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: rachel.simmonds@surrey.ac.uk; jinglese@mail.nih.gov;

ydu@uark.edu; demangel@pasteur.fr; stephen.high@manchester.ac.uk; ville.paavilainen@helsinki.fi;

weishi@uark.edu, shiwei43@gmail.com.

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

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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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18. Nebane, N. M.; Coric, T.; McKellip, S.; Woods, L.; Sosa, M.; Rasmussen, L.; Bjornsti, M.-A.;

White, E. L. J. Lab. Autom. 2016, 21, 198-203.

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Biol. Cell 2014, 25, 2828-2839.

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Academic Press: 2013; Vol. 118, p 85-103.

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Med. 2016, 213, 2885-2896.

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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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)