to a curve of variable slope (four parameters) using the least squares
fitting method in which the bottom and top plateaus of the curve were defined as 0% and 100%
respectively. The differences between the estimated IC50 values for the three compounds analysed were
statistically significant (****) according to the extra-sum-of-squares F test. Statistical analyses and IC50
estimates were performed using Prism 7.0d (GraphPad) with statistical significance given as n.s., non-
significant and ****, P < 0.0001.
U2-OS secNLuc-ATZ Secretion and Cell Viability Assays
The secNLuc-ATZ assay has been described elsewhere.18 Briefly, U2-OS cells stably expressing
secNLuc-ATZ were cultured in McCoy’s 5A supplemented with 10% bovine growth serum (Hyclone
SH30540.03), 50 units/mL penicillin, and 50 µg/mL streptomycin and plated into 1536-well black, clear-
bottom treatment plates (Aurora Microplates #ABI121000A) at 1200 cells/well in 5 µL volume using a
Multidrop Combi dispenser (ThermoFisher). Cells were returned to 37 ̊ C for 1 h to adhere and compound
or vehicle control was added to wells using a 1536-well pin-tool transfer device (Wako Automation, San
Diego). Cells were incubated for 24 h and secNLuc-ATZ-containing media was transferred using an
ATS-100 acoustic dispenser (EDC Biosystems, Fremont, CA) from the treatment plate to a white, solid-
bottom 1536-well recipient plate (Greiner Bio One #789175-F) containing 3 µL/well of 1X PBS. Next,
recipient plates received 2 µL/well Nano-Glo luciferase assay reagent by BioRAPTR FRD (Beckman
Coulter), incubated for 10 min then imaged on a ViewLux uHTS Microplate Imager (PerkinElmer).
For the multiplexed cytotoxicity assay (24 h), after acoustic media transfer 3 µL/well CellTiter-Glo
reagent (Promega) was added by BioRAPTR FRD to the black, clear-bottom treatment plate containing
S25
treated cells, incubated for 10 min, and then imaged with a ViewLux. Imaging conditions for the
multiplexed cytotoxicity assay were: clear filter with 10 sec integration, slow speed, high sensitivity, and
2X binning.
SH-SY5Y GLuc Secretion and Cell Viability Assays.
SH-SY5Y human neuroblastoma cells stably expressing a constitutively secreted variant of Gaussia
luciferase (GLuc-‘No Tag’) were previously described.19 Cells were cultured in DMEM (4.5 g/L D-
glucose, 1 mM sodium pyruvate) supplemented with 10% bovine growth serum (Hyclone SH30540.03)
20 mM Hepes, 50 units/mL penicillin, and 50 µg/mL streptomycin. Cells were plated into white 1536-
well tissue culture treated plates (Corning cat. #7464) at 1,000 cells per well in a 5 µL volume using a
Multidrop Combi dispenser. Cells were returned to 37 ˚C for 4 h to adhere and compound or vehicle
control was added to wells using a 1536-well pin-tool transfer device (Wako Automation, San Diego).
Cells were incubated at 37 ˚C for 24 h and secreted luciferase was detected by adding 1 µL of
coelenterazine substrate to each well using a BioRAPTR FRD workstation. The substrate was prepared
from a Pierce Gaussia Luciferase Glow Assay Kit (ThermoFisher) by adding 1 volume of coelenterazine
to 99 volumes of Gaussia Glow Assay Buffer (final concentration of coelenterazine = 0.17X).
Luminescence was measured using a ViewLux microplate imager equipped with clear filters.
For cell viability assays, cells were cultured, plated, and treated with test compounds as outlined for
the secretion assays. 48 h after treatment, viability was assessed using a CellTiter-Glo assay by adding 3
µL of reagent to each well, incubating 15 min at room temperature, and reading luminescence using a
ViewLux microplate imager.
Metabolic Labelling Assays in HCT116 Cells
HCT116 cells were counted using an automated cell counter (BioRad TC20) immediately prior to each
experiment. 3 ml of HCT116 cells at a density of 0.166x106 cells/mL were seeded to 6-well plates (0.5x106
cells/well) and then incubated at 37 ºC and 5% CO2 for 24 h. The compounds were dissolved in DMSO
to make drug stocks (10 mM). The stock solutions were diluted with DMEM high glucose culture medium
lacking methionine and cysteine to make a fresh working solution immediately prior to each test. Cells
were washed twice and 1ml of fresh DMEM lacking methionine and cysteine was added to each well.
Subsequently, the cells were treated with 250 µl of compound solutions in a total volume of 1,250 µl/well.
The plate was then incubated at 37 ºC and 5% CO2 for 30 min before 100 µCi 35S-labelled
methionine/cysteine was added (Expre35s35s protein labelling mix). Plates were incubated for a further 30
S26
min, then media collected and cells harvested by scraping after four washes in ice-cold PBS. Total protein
was acquired by RIPA extraction from the cell pellet, cytoplasmic fraction was acquired by partial
permeabilization of the outer cell membranes with 0.15% Digitonin followed by centrifugation, secreted
proteins were acquired by TCA precipitation of the media.
Pulse‐Chase Analysis of Protein Secretion in HepG2 Cells
The secretion assay was performed using a pulse–chase approach as described previously.20 HepG2 cells
were treated with DMSO, 100 nM compound 8, 100 nM Ipom-F or 31.3 ng/μl mycolactone for 13.5 h
before initiating the experiment, whereas pre-treatment with bortezomib 100 nM (Stratech) or 2.5 μg/ml
brefeldin A were performed for 2 h and 1 h, respectively. Cells were starved in Met‐ and Cys‐free DMEM
(Invitrogen) for 20 min at 37°C. Labeling was initiated by addition of fresh starvation medium containing
22 mCi/ml [35S]Met and [35S]Cys protein labeling mix (PerkinElmer; specific activity >1,000 Ci/mmol)
for 15 min. After washing with PBS, cells were incubated in serum‐free DMEM supplemented with
excess unlabeled L‐Met and L‐Cys (2 mM final concentration) for 2 h. The compounds were included
throughout the starvation, pulse and chase. Secreted proteins in the media were recovered by precipitation
with 13% trichloroacetic acid, washed in acetone and dissolved in 2x sample buffer. Cells were solubilized
with Triton X‐100 lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1% TX-100, 1 mM EDTA, 1 mM
EGTA) supplemented with protease inhibitor cocktail and phenylmethylsulfonyl fluoride. Equal amounts
of precipitated proteins in the media and clarified lysates were analyzed by SDS‐PAGE and
phosphorimaging (FLA‐3000; Fuji).
T Cell Assays
Jurkat E6.1 (ATCC TIB-152TM) T cells were cultured in RPMI GlutamaxTM (Life Technologies),
supplemented with 10% heat-inactivated fetal calf serum (FCS) (Invitrogen) and penicillin/streptomycin
(100 U/ml, 100 µg/ml). Cells were treated with Ipomoeassin F or mycolactone for 24 h, then pelleted and
stained with FITC-conjugated anti-human CD62L (clone DREG-56 (BioLegend #304804) at 500 ng/ml)
during 40 min at 4°C. Fluorescence signals were acquired on an Accuri CD (BD) and analyzed with the
FlowJo X software. Immunosuppressive activity on T cells was measured through the inhibition of IL-2
production by Jurkat T cells incubated with mycolactone for 1 h prior to 24 h of activation with
phytohemagglutinin-ionomycin, as described.21 In both assays, blanks corresponded to cells treated with
S27
the vehicle volume of the highest concentration of sample tested (DMSO for IpoF; EtOH for
mycolactone).
S28
Figures:
Cell Cytotoxicity Assay
A.
B.
S29
C.
1 1 0 1 0 0 1 0 0 0 1 0 0 0 0
2 0
4 0
6 0
8 0
1 0 0
H C T 1 1 6
D ru g n M
% c
ell
via
bil
ity
C T 8
8
1 5
1 4
Ip o m o e a s s in F
Figure S1. Cytotoxicity in MDA-MB-231 and HCT116 cells. (A) Cell viability curves of analogues 3,
5, 7 and ipomoeassin F (72 h). (B) Cell viability curves of analogues 10, 11, 12 and ipomoeassin F (72
h). (C) HCT116 colon carcinoma cells were treated with increasing concentrations of analogues 8, 14, 15,
CT8 and ipomoeassin F or carrier (0.1% DMSO) and cell viability was assessed at 72 h by the Alamar
Blue assay (mean ± S.D., n = 4).
S30
Activity Based Protein Profiling
A.
Figure S2. Click reaction performed after incubating cells with competitor and its corresponding
alkyne probe. (A) Fluorescent image by a Typhoon scanner. (B) Coomassie Blue stain of the gel. (The
arrow indicates a positive result for probe X, where a protein band appeared in lane 4 but was absent in
lanes 1 and 5.)
B.
S31
Biotin Affinity Pulldown
A.
C.
Figure S3. Biotin affinity pulldown with probes 5 and 7. Cells were incubated with ipomoeassin F and
probes, then lysed and subjected to biotin affinity pulldown with streptavidin beads. (A) SDS-PAGE and
silver staining analysis of pulldown samples from live cells. (B) SDS-PAGE analysis of pulldown samples
B.
1 2 3 4 5
Ipomoeassin F ─ ─ + ─ +
5 ─ + + ─ ─
7 ─ ─ ─ + +
S32
from live cells using fluorescently labeled streptavidin. (C) SDS-PAGE and silver staining analysis of
pulldown samples from cell lysates. Arrows indicate the ~40 kDa protein position.
A.
Figure S4. Biotin affinity pulldown with probe 7 or 9 in the presence of the competitor ipomoeassin
F or 8. (A) Pulldown experiment with 10 nM 7 and 50 nM ipomoeassin F. (B) Pulldown experiments
with either 20 nM 7 and 1 μM 8 or 500 nM 9 without competition. Arrows indicate the ~40 kDa protein
position.
B.
1 2 3 4
8 ─ ─ + ─
7 ─ + + ─
9 ─ ─ ─ +
40kd
100kd
S33
Cell Imaging
A.
12, 0.2 μM DAPI Merged
B.
12 (0.2 μM) + Ipom-F (20 μM) DAPI Merged
C.
12 (0.2 μM) + 8 (20 μM) DAPI Merged
Figure S5. Live cell imaging-based competition experiments. (A) Cells were treated with 1 μM 12. (B)
Cells were treated with ipomoeassin F (100-fold) and 12; (C) Cells were treated with 8 (100-fold) and 12.
S34
Pulldown in ER Microsomes.
A. B.
Figure S6. Biotin affinity pulldown with probe 7 in ER microsomes. (A) The proteins were visualized
by silver staining. The arrow indicates the positive result. (B) The proteins were transferred onto a
nitrocellulose membrane and visualized by western blot using an anti-Sec61α antibody.
Sec61α
S35
Figure S7. Membrane topology schematic.
S36
In Vitro Membrane Insertion Assays of a Model Type II Protein.
Figure S8. Comparison of the effectiveness of ipomoeassin F and related structural variants on the
membrane insertion of Ii. (A) Phosphorimage of the type II integral membrane protein Ii synthesized
in the presence of ipomoeassin F (Ipom-F), the open-chain analogue 13, inactive analogues 14 and 3 and
mycolactone (Myco). (B) Quantitative analysis of A whereby the efficiency of Ii membrane integration
was estimated using the ratio of signal intensity for the 2Gly/0Gly forms and expressed relative to the
control. (C) Ipom-F, 13 and Myco were included at 1000-5 nM concentrations during the translation of
Ii and (D) quantitative analysis of the efficiency of Ii membrane integration was performed as described
in B. IC50 values were estimated using non-linear regression as described in materials and methods.
Each phosphorimage depicts experiments performed using a translation master mix, to which different
compounds and Ii mRNA were then added prior to translation and analysis of the membrane-associated
fraction from three independent experiments (n=3).
S37
Pulse‐Chase Analysis of Protein Secretion in HepG2 Cells
Figure S9. Ipomoeassin-F inhibits protein secretion. HepG2 cells were treated with DMSO, 100 nM
compound 8, 100 nM Ipom-F, or 42 nM mycolactone (Myco) for 13.5 h. HepG2 cells were treated with
100 nM bortezomib (BZ) for 2 h or 2.5 μg/ml BFA for 1 h. Newly synthesized proteins were pulse-labeled
for 15 min and then chased in medium containing unlabeled Met and Cys for 2 h. The compounds were
included throughout the pulse and chase. At the end of chase, radiolabeled proteins in media (upper panel)
and whole cell lysates (middle panel) were resolved by SDS‐PAGE and visualized by phosphorimaging.
Whole cell lysates of treated cells were also analyzed by immunoblotting with anti-α-tubulin antibodies
(lower panel). Molecular masses are in kDa.
S38
Table S1. Spectral Counts for Proteins Identified in ~40 kDa Bands
Identified Proteins Accession
Number
Molecular
Weight
Spectral count DMSO Probe
7
7 +
Ipom-
F
delta
(Probe/probe
+ Ipom-F)
Vimentin VIME_HUMAN 54 kDa 108 88 126 0.70
Protein transport protein
Sec61 subunit alpha
isoform 1 S61A1_HUMAN 52 kDa 11 80 16 5.00
Pyruvate carboxylase,
mitochondrial PYC_HUMAN 130 kDa 97 68 109 0.62
Poly(rC)-binding protein
1 PCBP1_HUMAN 37 kDa 45 45 82 0.55
mRNA export factor RAE1L_HUMAN 41 kDa 50 41 53 0.77
Actin, cytoplasmic 1 ACTB_HUMAN 42 kDa 45 40 54 0.74
Stomatin-like protein 2,
mitochondrial STML2_HUMAN 39 kDa 42 37 68 0.54
Enoyl-CoA delta
isomerase 2,
mitochondrial ECI2_HUMAN 44 kDa 34 36 51 0.71
Plectin PLEC_HUMAN 532 kDa 30 29 65 0.45
Methylcrotonoyl-CoA
carboxylase subunit
alpha, mitochondrial MCCA_HUMAN 80 kDa 42 28 44 0.64
26S proteasome
regulatory subunit 10B PRS10_HUMAN 44 kDa 27 25 45 0.56
Acetyl-CoA
acetyltransferase,
mitochondrial THIL_HUMAN 45 kDa 24 25 37 0.68
Core histone macro-
H2A.1 H2AY_HUMAN 40 kDa 30 24 50 0.48
Heterogeneous nuclear
ribonucleoprotein A3 ROA3_HUMAN 40 kDa 35 24 47 0.51
Methylcrotonoyl-CoA
carboxylase beta chain,
mitochondrial MCCB_HUMAN 61 kDa 36 23 37 0.62
DnaJ homolog subfamily
B member 11 DJB11_HUMAN 41 kDa 25 21 28 0.75
Fructose-bisphosphate
aldolase A ALDOA_HUMAN 39 kDa 55 21 37 0.57
Succinate--CoA ligase
[GDP-forming] subunit
beta, mitochondrial SUCB2_HUMAN 47 kDa 25 21 28 0.75
Poly(rC)-binding protein
2 PCBP2_HUMAN 39 kDa 23 20 34 0.59
Cytosolic acyl coenzyme
A thioester hydrolase BACH_HUMAN 42 kDa 23 19 23 0.83
Transmembrane protein
43 TMM43_HUMAN 45 kDa 19 18 21 0.86
S39
Galactokinase GALK1_HUMAN 42 kDa 15 17 29 0.59
Leukocyte elastase
inhibitor ILEU_HUMAN 43 kDa 35 17 34 0.50
Acetyl-CoA carboxylase
1 ACACA_HUMAN 266 kDa 32 16 34 0.47
Caveolae-associated
protein 1 CAVN1_HUMAN 43 kDa 17 16 23 0.70
Perilipin-3 PLIN3_HUMAN 47 kDa 16 16 23 0.70
RNA-binding protein 4 RBM4_HUMAN 40 kDa 17 16 27 0.59
28S ribosomal protein
S31, mitochondrial RT31_HUMAN 45 kDa 14 15 21 0.71
Heterogeneous nuclear
ribonucleoprotein D0 HNRPD_HUMAN 38 kDa 23 15 36 0.42
Inhibitor of nuclear
factor kappa-B kinase-
interacting protein IKIP_HUMAN 39 kDa 14 15 19 0.79
Protein CYR61 CYR61_HUMAN 42 kDa 17 15 10 1.50
Tubulin beta chain TBB5_HUMAN 50 kDa 16 15 25 0.60
Tubulin beta-4B chain TBB4B_HUMAN 50 kDa 16 15 25 0.60
Erlin-2 ERLN2_HUMAN 38 kDa 16 14 20 0.70
Guanine nucleotide-
binding protein subunit
alpha-13 GNA13_HUMAN 44 kDa 15 14 18 0.78
Macrophage-capping
protein CAPG_HUMAN 38 kDa 22 14 24 0.58
Serpin B6 SPB6_HUMAN 43 kDa 20 14 28 0.50
Endophilin-B1 SHLB1_HUMAN 41 kDa 12 13 17 0.76
Mitogen-activated
protein kinase 1 MK01_HUMAN 41 kDa 16 13 20 0.65
Prelamin-A/C LMNA_HUMAN 74 kDa 19 13 25 0.52
Eukaryotic translation
initiation factor 3 subunit
M EIF3M_HUMAN 43 kDa 16 12 21 0.57
Mitochondrial import
receptor subunit TOM40
homolog TOM40_HUMAN 38 kDa 14 12 19 0.63
Protein FAM50A FA50A_HUMAN 40 kDa 12 12 22 0.55
Reticulocalbin-1 RCN1_HUMAN 39 kDa 6 12 16 0.75
RNA-binding protein 4B RBM4B_HUMAN 40 kDa 0 12 23 0.52
Septin-2 SEPT2_HUMAN 41 kDa 20 12 21 0.57
26S proteasome non-
ATPase regulatory
subunit 13 PSD13_HUMAN 43 kDa 12 11 28 0.39
28S ribosomal protein
S29, mitochondrial RT29_HUMAN 46 kDa 11 11 19 0.58
Elongation factor 1-
alpha 1
EF1A1_HUMAN
(+1) 50 kDa 11 11 21 0.52
Heterogeneous nuclear
ribonucleoproteins
A2/B1 ROA2_HUMAN 37 kDa 12 11 22 0.50
S40
Heterogeneous nuclear
ribonucleoproteins
C1/C2 HNRPC_HUMAN 34 kDa 13 11 17 0.65
Immunoglobulin-binding
protein 1 IGBP1_HUMAN 39 kDa 8 11 15 0.73
Nuclear pore complex
protein Nup153 NU153_HUMAN 154 kDa 11 11 21 0.52
Protein quaking QKI_HUMAN 38 kDa 10 11 17 0.65
28S ribosomal protein
S35, mitochondrial RT35_HUMAN 37 kDa 10 10 15 0.67
Activator of 90 kDa heat
shock protein ATPase
homolog 1 AHSA1_HUMAN 38 kDa 7 10 14 0.71
ATP synthase subunit
alpha, mitochondrial ATPA_HUMAN 60 kDa 20 10 28 0.36
Centrosomal protein of
170 kDa CE170_HUMAN 175 kDa 11 10 8 1.25
DNA-directed RNA
polymerases I and III
subunit RPAC1 RPAC1_HUMAN 39 kDa 14 10 18 0.56
Lamina-associated
polypeptide 2, isoform
alpha LAP2A_HUMAN 75 kDa 8 10 12 0.83
60 kDa heat shock
protein, mitochondrial CH60_HUMAN 61 kDa 6 9 16 0.56
Actin-related protein 2 ARP2_HUMAN 45 kDa 22 9 18 0.50
Aspartate
aminotransferase,
mitochondrial AATM_HUMAN 48 kDa 23 9 18 0.50
Developmentally-
regulated GTP-binding
protein 1 DRG1_HUMAN 41 kDa 13 9 15 0.60
DnaJ homolog subfamily
B member 1 DNJB1_HUMAN 38 kDa 5 9 16 0.56
DnaJ homolog subfamily
B member 6 DNJB6_HUMAN 36 kDa 10 9 10 0.90
Hemoglobin subunit
gamma-1
HBG1_HUMAN
(+1) 16 kDa 13 9 3 3.00
Lamina-associated
polypeptide 2, isoforms
beta/gamma LAP2B_HUMAN 51 kDa 7 9 12 0.75
Lysophospholipid
acyltransferase 7 MBOA7_HUMAN 53 kDa 7 9 10 0.90
Nuclear distribution
protein nudE homolog 1 NDE1_HUMAN 39 kDa 8 9 16 0.56
Hemoglobin subunit beta HBB_HUMAN 16 kDa 12 8 6 1.33
Heterogeneous nuclear
ribonucleoprotein M HNRPM_HUMAN 78 kDa 7 8 19 0.42
S41
HLA class I
histocompatibility
antigen, A-2 alpha chain 1A02_HUMAN 41 kDa 8 8 16 0.50
Mevalonate kinase KIME_HUMAN 42 kDa 10 8 15 0.53
Mitotic checkpoint
protein BUB3 BUB3_HUMAN 37 kDa 8 8 15 0.53
Nucleolar protein 7 NOL7_HUMAN 29 kDa 9 8 10 0.80
Nucleolysin TIAR TIAR_HUMAN 42 kDa 9 8 13 0.62
Protein KTI12 homolog KTI12_HUMAN 39 kDa 4 8 12 0.67
Protein SGT1 homolog SGT1_HUMAN 41 kDa 8 8 22 0.36
Transcriptional activator
protein Pur-alpha PURA_HUMAN 35 kDa 15 8 19 0.42
Tropomodulin-3 TMOD3_HUMAN 40 kDa 15 8 19 0.42
40S ribosomal protein
SA RSSA_HUMAN 33 kDa 7 7 13 0.54
Cytoskeleton-associated
protein 4 CKAP4_HUMAN 66 kDa 5 7 12 0.58
Erlin-1 ERLN1_HUMAN 39 kDa 8 7 9 0.78
Mitochondrial inner
membrane protein
OXA1L OXA1L_HUMAN 49 kDa 8 7 9 0.78
Polymerase delta-
interacting protein 3 PDIP3_HUMAN 46 kDa 8 7 11 0.64
Rab11 family-interacting
protein 5 RFIP5_HUMAN 70 kDa 7 7 10 0.70
T-complex protein 1
subunit delta TCPD_HUMAN 58 kDa 14 7 13 0.54
tRNA methyltransferase
10 homolog C TM10C_HUMAN 47 kDa 13 7 14 0.50
Ubiquitin-60S ribosomal
protein L40 RL40_HUMAN 15 kDa 7 7 12 0.58
V-type proton ATPase
subunit C 1 VATC1_HUMAN 44 kDa 10 7 15 0.47
28S ribosomal protein
S5, mitochondrial RT05_HUMAN 48 kDa 7 6 15 0.40
Activity-dependent
neuroprotector
homeobox protein ADNP_HUMAN 124 kDa 6 6 9 0.67
Aspartate
aminotransferase,
cytoplasmic AATC_HUMAN 46 kDa 21 6 16 0.38
COP9 signalosome
complex subunit 4 CSN4_HUMAN 46 kDa 7 6 9 0.67
Eukaryotic translation
initiation factor 3 subunit
H EIF3H_HUMAN 40 kDa 6 6 17 0.35
Ferrochelatase,
mitochondrial HEMH_HUMAN 48 kDa 9 6 13 0.46
Filamin-B FLNB_HUMAN 278 kDa 8 6 10 0.60
S42
Glutaminyl-peptide
cyclotransferase-like
protein QPCTL_HUMAN 43 kDa 7 6 6 1.00
Heat shock protein HSP
90-beta HS90B_HUMAN 83 kDa 14 6 15 0.40
Hemoglobin subunit
alpha HBA_HUMAN 15 kDa 10 6 2 3.00
Mannose-1-phosphate
guanyltransferase beta GMPPB_HUMAN 40 kDa 6 6 13 0.46
Nucleolin NUCL_HUMAN 77 kDa 5 6 6 1.00
Proline-rich protein 11 PRR11_HUMAN 40 kDa 11 6 14 0.43
Protein arginine N-
methyltransferase 1 ANM1_HUMAN 42 kDa 11 6 14 0.43
Short/branched chain
specific acyl-CoA
dehydrogenase,
mitochondrial ACDSB_HUMAN 47 kDa 4 6 11 0.55
SUMO-activating
enzyme subunit 1 SAE1_HUMAN 38 kDa 11 6 12 0.50
Ubiquitin-conjugating
enzyme E2 Z UBE2Z_HUMAN 38 kDa 8 6 10 0.60
Alpha-actinin-1 ACTN1_HUMAN 103 kDa 3 5 8 0.63
Alpha-actinin-4 ACTN4_HUMAN 105 kDa 11 5 11 0.45
Choline-phosphate
cytidylyltransferase A PCY1A_HUMAN 42 kDa 2 5 5 1.00
DnaJ homolog subfamily
B member 2 DNJB2_HUMAN 36 kDa 5 5 3 1.67
Fructose-bisphosphate
aldolase C ALDOC_HUMAN 39 kDa 21 5 13 0.38
GDP-mannose 4,6
dehydratase GMDS_HUMAN 42 kDa 9 5 11 0.45
Heat shock cognate 71
kDa protein HSP7C_HUMAN 71 kDa 14 5 19 0.26
Hsp70-binding protein 1 HPBP1_HUMAN 39 kDa 6 5 8 0.63
Matrin-3 MATR3_HUMAN 95 kDa 2 5 7 0.71
Minor histocompatibility
antigen H13 HM13_HUMAN 41 kDa 6 5 7 0.71
Nucleoside diphosphate
kinase 7 NDK7_HUMAN 42 kDa 9 5 10 0.50
Probable ATP-dependent
RNA helicase DDX5 DDX5_HUMAN 69 kDa 6 5 11 0.45
Probable
methyltransferase-like
protein 15 MET15_HUMAN 46 kDa 5 5 10 0.50
Propionyl-CoA
carboxylase alpha chain,
mitochondrial PCCA_HUMAN 80 kDa 11 5 13 0.38
Propionyl-CoA
carboxylase beta chain,
mitochondrial PCCB_HUMAN 58 kDa 7 5 6 0.83
S43
Replication factor C
subunit 2 RFC2_HUMAN 39 kDa 4 5 25 0.20
RNA 3'-terminal
phosphate cyclase RTCA_HUMAN 39 kDa 6 5 9 0.56
Serine/threonine-protein
kinase MARK2 MARK2_HUMAN 88 kDa 3 5 4 1.25
Serpin B9 SPB9_HUMAN 42 kDa 11 5 9 0.56
Twinfilin-2 TWF2_HUMAN 40 kDa 10 5 10 0.50
7-dehydrocholesterol
reductase DHCR7_HUMAN 54 kDa 3 4 2 2.00
Alpha-2-macroglobulin
receptor-associated
protein AMRP_HUMAN 41 kDa 2 4 4 1.00
Apoptosis-inducing
factor 2 AIFM2_HUMAN 41 kDa 5 4 13 0.31
ATPase ASNA1 ASNA_HUMAN 39 kDa 3 4 5 0.80
BRISC and BRCA1-A
complex member 1 BABA1_HUMAN 37 kDa 6 4 11 0.36
cAMP-dependent protein
kinase catalytic subunit
alpha KAPCA_HUMAN 41 kDa 16 4 13 0.31
Cdc42 effector protein 1 BORG5_HUMAN 40 kDa 6 4 9 0.44
Desmocollin-1 DSC1_HUMAN 100 kDa 0 4 0 #DIV/0!
Filamin-A FLNA_HUMAN 281 kDa 7 4 8 0.50
Flap endonuclease 1 FEN1_HUMAN 43 kDa 3 4 7 0.57
Glucose-fructose
oxidoreductase domain-
containing protein 1 GFOD1_HUMAN 43 kDa 4 4 5 0.80
HAUS augmin-like
complex subunit 4 HAUS4_HUMAN 42 kDa 1 4 11 0.36
Heat shock 70 kDa
protein 1A
HS71A_HUMAN
(+1) 70 kDa 6 4 7 0.57
HLA class I
histocompatibility
antigen, alpha chain E HLAE_HUMAN 40 kDa 5 4 4 1.00
HLA class I
histocompatibility
antigen, B-41 alpha
chain 1B41_HUMAN 41 kDa 2 4 4 1.00
Isovaleryl-CoA
dehydrogenase,
mitochondrial IVD_HUMAN 46 kDa 8 4 8 0.50
MAP7 domain-
containing protein 3 MA7D3_HUMAN 98 kDa 4 4 8 0.50
Monocarboxylate
transporter 4 MOT4_HUMAN 49 kDa 4 4 7 0.57
Muscleblind-like protein
1 MBNL1_HUMAN 42 kDa 3 4 5 0.80
Pinin PININ_HUMAN 82 kDa 2 4 5 0.80
S44
Polyhomeotic-like
protein 2 PHC2_HUMAN 91 kDa 4 4 4 1.00
RelA-associated
inhibitor IASPP_HUMAN 89 kDa 1 4 7 0.57
Serum albumin ALBU_HUMAN 69 kDa 4 4 4 1.00
Stress-70 protein,
mitochondrial GRP75_HUMAN 74 kDa 5 4 6 0.67
Synaptic vesicle
membrane protein VAT-
1 homolog VAT1_HUMAN 42 kDa 3 4 10 0.40
Testis-expressed protein
264 TX264_HUMAN 34 kDa 7 4 10 0.40
Transcription factor jun-
B JUNB_HUMAN 36 kDa 4 4 9 0.44
Transformer-2 protein
homolog beta TRA2B_HUMAN 34 kDa 5 4 6 0.67
UBX domain-containing
protein 1 UBXN1_HUMAN 33 kDa 4 4 5 0.80
S45
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download fileview on ChemRxivSupporting Info_1.pdf (1.76 MiB)
S1
Supporting Information for:
Ipomoeassin F Binds Sec61α to Inhibit Protein Translocation
Guanghui Zong,a,b,† Zhijian Hu,a,† Sarah O’Keefe,c,# Dale Tranter, d,e,# Michael J. Iannotti,f,# Ludivine
Baron,g,h,# Belinda Hall,i,# Katherine Corfield,i, # Anja O. Paatero,d,e,# Mark J. Henderson,f,# Peristera
Roboti,c,# Jianhong Zhou,j Xianwei Sun,a,k Mugunthan Govindarajan,a,l Jason Rohde,f Nicolas
Blanchard,m Rachel Simmonds,i,* James Inglese,f,* Yuchun Du,j,* Caroline Demangel,g,h,* Stephen
High,c,* Ville O. Paavilainen,d,e,* and Wei Q. Shia,*
a Department of Chemistry and Biochemistry, and j Department of Biological Sciences, J. William Fulbright College
of Arts & Science, University of Arkansas, Fayetteville, Arkansas, 72701, USA
b Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, United
States
c School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester,
M13 9PT, United Kingdom.
d University of Helsinki, HiLIFE, and e Institute of Biotechnology, Helsinki, Finland
f National Center for Advancing Translational Sciences, National Institutes of Health, Rockville,
Maryland 20850, USA.
g Institut Pasteur, Immunobiology of Infection Unit, 75015 and h INSERM, U1221, 75005 Paris, France
i Department of Microbial Sciences, School of Biosciences and Medicine, University of Surrey, Guildford, Surrey
GU2 7XH, United Kingdom
k Department of Radiology, Baylor College of Medicine, Houston, TX, 77030, USA
l Emory Institute for Drug Development, Emory University, 954 Gatewood Road, Atlanta, Georgia, 30329, USA
m Université de Haute-Alsace, Université de Strasbourg, CNRS, LIMA, UMR 7042, 68000 Mulhouse,
France
† Equal contribution as first-author
# Equal contribution as second-author
* Corresponding authors
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S5
S6
S7
S8
S9
S10
xws-cl-2018-01-22f 400 H-NMR 400
8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm
2.494
4.023
6.015
6.806
7.270
7.666
3.27
3.13
0.68
1.00
0.82
S11
xws-cl-2018-01-22f in CDCl3
240 220 200 180 160 140 120 100 80 60 40 20 0 ppm
30.270
56.740
76.681
76.998
77.316
108.578
110.704
131.862
146.606
150.980
199.832
S12
xws-cl-2016-10-20c 400 H-NMR 400
8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm
0.088
0.885
2.489
3.932
3.937
3.952
4.018
4.030
4.043
4.187
4.200
4.213
6.753
7.270
7.693
7.76
13.47
3.19
4.32
2.82
2.79
1.00
1.05
S13
xws-cl-2016-10-23a 400 H-NMR 400
9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm
0.090
0.095
0.889
1.767
1.783
3.996
4.012
4.025
4.038
4.159
4.172
4.185
6.512
6.528
6.544
6.560
7.114
7.333
7.356
7.681
8.235
8.258
6.47
10.89
2.85
3.50
1.74
5.93
2.32
1.00
1.09
2.07
1.06
2.92
S14
xws-cl-2017-10-17b 400 H-NMR 400
8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm
0.092
0.098
0.896
1.657
1.673
3.008
3.288
3.358
3.379
3.391
3.403
3.446
3.462
3.470
3.479
3.524
3.541
3.558
3.569
3.578
3.649
3.660
3.669
3.675
3.684
3.689
3.696
3.707
3.718
3.958
4.005
4.018
4.031
4.144
4.157
4.170
4.213
4.226
4.230
4.241
4.251
4.261
4.272
4.285
4.303
6.370
6.385
6.401
6.417
6.496
6.509
7.070
7.270
7.661
8.238
5.88
10.47
3.66
7.62
2.05
5.45
3.19
23.74
3.34
2.28
2.32
4.19
1.00
2.65
1.03
1.02
2.80
S15
xws-cl-2017-10-17d 400 H-NMR 400
8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm
1.658
1.674
1.753
2.323
2.338
2.353
3.375
3.387
3.400
3.639
3.653
3.672
3.685
3.701
3.714
3.971
3.988
4.002
4.012
4.170
4.181
4.192
4.206
4.218
4.236
4.248
4.262
4.275
4.292
6.363
6.379
6.395
6.411
7.092
7.270
7.634
3.38
1.00
2.19
14.24
3.23
2.35
2.21
2.48
0.94
1.00
1.02
S16
xws-cl-2017-10-17d 13C in CDCl3
220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm
22.080
50.655
56.440
60.960
67.049
68.849
70.020
70.505
70.625
70.652
71.062
72.093
76.682
77.000
77.318
108.197
109.721
133.289
139.665
147.194
154.043
154.278
S17
xws-cl-2017-10-18b 400 H-NMR 400
8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm
1.452
1.471
1.489
1.507
1.631
1.650
1.667
1.687
1.703
1.732
1.749
1.766
2.243
2.262
2.280
2.729
2.760
2.942
2.954
2.973
2.986
3.228
3.239
3.249
3.353
3.392
3.399
3.405
3.417
3.430
3.564
3.577
3.589
3.645
3.665
3.686
3.699
3.711
3.721
3.726
3.739
4.007
4.263
4.275
4.285
4.297
4.329
4.340
4.349
4.360
4.450
4.519
4.531
4.539
4.550
6.301
6.317
7.204
7.696
2.59
9.09
2.29
1.22
1.27
1.41
5.11
4.45
23.60
3.36
2.16
3.27
2.07
1.16
1.00
1.08
0.98
0.48
S18
xws-cl-2017-10-18b 13C in CDCl3
210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm
29.649
29.892
36.883
40.432
41.202
41.878
48.510
48.723
48.936
49.149
49.362
49.574
49.787
51.934
57.122
57.224
61.757
63.482
64.368
68.412
69.442
70.143
70.868
71.209
71.298
71.330
71.590
71.601
71.666
71.734
73.319
109.868
110.944
134.178
141.428
148.876
155.860
155.916
158.876
166.162
176.288
S19
ZGH-Ipom-3-290-171022-A in CDCl3
0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0 ppm
0.872
0.890
0.906
1.163
1.179
1.243
1.260
1.278
1.290
1.303
1.318
1.435
1.513
1.639
1.655
1.675
1.691
1.724
1.749
2.171
2.726
2.757
3.420
3.433
3.565
3.571
3.582
3.591
3.598
3.605
3.627
3.651
3.663
3.674
3.864
3.876
3.888
3.944
4.276
4.456
4.464
4.544
4.556
5.128
5.151
5.228
6.178
6.218
6.977
6.999
7.061
7.270
7.430
7.452
7.625
7.842
3.43
3.48
21.21
17.25
1.78
5.67
8.15
1.31
1.34
4.99
31.91
3.51
3.15
1.18
5.88
1.32
5.46
2.21
1.04
0.95
2.29
3.26
1.16
0.83
1.00
1.00
0.97
0.86
1.04
2.12
1.03
2.08
1.05
1.05
0.93
S20
ZGH-Ipom-3-290-171022-A 13C in CDCl3
2030405060708090100110120130140150160170180190200210 ppm
11.975
14.077
14.545
16.336
20.949
22.046
22.628
23.502
24.501
24.734
25.440
28.111
28.318
29.146
31.901
33.207
34.376
37.568
39.099
40.455
41.824
50.341
55.371
56.498
60.140
61.758
62.003
66.979
68.793
68.842
69.351
70.050
70.196
70.386
70.480
72.072
72.358
72.580
76.683
77.000
77.202
77.318
100.371
115.144
124.157
127.237
127.645
129.972
139.445
139.653
143.330
154.046
160.261
163.474
171.667
171.701
S21
1.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0 ppm
1
2
3
4
5
6
7
8
ppmCurrent Data ParametersNAME ZGH-Ipom-3-290-171022-AEXPNO 13PROCNO 1
F2 - Acquisition ParametersDate_ 20171029Time 1.19INSTRUM spectPROBHD 5 mm PABBO BB-PULPROG cosygpqfTD 2048SOLVENT CDCl3NS 64DS 8SWH 8012.820 HzFIDRES 3.912510 HzAQ 0.1277952 secRG 181DW 62.400 usecDE 6.50 usecTE 295.4 KD0 0.00000300 secD1 1.50000000 secD13 0.00000400 secD16 0.00020000 secIN0 0.00012480 sec
======== CHANNEL f1 ========SFO1 400.1520008 MHzNUC1 1HP0 12.50 usecP1 12.50 usecPLW1 20.00000000 W
====== GRADIENT CHANNEL =====GPNAM[1] SMSQ10.100GPZ1 10.00 %P16 1000.00 usec
F1 - Acquisition parametersTD 128SFO1 400.152 MHzFIDRES 62.600159 HzSW 20.024 ppmFnMODE QF
F2 - Processing parametersSI 1024SF 400.1500069 MHzWDW QSINESSB 0LB 0 HzGB 0PC 1.00
F1 - Processing parametersSI 1024MC2 QFSF 400.1500054 MHzWDW QSINESSB 0LB 0 HzGB 0
ZGH-Ipom-3-290-171022-A_2 COSY in CDCL3
S22
1.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0 ppm
20
40
60
80
100
120
140
ppmCurrent Data ParametersNAME ZGH-Ipom-3-290-171022-AEXPNO 14PROCNO 1
F2 - Acquisition ParametersDate_ 20171029Time 5.04INSTRUM spectPROBHD 5 mm PABBO BB-PULPROG hsqcetgpsiTD 1024SOLVENT CDCl3NS 56DS 16SWH 5197.505 HzFIDRES 5.075689 HzAQ 0.0985088 secRG 2050DW 96.200 usecDE 6.50 usecTE 295.5 KCNST2 145.0000000D0 0.00000300 secD1 1.50000000 secD4 0.00172414 secD11 0.03000000 secD16 0.00020000 secD24 0.00110000 secIN0 0.00003000 secZGOPTNS
======== CHANNEL f1 ========SFO1 400.1524058 MHzNUC1 1HP1 12.50 usecP2 25.00 usecP28 1000.00 usecPLW1 20.00000000 W
======== CHANNEL f2 ========SFO2 100.6253021 MHzNUC2 13CCPDPRG[2 garpP3 10.00 usecP4 20.00 usecPCPD2 80.00 usecPLW2 65.00000000 WPLW12 1.01559997 W
====== GRADIENT CHANNEL =====GPNAM[1] SMSQ10.100GPNAM[2] SMSQ10.100GPZ1 80.00 %GPZ2 20.10 %P16 1000.00 usec
F1 - Acquisition parametersTD 128SFO1 100.6253 MHzFIDRES 130.208328 HzSW 165.631 ppmFnMODE Echo-Antiecho
F2 - Processing parametersSI 1024SF 400.1500000 MHzWDW QSINESSB 2LB 0 HzGB 0PC 1.40
F1 - Processing parametersSI 1024MC2 echo-antiechoSF 100.6177975 MHzWDW QSINESSB 2LB 0 HzGB 0
ZGH-Ipom-3-290-171022-A_2 HSQC in CDCl3
S23
ZGH-Ipom-3-218-170605-A in CDCl3
7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm
1.222
1.240
1.258
2.512
2.518
2.524
2.574
2.593
2.613
2.886
2.906
2.925
4.103
4.121
4.139
4.157
4.669
4.675
6.900
6.916
6.921
7.131
7.153
7.270
3.00
0.70
2.21
2.27
2.02
2.18
1.96
1.99
S24
ZGH-Ipom-3-218-170605-A 13C in CDCl3
180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm
14.175
30.094
36.102
55.819
60.338
75.357
76.680
77.000
77.317
78.665
114.889
129.247
133.636
156.008
172.883
S25
1.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0 ppm
1
2
3
4
5
6
7
8
ppmCurrent Data ParametersNAME ZGH-Ipom-3-218-170605-AEXPNO 3PROCNO 1
F2 - Acquisition ParametersDate_ 20170607Time 17.18INSTRUM spectPROBHD 5 mm PABBO BB-PULPROG cosygpqfTD 2048SOLVENT CDCl3NS 16DS 8SWH 8012.820 HzFIDRES 3.912510 HzAQ 0.1277952 secRG 90.5DW 62.400 usecDE 6.50 usecTE 294.9 KD0 0.00000300 secD1 1.50000000 secD13 0.00000400 secD16 0.00020000 secIN0 0.00012480 sec
======== CHANNEL f1 ========SFO1 400.1520008 MHzNUC1 1HP0 12.50 usecP1 12.50 usecPLW1 20.00000000 W
====== GRADIENT CHANNEL =====GPNAM[1] SMSQ10.100GPZ1 10.00 %P16 1000.00 usec
F1 - Acquisition parametersTD 47SFO1 400.152 MHzFIDRES 170.485550 HzSW 20.024 ppmFnMODE QF
F2 - Processing parametersSI 1024SF 400.1500070 MHzWDW QSINESSB 0LB 0 HzGB 0PC 1.00
F1 - Processing parametersSI 1024MC2 QFSF 400.1500052 MHzWDW QSINESSB 0LB 0 HzGB 0
ZGH-Ipom-3-218-170605-A in CDCL3
S26
ZGH-Ipom-3-220-170607-A in CDCl3
123456789101112 ppm
2.520
2.525
2.531
2.648
2.667
2.687
2.903
2.923
2.942
4.680
4.686
6.915
6.936
7.145
7.166
7.270
11.449
0.58
2.00
2.01
1.90
1.79
1.82
0.57
111213 ppm
11.449
S27
ZGH-Ipom-3-220-170607-A 13C in CDCl3
2030405060708090100110120130140150160170180190 ppm
29.713
35.749
55.848
75.421
76.681
77.000
77.317
78.650
114.996
129.247
133.202
156.123
179.011
S28
234567891011 ppm
40
60
80
100
120
140
ppmCurrent Data ParametersNAME ZGH-Ipom-3-220-170607-AEXPNO 4PROCNO 1
F2 - Acquisition ParametersDate_ 20170608Time 21.38INSTRUM spectPROBHD 5 mm PABBO BB-PULPROG hsqcetgpsiTD 1024SOLVENT CDCl3NS 8DS 16SWH 5197.505 HzFIDRES 5.075689 HzAQ 0.0985088 secRG 2050DW 96.200 usecDE 6.50 usecTE 295.4 KCNST2 145.0000000D0 0.00000300 secD1 1.50000000 secD4 0.00172414 secD11 0.03000000 secD16 0.00020000 secD24 0.00110000 secIN0 0.00003000 secZGOPTNS
======== CHANNEL f1 ========SFO1 400.1524058 MHzNUC1 1HP1 12.50 usecP2 25.00 usecP28 1000.00 usecPLW1 20.00000000 W
======== CHANNEL f2 ========SFO2 100.6253021 MHzNUC2 13CCPDPRG[2 garpP3 10.00 usecP4 20.00 usecPCPD2 80.00 usecPLW2 65.00000000 WPLW12 1.01559997 W
====== GRADIENT CHANNEL =====GPNAM[1] SMSQ10.100GPNAM[2] SMSQ10.100GPZ1 80.00 %GPZ2 20.10 %P16 1000.00 usec
F1 - Acquisition parametersTD 52SFO1 100.6253 MHzFIDRES 320.512817 HzSW 165.631 ppmFnMODE Echo-Antiecho
F2 - Processing parametersSI 1024SF 400.1500000 MHzWDW QSINESSB 2LB 0 HzGB 0PC 1.40
F1 - Processing parametersSI 1024MC2 echo-antiechoSF 100.6177975 MHzWDW QSINESSB 2LB 0 HzGB 0
ZGH-Ipom-3-220-170607-A HSQC in CDCl3
S29
ZGH-Ipom-3-222-170610-A_2 in CDCl3
0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.5 ppm
0.848
0.866
0.889
0.905
1.053
1.070
1.085
1.103
1.168
1.184
1.236
1.279
1.290
1.299
1.315
1.335
1.456
1.475
1.490
1.503
1.602
1.620
1.638
1.654
1.673
2.173
2.518
2.524
2.531
2.538
2.551
2.568
2.587
2.794
2.804
2.814
3.591
3.616
3.634
3.665
3.681
4.374
4.393
4.511
4.531
4.664
4.670
5.081
5.093
5.107
5.116
6.876
6.897
7.091
7.111
7.270
6.71
3.19
26.60
2.98
12.45
5.70
1.20
1.14
2.17
1.06
2.00
3.05
1.95
1.96
S30
ZGH-Ipom-3-222-170610-A_2 13C in CDCl3
210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm
11.465
11.493
14.074
16.293
16.383
16.461
20.902
22.623
23.526
24.542
24.692
26.456
26.538
28.303
28.972
29.149
29.605
29.627
31.860
33.071
34.385
35.605
35.618
37.609
40.978
41.181
41.848
55.819
61.275
68.753
72.521
72.709
72.732
72.851
75.425
76.683
77.000
77.318
78.655
79.461
82.588
100.032
105.687
114.940
114.949
129.212
129.218
133.186
133.204
156.054
171.447
171.504
171.684
171.692
171.979
209.981
S31
1.01.52.02.53.03.54.04.55.05.56.06.57.07.5 ppm
1
2
3
4
5
6
7
ppmCurrent Data ParametersNAME ZGH-Ipom-3-222-170610-A_2EXPNO 3PROCNO 1
F2 - Acquisition ParametersDate_ 20170610Time 8.13INSTRUM spectPROBHD 5 mm PABBO BB-PULPROG cosygpqfTD 2048SOLVENT CDCl3NS 16DS 8SWH 8012.820 HzFIDRES 3.912510 HzAQ 0.1277952 secRG 144DW 62.400 usecDE 6.50 usecTE 295.8 KD0 0.00000300 secD1 1.50000000 secD13 0.00000400 secD16 0.00020000 secIN0 0.00012480 sec
======== CHANNEL f1 ========SFO1 400.1520008 MHzNUC1 1HP0 12.50 usecP1 12.50 usecPLW1 20.00000000 W
====== GRADIENT CHANNEL =====GPNAM[1] SMSQ10.100GPZ1 10.00 %P16 1000.00 usec
F1 - Acquisition parametersTD 128SFO1 400.152 MHzFIDRES 62.600159 HzSW 20.024 ppmFnMODE QF
F2 - Processing parametersSI 1024SF 400.1500070 MHzWDW QSINESSB 0LB 0 HzGB 0PC 1.00
F1 - Processing parametersSI 1024MC2 QFSF 400.1500052 MHzWDW QSINESSB 0LB 0 HzGB 0
ZGH-Ipom-3-222-170610-A_2 in CDCL3
S32
1.01.52.02.53.03.54.04.55.05.56.06.57.07.5 ppm
20
30
40
50
60
70
80
90
100
110
120
130
ppm
Current Data ParametersNAME ZGH-Ipom-3-222-170610-A_2EXPNO 4PROCNO 1
F2 - Acquisition ParametersDate_ 20170610Time 9.09INSTRUM spectPROBHD 5 mm PABBO BB-PULPROG hsqcetgpsiTD 1024SOLVENT CDCl3NS 8DS 16SWH 5197.505 HzFIDRES 5.075689 HzAQ 0.0985088 secRG 2050DW 96.200 usecDE 6.50 usecTE 295.0 KCNST2 145.0000000D0 0.00000300 secD1 1.50000000 secD4 0.00172414 secD11 0.03000000 secD16 0.00020000 secD24 0.00110000 secIN0 0.00003000 secZGOPTNS
======== CHANNEL f1 ========SFO1 400.1524058 MHzNUC1 1HP1 12.50 usecP2 25.00 usecP28 1000.00 usecPLW1 20.00000000 W
======== CHANNEL f2 ========SFO2 100.6253021 MHzNUC2 13CCPDPRG[2 garpP3 10.00 usecP4 20.00 usecPCPD2 80.00 usecPLW2 65.00000000 WPLW12 1.01559997 W
====== GRADIENT CHANNEL =====GPNAM[1] SMSQ10.100GPNAM[2] SMSQ10.100GPZ1 80.00 %GPZ2 20.10 %P16 1000.00 usec
F1 - Acquisition parametersTD 128SFO1 100.6253 MHzFIDRES 130.208328 HzSW 165.631 ppmFnMODE Echo-Antiecho
F2 - Processing parametersSI 1024SF 400.1500000 MHzWDW QSINESSB 2LB 0 HzGB 0PC 1.40
F1 - Processing parametersSI 1024MC2 echo-antiechoSF 100.6177975 MHzWDW QSINESSB 2LB 0 HzGB 0
ZGH-Ipom-3-222-170610-A_2 HSQC in CDCl3
S33
xws-cl-2018-01-29a 400 H-NMR 400
8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm
1.596
1.612
2.178
3.353
3.357
3.364
3.387
3.399
3.411
3.537
3.544
3.622
3.635
3.651
3.666
3.682
3.702
3.965
3.991
4.002
4.014
4.166
4.177
4.188
5.342
6.357
6.373
6.388
6.404
7.038
7.270
7.630
3.59
4.28
4.81
2.60
12.96
3.34
2.56
2.45
0.97
1.00
1.09
1.12
S34
xws-cl-2018-01-29b 400 H-NMR 400
9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm
1.580
1.596
3.299
3.310
3.323
3.335
3.341
3.346
3.369
3.381
3.393
3.522
3.609
3.621
3.638
3.667
3.685
3.961
4.353
4.364
4.375
4.663
4.671
4.674
4.685
5.372
5.384
6.336
6.351
6.367
6.382
7.050
7.381
7.404
7.628
8.257
8.275
8.280
3.38
4.50
2.38
11.33
3.39
2.12
2.15
0.95
1.00
1.07
1.89
1.04
1.91
S35
xws-cl-2018-01-29b in CDCl3
210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm
22.096
40.722
50.562
56.335
66.856
66.953
68.743
69.861
69.960
70.151
70.484
70.522
70.575
76.681
76.999
77.317
108.439
109.948
115.467
121.681
125.225
125.936
135.258
139.397
145.368
146.326
152.278
154.177
155.325
S36
xws-cl-2018-01-29ggg 400 H-NMR 400
8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm
1.408
1.427
1.445
1.462
1.477
1.582
1.597
1.623
1.640
1.656
1.673
1.704
1.721
1.737
2.197
2.215
2.233
2.680
2.712
2.894
2.907
2.926
2.939
3.246
3.259
3.302
3.306
3.310
3.314
3.345
3.357
3.371
3.387
3.399
3.411
3.481
3.495
3.514
3.528
3.541
3.593
3.621
3.631
3.656
3.668
3.963
4.279
4.290
4.299
4.310
4.403
4.469
4.481
4.500
4.870
6.265
6.281
7.198
7.655
2.99
8.85
2.41
0.95
1.06
13.29
30.35
3.57
3.32
2.25
1.07
1.00
1.04
0.43
1.73
S37
xws-cl-2018-01-29ggg in CDCl3
220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm
29.492
29.726
36.724
40.273
41.050
41.624
41.744
48.360
48.573
48.786
48.999
49.212
49.425
49.637
51.807
56.976
57.039
61.617
63.336
64.223
69.307
69.735
70.749
70.997
71.118
71.199
71.232
71.415
71.460
71.507
109.835
110.916
119.746
128.012
129.915
130.037
133.018
133.119
133.758
133.785
140.978
148.399
155.783
157.923
166.040
176.119
S38
ZGH-Ipom-4-26-180201-A in CDCl3
0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0 ppm
0.860
0.870
0.878
0.888
0.897
0.904
1.046
1.064
1.080
1.097
1.160
1.176
1.237
1.263
1.280
1.289
1.299
1.314
1.493
1.504
1.574
1.589
1.615
1.633
1.650
1.668
2.167
2.549
2.557
2.570
2.799
3.433
3.572
3.591
3.603
3.631
3.657
3.881
3.893
3.905
3.936
4.449
4.541
4.552
4.566
5.070
5.175
5.304
6.883
6.904
7.030
7.070
7.091
7.270
7.625
7.817
6.00
3.12
3.17
32.09
5.02
7.32
6.19
1.14
35.96
7.44
4.18
1.22
8.48
3.11
2.89
0.83
0.91
0.88
0.95
0.95
0.71
2.02
2.98
0.93
0.83
S39
ZGH-Ipom-4-26-180201-A 13C in CDCl3
210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm
11.477
11.525
14.078
16.310
16.399
16.462
20.951
22.219
22.624
23.606
24.549
24.709
25.411
26.437
26.523
28.050
28.334
28.985
29.191
29.590
31.869
33.165
34.415
35.656
37.629
39.096
40.957
41.176
41.823
68.746
69.392
69.953
70.044
70.191
70.364
70.386
70.435
70.464
70.521
72.428
72.967
74.637
76.682
77.000
77.203
77.317
79.614
82.473
100.252
114.799
129.252
156.763
171.655
171.665
171.900
209.989
S40
1.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0 ppm
1
2
3
4
5
6
7
ppm
Current Data ParametersNAME ZGH-Ipom-4-26-180201-AEXPNO 3PROCNO 1
F2 - Acquisition ParametersDate_ 20180205Time 21.33INSTRUM spectPROBHD 5 mm PABBO BB-PULPROG cosygpqfTD 2048SOLVENT CDCl3NS 20DS 8SWH 8012.820 HzFIDRES 3.912510 HzAQ 0.1277952 secRG 287DW 62.400 usecDE 6.50 usecTE 295.2 KD0 0.00000300 secD1 1.50000000 secD13 0.00000400 secD16 0.00020000 secIN0 0.00012480 sec
======== CHANNEL f1 ========SFO1 400.1520008 MHzNUC1 1HP0 12.50 usecP1 12.50 usecPLW1 20.00000000 W
====== GRADIENT CHANNEL =====GPNAM[1] SMSQ10.100GPZ1 10.00 %P16 1000.00 usec
F1 - Acquisition parametersTD 128SFO1 400.152 MHzFIDRES 62.600159 HzSW 20.024 ppmFnMODE QF
F2 - Processing parametersSI 1024SF 400.1500070 MHzWDW QSINESSB 0LB 0 HzGB 0PC 1.00
F1 - Processing parametersSI 1024MC2 QFSF 400.1500052 MHzWDW QSINESSB 0LB 0 HzGB 0
ZGH-Ipom-4-26-180201-A in CDCL3
S41
0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0 ppm
20
40
60
80
100
120
140
ppm Current Data ParametersNAME ZGH-Ipom-4-26-180201-AEXPNO 4PROCNO 1
F2 - Acquisition ParametersDate_ 20180205Time 22.44INSTRUM spectPROBHD 5 mm PABBO BB-PULPROG hsqcetgpsiTD 1024SOLVENT CDCl3NS 12DS 16SWH 5197.505 HzFIDRES 5.075689 HzAQ 0.0985088 secRG 2050DW 96.200 usecDE 6.50 usecTE 295.2 KCNST2 145.0000000D0 0.00000300 secD1 1.50000000 secD4 0.00172414 secD11 0.03000000 secD16 0.00020000 secD24 0.00110000 secIN0 0.00003000 secZGOPTNS
======== CHANNEL f1 ========SFO1 400.1524058 MHzNUC1 1HP1 12.50 usecP2 25.00 usecP28 1000.00 usecPLW1 20.00000000 W
======== CHANNEL f2 ========SFO2 100.6253021 MHzNUC2 13CCPDPRG[2 garpP3 10.00 usecP4 20.00 usecPCPD2 80.00 usecPLW2 65.00000000 WPLW12 1.01559997 W
====== GRADIENT CHANNEL =====GPNAM[1] SMSQ10.100GPNAM[2] SMSQ10.100GPZ1 80.00 %GPZ2 20.10 %P16 1000.00 usec
F1 - Acquisition parametersTD 128SFO1 100.6253 MHzFIDRES 130.208328 HzSW 165.631 ppmFnMODE Echo-Antiecho
F2 - Processing parametersSI 1024SF 400.1500000 MHzWDW QSINESSB 2LB 0 HzGB 0PC 1.40
F1 - Processing parametersSI 1024MC2 echo-antiechoSF 100.6177975 MHzWDW QSINESSB 2LB 0 HzGB 0
ZGH-Ipom-4-26-180201-A HSQC in CDCl3
S42
S43
S44
S45
S46
S47
ZGH-Ipom-3-231-170705-A in CDCl3
1.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5 ppm
0.853
0.870
0.887
1.146
1.162
1.223
1.240
1.258
1.275
1.296
1.314
1.334
1.493
1.502
1.516
1.704
1.729
2.026
2.147
3.564
3.572
3.579
3.595
3.613
3.621
3.627
3.637
3.658
3.676
3.775
3.788
3.885
3.897
3.909
4.092
4.110
4.375
4.543
4.555
4.567
5.118
5.125
5.148
5.199
6.150
6.159
6.181
6.189
6.930
6.952
7.385
7.407
7.507
7.547
8.417
8.439
2.99
3.10
14.33
6.14
5.92
2.07
0.81
2.84
5.76
14.06
3.11
3.07
2.21
0.82
1.00
0.96
1.99
1.01
0.80
3.97
0.96
1.88
0.89
1.90
0.89
1.89
0.89
0.82
0.78
S48
ZGH-Ipom-3-231-170705-A 13C in CDCl3
210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm
11.906
14.014
14.104
14.489
16.261
20.853
20.953
22.560
23.464
24.444
24.649
28.241
28.997
29.088
31.824
33.123
34.312
37.545
41.748
50.334
60.307
61.902
67.334
68.130
68.730
69.270
70.399
70.427
70.440
70.499
72.316
72.559
72.672
73.884
75.663
76.682
77.000
77.318
79.701
82.686
100.221
105.669
114.480
115.025
127.196
127.556
129.881
136.453
139.466
144.238
145.360
160.114
165.532
168.566
171.627
171.658
210.066
S49
1.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5 ppm
1
2
3
4
5
6
7
8
ppmCurrent Data ParametersNAME ZGH-Ipom-3-231-170705-AEXPNO 4PROCNO 1
F2 - Acquisition ParametersDate_ 20170707Time 3.58INSTRUM spectPROBHD 5 mm PABBO BB-PULPROG cosygpqfTD 2048SOLVENT CDCl3NS 16DS 8SWH 8012.820 HzFIDRES 3.912510 HzAQ 0.1277952 secRG 71.8DW 62.400 usecDE 6.50 usecTE 295.8 KD0 0.00000300 secD1 1.50000000 secD13 0.00000400 secD16 0.00020000 secIN0 0.00012480 sec
======== CHANNEL f1 ========SFO1 400.1520008 MHzNUC1 1HP0 12.50 usecP1 12.50 usecPLW1 20.00000000 W
====== GRADIENT CHANNEL =====GPNAM[1] SMSQ10.100GPZ1 10.00 %P16 1000.00 usec
F1 - Acquisition parametersTD 128SFO1 400.152 MHzFIDRES 62.600159 HzSW 20.024 ppmFnMODE QF
F2 - Processing parametersSI 1024SF 400.1500069 MHzWDW QSINESSB 0LB 0 HzGB 0PC 1.00
F1 - Processing parametersSI 1024MC2 QFSF 400.1500054 MHzWDW QSINESSB 0LB 0 HzGB 0
ZGH-Ipom-3-231-170705-A in CDCL3
S50
1.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5 ppm
20
40
60
80
100
120
140
ppm Current Data ParametersNAME ZGH-Ipom-3-231-170705-AEXPNO 2PROCNO 1
F2 - Acquisition ParametersDate_ 20170706Time 22.56INSTRUM spectPROBHD 5 mm PABBO BB-PULPROG hsqcetgpsiTD 1024SOLVENT CDCl3NS 8DS 16SWH 5197.505 HzFIDRES 5.075689 HzAQ 0.0985088 secRG 2050DW 96.200 usecDE 6.50 usecTE 295.3 KCNST2 145.0000000D0 0.00000300 secD1 1.50000000 secD4 0.00172414 secD11 0.03000000 secD16 0.00020000 secD24 0.00110000 secIN0 0.00003000 secZGOPTNS
======== CHANNEL f1 ========SFO1 400.1524058 MHzNUC1 1HP1 12.50 usecP2 25.00 usecP28 1000.00 usecPLW1 20.00000000 W
======== CHANNEL f2 ========SFO2 100.6253021 MHzNUC2 13CCPDPRG[2 garpP3 10.00 usecP4 20.00 usecPCPD2 80.00 usecPLW2 65.00000000 WPLW12 1.01559997 W
====== GRADIENT CHANNEL =====GPNAM[1] SMSQ10.100GPNAM[2] SMSQ10.100GPZ1 80.00 %GPZ2 20.10 %P16 1000.00 usec
F1 - Acquisition parametersTD 128SFO1 100.6253 MHzFIDRES 130.208328 HzSW 165.631 ppmFnMODE Echo-Antiecho
F2 - Processing parametersSI 1024SF 400.1500000 MHzWDW QSINESSB 2LB 0 HzGB 0PC 1.40
F1 - Processing parametersSI 1024MC2 echo-antiechoSF 100.6177975 MHzWDW QSINESSB 2LB 0 HzGB 0
ZGH-Ipom-3-231-170705-A HSQC in CDCl3
S51
1.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5 ppm
20
40
60
80
100
120
140
160
ppmCurrent Data ParametersNAME ZGH-Ipom-3-231-170705-AEXPNO 5PROCNO 1
F2 - Acquisition ParametersDate_ 20170707Time 4.56INSTRUM spectPROBHD 5 mm PABBO BB-PULPROG hmbcgplpndqfTD 4096SOLVENT CDCl3NS 60DS 16SWH 5208.333 HzFIDRES 1.271566 HzAQ 0.3932160 secRG 2050DW 96.000 usecDE 6.50 usecTE 295.2 KCNST2 145.0000000CNST13 10.0000000D0 0.00000300 secD1 1.50000000 secD2 0.00344828 secD6 0.05000000 secD16 0.00020000 secIN0 0.00002240 sec
======== CHANNEL f1 ========SFO1 400.1516006 MHzNUC1 1HP1 12.50 usecP2 25.00 usecPLW1 20.00000000 W
======== CHANNEL f2 ========SFO2 100.6258470 MHzNUC2 13CP3 10.00 usecPLW2 65.00000000 W
====== GRADIENT CHANNEL =====GPNAM[1] SINE.100GPNAM[2] SINE.100GPNAM[3] SINE.100GPZ1 50.00 %GPZ2 30.00 %GPZ3 40.10 %P16 1000.00 usec
F1 - Acquisition parametersTD 128SFO1 100.6258 MHzFIDRES 174.386154 HzSW 221.826 ppmFnMODE QF
F2 - Processing parametersSI 2048SF 400.1500062 MHzWDW SINESSB 0LB 0 HzGB 0PC 4.00
F1 - Processing parametersSI 1024MC2 QFSF 100.6177921 MHzWDW SINESSB 0LB 0 HzGB 0
ZGH-Ipom-3-231-170705-A HMBC in CDCl3
S52
S53
S54
S55
S56
download fileview on ChemRxivSupporting Info_2.pdf (4.85 MiB)