Concise Chemoenzymatic Total Synthesis and Identification of Cellular Targets of Cepafungin I

Brief Communication

Concise Chemoenzymatic Total Synthesis and

Identiﬁcation of Cellular Targets of Cepafungin I

Graphical Abstract

Authors

Alexander Amatuni, Anton Shuster,

Alexander Adibekian, Hans Renata

Correspondence

aadibeki@scripps.edu (A.A.),

hrenata@scripps.edu (H.R.)

In Brief

Amatuni et al. established a concise

chemoenzymatic synthesis of cepafungin

I. The route enabled access to a

chemoproteomic probe, revealing high

selectivity for proteasome subunits b5/2.

Potent inhibition was associated with the

macrocyclic hydroxyl group and lipid tail.

Cepafungin I exhibited similar mode of

action with the clinical drug bortezomib.

Highlights

d

A chemoenzymatic strategy provides rapid access to

cepafungin I and analogs

Chemoproteomics reveals high selectivity of cepafungin I for

proteasome subunits b5/2

Preliminary SAR study indicates essential motifs for potent

proteasome inhibition

Cepafungin I and bortezomib share a similar mode of action

Amatuni et al., 2020, Cell Chemical Biology 27, 1–9

October 15, 2020 ª 2020 Elsevier Ltd.

https://doi.org/10.1016/j.chembiol.2020.07.012

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ll

Brief Communication

Concise Chemoenzymatic Total Synthesis

and Identiﬁcation of Cellular

Targets of Cepafungin I

Alexander Amatuni, Anton Shuster, Alexander Adibekian, * and Hans Renata¹^,²^,*

1

1,

1

Department of Chemistry, The Scripps Research Institute, 130 Scripps Way, Jupiter, FL 33458, USA

²Lead Contact

*Correspondence: aadibeki@scripps.edu (A.A.), hrenata@scripps.edu (H.R.)

https://doi.org/10.1016/j.chembiol.2020.07.012

SUMMARY

The natural product cepafungin I was recently reported to be one of the most potent covalent inhibitors of the

2

0S proteasome core particle through a series of in vitro activity assays. Here, we report a short chemoen-

zymatic total synthesis of cepafungin I featuring the use of a regioselective enzymatic oxidation to prepare a

key hydroxylated amino acid building block in a scalable fashion. The strategy developed herein enabled ac-

cess to a chemoproteomic probe, which in turn revealed the exceptional selectivity and potency of cepafun-

gin I toward the b2 and b5 subunits of the proteasome. Further structure-activity relationship studies suggest

the key role of the hydroxyl group in the macrocycle and the identity of the lipid tail in modulating the potency

of this natural product family. This study lays the groundwork for further medicinal chemistry exploration to

fully realize the anticancer potential of cepafungin I.

INTRODUCTION

ment of multiple myeloma. However, both BTZ and carﬁlzomib

have been reported to exhibit several side effects, such as

Cepafungin I and glidobactin A are two macrolactams belonging thrombocytopenia, and high occurrence of relapse and chemo-

to a larger family of natural products called the syrbactins (Fig- resistance have also been reported (Guerrero-Garcia et al.,

ure 1A). Members of this family share a common 12-membered 2018). Thus, there is still an urgent need to advance new protea-

macrolactam core consisting of a vinylogous amino acid and a some inhibitors as drug candidates that will address these

lysine residue that may exist in various oxidation states. Further issues.

diversity can be found within the tail portion of the natural prod-

Similar to BTZ, the syrbactins trigger apoptosis by eliciting p53

ucts: while the cepafungins and the glidobactins possess unsat- accumulation and inhibiting nuclear factor kB activity. Pioneering

urated fatty acid tails, all syrbactins contain a Val-Val unit that is studies by Bachmann and co-workers using a syrbactin deriva-

linked via a unique ureido moiety (Krahn et al., 2011). Initial bio- tive additionally showed that, while this compound exhibited

logical evaluation suggested that the glidobactins and the cepa- cytotoxic effects on cancer cell lines, the effect was most pro-

fungins exhibit moderate antifungal activity and potent antitumor nounced in multiple myeloma (Archer et al., 2012). Recent

activity against P388 leukemia in mice (Oka et al., 1988a, 1988b, research has begun to elucidate certain resistance mechanisms

1

988c; Shoji et al., 1990). More recently, it has been determined in multiple myeloma. While current proteasome inhibitors primar-

that the latter activity arises from inhibition of the 20S protea- ily inhibit the b5 subunit, it has been shown that co-inhibition of

some core particle (CP) via covalent engagement of two distinct the b2 subunit prevents recovery of proteasome activity by

g

catalytically active Thr1O residues by the unsaturated lactam causing aggregation/inactivation of the proteasomal transcrip-

motif of the macrocycle (Groll et al., 2008). Cepafungin I, in tion factor Nrf1. Notably, the cytotoxicities of BTZ and carﬁlzo-

particular, exhibits remarkably strong inhibitory activity relative mib toward solid tumor cells (e.g., triple-negative breast cancer)

to all known proteasome inhibitors to date (half maximal inhibi- are signiﬁcantly enhanced in this manner, whereas neither drug

tory concentration [IC50 ] = 4 nM against the b5 subunit of yeast had otherwise shown clinical efﬁcacy on its own. Moreover,

CP [Stein et al., 2012]). The proteasome is a multiprotein com- co-inhibition of b5 with b2 provides a stronger antineoplastic ef-

plex that plays a critical role in protein degradation. Given the fect than with b1, highlighting the importance of advancing new

centrality of the proteasome in the regulation of cell cycle and proteasome inhibitors with equipotent activity for these subunits

apoptosis, its inhibition constitutes a promising modality for can- (Weyburne et al., 2017; Oerlemans et al., 2008). Cepafungin I,

cer therapy. In support of this notion, three proteasome inhibi- with its low nanomolar activity toward b5 and b2 (Figure 1A)

tors, bortezomib (BTZ), carﬁlzomib, and ixazomib, have been (IC50 = 4 and 24 nM, respectively), stands as an attractive candi-

approved by the US Food and Drug Administration for the treat- date for such purpose.

Cell Chemical Biology 27, 1–9, October 15, 2020 ª 2020 Elsevier Ltd.

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Brief Communication

A

B

Figure 1. Select Syrbactin Structures, Biological Activity, and Optimization of Preparative Scale Hydroxylation of 4 with GlbB

(

A) Molecular structures of cepafungin I and related natural products. In vitro yeast CP IC50 values from Stein et al. (2012).

B) Co-expression of chaperones GroES/GroEL was found to improve the soluble expression of GlbB, resulting in ca. 5-fold improvement in reaction conversion.

#

Lanes (I) and (III), soluble fraction; lanes (II) and (IV), insoluble fraction; lane (V), protein ladder. Reaction conducted at 6-g scale.

A seminal work by Schmidt et al. (1992) established the ﬁrst line. Substrate scope examination of GlbB revealed that, while

total synthesis of glidobactin A. Starting from malic acid, the the enzyme exhibits a narrow substrate speciﬁcity, it is able to

synthesis of the natural product was achieved in 21 steps. While hydroxylate lysine with remarkably high catalytic efﬁciency.

being a landmark achievement, this approach suffers from high Large-scale biocatalytic hydroxylation with puriﬁed GlbB, how-

step count due to inefﬁcient functional group interconversions ever, was hampered by poor scalability as the reaction gave

and extraneous protecting group manipulations. For example, poor conversion when conducted at > 100 mg scale. While this

the synthesis of the key 4-hydroxylysine moiety took place in issue could be solved by conducting the reaction with clariﬁed

1

2 steps from malic acid, featuring Horner-Wadsworth-Emmons lysate of cells expressing GlbB, the poor soluble expression of

homologation, asymmetric hydrogenation, and displacement of GlbB led to sub-optimal titer (Figure 1B). Previous reports from

the terminal alcohol with NaN . Efforts by Ichikawa and col- our laboratory and others have shown the beneﬁts of co-ex-

3

leagues (Chiba et al., 2014; Kitahata et al., 2016, 2017), Pirrung pressing molecular chaperones, especially GroES/EL, in

et al. (2010), Stephenson and colleague (Dai and Stephenson, improving heterologous production of enzymes from secondary

2

viable synthetic routes to the syrbactins. However, these routes et al., 2019). Indeed, co-expression of chaperones GroES/EL

010), and others (Clerc et al., 2009, 2010b) have established metabolite pathways (Payne et al., 2013; Zhang et al., 2018; Li

ꢀ

would not be amenable to the introduction of the key 2 alcohol was found to increase soluble expression of GlbB, which trans-

within the embedded lysine unit.

lates to ca. 5-fold improvement in reaction conversion and yield

on small scale. For large-scale reactions, ca. 6–7 g of lysine

could be fully converted to its C4-hydroxylated counterpart in

RESULTS AND DISCUSSION

a single pass with 1 L of clariﬁed cell lysate (ﬁnal OD600

=

We recognized that an efﬁcient route to 4-hydroxylysine or its 12.5), corresponding to a titer of ca. 6–7 g/L based on the original

synthetic equivalent would hold the key to the development of volume of the expression culture (Figure 1B).

a practical synthesis of cepafungin I. If such a route could be

Having solved the material throughput issue for the production

identiﬁed, iterative condensations with alanine and an acetate of 5, we turned our attention to its conversion to the macrocyclic

equivalent would rapidly generate the macrocyclic core of the core of 1. We have previously described the conversion of 5 to

natural product. Previous approaches to either diastereomers intermediate 6 by way of coupling with alaninol (Amatuni and Re-

ꢀ

of protected 4-hydroxylysine suffer from high step count arising nata, 2019). However, attempts to selectively oxidize the 1

from protecting group and functional group manipulations (Izu- alcohol of 6 were beset by low yields and poor selectivity (Fig-

miya et al., 1965; Schmidt et al., 1992; Marin et al., 2004). Of ure 2A). Efforts to effect differential protections of the a- and

these three approaches, only that by Witkop and colleagues (Izu- ε-amine also proved problematic due to the presence of the

miya et al., 1965) described the production of protected 5 while C4-OH. Finally, aminolysis (Sund et al., 2011; Sabot et al.,

the other two produced only its C4 diastereomer. Witkop’s route 2007; Foley and Jamison, 2010) of lactone 7 with protected

to protected 5, however, proceeded through the intermediacy of (4S)-amino-2(E)-pentenoic acid derivatives (8) led to only non-

its C4 diastereomer, required the use of harsh photolysis in the productive decomposition of 8. All of these setbacks led to the

presence of Cl

2

gas, and involved an inefﬁcient stereoinversion non-obvious tactical development involving the use of Weinreb

-assisted coupling with lactone 7 (Figure 2B).

process at the C4 stereocenter. We have previously established amide 9 in an AlMe

3

the biosynthetic origin of the 4-hydroxylysine moiety of glidobac- This protocol reliably afforded dipeptide 10 in 90% yield on a

tin A (Amatuni and Renata, 2019). Within the pathway, a multi-gram scale without any observable side reactions (e.g.,

nonheme dioxygenase, GlbB, catalyzes the direct C-H hydroxyl- polymerization of 9, epimerization or re-lactonization of 10). To

ation of free-standing lysine at the C4 position before loading of the best of our knowledge, this is the ﬁrst report of generating

the product onto the nonribosomal peptide synthetase assembly an aluminum amide reagent in situ that itself contains a Weinreb

2

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Brief Communication

A

B

C

Figure 2. Chemoenzymatic Synthesis of Cepafungin I

(

A) Initial strategies for elaborating 5 to the macrocyclic core of 1.

B) Optimized chemoenzymatic synthesis of cepafungin I.

C) Modular synthesis of tail fragment 14. See the STAR Methods for detailed reaction conditions and full characterization. Boc

2

O, di-tert-butyl carbonate; EDC,

1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; TFA, triﬂuoroacetic acid; HATU, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide

hexaﬂuorophosphate; DMTMMT, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium tetraﬂuoroborate; DEPBT, 3-(diethoxyphosphoryloxy)-1,2,3-

benzotriazin-4(3H)-one; DIPEA, N,N-diisopropylethylamine.

4

amide moiety. Treatment of 10 with LiAlH effected a clean absence of any protecting groups to maximize step economy

reduction of the Weinreb amide moiety to the corresponding and synthetic ideality (Gaich and Baran, 2010). Several side re-

ꢀ

aldehyde. This transformation was regarded as a risky endeavor actions, including oligomerization, lactonization by the 2

at the outset due to the presence of several carbonyl groups in alcohol, or alternative macrolactamization by the lysyl a-amino

1

0. Nevertheless, we observed remarkable chemoselectivity as group could have derailed this approach. Successful execution

no over-reduction or side reactivity with any of the carbonyl of our coupling strategy was made possible likely due to a com-

groups in the molecule could be detected. Oleﬁnation with Wittig bination of substrate conformational bias and the intrinsic differ-

reagent 11 furnished enoate 12, which constitutes the protected ence in rate of ring formation among medium ring sizes (Illuminati

linear form of the target macrocyclic core. As a testament to the and Mandolini, 1981). A synthetic route to the fully elaborated tail

robustness of the route, more than 1.5 g of 12 could be prepared fragment 14 was pursued in parallel (Figure 2C). Given the lack of

in a single pass.

commercial availability of the 7-methyloctan-1-ol precursor (16),

While global deprotection of 12 could be achieved cleanly un- we developed a modular route to access it via a Kochi coupling

der acidic conditions, the subsequent macrolactamization step (Tamura and Kochi, 1971) of bromoalcohol 15 and iPrMgBr. Sub-

required extensive optimization (see Table S1 for optimization). sequent oxidation, Horner-Wadsworth-Emmons homologation,

This observation was in line with previous efforts in the total syn- peptide coupling, and deprotection completed the synthesis of

thesis of glidobactin A and the syrbactins, where late-stage mac- 14. Due to the high polarity of 13, we elected to bypass any pu-

rolactamization typically proceeded in less than 30% yield. riﬁcation step and used the compound in a crude form for subse-

Eventually, DMTMMT (Kami nꢀ ski et al., 2005) was identiﬁed as quent coupling with 14. Extensive screening of peptide-coupling

the optimal coupling reagent, providing ꢁ60% NMR yield of conditions (Table S2) eventually identiﬁed DEPBT as an optimal

the desired macrolactam. In contrast to other approaches to coupling reagent for this step, leading to the formation of the

the syrbactins, we conducted this macrocyclization step in the target natural product in 19% yield from 12. Overall, our

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Brief Communication

Figure 3. Cytotoxicity and Cellular Target

Identiﬁcation of Cepafungin I

(

A) Cytotoxicity curves of RPMI 8226 and MM1.R

cells treated with the indicated concentrations of 1

for 24 or 48 h. Quantiﬁcation was performed using

the WST-1 assay (relative values ±SD and EC50

values; n = 3).

(

B) Gel-based competitive in situ proﬁling of tar-

gets of 1. RPMI 8226 cells were treated with

various concentrations of 1 for 6 h, lysed, and

treated with 10 mM 20.

(

2

C) Log label-free quantiﬁcation (LFQ) ratios of

proteins identiﬁed from the in situ competitive pull-

down experiment with 100 nM 1 or DMSO and with

1

0 mM 20 (n = 6, three biologicals and two techni-

cals each).

D) Cellular targets of 1 (>50% competition) in

(

RPMI 8226 cells. Quantiﬁcation was performed

using the LFQ method (n = 6).

drug Velcade (bortezomib) in 2003 (Mana-

sanch and Orlowski, 2017). Accordingly,

we started our biological investigations

with cytotoxicity measurements of 1 in

the commonly used MM cell lines RPMI

8226 and MM1.R to evaluate its potential

as a cancer drug candidate. Cells were

treated with various concentrations of 1

for 24 or 48 h and the percentage of viable

cells was determined via the WST-1

assay (Figure 3A). Indeed, 1 proved

potently toxic and the measured cytotox-

icity after 48 h treatment (half maximal

effective concentration [EC50] = of 31 nM

for RPMI 8226 and 23 nM for MM1.R)

aligned well with the previously reported

data for clinical proteasome inhibitor

drugs BTZ (30 nM for RPMI 8226; 3 nM

for MM1.R, Hideshima et al., 2001) and

carﬁlzomib (5 nM for RPMI 8226, Kuhn

et al., 2007). Although 1 and structurally

related natural products have previously

been shown to inhibit proteasomal sub-

chemoenzymatic synthesis of cepafungin I proceeded in nine units PSMB2 (b2) and PSMB5 (b5) in puriﬁed yeast (Stein et al.,

steps (longest linear sequence) and 7.9% overall yield from 2012) and mammalian (Pawar et al., 2020) proteasomes, direct

lysine. By virtue of this chemoenzymatic strategy, our route pro- target engagement of proteasomes by the syrbactins in

vides exceptionally rapid access to the key hydroxylysine resi- mammalian cells has not yet been shown. Accordingly, we

due and overcomes longstanding challenges associated with decided to perform a deep proﬁling of cellular targets of 1 using

ꢀ

synthetic access to syrbactins bearing 2 alcohol at the L-lysine a classical chemoproteomics approach. For this purpose, we

fragment. This route also highlights DMTMMT as a superior mac- synthesized covalent probe 20, an alkyne-tagged derivative of

rolactamization reagent and the use of substrate conformational 1 (Figure 3). RPMI 8226 lysates were treated with increasing con-

bias to preferentially construct the strained 12-membered ring centrations of 20 for 1 h at room temperature and the proteins

from a ‘‘protection-free’’ linear precursor. Finally, the modularity labeled by the covalent probe were then conjugated to TAMRA

of this route enables the rapid construction of synthetic analogs azide using copper(I)-catalyzed alkyne-azide cycloaddition

and chemoproteomic probes for further biological evaluation (CuAAC) (Speers and Cravatt, 2004), separated by SDS-PAGE,

(

vide infra).

and visualized by in-gel ﬂuorescence scanning (Figure S1A).

Multiple myeloma (MM) has remained the main indication for The gel proﬁle revealed only a few bands, thus suggesting low

clinical trials of novel proteasome inhibitor drug candidates since proteomic promiscuity of 20. To examine whether 20 can be

the Food and Drug Administration approval of the ﬁrst-in-class used as a clickable analog of 1, we performed a gel-based

4

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Brief Communication

Figure 4. Validation of Target Engagement,

Structure-Activity Relationship, and Down-

stream Effects of 1

(

A) Labeling of mock and overexpressed PSMB2-

FLAG and PSMB5-FLAG in HEK293T lysate with

0 mM 20. Shown are the TAMRA ﬂuorescence

3

proﬁle of 20 (top), western blot membrane probed

for FLAG (middle), and a-tubulin as a loading

control (bottom). See also Figure S1C.

(

B) In situ inhibition of chymotrypsin-like (b5) pro-

teasome activity by 1 and its derivatives. RPMI

226 cells were treated with various concentra-

tions of compounds for 6 h, lysed, treated with

8

1

00 mM ﬂuorogenic substrate Suc-LLVY-AMC,

ꢀ

and incubated for 2 h at 37 C. IC50 values were

quantiﬁed based on

A360ex/A460em readouts

(

n = 3).

C) Western blotting of lysates obtained from RPMI

226 cells treated with various concentrations of 1

8

for 6 h. Membranes were probed for mono- and

poly-ubiquitin, PARP-1, and a-tubulin as a loading

control.

(

D) Venn diagram representing the overlap in

signiﬁcantly upregulated proteins (false discovery

rate = 0.05; S = 0.01) upon 14 h treatment of RPMI

226 cells with 100 nM 1 or 2.5 nM BTZ versus

0

8

vehicle (see Figures 3A and S2A). Quantiﬁcation

was performed using LFQ method (n = 6).

philic a,b-unsaturated amides that could

potentially engage hundreds of reactive

cysteines (Abegg et al., 2015).

We chose to further validate the

engagement of PSMB2 (proteasome

2

0S subunit b2) and PSMB5 (proteasome

0S subunit b5) by overexpression and la-

beling by 1, because their chemical co-in-

hibition has been shown to be particularly

cytotoxic in proteasome inhibitor-resis-

tant MM cells and has therefore emerged

as a promising therapeutic strategy

(Besse et al., 2019). While single isolated

proteasome subunits do not display enzy-

competitive proﬁling experiment. RPMI 8226 cells were treated matic activity and can thus not be labeled by 1, successful incor-

in situ with various concentrations of 1 for 6 h, lysed, and then poration of various overexpressed tagged and untagged protea-

treated with 10 mM 20 for 1 h, followed by TAMRA conjugation some subunits into functional proteasome has previously been

and protein visualization, as described above (Figures 3B and documented in yeast and mammalian cells (Howell et al., 2019;

S1B). Excitingly, only a few bands were successfully competed Chondrogianni et al., 2005). We therefore hypothesized that

at nanomolar concentrations, suggesting high potency and overexpression of b2 and b5 subunits would result in their partial

selectivity of 1 toward its targets. To identify the competed incorporation into the proteolytic core (CP) where they can be

protein targets, we performed an in situ competitive liquid chro- labeled by our probe. Both proteins were cloned as FLAG-

matography-tandem mass spectrometry (LC-MS/MS)-based tagged versions and overexpressed in HEK293T cells. PSMB2

experiment. In brief, RPMI 8226 cells were treated with 100 nM and PSMB5-expressing cells were lysed and the lysates labeled

1

for 6 h, lysed, then treated with 10 mM 20 followed by with 30 mM 20 for 1 h, followed by conjugation to TAMRA azide,

CuAAC-mediated conjugation of biotin azide, enrichment with separation by SDS-PAGE, and visualization of probe-bound pro-

streptavidin beads, trypsin digestion, and LC-MS/MS analysis. teins by in-gel ﬂuorescence scanning (Figures 4A and S1C).

Out of 764 proteins enriched by 20, only 5 were >50% competed Indeed, a new labeled band was clearly seen in the overex-

by 1 (Figures 3C and 3D; Table S4). Strikingly, all 5 identiﬁed tar- pressed lane of both gels, indicating successful labeling of

gets were 20S proteasome subunits: PSMB5, PSMB10, PSMA5, both overexpressed proteins, PSMB2-F and PSMB5-F.

PSMB1, and PSMB2. Such high selectivity is remarkable, espe-

Having conﬁrmed both proteins as targets of cepafungin I, we

cially considering that the structure of 1 contains two electro- successfully assigned the ﬂuorescence bands in the gel proﬁle of

Cell Chemical Biology 27, 1–9, October 15, 2020

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Brief Communication

2

0 to respective proteasome subunits as described previously unsaturation in the lipid tail for proteasomal inhibition. While the

(

Chondrogianni et al., 2005; Altun et al., 2005; Britton et al., lipid tail occupies a hydrophobic patch in the binding pocket, it is

009) and quantiﬁed the in situ IC50 values of 1 for the endoge- unclear at the outset whether its rigidity will have any contribution

nously expressed proteasome subunits, yielding IC50 values of to activity. Indeed, 22 (saturated cepafungin) demonstrated a 14-

.5 nM for PSMB5 and 35 nM for PSMB2 (Figures S1D and fold loss in potency compared with 1, suggesting that the struc-

2

7

S1E). Interestingly, these values only slightly deviate from previ- tural rigidity conferred by the lipid tail unsaturation is important

ously reported IC50 values for inhibition of puriﬁed yeast protea- for potent proteasome engagement. Taken together, these re-

some subunits by 1 (PSMB5, 4 nM; PSMB2, 24 nM, Stein et al., sults suggest that the secondary alcohol on the macrocycle

2

012). To gain more insight into the structure-activity relationship and the 2,4-dienamide motif on the tail region of 1 are critical

between 1 and PSMB5, we synthesized several derivatives of 1 for the b5 inhibitory activity.

Figure 4) and compared their ability to inhibit b5 activity in RPMI Next, we sought to investigate if 1 mediates the expected bio-

226 cells. These derivatives were designed to assess the contri- logical downstream response similar to other proteasome inhib-

(

8

bution of various portions of the scaffold to its inhibitory activity, itors. One of the key cellular responses of proteasome inhibition

namely the macrocyclic secondary alcohol, the 2,4-dienamide is the accumulation of polyubiquitinated proteins, gradually lead-

portion of the tail and the terminal methyl branching. Previous ing to dysfunction and apoptosis (Adams, 2004). Indeed, west-

medicinal chemistry explorations of the natural product family ern blot of lysates from 1-treated RPMI 8226 cells using a

have focused on the syringolin series, which contain an addi- mono-/poly-ubiquitin-conjugate-speciﬁc antibody showed a

tional degree of unsaturation in the lysine unit instead of C4 hy- concentration-dependent increase in ubiquitinated proteins

droxylation. Within this series, independent studies by Pirrung (Figure 4C). Next, we probed the same lysates by western blot

and colleagues (Ibarra-Rivera et al., 2011) and by Ichikawa and for poly(ADP-ribose) polymerase (PARP-1) and observed

colleagues (Chiba et al., 2014; Kitahata et al., 2016, 2017) have PARP-1 cleavage in 1-treated samples, indicating that 1 induced

shed light on the importance of the macrocyclic ring size and apoptotic cell death (Figure 4C, Kaufmann et al., 1993). Finally,

the location of the double bond, as well as the need for a hydro- we performed a global proteomics experiment to identify pro-

phobic tail for high inhibitory activity. In contrast, the cepafungin/ teins that accumulate or are upregulated in RPMI 8226 cells

glidobactin series have been less explored for analog synthesis upon 1 treatment in comparison with the treatment with the clin-

and structure-activity relationship studies. Oka et al. had semi- ical MM drug BTZ, which is known to covalently inhibit the 20S

synthetically prepared various derivatives of glidobactin with proteasome (Groll et al., 2006). An earlier gel-based ABPP study

modiﬁcations in the tail portion of the molecule. These com- with vinyl sulfone-based proteasome probes showed that BTZ

pounds were tested for antifungal activity and in vivo cytotoxicity engages the proteasome subunits b5, b1, and b2 (Berkers

against B16 melanoma and P388 leukemia cells in mice but were et al., 2005). Therefore, we anticipated that 1 and BTZ-treated

not studied in the context of proteasome inhibition.

myeloma cells may share many upregulated protein targets.

To assess the inhibitory activity of the synthesized derivatives, Cells were treated for 14 h with either 1 (100 nM) or BTZ

cells were treated with various concentrations of compounds for (2.5 nM), at concentrations matching their corresponding EC50

6

h, then lysed and treated with the b5 subunit-selective ﬂuoro- values at the 24-h time point to avoid protein expression

genic substrate Suc-LLVY-AMC (Maher, 2014). Release of the changes linked to cell death (Figures 3A and S2A). Cells were

-amino-4-methylcoumarin (AMC) ﬂuorophore via proteolytic lysed, proteins digested, and peptides analyzed by LC-MS/

cleavage by b5 was followed over time and quantiﬁed to obtain MS. Out of 3,700 quantiﬁed proteins, 88 proteins were signiﬁ-

IC50 values (Figure 4B). From all compounds tested, 1 most cantly upregulated (false discovery rate = 0.05; S = 0.01) in

7

0

potently inhibited the b5 subunit activity. In agreement with pre- 1-treated samples and 25 in BTZ-treated samples compared

vious work (Stein et al., 2012), terminal methyl branching in 1 with DMSO control samples (Figures 4D and S2B; Table S5).

leads to a nearly 5-fold increase in b5 inhibitory activity Indeed, 19 of 25 proteins upregulated in BTZ-treated samples

compared with 2 (glidobactin A). Based on previous structural were also upregulated in proteomic samples from 1-treated

data (Stein et al., 2012), the lipid tail occupies a hydrophobic cells, thus conﬁrming a highly similar mode of action for these

patch formed by the adjacent b6 subunit in the active site. two compounds. We believe that even better overlap can be

Thus, it is likely that the improved activity observed with addi- achieved if factors, such as differential cell permeability, binding

tional terminal branching arises from increased van der Waals kinetics, and metabolic stability, are taken into account. Notably,

interaction in this region. The difference in potency between 21 11 out of these 19 common targets have previously been re-

(

desoxycepafungin) and 1, over 10-fold loss, is in agreement ported to be upregulated (Hong et al., 2007a, 2007b; Fabre

with a recently reported potency difference between 2 and its et al., 2019; Blanchard et al., 2002; Matondo et al., 2017; Mertins

corresponding desoxy-macrocycle variant luminmycin A (Pawar et al., 2013; Tatsumi et al., 2003; Yew et al., 2005; Kwan et al.,

et al., 2020). While the origin of this difference cannot be readily 2015; Chen et al., 2014) upon proteasome inhibition in general

explained by the available structural data, it is well established (HERPUD1, KIAA0101, RRM2, CDC6, CCNB1, NUSAP1,

that the b5 subunit prefers hydrophilic residues at its S3 site ORC1, CYBA, CKAP2, KRT18, and TPX2) and 5 more targets

(

Clerc et al., 2010a). Thus, it is possible that the 4-hydroxylysine (BAG3, SRXN1, HMOX1, SQSMT1, and HSPA1A/B) speciﬁcally

residue increases the hydrophilicity of the macrocycle for more upon bortezomib treatment (Liu et al., 2009; Starheim et al.,

potent binding. Ichikawa and colleagues have previously re- 2016; Shah et al., 2016). Altogether, our data show that 1 is a

ported the bioactivity of several syringolin derivatives bearing potent and highly selective covalent inhibitor of the 20S protea-

alternative alkyl tails. However, no direct head-to-head compar- some that mediates downstream effects similar to the clinical

ison has been done to investigate the relative importance of the drug BTZ in MM cells.

6

Cell Chemical Biology 27, 1–9, October 15, 2020

Please cite this article in press as: Amatuni et al., Concise Chemoenzymatic Total Synthesis and Identiﬁcation of Cellular Targets of Cepafungin I, Cell

Chemical Biology (2020), https://doi.org/10.1016/j.chembiol.2020.07.012

ll

Brief Communication

SIGNIFICANCE

B MaxQuant Analysis

B Transfection and Western Blot

By harnessing the ability of GlbB to hydroxylate L-lysine in a

selective and efﬁcient fashion, we have developed a concise

chemoenzymatic synthesis of cepafungin I. Despite chal-

lenges in the late-stage macrocyclization and amide

coupling steps, cepafungin I could be obtained in ca. 8%

yield over nine steps (longest linear sequence). The modu-

larity of this route allowed the development of a chemopro-

teomic probe to interrogate the cellular targets of cepafun-

gin I. Our chemoproteomics studies revealed that

cepafungin I is able to covalently engage 20S proteasome

subunits PSMB2 and PSMB5 with exceptional selectivity

and that cepafungin I elicits many similar downstream bio-

logical responses to the clinically approved proteasome in-

hibitor drug bortezomib. Contemporaneous to our efforts,

B o¨ ttcher and co-workers reported the development of an

activity-based probe by derivatization of syringolin A that

was obtained from its native producer and showed the abil-

ity of the syrbactins to coinhibit PSMB2 and PSMB5 with

high potency. Complementary to B o¨ ttcher’s ﬁndings, our

work provides the ﬁrst proteome-wide identiﬁcation of syr-

bactin protein targets in cancer cells and validation of their

downstream biological effects via in-depth quantitative pro-

teomics analyses. Moreover, the synthetic strategy devel-

oped herein enabled access to both natural and unnatural

cepafungin analogs for initial structure-activity relationship

studies. Especially notable in this regard is the surprising

observation that the macrocyclic secondary alcohol, as

well as the degree of unsaturation and the terminal branch-

ing of the lipid tail, are critical for high inhibitory potency. In

combination with previous structural data of the 20S protea-

some:cepafungin complex, our synthetic strategy facilitates

structure-guided design of further analogs featuring alter-

native macrocycle and lipid functionalities. Such studies

are ongoing in our laboratory.

B In Situ b5 Proteasome Activity Assay

B Western Blot for Mono-/Poly-Ub and PARP-1

B Global MS Proteomics Proﬁling of 1 and BTZ

d QUANTIFICATION AND STATISTICAL ANALYSIS

SUPPLEMENTAL INFORMATION

Supplemental Information can be found online at https://doi.org/10.1016/j.

chembiol.2020.07.012 .

ACKNOWLEDGMENTS

This work is supported, in part, by the NIH grant GM128895 (to H.R.). We

acknowledge the Bannister lab and the Shen lab for generous access to their

instrumentation. We acknowledge Dr. D. Abegg for valuable discussions and

proofreading of the manuscript. We thank P. Dickson and the Kodadek lab for

providing the cell lines used in this study and for their valuable suggestions.

AUTHOR CONTRIBUTIONS

Conceptualization, H.R., A.Amatuni, and A.Adibekian; Experiments, A.Ama-

tuni and A.S.; Writing, H.R., A.Amatuni, A.Adibekian, and A.S.; Supervision,

H.R. and A.Adibekian.

DECLARATION OF INTERESTS

A.Amatuni, A.S., A.Adibekian, and H.R. have applied for a provisional patent

for this work.

Received: April 22, 2020

Revised: May 22, 2020

Accepted: July 17, 2020

Published: August 6, 2020

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Chemical Biology (2020), https://doi.org/10.1016/j.chembiol.2020.07.012

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STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE

Antibodies

SOURCE

IDENTIFIER

Rabbit-polyclonal antibody against

DYKDDDDK (FLAG)

Cell Signaling Technology

Cat#2368S; RRID: AB_2217020

Rabbit-polyclonal antibody against a-tubulin

Cell Signaling Technology

Cat#2144S; UniProt: P68363; RRID: AB_2210548

Cat#711-605-152, RRID: AB_2492288

Alexa Fluor 647-AfﬁniPure Donkey Anti-Rabbit

IgG (H+L)

Jackson ImmunoResearch Inc.

Rabbit-monoclonal anti-PARP-1

Cell Signaling Technology

Cat#9532; UniProt: P09874; RRID: AB_659884

Mouse-monoclonal polyubiquitinylated conjugates Enzo Life Sciences

antibody (FK1)

Cat#BML-PW8805; UniProt: P0CG47

(UBB), P0CG48 (UBC), P62979 (RPS27A),

P62987 (UBA52); RRID: AB_10541434

Alexa Fluor 488-AfﬁniPure Donkey Anti-Mouse IgG Jackson ImmunoResearch Inc.

Chemicals, Peptides, and Recombinant Proteins

Cat#715-545-150; RRID: AB_2340846

RPMI-1640

Fisher Scientiﬁc

Mediatech Inc

Life Technologies

Sigma-Aldrich

Fisher Scientiﬁc

VWR

Cat#11-875-093

DMEM

Cat#10-013-CV

Fetal bovine serum

Penicillin-Streptomycin

MEM Non-essential Amino Acid

Chloramphenicol

Cat#10437028

Cat#P4333

Cat#M7145

AC227920250, CAS 56-75-7

A4789.0010; CAS 25389-94-0

Cat#15300; CAS 367-93-1

Cat#QA-6576; CAS 5328-37-0

N/A

Kanamycin sulfate

IPTG

Cayman Chemical

Combi Blocks

This paper

L-(+)-arabinose

Compound 1

Bortezomib

Cayman Chemical

This paper

Cat#10008822; CAS 179324-69-7

N/A

Compound 20

TAMRA azide

Click Chemistry Tools

Alfa Aesar

Cat# AZ109

tris(2-carboxyethyl)phosphine hydrochloride

tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine

Biotin azide

Cat#J60316; CAS 51805-45-9

Cat#678937; 510758-28-8

Cat#762024; CAS 875770-34-6

Cat#I1149; CAS 144-48-9

Cat#G4108

Sigma Aldrich

Sigma-Aldrich

ProteoChem

Iodoacetamide

Streptavidin agarose

Pierceꢀ Trypsin Protease, MS Grade

PEI MAX 40000

Thermo Scientiﬁc

Polysciences Inc.

Thermo Fisher

This paper

Cat#90058

Cat#24765; CAS 49553-93-7

Cat#11058021

Opti-MEM

Compound 2

N/A

Compound 21

This paper

N/A

Compound 22

This paper

N/A

Suc-LLVY-AMC

Enzo Life Sciences

Cat#BML-P802-0005; CAS 94367-21-2

Protease Inhibitor Cocktail III, Mammalian

Critical Commercial Assays

Bradford Assay

Research Products International Corp. Cat#P50700-1

Bio-Rad

Cat#5000006

Cat#CELLPRO-RO

Cat#23225

WST-1 assay

Roche

BCA protein assay

Experimental Models: Cell Lines

HEK293T

Thermo Fisher Scientiﬁc

ATCC

Cat#ACS-4500

Cat#CCL-155

RPMI 8226

(Continued on next page)

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ll

Brief Communication

Continued

REAGENT or RESOURCE

MM.1R

SOURCE

ATCC

IDENTIFIER

Cat#CRL-2975

Experimental Models: Organisms/Strains

E. coli BL21(DE3)

Lucigen

Cat#60300-2

Recombinant DNA

PSMB5 (NM_002797) Human Tagged ORF Clone

PSMB2 (NM_002794) Human Tagged ORF Clone

pET-28a(+)-GlbB

Origene

Cat#RC209326

Cat#RC210452

N/A

Origene

(Amatuni and Renata, 2019)

Takara Bio

pGro7

Cat#3340

Software and Algorithms

GraphPad Prism (V7.02)

GraphPad

https://www.graphpad.com/scientiﬁc-

software/prism/

MaxQuant (V1.6.1.0)

ImageJ

MaxQuant

ImageJ

https://www.maxquant.org/

https://imagej.nih.gov/ij/

Perseus (V1.6.0.2)

Perseus

https://maxquant.net/perseus/

RESOURCE AVAILABILITY

Lead Contact

Further information and requests for resources and reagents should be directed to and will be fulﬁlled by the Lead Contact, Hans

Renata. Email: hrenata@scripps.edu .

Materials Availability

All unique/stable reagents generated in this study are available from the Lead Contact with a completed Materials Transfer

Agreement.

Data and Code Availability

The published article includes all datasets generated and analyzed during this study. This study did not generate any code.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Cell Culture and Lysate Preparation

RPMI 8226 and MM1.R cells were maintained in RPMI-1640 media. HEK293T cells were maintained in DMEM. All media were sup-

ꢀ

plemented with 10% fetal bovine serum (FBS), non-essential amino acids and penicillin/streptomycin. Cells were grown at 37 C un-

der 5% CO

centrifugation at 300 x g at 4 C and resuspended in PBS. Cells were lysed by needle sonication to obtain cell lysates and protein

concentration was determined using the Bradford assay.

2

atmosphere. Cells were allowed to grow to conﬂuence, harvested by pipeting, then washed 2 times in PBS by brief

ꢀ

METHOD DETAILS

Chemistry Materials

Unless otherwise noted, all chemicals and reagents for chemical reactions were purchased at the highest commercial quality and

used without further puriﬁcation. Reactions were monitored by thin layer chromatography (TLC) and liquid chromatography/mass

spectrometry (LC/MS). TLC was performed with 0.25 mm E. Merck silica plates (60F-254) using short-wave UV light as the visualizing

agent, and ninhydrin or KMnO4 and heat as developing agents. Sonication was performed using a Qsonica Q500 sonicator. LC/MS

was performed with an Agilent 1260 Inﬁnity System equipped with Poroshell 120 EC-C18 column (3.0 x 50 mm, 2.7 mm). HRMS(ESI-

TOF) was performed with an Agilent 1260 Inﬁnity System equipped with Poroshell 120 EC-C18 column (4.6 x 50 mm, 2.7 mm) and an

Agilent G6230B TOF LC/MS. Preparative HPLC was performed on an Agilent 1260 Inﬁnity II system equipped with a Dr. Maisch Re-

prosil 100 C18 column (10 x 250 mm, 5 mm), or on an Agilent 1260 Inﬁnity system equipped with a Varian Dynamax C18 column (21.4 x

2

50 mm, 10 mm). NMR spectra were recorded on a Bruker AVANCE AV400 (400 MHz and 100 MHz) or Bruker AVANCE AV600 (600

ꢀ

MHz and 151 MHz) at 23 C and calibrated using residual non-deuterated solvent. NMR spectra can be found under Data S1. Speciﬁc

rotations were measured on Autopol IV polarimeter (Rudolph Research Analytical).

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Brief Communication

Bacterial Strain Construction

pET-28a(+)-GlbB expression vector was obtained from Genscript as described previously (Amatuni and Renata, 2019). E. coli

BL21(DE3) harboring pET-28a(+)-GlbB and pGro7 (Takara Bio, Cat#3340) was constructed by transforming electrocompetent

E. coli BL21(DE3) (Lucigen, Cat#60300-2) with the appropriate plasmids.

Chemical Synthesis and Characterization

Chemoenzymatic Synthesis of Cepafungin I

(

2S,4S)-2,6-diamino-4-hydroxyhexanoic acid (5). 200 mL of TB media containing 25 mg/mL chloramphenicol and 50 mg/mL kana-

mycin was inoculated with 1 mL of an overnight culture of E. coli BL21(DE3) cells harboring pET-28a(+)-GlbB and pGro7. The culture

ꢀ

was shaken at 250 rpm at 36 C for approximately 2.5 hours or until an optical density of 0.6 was reached. The culture was then cooled

on ice (20 minutes) and induced by adding IPTG and arabinose to a ﬁnal concentration of 0.025 mM and 1 mg/mL, respectively. The

ꢀ

culture was allowed to continue for another 20 hours at 23 C and shaking at 250 rpm. Cells were harvested by centrifugation at

ꢀ

4

by sonication for 3 minutes (1 second on, 4 seconds off) at 0 C and 50% amplitude. Cell debris was pelleted by centrifugation

200 rpm at 4 C for 15 minutes, then resuspended in pH = 8 kPi buffer (150 mL) to an optical density of 25. The cells were lysed

ꢀ

and the supernatant was diluted to 250 mL with pH = 8 kPi buffer into a 1L Erlenmeyer ﬂask containing L-lysine (4) (1.46 g,

1

0 mmol, 1 eq, 40 mM ﬁnal concentration), a-ketoglutaric acid (2.83 g, 12.5 mmol, 1.25 eq, 50 mM ﬁnal concentration), ascorbic

acid (440 mg, 2.5 mmol, 0.25 eq, 10 mM ﬁnal concentration) and FeSO

ꢀ

4

,7H

The mixture was shaken at 200 rpm at 23 C overnight or until completion as judged by LC-MS.

2

O (139 mg, 0.5 mmol, 0.05 eq, 2 mM ﬁnal concentration).

ꢀ

The reaction mixture was then acidiﬁed to pH = 2 with concentrated HCl and centrifugated at 4200 rpm at 4 C for 15 minutes. The

supernatant containing crude 4-hydroxylysine 5 was carried forward to the next step without further puriﬁcation. For characterization

purposes, the crude product was puriﬁed by ion exchange chromatography with Dowex 50WX8 resin.

1

2

H NMR (600 MHz, D O with 1% TFA): d 4.09 – 3.99 (m, 1H), 3.89 – 3.78 (m, 1H), 3.21 – 3.08 (m, 2H), 2.18 – 2.09 (m, 1H), 1.98 – 1.91

(

m, 1H), 1.91 – 1.80 (m, 2H).

1

3

C NMR (151 MHz, D

2

O with 1% TFA): d 174.4, 67.7, 53.5, 37.1, 36.6, 34.0.

2

0

½

aꢂ = 3.7 (c = 0.5, H

2

O)

D

+

6 16 2 3

HRMS (ESI-TOF): calculated for C H N O ([M+H] ) 163.1083, found 163.1078.

tert-butyl (2-((2S,4S)-4-((tert-butoxycarbonyl)amino)-5-oxotetrahydrofuran-2-yl)ethyl)carbamate (7). The above supernatant was

O (8.73 g, 40 mmol, 4 eq) in 17 mL ethanol was added and

concentrated to ꢁ33 mL then basiﬁed to pH 11 with 2 M NaOH. Boc

2

the mixture was allowed to stir at room temperature. The mixture was re-basiﬁed to pH 11 after 3.5 hours and allowed to continue

stirring overnight or until completion as indicated by LC-MS. Ethanol was removed in vacuo and the mixture was diluted with 50 mL

ethyl acetate. The pH of the mixture was adjusted to 2 with 6 M HCl and the aqueous layer was separated and further extracted with

ethyl acetate (3 x 50 mL). The combined organic layers were washed with brine, dried over Na

provide 4.29 g crude di-Boc-4-hydroxylysine that was used without further puriﬁcation.

2

SO

4

and concentrated in vacuo to

ꢀ

The crude Boc-protected hydroxylysine (4.29 g) was dissolved in 50 mL anhydrous DCM, cooled to 0 C, then treated with

ꢀ

EDC,HCl (1.92 g, 10 mmol, 1 eq) followed by triethylamine (1.39 mL, 10 mmol, 1 eq). The reaction was stirred under argon at 0 C

for 2 hours or until completion as indicated by TLC. The reaction was concentrated to dryness. The resulting residue was dissolved

2 4

in 100 mL ethyl acetate and washed with water (3 x 20 mL) and brine, then dried over Na SO . Puriﬁcation by silica ﬂash chroma-

tography (step gradient 35-40-50% ethyl acetate in hexanes) provided pure lactone 7 (2.39 g, 6.93 mmol, 69% over 3 steps).

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Brief Communication

Note: the above 3-step sequence can be scaled up to 6.90 grams of L-lysine (47.2 mmol) to provide 9.53 grams of lactone 7

59% yield).

(

1

3 2

H NMR (400 MHz, (CD ) SO): d 7.45 (d, J = 8.3 Hz, 1H), 6.89 (t, J = 5.7 Hz, 1H), 4.62 – 4.48 (m, 1H), 4.28 (q, J = 9.1 Hz, 1H), 3.07 –

2

.90 (m, 2H), 2.32 – 2.09 (m, 2H), 1.82 – 1.60 (m, 2H), 1.38 (s, 9H), 1.37 (s, 9H).

13

3 2

C NMR (101 MHz, (CD ) SO): d 175.2, 155.6, 155.1, 78.6, 77.6, 75.4, 48.5, 36.4, 35.1, 32.5, 28.2, 28.1.

2

D

0

½

aꢂ = 26.7 (c = 0.5, CHCl

3

)

+

29 2 6

HRMS (ESI-TOF): calculated for C16H N O ([M+H] ) 345.2026, found 345.2026.

tert-butyl (S)-(1-(methoxy(methyl)amino)-1-oxopropan-2-yl)carbamate (S2). Boc-L-alanine (S1, 9.10 g, 48.1 mmol, 1.0 eq), N,O-di-

methylhydroxylamine hydrochloride (5.16 g, 52.9 mmol, 1.1 eq) and N-methylmorpholine (5.82 mL, 52.9 mmol, 1.1 eq) were dissolved

ꢀ

in 120 mL anhydrous DCM. The mixture was cooled to 0 C, and EDC,HCl (9.22 g, 48.1 mmol, 1.0 eq) was added portionwise. The

ꢀ

reaction was stirred at 0 C for two hours, and then at room temperature overnight. Upon complete consumption of S1 by TLC, the

reaction was diluted with 75 mL water and the pH of the mixture was adjusted to 2 with 1 M HCl. The layers were separated, and the

aqueous phase extracted twice with 20 mL DCM. The combined organic layers were washed with 30 mL water, saturated NaHCO

brine, then dried over Na SO and concentrated in vacuo to provide pure S2 as a crystalline white solid (10.7 g, 46.1 mmol, 96%).

H NMR (600 MHz, CD OD): d 4.60 – 4.53 (m, 1H), 3.81 (s, 3H), 3.20 (s, 3H), 1.44 (s, 9H), 1.26 (d, J = 7.1 Hz, 3H).

OD): d 175.7, 157.6, 80.4, 62.0, 48.0, 32.4, 28.7, 17.4.

3

,

2

4

1

3

1

3

C NMR (151 MHz, CD

3

2

½

aꢂ = –26.7 (c 2.45, MeOH)

D

+

20 2 4

HRMS (ESI-TOF): calculated for C10H N O Na ([M+Na] ) 255.1315, found 255.1315.

(

S)-2-amino-N-methoxy-N-methylpropanamide hydrochloride (9). Boc-L-alanine amide S2 (7.00 g, 29.4 mmol, 1 eq) was dissolved

in 49 mL of 4 M HCl in dioxane at room temperature. Upon complete consumption of S2 by TLC, the reaction was concentrated to

dryness, then evaporated twice from 50 mL 1:1 methanol:toluene to provide 9 as a white solid (5.72 g, quantitative).

1

H NMR (600 MHz, CD

3

OD): d 4.35 (q, J = 7.0 Hz, 1H), 3.81 (s, 3H), 3.25 (s, 3H), 1.50 (d, J = 7.0 Hz, 3H).

3

OD): d 170.9, 62.4, 48.7, 32.5, 16.3.

13

C NMR (151 MHz, CD

2

4

½

aꢂ = –27.7 (c 2.23, MeOH)

D

+

5 13 2 2

HRMS (ESI-TOF): calculated for C H N O ([M+H] ) 133.0972, found 133.0974.

di-tert-butyl ((3S,5S)-3-hydroxy-6-(((S)-1-(methoxy(methyl)amino)-1-oxopropan-2-yl)amino)-6-oxohexane-1,5-diyl)dicarbamate

(

to –20 C, then a 2 M solution of trimethylaluminum in anhydrous toluene was added dropwise (16.8 mL, 4.95 eq). The reaction was

10). Amine hydrochloride 9 (5.71g, 33.9 mmol, 5 eq) was set stirring (undissolved) in 85 mL anhydrous THF. The mixture was cooled

ꢀ

stirred at –20 C for 30 minutes under argon to provide a clear, light yellow solution. To this mixture was added lactone 7 in 17 mL

ꢀ

anhydrous THF (2.33 g, 6.77 mmol, 1 eq) dropwise at –20 C. The reaction was stirred to room temperature overnight, until completion

ꢀ

by TLC. After 28 hours, the reaction was cooled to 0 C and quenched by the careful addition of 20 mL 2 M NaHSO

4

solution dropwise.

The pH of the mixture was adjusted to 1 with 50 mL 1 M HCl, then concentrated in vacuo to remove most of the organic solvent. The

remaining aqueous mixture was extracted with ethyl acetate (5 x 50 mL). The combined organic layers were then washed with water,

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Brief Communication

3 2 4

saturated NaHCO , water, brine and ﬁnally dried over Na SO . Concentration in vacuo provided 3.15 g of crude dipeptide 10 that was

further puriﬁed by silica ﬂash chromatography (60:30:10 hexanes:ethyl acetate:methanol) to provide pure 10 as a white solid (2.90 g,

6

3

.09 mmol, 90 %).

1

3

H NMR (400 MHz, CD OD): d 6.52 (s, 1H), 4.86 – 4.77 (m, 1H), 4.17 (t, J = 7.3 Hz, 1H), 3.82 (s, 3H), 3.80 – 3.73 (m, 1H), 3.21 (s, 3H),

.19 – 3.13 (m, 2H), 1.90 – 1.75 (m, 1H), 1.72 – 1.61 (m, 2H), 1.60 – 1.49 (m, 1H), 1.44 (s, 9H), 1.43 (s, 9H), 1.34 (d, J = 7.1 Hz, 3H).

1

3

C NMR (151 MHz, CD OD): d 174.6, 174.5, 158.6, 157.6, 80.6, 80.0, 67.2, 62.1, 53.5, 47.1, 40.8, 38.4, 38.1, 32.5, 28.8, 28.7, 17.1.

2

4

½

aꢂ = –28.8 (c 1.01, MeOH)

D

+

41 4 8

HRMS (ESI-TOF): calculated for C21H N O ([M+H] ) 477.2919, found 477.2917.

di-tert-butyl ((3S,5S)-3-hydroxy-6-oxo-6-(((S)-1-oxopropan-2-yl)amino)hexane-1,5-diyl)dicarbamate (S3). In a procedure adapted

from Fehrentz et al. (1985), dipeptide 10 (100 mg, 0.210 mmol, 1 eq) was dissolved in 2.1 mL anhydrous diethyl ether and cooled

ꢀ

to 0 C. A solution of lithium aluminum hydride (1.05 M in Et

2

O, 0.50 mL, 2.5 eq) was then added dropwise at 0 C. Upon complete

. The layers were separated and

and brine,

. Concentration in vacuo provided aldehyde S3 as a white foam, which was used without further puriﬁcation. A

ꢀ

addition, the reaction was stirred for 30 minutes at 0 C then quenched with 2 mL 2M NaHSO

4

the aqueous phase was extracted with Et

then dried over Na SO

2 3

O (2 x 2 mL). The combined organic layers were washed with saturated NaHCO

2

4

sample of the material was puriﬁed by silica ﬂash chromatography (9:0.9:0.1 ethyl acetate:dichloromethane:triethylamine) to aid

1

characterization by H NMR.

1

3

H NMR (400 MHz, CDCl ): d 9.53 (d, J = 2.4 Hz, 1H), 7.23 – 6.99 (m, 1H), 5.82 – 5.55 (m, 1H), 4.91 – 4.73 (m, 1H), 4.50 – 4.36 (m, 1H),

4

2

.35 – 4.18 (m, 2H), 3.86 – 3.68 (m, 1H), 3.55 – 3.36 (m, 1H), 3.18 – 3.00 (m, 1H), 2.03 – 1.91 (m, 1H), 1.85 – 1.71 (m, 1H), 1.63 – 1.53 (m,

H), 1.45 – 1.41 (m, 18H), 1.35 (d, J = 7.3 Hz, 2H).

+

36 3 7

HRMS (ESI-TOF): calculated for C19H N O ([M+H] ) 418.2548, found 418.2548.

tert-butyl (S,E)-4-((2S,4S)-2,6-bis((tert-butoxycarbonyl)amino)-4-hydroxyhexanamido)pent-2-enoate (12). Ylide 11 (106 mg,

0

was added dropwise at room temperature. The reaction was reﬂuxed for 2 hours at 60 C, then concentrated to dryness in vacuo.

.281 mmol, 1.25 eq) was dissolved in 0.5 mL anhydrous acetonitrile, then a solution of aldehyde S3 (94 mg) in 3 mL acetonitrile

ꢀ

Puriﬁcation by silica ﬂash chromatography (step gradient 70:29.5:0.5 to 70:27:3 dichloromethane:ethyl acetate:methanol) provided

1

2 as an off-white solid (97 mg, 0.188 mmol, 67% over 2 steps).

1

H NMR (600 MHz, CD

4.03 (m, 1H), 3.73 – 3.62 (m, 1H), 3.16 (t, J = 6.9 Hz, 2H), 1.89 – 1.77 (m, 1H), 1.75 – 1.64 (m, 2H), 1.58 – 1.51 (m, 1H), 1.47 (s, 9H), 1.45

3

OD): d 6.78 (dd, J = 15.7, 5.0 Hz, 1H), 5.84 (dd, J = 15.7, 1.8 Hz, 1H), 4.59 (qdd, J = 7.0, 4.9, 1.7 Hz, 1H), 4.16

–

(

s, 9H), 1.43 (s, 9H), 1.28 (d, J = 7.0 Hz, 3H).

1

3

C NMR (151 MHz, CD

8.8, 28.4, 19.9.

3

OD): d 174.4, 167.4, 158.7, 157.7, 149.1, 122.9, 81.7, 80.7, 80.0, 67.7, 54.4, 47.0, 40.7, 38.4, 38.1, 28.8,

2

D

4

½

aꢂ = –23.5 (c 0.72)

+

46 3 8

HRMS (ESI-TOF): calculated for C25H N O ([M+H] ) 516.3279, found 516.3299.

di-tert-butyl ((3S,5S)-3-hydroxy-6-oxo-6-(((S)-1-oxopropan-2-yl)amino)hexane-1,5-diyl)dicarbamate (S3, Gram Scale). Dipeptide

ꢀ

1

0 (2.84 g, 5.96 mmol, 1 eq) was dissolved in 60 mL anhydrous diethyl ether and cooled to 0 C. A solution of lithium aluminum hydride

ꢀ

(1 M in Et

then quenched with 30 mL 2 M NaHSO

2

O, 14.9 mL, 2.5 eq) was then added dropwise at 0 C. Upon complete addition, the reaction was stirred for 30 minutes at 0 C

. The layers were separated and the aqueous phase was extracted with Et O (4 x 25 mL). The

4

2

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Brief Communication

combined organic layers were washed with water, saturated NaHCO

3 2 4

and brine, then dried over Na SO . Concentration in vacuo

provided aldehyde S3 as a white foam that was used for the next step without further puriﬁcation (2.15 g).

tert-butyl (S,E)-4-((2S,4S)-2,6-bis((tert-butoxycarbonyl)amino)-4-hydroxyhexanamido)pent-2-enoate (12, Gram Scale). Ylide 11

2.42 g, 6.42 mmol, 1.25 eq) was dissolved in 63 mL anhydrous acetonitrile, then a solution of aldehyde S3 (2.15 g) in 10 mL aceto-

(

1

nitrile was added dropwise at room temperature. The reaction was stirred for 30 minutes or until completion by H NMR or TLC, and

then concentrated to dryness. Puriﬁcation by silica ﬂash chromatography (100% diethyl ether) provided 12 as an off-white solid (1.59

g, 3.08 mmol, 52% over 2 steps).

(

S,E)-4-((2S,4S)-2,6-diamino-4-hydroxyhexanamido)pent-2-enoic acid–2,2,2-triﬂuoroacetic acid (1/2) (S4). Dipeptide 12 (500 mg,

.970 mmol, 1 eq) was dissolved in 24.2 mL of a freshly prepared solution of ‘‘Reagent B’’ (88:5:5:2 triﬂuoroacetic acid:phenol:wa-

0

ter:triisopropylsilane). After stirring for 1 hour at room temperature, the reaction was diluted with 30 mL toluene and concentrated in

vacuo to dryness, followed by an additional evaporation from 30 mL toluene. The residue was then dissolved in 2 mL methanol and

ꢀ

added dropwise to 100 mL diethyl ether at 0 C. The solids were collected by centrifugation at 4200 RPM for 10 minutes at 4 C, then

ꢀ

precipitated again into 50 mL diethyl ether at 0 C from 1.5 mL methanol. The solids were collected by centrifugation as above, then

dried from methanol to provide S4 as an off-white solid (442 mg, 0.907 mmol, 94 %).

1

3

H NMR (600 MHz, CD OD): d 6.82 (dd, J = 15.7, 5.4 Hz, 1H), 5.89 (dd, J = 15.7, 1.6 Hz, 1H), 4.67 – 4.61 (m, 1H), 4.04 – 3.97 (m, 1H),

3

.96 – 3.87 (m, 1H), 3.13 – 3.01 (m, 2H), 2.06 – 2.00 (m, 1H), 1.94 – 1.84 (m, 2H), 1.84 – 1.74 (m, 1H), 1.33 (d, J = 7.0 Hz, 3H).

13

3

C NMR (151 MHz, CD OD): d 170.3, 169.4, 148.9, 122.9, 68.3, 53.6, 47.4, 39.2, 37.8, 36.0, 19.7.

2

D

4

½

aꢂ = –1.8 (c 4.03, MeOH)

+

22 3 4

HRMS (ESI-TOF): calculated for C11H N O ([M+H] ) 260.1605, found 260.1605.

(5S,8S,10S,E)-8-amino-10-hydroxy-5-methyl-1,6-diazacyclododec-3-ene-2,7-dione (13). Linear macrocycle precursor S4

(366 mg, 0.751 mmol, 1 eq) was dissolved in 750 mL anhydrous DMF. The solution was treated with diisopropylethylamine

(0.523 mL, 3.00 mmol, 4 eq), followed by solid 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium tetraﬂuoroborate

(DMTMMT) (369 mg, 1.13 mmol, 1.50 eq) at room temperature. The reaction was stirred under argon for 36 hours, then

quenched with 20 mL water and concentrated in vacuo to dryness. The residue was dissolved in 3 mL methanol and precipitated

ꢀ

into 50 mL diethyl ether with rapid stirring. The solids were collected by centrifugation at 4200 RPM for 15 minutes at 4 C to yield

1

crude 13 as a tan solid (540 mg, 0.473 mmol, 63% from S4, 59% from 12). Yield was determined by H NMR analysis of a 7.4 mg

crude sample with 4-toluenesulfonamide (7.8 mg, 0.0465 mmol) added as internal standard. See Table S1 for macrolactamization

optimization.

1

3

H NMR (400 MHz, CD OD): 4-toluenesulfonamide: d 7.82 – 7.72 (m, 2H), 7.40 – 7.27 (m, 2H), 2.42 (s, 3H). Compound 13: 6.63 (dd,

J = 16.0, 6.1 Hz, 1H), 6.38 (dd, J = 16.0, 1.1 Hz, 1H), 4.73 – 4.64 (m, 1H), 3.83 – 3.76 (m, 1H), 2.27 – 2.10 (m, 1H), 2.01 – 1.92 (m, 1H),

.92 – 1.79 (m, 0H), 1.75 – 1.64 (m, 2H).

1

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ll

Brief Communication

+

20 3 3

HRMS (ESI-TOF): calculated for C11H N O ([M+H] ) 242.1499, found 242.1495.

7

-methyloctan-1-ol (16). Following a procedure adapted from Melaugh et al. (2014), lithium chloride (6.66 g, 157 mmol, 2.05 eq) was

ﬂame-dried in a 250 mL round-bottom ﬂask under high vacuum. Solid copper(II) chloride (10.3 g, 76.6 mmol, 1 eq) was added under

argon. The combined reagents were then dissolved in 105 mL anhydrous THF to provide a dark red solution of Li CuCl

2

4

.

A ﬂame-dried 3-neck 1L ﬂask ﬁtted with a reﬂux condenser was charged with magnesium turnings (12.9 g, 531 mmol, 1.18 eq with

respect to 2-bromopropane) and 200 mL anhydrous THF. With rapid stirring, 2-bromopropane (42.3 mL, 451 mmol, 6.5 eq with

respect to 15) was added dropwise until reﬂuxing began, at which point the ﬂask was lowered into a water bath. Upon complete addi-

tion, the water bath was removed and the reaction was allowed to stir under argon for 30 minutes to produce a dark gray suspension

ꢀ

of S5. The mixture was cooled to –78 C, and 6-bromohexan-1-ol 15 (12.6 g, 69.6 mmol, 1 eq) was added in 10 mL dry THF, followed

by the above solution of Li

removed from its cooling bath and allowed to warm to room temperature. Reaction progress was monitored by TLC until completion

2

CuCl

4

(ꢁ0.7 M in THF, 105 mL, 1.1 eq with respect to 15). Upon complete addition, the reaction was

ꢀ

at 1 hour. The reaction was then carefully quenched at 0 C with 50 mL saturated NH

4

Cl solution, ﬁltered through a sintered glass

funnel, and the ﬁltrate was concentrated in vacuo to remove most of the THF. The mixture was diluted with 100 mL ethyl acetate

and 100 mL water. The layers were separated, and the aqueous phase was extracted with ethyl acetate (3 x 200 mL). The combined

organic layers were washed with 50 mL each of saturated aqueous NaHCO

3 2 4

and brine, then dried over Na SO . Puriﬁcation by silica

ﬂash chromatography (90:10 hexanes:ethyl acetate) provided pure 16 as a colorless liquid (8.38 g, 58.1 mmol, 84%).

1

H NMR (600 MHz, CDCl

.85 (d, J = 6.7 Hz, 6H).

3

): d 3.63 (t, J = 6.7 Hz, 2H), 1.60 – 1.45 (m, 3H), 1.38 – 1.31 (m, 2H), 1.31 – 1.22 (m, 4H), 1.20 – 1.10 (m, 2H),

): d 63.2, 39.1, 32.9, 29.8, 28.1, 27.5, 25.9, 22.8.

0

1

3

C NMR (151 MHz, CDCl

3

+

HRMS (ESI-TOF): calculated for C11H24NO ([M+MeCN+H] ) 186.1852, found 186.1852.

ꢀ

7

solution of DMSO (1.48 mL, 20.8 mmol, 3 eq) in 3 mL DCM was added dropwise and the resulting mixture was stirred at –78 C for

-methyloctanal (S6). Oxalyl chloride (893 mL, 10.4 mmol, 1.5 eq) was dissolved in 8 mL anhydrous DCM at –78 C. An anhydrous

ꢀ

1

nutes. The mixture was stirred at –78 C for 1 hour, and treated with triethylamine (4.83 mL, 34.7 mmol, 5 eq) dropwise. The resulting

5 minutes. A solution of branched alcohol 16 (1.00 g, 6.93 mmol, 1 eq) in 6 mL anhydrous DCM was then added dropwise over 10 mi-

ꢀ

thick slurry was diluted with 10 mL DCM to aid stirring, and reaction progress was monitored by TLC until completion at 1 hour. The

ꢀ

reaction was quenched at –78 C with 20 mL water and allowed to warm to room temperature. The layers were separated, and the

aqueous layer was extracted with DCM (3 x 10 mL). The combined organic layers were washed with water (twice) and brine, then

dried over Na

2

SO

H NMR (400 MHz, CDCl

m, 4H), 1.20 – 1.10 (m, 2H), 0.85 (d, J = 6.6 Hz, 6H).

4

. Concentration in vacuo provided S6 as a pale yellow oil, which was used without further puriﬁcation (1.19 g).

1

3

): d 9.75 (t, J = 1.9 Hz, 1H), 2.41 (td, J = 7.3, 1.9 Hz, 2H), 1.66 – 1.58 (m, 2H), 1.56 – 1.44 (m, 1H), 1.34 – 1.24

(

+

9

HRMS (ESI-TOF): calculated for C H19O ([M+H] ) 143.1430, found 143.1431.

Ethyl (2E,4E)-11-methyldodeca-2,4-dienoate (18). In a procedure adapted from Imker et al. (2010), ethyl (E)-4-(diethoxyphosphoryl)

ꢀ

but-2-enoate 17 (2.60 g, 10.4 mmol, 1.5 eq) in 20 mL anhydrous THF was cooled to –78 C. A solution of lithium hexamethyldisilazide

ꢀ

(

in 5 mL THF was added dropwise at –78 C, and the mixture was allowed to stir to room temperature overnight. Upon completion by

1 M THF, 9.91 mL, 1.4 eq) was then added dropwise and allowed to stir for an additional 1 hour at –78 C. Crude aldehyde S6 (1.19 g)

ꢀ

TLC, the reaction was cooled to 0 C and quenched with 20 mL saturated NH

was extracted with ethyl acetate (3 x 30 mL). The combined organic layers were washed with 20 mL brine and dried over Na

4

Cl. The layers were separated, and the aqueous layer

4

SO .

2

Puriﬁcation by silica ﬂash chromatography (97:3 hexanes:ethyl acetate) provided 18 as a yellow oil (957 mg, 4.01 mmol, 58%

from 16).

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Brief Communication

1

3

H NMR (600 MHz, CDCl ): d 7.26 (dd, J = 18.1, 15.4 Hz, 1H), 6.20 – 6.07 (m, 2H), 5.78 (d, J = 15.4 Hz, 1H), 4.19 (q, J = 7.2 Hz, 2H),

2

.16 (td, J = 7.5, 6.1 Hz, 2H), 1.55 – 1.48 (m, 1H), 1.46 – 1.38 (m, 2H), 1.29 (t, J = 7.1 Hz, 3H), 1.30 – 1.24 (m, 4H), 1.18 – 1.12 (m, 2H),

.86 (d, J = 6.6 Hz, 6H).

0

13

C NMR (151 MHz, CDCl

3

): d 167.5, 145.3, 144.9, 128.5, 119.3, 60.3, 39.0, 33.1, 29.6, 28.9, 28.1, 27.3, 22.8, 14.5.

+

HRMS (ESI-TOF): Calculated for C15

27 2

H O ([M+H] ) 239.2006, found 239.2004.

(

2E,4E)-11-methyldodeca-2,4-dienoic acid (S7). Ester 18 (450 mg, 1.89 mmol, 1 eq) was dissolved in 9.5 mL methanol. With rapid

ꢀ

stirring, a solution of lithium hydroxide (226 mg, 9.44 mmol, 5 eq) in 9.5 mL water was added. The reaction was reﬂuxed at 80 C for 5

hours or until completion by TLC. Methanol was evaporated in vacuo, then the mixture was adjusted to pH 1 with 1 M HCl and diluted

with 20 mL DCM. The layers were separated, and the aqueous layer was extracted with DCM (3 x 20 mL). The combined organic

layers were washed with 10 mL of brine then dried over Na

(

2

SO

4

. Concentration in vacuo provided pure S7 as a waxy white solid

379 mg, 1.80 mmol, 95%).

1

H NMR (600 MHz, CDCl

3

): d 7.38 – 7.31 (m, 1H), 6.24 – 6.14 (m, 2H), 5.78 (d, J = 15.3 Hz, 1H), 2.18 (td, J = 7.4, 5.5 Hz, 2H), 1.56 –

1

.48 (m, 1H), 1.46 – 1.40 (m, 2H), 1.32 – 1.26 (m, 4H), 1.19 – 1.12 (m, 2H), 0.86 (d, J = 6.6 Hz, 6H).

13

3

C NMR (151 MHz, CDCl ): d 172.8, 147.7, 146.5, 128.3, 118.3, 39.0, 33.2, 29.6, 28.8, 28.1, 27.3, 22.8.

+

23 2

HRMS (ESI-TOF): calculated for C13H O ([M+H] ) 211.1693, found 211.1695.

tert-butyl O-(tert-butyl)-N-((2E,4E)-11-methyldodeca-2,4-dienoyl)-L-threoninate (S8). Fatty acid S7 (125 mg, 0.594 mmol, 1 eq) was

combined with tert-butyl O-(tert-butyl)-L-threoninate 19 (206 mg, 0.892 mmol, 1.5 eq) in 3 mL anhydrous DMF. Diisopropylethylamine

was added (0.311 mL, 1.78 mmol, 3 eq), followed by HATU (237 mg, 0.624 mmol, 1.05 eq). The reaction was stirred at room temper-

ature under argon for 4 hours or until completion by LCMS. The reaction was then quenched with 0.3 mL water and concentrated in

vacuo. The residue was dissolved in 50 mL ethyl acetate and washed with 1M HCl (twice), water, NaHCO

Na SO . Concentration in vacuo followed by silica ﬂash chromatography (90:10 hexanes:ethyl acetate) provided S8 as a colorless oil

203 mg, 0.479 mmol, 81%).

3

and brine, then dried over

2

4

(

1

3

H NMR (600 MHz, CDCl ): d 7.21 (dd, J = 15.0, 10.6 Hz, 1H), 6.19 – 6.11 (m, 2H), 6.06 (dt, J = 15.1, 6.8 Hz, 1H), 5.87 (d, J = 15.1 Hz,

1

H), 4.49 (dd, J = 9.2, 2.2 Hz, 1H), 4.21 (qd, J = 6.3, 2.2 Hz, 1H), 2.17 – 2.11 (m, 2H), 1.55 – 1.47 (m, 1H), 1.45 (s, 9H), 1.43 – 1.38 (m, 2H),

.30 – 1.23 (m, 4H), 1.17 (s, 9H), 1.16 (d, J = 6.3 Hz, 3H), 1.15 – 1.12 (m, 2H), 0.86 (d, J = 6.6 Hz, 6H).

1

13

C NMR (151 MHz, CDCl

8.1, 27.3, 22.8, 21.0.

3

): d 170.2, 166.6, 143.6, 141.9, 128.4, 121.6, 82.0, 73.9, 67.6, 58.4, 39.0, 33.1, 29.5, 29.0, 28.9, 28.3,

2

4

½aꢂ = 2.8 (c 2.01, MeOH)

D

+

4

HRMS (ESI-TOF): calculated for C25H46NO ([M+H] ) 424.3421, found 424.3423.

(

(2E,4E)-11-methyldodeca-2,4-dienoyl)-L-threonine (14). Amide S8 (170 mg, 0.401 mmol, 1 eq) was dissolved in 4 mL triﬂuoroace-

ꢀ

tic acid at 0 C, then stirred at room temperature for 1 hour. The reaction was then diluted with 10 mL toluene and concentrated in

vacuo to dryness. The residue was evaporated twice more from 10 mL toluene to provide 14 as a colorless resin (138 mg,

quantitative).

1

3

H NMR (600 MHz, CDCl ): d 7.22 (dd, J = 15.0, 9.5 Hz, 1H), 7.04 (d, J = 8.4 Hz, 1H), 6.18 – 6.07 (m, 2H), 5.93 (d, J = 15.1 Hz, 1H),

4

.67 – 4.59 (m, 1H), 4.53 – 4.43 (m, 1H), 2.21 – 2.09 (m, 2H), 1.55 – 1.47 (m, 1H), 1.46 – 1.37 (m, 2H), 1.30 – 1.24 (m, 4H), 1.21 (d, J =

.3 Hz, 3H), 1.18 – 1.12 (m, 2H), 0.86 (d, J = 6.6 Hz, 6H).

6

13

C NMR (151 MHz, CDCl

3

): d 173.6, 168.6, 145.3, 143.7, 128.2, 120.3, 67.8, 57.8, 39.1, 33.3, 29.7, 29.0, 28.1, 27.3, 22.8, 19.3.

2

4

½

aꢂ = 6.2 (c 2.66, MeOH)

D

Cell Chemical Biology 27, 1–9.e1–e18, October 15, 2020 e8

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ll

Brief Communication

+

4

HRMS (ESI-TOF): calculated for C17H30NO ([M+H] ) 312.2169, found 312.2170.

(

2E,4E)-N-((2S,3R)-3-hydroxy-1-(((5S,8S,10S,E)-10-hydroxy-5-methyl-2,7-dioxo-1,6-diazacyclododec-3-en-8-yl)amino)-1-oxobu-

tan-2-yl)-11-methyldodeca-2,4-dienamide (1). Acid 14 (38 mg, 0.122 mmol, 2 eq) and crude glidobamine 13 (75 mg, ꢁ0.0610 mmol

1

as judged by H NMR analysis, 1 eq) were dissolved in 1.5 mL anhydrous DMF and cooled to 0 C. To the mixture was added diiso-

ꢀ

propylethylamine (64 mL, 0.366 mmol, 6 eq), followed by solid DEPBT (37 mg, 0.122 mmol, 2 eq). The reaction was warmed to room

ꢀ

temperature and stirred overnight, and the reaction progress was monitored by LCMS. After 12 hours, the reaction was cooled to 0 C

ꢀ

and quenched with 0.5 mL water, then concentrated in vacuo to dryness. The residue was precipitated into 50 mL diethyl ether at 0 C

ꢀ

from 0.9 mL methanol, and the resulting solids were collected by centrifugation at 4200 RPM at 4 C for 15 minutes. The solids were

dissolved in methanol, ﬁltered through a 0.2 mm PTFE membrane ﬁlter, then puriﬁed by reversed-phase HPLC (gradient 30–60%

acetonitrile in water over 35 minutes, 0.1% formic acid added) to provide cepafungin I (1) as a ﬂuffy white powder after lyophilization

(10.9 mg, 0.0204 mmol, 33%, 20% from 12). See Table S2 for coupling optimization. See Table S3 for comparison of literature NMR

data with synthetic cepafungin I.

1

H NMR (600 MHz, (CD

5.1, 10.8 Hz, 1H), 6.40 (d, J = 14.4 Hz, 1H), 6.22 – 6.04 (m, 4H), 5.03 – 4.80 (m, 1H), 4.80 – 4.56 (m, 1H), 4.46 – 4.30 (m, 2H), 4.28 (dd,

J = 8.9, 4.1 Hz, 1H), 4.01 – 3.90 (m, 1H), 3.63 – 3.51 (m, 1H), 3.09 – 2.91 (m, 2H), 2.16 – 2.09 (m, 2H), 1.93 – 1.73 (m, 1H), 1.58 (d, J =

3 2

) SO ): d 8.79 – 8.53 (m, 1H), 7.99 – 7.91 (m, 1H), 7.75 (d, J = 7.7 Hz, 1H), 7.46 – 7.36 (m, 1H), 7.00 (dd, J =

1

1.1 Hz, 1H), 1.53 – 1.46 (m, 1H), 1.46 – 1.41 (m, 1H), 1.41 – 1.34 (m, 3H), 1.29 – 1.18 (m, 7H), 1.16 – 1.11 (m, 2H), 1.00 (d, J = 6.3 Hz,

H), 0.84 (d, J = 6.6 Hz, 6H).

3

1

3

C NMR (151 MHz, (CD

2.5, 38.4, 32.3, 28.9, 28.4, 27.4, 26.6, 22.5, 19.9, 18.5.

3 2

) SO): d 171.1, 169.4, 167.6, 165.4, 143.1, 142.1, 139.7, 128.6, 123.3, 123.1, 66.9, 66.7, 58.2, 51.2, 44.7,

4

2

D

4

ꢀ

½

aꢂ = –106.5 (c 0.20, MeOH)

+

47 4 6

HRMS (ESI-TOF): calculated for C28H N O ([M+H] ) 535.3490, found 535.3491.

Synthesis of Cepafungin Analogs

Synthesis of Cepafungin Alkyne (20)

8

eq) was dissolved in 21 mL anhydrous THF, cooled to –78 C and treated with n-butyllithium (2.5 M in hexanes, 12.5 mL, 2.82 eq)

-(trimethylsilyl)oct-7-yn-1-ol (S10). In a procedure adapted from Ye et al. (2018), ethynyltrimethylsilane S9 (2.95 g, 30.0 mmol, 2.72

ꢀ

dropwise. The mixture was stirred at –78 C for 1 hour, then treated dropwise with a solution of 6-bromohexan-1-ol 15 (2.00 g,

ꢀ

1

room temperature overnight or until completion by TLC analysis. The reaction was quenched at –20 C with 20 mL saturated

1.0 mmol, 1 eq) in 17 mL anhydrous THF, followed by 8.4 mL HMPA. The reaction was allowed to stir under argon at –78 C to

ꢀ

NH

5 mL). The combined organic layers were washed with 20 mL water, brine and dried over Na

by silica ﬂash chromatography (85:15 hexanes:ethyl acetate) provided S10 as a colorless oil (1.37 g, 6.91 mmol, 63%).

4

Cl, diluted with 20 mL ethyl acetate and the layers were separated. The aqueous layer was extracted with ethyl acetate (3 x

2

SO . Concentration in vacuo followed

4

1

H NMR (600 MHz, CDCl

3

): d 3.63 (t, J = 6.6 Hz, 2H), 2.21 (t, J = 7.1 Hz, 2H), 1.60 – 1.48 (m, 4H), 1.43 – 1.32 (m, 4H), 0.13 (s, 9H).

C NMR (151 MHz, CDCl ): d 107.7, 84.5, 63.0, 32.7, 28.6, 28.6, 25.3, 19.9, 0.3.

HRMS (ESI-TOF): calculated for C11

1

3

+

H

23OSi ([M+H] ) 198.1513, found 199.1517.

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Brief Communication

ꢀ

8

anhydrous solution of DMSO (727 mL, 10.2 mmol, 3 eq) in 1 mL DCM was added dropwise and the resulting mixture was stirred at –78

-(trimethylsilyl)oct-7-ynal (S11). Oxalyl chloride (439 mL, 5.12 mmol, 1.5 eq) was dissolved in 4 mL anhydrous DCM at –78 C. An

ꢀ

C for 15 minutes. A solution of alkynyl alcohol S10 (677 mg, 3.41 mmol, 1 eq) in 4 mL anhydrous DCM was then added dropwise. The

ꢀ

mixture was stirred at –78 C for 30 minutes, then treated with triethylamine (2.38 mL, 17.1 mmol, 5 eq) dropwise. Reaction progress

ꢀ

was monitored by TLC until completion at 1.5 hours. The reaction was quenched at –78 C with 20 mL water and allowed to warm to

room temperature. The layers were separated, and the aqueous layer was extracted with DCM (3 x 10 mL). The combined organic

2 4

layers were washed with water (twice) and brine, then dried over Na SO . Concentration in vacuo provided S11 as a pale yellow oil

which was used without further puriﬁcation (975 mg).

1

3

H NMR (600 MHz, CDCl ): d 9.76 (t, J = 1.8 Hz, 1H), 2.43 (td, J = 7.4, 1.8 Hz, 2H), 2.22 (t, J = 7.1 Hz, 2H), 1.64 (p, J = 7.4 Hz, 2H),

1

.57 – 1.48 (m, 2H), 1.46 – 1.40 (m, 2H), 1.27 – 1.17 (m, 1H), 0.13 (s, 9H).

Ethyl (2E,4E)-12-(trimethylsilyl)dodeca-2,4-dien-11-ynoate (S12). Ethyl (E)-4-(diethoxyphosphoryl)but-2-enoate 17 (1.28 g,

ꢀ

5

.12 mmol, 1.5 eq) in 17 mL anhydrous THF was cooled to –78 C. A solution of lithium hexamethyldisilazide (1 M THF, 4.78 mL,

ꢀ

1.4 eq) was then added dropwise and allowed to stir for 30 minutes at –78 C. Crude aldehyde S11 (975 mg) in 2 mL THF was added

dropwise at –78 C, and the mixture was allowed to stir to room temperature overnight. Upon its completion as judged by TLC, the

ꢀ

reaction was quenched with 20 mL saturated NH

with 30 mL water, 30 mL ethyl acetate and minimal brine to aid separation. The aqueous layer was extracted with ethyl acetate (3 x

0 mL). The combined organic layers were washed with 20 mL water and brine, then dried over Na SO . Puriﬁcation by silica ﬂash

chromatography (97:3 hexanes:ethyl acetate) provided S12 as a yellow oil (498 mg, 1.70 mmol, 50% from S10).

4

Cl at room temperature. After evaporation of THF in vacuo, the mixture was diluted

4

2

4

1

H NMR (600 MHz, CDCl

.21 (t, J = 7.1 Hz, 2H), 2.17 (q, J = 7.4, 7.0 Hz, 2H), 1.51 (p, J = 7.3 Hz, 2H), 1.47 – 1.36 (m, 4H), 1.28 (t, J = 7.1 Hz, 3H), 0.13 (s, 9H).

3

): d 7.24 (dd, J = 15.4, 10.5 Hz, 1H), 6.21 – 6.04 (m, 2H), 5.77 (d, J = 15.4 Hz, 1H), 4.18 (q, J = 7.1 Hz, 2H),

2

13

3

C NMR (151 MHz, CDCl ): d 167.4, 145.1, 144.4, 128.6, 119.4, 107.5, 84.6, 60.3, 32.9, 28.5, 28.4, 28.3, 19.9, 14.4, 0.3.

(

2E,4E)-dodeca-2,4-dien-11-ynoic acid (S13). Ester S12 (374 mg, 1.28 mmol, 1 eq) was dissolved in 6.4 mL methanol. With rapid

ꢀ

stirring, a solution of lithium hydroxide (153 mg, 6.40 mmol, 5 eq) in 6.4 mL water was added. The reaction was heated at 60 C over-

night or until completion as judged by TLC. Methanol was evaporated in vacuo. The mixture was diluted with 10 mL water and the

mixture was adjusted to pH 1 with 1 M HCl and diluted with 10 mL DCM. The layers were separated, and the aqueous layer was ex-

2 4

tracted with DCM (3 x 10 mL). The combined organic layers were washed with 10 mL brine then dried over Na SO . Concentration in

vacuo provided pure S13 as a white solid (221 mg, 1.15 mmol, 90%).

1

3

H NMR (400 MHz, CDCl ): d 7.34 (dd, J = 15.2, 9.5 Hz, 1H), 6.27 – 6.10 (m, 2H), 5.79 (d, J = 15.3 Hz, 1H), 2.23 – 2.16 (m, 4H), 1.95 (t,

J = 2.6 Hz, 1H), 1.54 (p, J = 7.0 Hz, 2H), 1.49 – 1.39 (m, 4H).

1

3

C NMR (101 MHz, CDCl

3

): d 172.5, 147.6, 146.0, 128.5, 118.4, 84.6, 68.5, 33.0, 28.4, 28.3, 28.2, 18.4.

–

HRMS (ESI-TOF): calculated for C12

15 2

H O ([M-H] ) 191.1077, found 191.1078.

tert-butyl O-(tert-butyl)-N-((2E,4E)-dodeca-2,4-dien-11-ynoyl)-L-threoninate (S14). Alkynyl fatty acid S13 (216 mg, 1.12 mmol, 1 eq)

was combined with diisopropylethylamine (880 mL, 5.05 mmol, 4.5 eq) and HATU (427 mg, 1.12 mmol, 1 eq) in 5 mL anhydrous DMF

and allowed to stir for 5 minminutes. tert-butyl O-(tert-butyl)-L-threoninate 19 (390 mg, 1.69 mmol, 1.5 eq) dissolved in 0.6 mL DMF

was then added and the reaction was stirred under argon for 2.5 hours or until completion by TLC or LCMS. The reaction was then

ꢀ

quenched at 0 C with 20 mL saturated NH

4

Cl, diluted with 15 mL ethyl acetate and the resulting layers were separated. The aqueous

layer was extracted with ethyl acetate (4 x 15 mL). The combined organic layers were washed with water (2 x 10 mL) and 10 mL brine,

then dried over Na SO . Concentration in vacuo followed by silica ﬂash chromatography (85:15 hexanes:ethyl acetate) provided S14

as a yellow oil (410 mg, 1.01 mmol, 90%).

2

4

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Brief Communication

1

3

H NMR (600 MHz, CDCl ): d 7.19 (dd, J = 15.0, 10.8 Hz, 1H), 6.21 – 6.10 (m, 2H), 6.08 – 6.01 (m, 1H), 5.87 (d, J = 15.0 Hz, 1H), 4.48

(

dd, J = 9.2, 2.1 Hz, 1H), 4.20 (qd, J = 6.3, 2.2 Hz, 1H), 2.16 (dtd, J = 14.1, 7.0, 1.9 Hz, 4H), 1.93 (t, J = 2.7 Hz, 1H), 1.55 – 1.49 (m, 2H),

.44 (s, 9H), 1.43 – 1.38 (m, 4H), 1.16 (s, 9H), 1.15 (d, J = 6.3 Hz, 3H).

1

3

C NMR (151 MHz, CDCl

8.3, 28.2, 21.0, 18.4.

3

): d 170.2, 166.6, 143.0, 141.8, 128.6, 121.8, 84.6, 82.0, 73.9, 68.4, 67.6, 58.4, 32.9, 28.8, 28.4, 28.4,

2

3

½aꢂ = 5.0 (c 0.680, MeOH)

D

–

4

HRMS (ESI-TOF): calculated for C24H39NO Cl ([M+Cl] ) 440.2573, found 440.2573.

(

(2E,4E)-dodeca-2,4-dien-11-ynoyl)-L-threonine (S15). Alkynyl fatty acid amide S14 (50 mg, 0.123 mmol, 1 eq) was dissolved in

ꢀ

1

.2 mL triﬂuoroacetic acid at 0 C, then stirred at room temperature for 1 hour. The reaction was then diluted with 10 mL toluene

and concentrated in vacuo to dryness to provide S15 as a pale yellow resin (50 mg, quant.).

1

H NMR (600 MHz, CDCl

J = 15.0 Hz, 1H), 4.63 (d, J = 8.5 Hz, 1H), 4.46 (s, 1H), 2.21 – 2.13 (m, 4H), 1.95 (t, J = 2.6 Hz, 1H), 1.56 – 1.49 (m, 2H), 1.46 – 1.37 (m, 4H),

.20 (d, J = 6.3 Hz, 3H).

3

): d 7.21 (dd, J = 15.0, 10.1 Hz, 1H), 7.05 (d, J = 8.1 Hz, 1H), 6.12 (dtd, J = 21.8, 15.1, 8.4 Hz, 2H), 5.94 (d,

1

3

C NMR (151 MHz, CDCl ): d 173.7, 168.3, 144.6, 143.4, 128.5, 120.6, 84.6, 68.5, 67.8, 57.8, 33.0, 28.4, 28.4, 28.4, 19.4, 18.5.

2

3

½

aꢂ = 8.3 (c 2.14, MeOH)

D

–

45 2 8

HRMS (ESI-TOF): calculated for C32H N O ([2M-H] ) 585.3181, found 585.3192

(

2E,4E)-N-((2S,3R)-3-hydroxy-1-(((5S,8S,10S,E)-10-hydroxy-5-methyl-2,7-dioxo-1,6-diazacyclododec-3-en-8-yl)amino)-1-oxobu-

tan-2-yl)dodeca-2,4-dien-11-ynamide (20). Acid S15 (38 mg, 0.128 mmol, 2 eq) and crude glidobamine 13 (82 mg, ꢁ0.0641 mmol

1

as judged by H NMR analysis, 1 eq) were dissolved in 3.2 mL anhydrous DMF and cooled to 0 C. To the mixture was added dii-

ꢀ

sopropylethylamine (34 mL, 0.192 mmol, 3 eq), followed by solid DEPBT (38 mg, 0.128 mmol, 2 eq). The reaction was stirred to

ꢀ

room temperature overnight, and reaction progress was monitored by LCMS. At 12 hours, the reaction was cooled to 0 C and

ꢀ

quenched with 0.5 mL water, then concentrated in vacuo to dryness. The residue was precipitated into 50 mL diethyl ether at 0 C

ꢀ

from 1.2 mL methanol, and the resulting solids were collected by centrifugation at 4200 RPM at 4 C for 15 minutes. The solids

were dissolved in methanol, ﬁltered through a 0.2 mm PTFE membrane ﬁlter, then puriﬁed by reversed-phase HPLC (gradient 30–

6

0% acetonitrile in water over 35 minutes, 0.1% formic acid added) to provide cepafungin alkyne (20) as a ﬂuffy white powder after

lyophilization (8.1 mg, 0.0157 mmol, 14% from 12).

1

H NMR (600 MHz, (CD

dd, J = 15.2, 10.8 Hz, 1H), 6.40 (d, J = 13.2 Hz, 1H), 6.24 – 6.03 (m, 4H), 4.87 (s, 1H), 4.67 (s, 1H), 4.50 – 4.30 (m, 2H), 4.28 (dd, J = 8.9,

3 2

) SO ): d 8.77 – 8.46 (m, 1H), 7.91 (d, J = 9.1 Hz, 1H), 7.74 (d, J = 7.8 Hz, 1H), 7.41 (t, J = 6.3 Hz, 1H), 7.00

(

4

1

.1 Hz, 1H), 4.03 – 3.88 (m, 1H), 3.65 – 3.49 (m, 1H), 3.08 – 2.92 (m, 2H), 2.74 (t, J = 2.6 Hz, 1H), 2.19 – 2.06 (m, 4H), 1.93 – 1.74 (m, 1H),

.58 (d, J = 13.5 Hz, 1H), 1.48 – 1.41 (m, 3H), 1.41 – 1.32 (m, 5H), 1.29 – 1.11 (m, 3H), 1.00 (d, J = 6.3 Hz, 3H).

1

3

C NMR (151 MHz, (CD

1.2, 44.7, 42.5, 32.1, 27.8, 27.8, 27.7, 19.9, 18.5, 17.6.

3 2

) SO ): d 171.0, 169.3, 167.6, 165.4, 143.1, 141.9, 139.7, 128.6, 123.3, 123.1, 84.5, 71.2, 67.0, 66.7, 58.1,

5

2

D

3

½

aꢂ = –74.2 (c 0.260, MeOH)

–

41 4 8

HRMS (ESI-TOF): calculated for C28H N O ([M+HCOOH–H] 561.2930, found 561.2936.

Synthesis of desoxycepafungin (21)

2

6

di-tert-butyl ((S)-6-(((S)-1-(methoxy(methyl)amino)-1-oxopropan-2-yl)amino)-6-oxohexane-1,5-diyl)dicarbamate (S17). N ,N -bis(-

tert-butoxycarbonyl)-L-lysine S16 (2.38 g, 6.86 mmol, 1 eq) and amine hydrochloride 9 (1.45 g, 8.57 mmol, 1.25 eq) were combined

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Brief Communication

in 34 mL anhydrous DMF. The mixture was treated with diisopropylethylamine (5.37 mL, 30.9 mmol, 4.5 eq) followed by HATU

(

2.61 g, 6.86 mmol, 1 eq) and allowed to stir at room temperature under argon for 1.5 hours or until completion by TLC. The

ꢀ

reaction was then quenched at 0 C with 5 mL saturated aqueous NH

4

Cl and concentrated to dryness. The residue was diluted

and brine. The combined aqueous layers were back-

extracted twice with 30 mL ethyl acetate. The combined organic layers were then dried over Na SO , concentrated in vacuo, and

puriﬁed by ﬂash silica chromatography (55:40:5 hexanes:ethyl acetate:methanol) to provide S17 as a colorless foam (3.10 g,

with 200 mL ethyl acetate and washed with 30 mL 1 M HCl, water, NaHCO

3

2

4

6

.73 mmol, 98%).

1

3

H NMR (600 MHz, CD OD): d 4.85 – 4.78 (m, 1H), 4.05 – 3.91 (m, 1H), 3.82 (s, 3H), 3.20 (s, 3H), 3.04 (h, J = 6.8 Hz, 2H), 1.80 – 1.66

(

m, 1H), 1.63 – 1.52 (m, 1H), 1.52 – 1.45 (m, 3H), 1.44 (s, 9H), 1.43 (s, 9H), 1.42 – 1.36 (m, 2H), 1.32 (d, J = 7.0 Hz, 3H).

13

3

C NMR (151 MHz, CD OD): d 174.8, 174.6, 158.6, 157.9, 80.6, 79.8, 62.1, 55.7, 46.9, 41.0, 33.0, 32.4, 30.6, 28.8, 28.7, 24.0, 17.3.

2

D

3

½

aꢂ = –20.0 (c 1.45, MeOH)

–

41 4 9

HRMS (ESI-TOF): calculated for C22H N O ([M+HCOOH–H] ) 505.2879, found 505.2880.

di-tert-butyl ((S)-6-oxo-6-(((S)-1-oxopropan-2-yl)amino)hexane-1,5-diyl)dicarbamate (S18). Dipeptide S17 (1.00 g, 2.17 mmol, 1 eq)

ꢀ

was dissolved in 22 mL anhydrous diethyl ether and cooled to 0 C. A solution of lithium aluminum hydride (1 M in Et

2

O, 8.14 mL, 3.75

ꢀ

eq) was then added dropwise at 0 C. Upon complete addition, the reaction was stirred for 30 minutes at 0 C then quenched with

6 mL 2 M NaHSO . The layers were separated and the aqueous phase was extracted with Et O (3 x 25 mL). The combined organic

layers were washed with water, saturated NaHCO and brine, then dried over Na SO . Concentration in vacuo provided aldehyde

S18 as a white foam that was used for the next step without further puriﬁcation (0.892 g).

1

4

2

3

2

4

1

3

H NMR (600 MHz, CDCl ): d 9.44 (s, 1H), 6.84 (d, J = 6.3 Hz, 1H), 5.17 (d, J = 7.5 Hz, 1H), 4.65 – 4.53 (m, 1H), 4.41 – 4.32 (m, 1H),

4

7

.07 – 3.98 (m, 1H), 3.06 – 2.94 (m, 2H), 1.81 – 1.67 (m, 1H), 1.63 – 1.48 (m, 1H), 1.47 – 1.37 (m, 4H), 1.34 (s, 9H), 1.34 (s, 9H), 1.26 (d, J =

.4 Hz, 3H).

–

34 3 6

HRMS (ESI-TOF): calculated for C19H N O ([M–H] ) 400.2453, found 400.2457.

tert-butyl (S,E)-4-((S)-2,6-bis((tert-butoxycarbonyl)amino)hexanamido)pent-2-enoate (S19). tert-Butyl(triphenylphosphoranylidene)

acetate 11 (1.02 g, 2.71 mmol, 1.25 eq) was dissolved in 21 mL anhydrous acetonitrile, then a solution of crude aldehyde S18 (0.892 g)

1

in 10 mL acetonitrile was added dropwise at room temperature. The reaction was stirred for 30 minutes or until completion by H NMR

or TLC, and then concentrated to dryness. Puriﬁcation by silica ﬂash chromatography (60:40 hexanes:ethyl acetate) provided S19 as

a white foam (0.863 g, 1.73 mmol, 80% from S17).

1

3

H NMR (600 MHz, CD OD): d 6.77 (dd, J = 15.7, 5.0 Hz, 1H), 5.80 (dd, J = 15.7, 1.8 Hz, 1H), 4.59 (qdd, J = 6.9, 4.9, 1.7 Hz, 1H), 4.03

–

3.84 (m, 1H), 3.03 (td, J = 6.8, 2.8 Hz, 2H), 1.78 – 1.66 (m, 1H), 1.66 – 1.56 (m, 1H), 1.54 – 1.48 (m, 2H), 1.47 (s, 9H), 1.45 (s, 9H), 1.43

s, 9H), 1.40 – 1.31 (m, 2H), 1.28 (d, J = 7.0 Hz, 3H).

(

13

C NMR (151 MHz, CD

8.4, 24.2, 19.9.

3

OD): d 174.6, 167.3, 158.6, 157.8, 149.2, 122.8, 81.7, 80.6, 79.8, 56.3, 46.9, 41.0, 33.1, 30.6, 28.8, 28.8,

2

3

½aꢂ = –17.6 (c 1.46, MeOH)

D

–

46 3 9

HRMS (ESI-TOF): calculated for C26H N O ([M+HCOOH–H] ) 544.3240, found 544.3246.

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Brief Communication

(

S,E)-4-((S)-2,6-diaminohexanamido)pent-2-enoic acid–2,2,2-triﬂuoroacetic acid (1/2) (S20). Dipeptide S19 (431 mg, 0.863 mmol, 1

eq) was dissolved in 22 mL of a freshly prepared solution of ‘‘Reagent B’’ (88:5:5:2 triﬂuoroacetic acid:phenol:water:triisopropylsi-

lane). After stirring for 1 hour at room temperature, the reaction was diluted with 30 mL toluene and concentrated in vacuo to dryness,

followed by an additional evaporation from 30 mL toluene. The residue was then dissolved in 2 mL methanol and added dropwise to

ꢀ

1

into 50 mL diethyl ether at 0 C from 1.5 mL methanol. The solids were collected by centrifugation as above, then dried from methanol

00 mL diethyl ether at 0 C. The solids were collected by centrifugation at 4200 RPM for 10 minutes at 4 C, then precipitated again

ꢀ

to provide S20 as an off-white solid (356 mg, 0.755 mmol, 88%).

1

3

H NMR (600 MHz, CD OD): d 6.83 (dd, J = 15.7, 5.4 Hz, 1H), 5.86 (dd, J = 15.7, 1.6 Hz, 1H), 4.69 – 4.59 (m, 1H), 3.87 (t, J = 6.4 Hz,

1

H), 2.98 – 2.89 (m, 2H), 1.99 – 1.82 (m, 2H), 1.76 – 1.66 (m, 2H), 1.54 – 1.42 (m, 2H), 1.33 (d, J = 7.0 Hz, 3H).

3

1

3

C NMR (151 MHz, CD OD): d 170.1, 169.3, 149.0, 122.7, 54.2, 47.4, 40.3, 32.1, 28.2, 22.9, 19.7.

2

D

3

½

aꢂ = –13.6 (c 0.530, MeOH)

–

20 3 3

HRMS (ESI-TOF): calculated for free base C11H N O ([M–H] ) 242.2521, found 242.1511.

(

5S,8S,E)-8-amino-5-methyl-1,6-diazacyclododec-3-ene-2,7-dione (S21). Macrocycle precursor S20 (200 mg, 0.424 mmol, 1 eq)

was dissolved in 424 mL anhydrous DMF. The solution was treated with diisopropylethylamine (296 mL, 1.70 mmol, 4 eq), followed

by solid 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium tetraﬂuoroborate (DMTMMT) (209 mg, 0.637 mmol, 1.5 eq) at

room temperature. The reaction was stirred under argon for 27 hours, then quenched with 10 mL water and concentrated in vacuo

to dryness. The residue was dissolved in 2 mL methanol and precipitated into 50 mL diethyl ether with rapid stirring. The solids were

ꢀ

collected by centrifugation at 4200 RPM for 15 minutes at 4 C to yield crude S21 as a tan solid (276 mg, 0.187 mmol, 44% from S20,

1

3

0

9% from S19). Yield was determined by H NMR analysis of a 6.2 mg crude sample with 4-toluenesulfonamide (7.7 mg,

.0450 mmol) added as internal standard.

1

3

H NMR (400 MHz, CD OD): 4-toluenesulfonamide: d 7.82 – 7.72 (m, 2H), 7.40 – 7.30 (m, 2H), 2.42 (s, 3H). Compound S21: 7.03

(

dd, J = 15.4, 4.9 Hz, 1H), 6.35 (dd, J = 15.5, 1.4 Hz, 1H), 4.66 – 4.58 (m, 1H), 4.16 (t, J = 3.7 Hz, 1H), 3.58 – 3.44 (m, 2H), 2.36 – 2.24 (m,

H), 1.98 – 1.77 (m, 2H), 1.77 – 1.62 (m, 2H).

HRMS (ESI-TOF): calculated for C11

1

–

18 3 2

H N O ([M–H] ) 224.1404, found 224.1406.

(

2E,4E)-N-((2S,3R)-3-hydroxy-1-(((5S,8S,E)-5-methyl-2,7-dioxo-1,6-diazacyclododec-3-en-8-yl)amino)-1-oxobutan-2-yl)-11-meth-

yldodeca-2,4-dienamide (21). Acid 14 (42 mg, 0.134 mmol, 2 eq) and crude desoxyglidobamine S21 (100 mg, ꢁ0.0670 mmol as

1

judged by H NMR analysis, 1 eq) were dissolved in 3.4 mL anhydrous DMF and cooled to 0 C. To the mixture was added diisopro-

ꢀ

pylethylamine (35 mL, 0.201 mmol, 3 eq), followed by solid DEPBT (40 mg, 0.134 mmol, 2 eq). The reaction was warmed to room tem-

ꢀ

perature and stirred overnight, and the reaction progress was monitored by LCMS. At 24 hours, the reaction was cooled to 0 C and

quenched with 2 mL saturated NH

bined organic layers were washed with brine, dried over Na

.2 mm PTFE membrane ﬁlter in methanol, then puriﬁed by reversed-phase HPLC (gradient 30–60% acetonitrile in water over 35 mi-

nutes, 0.1% formic acid added) to provide desoxycepafungin I (21) as a ﬂuffy white powder after lyophilization (7.9 mg, 0.0152 mmol,

4

Cl. The mixture was extracted with ethyl acetate (4 x 4 mL), then n-butanol (3 x 4 mL). The com-

2

SO , and concentrated in vacuo. The residue was ﬁltered through a

4

0

2

3%, 9% from S24).

1

3 2

H NMR (600 MHz, (CD ) SO): d 8.44 (d, J = 7.9 Hz, 1H), 7.99 (d, J = 8.5 Hz, 1H), 7.62 (d, J = 7.2 Hz, 1H), 7.35 (dd, J = 8.0, 6.1 Hz,

1H), 7.00 (dd, J = 15.1, 10.8 Hz, 1H), 6.78 (dd, J = 15.3, 4.7 Hz, 1H), 6.23 (dd, J = 15.4, 1.4 Hz, 1H), 6.21 – 6.15 (m, 2H), 6.09 (dt, J = 14.8,

6.9 Hz, 1H), 4.54 – 4.48 (m, 1H), 4.44 – 4.36 (m, 1H), 4.32 (dd, J = 8.5, 3.9 Hz, 1H), 4.02 – 3.96 (m, 1H), 3.80 (s, 1H), 3.32 – 3.23 (m, 1H),

2.98 – 2.91 (m, 1H), 2.13 (q, J = 7.2 Hz, 2H), 2.10 – 2.03 (m, 1H), 1.74 – 1.64 (m, 1H), 1.50 (hept, J = 6.7 Hz, 1H), 1.46 – 1.41 (m, 1H), 1.41

– 1.35 (m, 2H), 1.35 – 1.29 (m, 1H), 1.29 – 1.22 (m, 5H), 1.20 (d, J = 7.0 Hz, 3H), 1.17 – 1.11 (m, 2H), 1.02 (d, J = 6.3 Hz, 3H), 0.98 – 0.90

(

m, 1H), 0.85 (d, J = 6.6 Hz, 6H).

1

3

3 2

C NMR (151 MHz, (CD ) SO): d 170.5, 169.4, 165.8, 165.5, 146.7, 142.1, 139.8, 128.6, 122.9, 118.4, 66.6, 58.1, 51.5, 45.6, 38.4,

7.9, 32.2, 30.2, 29.6, 28.8, 28.4, 27.4, 26.6, 22.5, 19.6, 18.3, 17.1.

3

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Brief Communication

2

D

3

½

aꢂ = –14.4 (c = 0.160, MeOH)

+

47 4 5

HRMS (ESI-TOF): calculated for C28H N O ([M+H] ) 519.3541, found 519.3564.

Synthesis of Cepafungin Analog with Saturated Lipid Tail (22)

1

1-methyldodecan-1-ol (S23). Following a procedure adapted from Melaugh et al. (2014), lithium chloride (1.61 g, 42.4 mmol, 2.05

eq) was ﬂame-dried in a 250 mL round-bottom ﬂask under high vacuum. Solid copper(II) chloride (2.50 g, 18.6 mmol, 1 eq) was added

under argon. The combined reagents were then dissolved in 25 mL anhydrous THF to provide a dark red solution of Li CuCl

2

4

.

A ﬂame-dried 3-neck 500 mL ﬂask ﬁtted with a reﬂux condenser was charged with magnesium turnings (3.14 g, 129 mmol, 1.18 eq

with respect to 2-bromopropane) and 50 mL anhydrous THF. With rapid stirring, 2-bromopropane (10.3 mL, 110 mmol, 6.5 eq with

respect to S22) was added dropwise until reﬂuxing began, at which point the ﬂask was lowered into a water bath. Upon complete

addition, the water bath was removed and the reaction was allowed to stir under argon for 30 minutes to produce a dark gray sus-

ꢀ

pension of S5. The mixture was cooled to –78 C, and 10-bromodecan-1-ol S22 (4.00 g, 16.9 mmol, 1 eq) was added in 4.2 mL dry

THF, followed by the above solution of Li

reaction was removed from its cooling bath and allowed to warm to room temperature. Reaction progress was monitored by TLC

2

CuCl

4

(ꢁ0.7 M in THF, 25 mL, 1.1 eq with respect to S22). Upon complete addition, the

ꢀ

until completion at 1 hour. The reaction was then carefully quenched at 0 C with 50 mL saturated NH

4

Cl solution, ﬁltered through

a sintered glass funnel, and the ﬁltrate concentrated in vacuo to remove most of the THF. The mixture was diluted with 100 mL ethyl

acetate and 100 mL 1M HCl. The layers were separated, and the aqueous phase was extracted with ethyl acetate (3 x 50 mL). The

combined organic layers were washed with 50 mL each of water, saturated aqueous NaHCO

3 2 4

and brine, then dried over Na SO .

Puriﬁcation by silica ﬂash chromatography (90:10 hexanes:ethyl acetate) provided pure S23 as a colorless oil (3.22 g,

1

2

6.1 mmol, 95%).

1

3

H NMR (600 MHz, CDCl ): d 3.63 (t, J = 6.7 Hz, 2H), 1.59 – 1.54 (m, 2H), 1.53 – 1.47 (m, 1H), 1.36 – 1.22 (m, 14H), 1.19 – 1.10 (m,

H), 0.85 (d, J = 6.7 Hz, 6H).

13

C NMR (151 MHz, CDCl

3

): d 63.2, 39.2, 32.9, 30.1, 29.8, 29.8, 29.7, 29.6, 28.1, 27.6, 25.9, 22.8.

+

HRMS (ESI-TOF): calculated for C14

32 1 1 3

H O N ([M+CH CN+H] ) 242.2478, found 242.2475.

ꢀ

1

1-methyldodecanoic acid (S24). Alcohol S23 (1.60 g, 7.96 mmol, 1 eq) was dissolved in 50 mL acetone and cooled to 0 C. A so-

lution of Jones’ reagent (CrO

3

/H

2

SO

4

/H

2

O) (6.52 mL, 19.9 mmol, 2.5 eq) was then added dropwise and allowed to stir at 0 C. Upon

completion by TLC in ﬁve minutes, the reaction was quenched with 3 mL isopropanol, ﬁltered through a plug of celite and concen-

trated in vacuo to remove the organic solvent. The remaining aqueous mixture was diluted with 25 mL 1 M HCl and extracted with

2 4

ethyl acetate (4 x 25 mL). The combined organic layers were washed with 25 mL water and brine, then dried over Na SO and concen-

trated in vacuo to provide pure S24 (1.58 g, 7.37 mmol, 93%).

1

3

H NMR (600 MHz, CDCl ): d 2.34 (t, J = 7.5 Hz, 2H), 1.67 – 1.59 (m, 2H), 1.56 – 1.46 (m, 1H), 1.36 – 1.21 (m, 12H), 1.19 – 1.11 (m,

2

H), 0.86 (d, J = 6.6 Hz, 6H).

13

C NMR (151 MHz, CDCl

3

): d 180.5, 39.3, 34.4, 30.1, 29.9, 29.7, 29.5, 29.3, 28.2, 27.6, 24.9, 22.9.

–

HRMS (ESI-TOF): calculated for C13

25 2

H O ([M–H] ) 213.1860, found 213.1861.

tert-butyl O-(tert-butyl)-N-(11-methyldodecanoyl)-L-threoninate (S25). Fatty acid S24 (636 mg, 2.97 mmol, 1 eq) was dissolved in

ꢀ

1

4

4 mL anhydrous DMF at 0 C. Diisopropylethylamine was added (2.33 mL, 13.4 mmol, 4.5 eq), followed by HATU (1.13 g, 2.97 mmol,

ꢀ

eq). The mixture was stirred at 0 C under argon for ﬁve minutes before adding tert-butyl O-(tert-butyl)-L-threoninate 19 (1.03 g,

.45 mmol, 1.5 eq) in 1 mL DMF. The reaction was stirred to room temperature under argon for 4 hours or until completion by

ꢀ

LCMS. The reaction was then diluted at 0 C with 50 mL saturated NH

4

Cl and ethyl acetate. The pH of the mixture was adjusted

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Brief Communication

to 7 with 1 M HCl, and the aqueous layer was extracted with ethyl acetate (3 x 50 mL). The combined organic layers were washed with

3

0 mL 1M HCl, water, NaHCO

3 2 4

and brine, then dried over Na SO . Concentration in vacuo followed by silica ﬂash chromatography

(

85:15 hexanes:ethyl acetate) provided S25 as a colorless oil (955 mg, 2.23 mmol, 75%).

1

3

H NMR (600 MHz, CDCl ): d 6.12 (d, J = 9.2 Hz, 1H), 4.39 (dd, J = 9.2, 2.1 Hz, 1H), 4.19 (qd, J = 6.2, 2.1 Hz, 1H), 2.26 (d, J = 7.5 Hz,

H), 1.69 – 1.61 (m, 2H), 1.51 (dq, J = 13.3, 6.6 Hz, 1H), 1.45 (s, 9H), 1.38 – 1.21 (m, 14H), 1.16 (s, 9H), 1.15 (d, J = 6.3 Hz, 3H), 0.85 (d,

2

J = 6.6 Hz, 6H).

1

3

C NMR (151 MHz, CDCl

5.8, 22.8, 21.1.

3

): d 173.5, 170.3, 81.9, 73.9, 67.4, 58.3, 39.2, 36.9, 30.0, 29.8, 29.6, 29.5, 29.4, 28.9, 28.3, 28.1, 27.5,

2

3

½aꢂ = –4.4 (c 2.09, MeOH)

D

+

4

HRMS (ESI-TOF): calculated for C25H50NO ([M+H] ) 428.3735, found 428.3748.

(

acid at 0 C, then stirred at room temperature for 1 hour. The solution was then diluted with 10 mL toluene and concentrated in vacuo

11-methyldodecanoyl)-L-threonine (S26). Fatty acid amide S25 (100 mg, 0.234 mmol, 1 eq) was dissolved in 2.3 mL triﬂuoroacetic

ꢀ

to dryness, followed by an additional evaporation from 10 mL toluene to provide S26 as a colorless oil (91 mg, quantitative).

1

3

H NMR (600 MHz, CDCl ): d 6.99 (s, 1H), 4.76 – 4.10 (m, 2H), 2.28 (s, 2H), 1.62 (s, 2H), 1.53 – 1.46 (m, 1H), 1.35 – 1.22 (m, 14H),

1

.19 (s, 1H), 1.17 – 1.11 (m, 2H), 0.86 (d, J = 6.6 Hz, 6H).

3

1

3

C NMR (151 MHz, CDCl ): d 175.5, 39.2, 36.6, 32.0, 30.1, 29.9, 29.8, 29.6, 29.5, 28.1, 27.6, 26.3, 26.0, 22.8, 19.5, 14.3.

2

D

3

½

aꢂ = 4.6 (c 1.40, MeOH)

–

4

HRMS (ESI-TOF): calculated for C17H32NO ([M–H] ) 314.2337, found 314.2339

N-((2S,3R)-3-hydroxy-1-(((5S,8S,10S,E)-10-hydroxy-5-methyl-2,7-dioxo-1,6-diazacyclododec-3-en-8-yl)amino)-1-oxobutan-2-yl)-

1

1-methyldodecanamide (22). Acid S26 (33 mg, 0.104 mmol, 2 eq) and crude glidobamine 13 (62 mg, ꢁ0.0521 mmol as judged by

1

ꢀ

H NMR analysis, 1 eq) were dissolved in 2.6 mL anhydrous DMF and cooled to 0 C. To the mixture was added diisopropylethyl-

amine (27 mL, 0.156 mmol, 3 eq), followed by solid DEPBT (31 mg, 0.104 mmol, 2 eq). The reaction was stirred to room temperature

ꢀ

overnight, and reaction progress was monitored by LCMS. At 12 hours, the reaction was cooled to 0 C and quenched with 0.5 mL

ꢀ

water, then concentrated in vacuo to dryness. The residue was precipitated into 50 mL diethyl ether at 0 C from 0.75 mL methanol,

ꢀ

and the resulting solids were collected by centrifugation at 4200 RPM at 4 C for 15 minutes. The solids were dissolved in methanol,

ﬁltered through a 0.2 mm PTFE membrane ﬁlter, then puriﬁed by reversed-phase HPLC (gradient 30–100% acetonitrile in water over

2

0

0 minutes, 0.1% formic acid added) to provide saturated cepafungin (22) as a ﬂuffy white powder after lyophilization (9.6 mg,

.0178 mmol, 34%, 20% from 12).

1

3 2

H NMR (600 MHz, (CD ) SO): d 8.66 (s, 1H), 7.69 (d, J = 8.8 Hz, 1H), 7.64 (d, J = 7.9 Hz, 1H), 7.41 (t, J = 6.3 Hz, 1H), 6.40 (d, J =

1

3

4

5.9 Hz, 1H), 6.18 (dd, J = 15.8, 1.2 Hz, 1H), 4.85 – 4.74 (m, 1H), 4.73 – 4.60 (m, 1H), 4.48 – 4.25 (m, 2H), 4.18 (dd, J = 8.8, 3.9 Hz, 1H),

.99 – 3.88 (m, 1H), 3.63 – 3.50 (m, 1H), 3.09 – 2.91 (m, 2H), 2.24 – 2.09 (m, 2H), 1.92 – 1.74 (m, 1H), 1.64 – 1.54 (m, 1H), 1.54 – 1.41 (m,

H), 1.41 – 1.33 (m, 1H), 1.27 – 1.18 (m, 15H), 1.17 – 1.10 (m, 2H), 0.99 (d, J = 6.3 Hz, 3H), 0.84 (d, J = 6.6 Hz, 6H).

1

3

C NMR (151 MHz, (CD

9.0, 28.8, 28.6, 27.4, 26.8, 25.4, 22.5, 19.9, 18.5.

3 2

) SO): d 172.5, 171.0, 169.4, 167.6, 142.7, 123.5, 67.0, 66.6, 57.9, 51.2, 44.7, 42.5, 38.5, 35.2, 29.3, 29.1,

2

D

3

½

aꢂ = –76.8 (c 0.190, MeOH)

–

49 4 6

HRMS (ESI-TOF): calculated for C28H N O ([M–H] ) 537.3657, found 537.3657.

Synthesis of Glidobactin A (2)

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Brief Communication

(

dissolved in 22.5 mL methanol. With rapid stirring, a solution of lithium hydroxide (534 mg, 22.3 mmol, 5 eq) in 22.5 mL water was

2E,4E)-dodeca-2,4-dienoic acid (S30). Ethyl (2E,4E)-dodeca-2,4-dienoate S29 (Imker et al., 2010) (1.00 g, 4.46 mmol, 1 eq) was

ꢀ

added. The reaction was heated at 80 C for 2.5 hours or until completion as judged by TLC. Methanol was evaporated in vacuo.

The mixture was adjusted to pH 1 with 1 M HCl and diluted with 50 mL DCM. The layers were separated, and the aqueous layer

2 4

was extracted with DCM (3 x 20 mL). The combined organic layers were washed with 20 mL brine then dried over Na SO . Concen-

tration in vacuo provided pure S30 as a waxy white solid (764 mg, 3.89 mmol, 87%).

1

3

H NMR (600 MHz, CDCl ): d 7.39 – 7.29 (m, 1H), 6.25 – 6.11 (m, 2H), 5.78 (d, J = 15.3 Hz, 1H), 2.24 – 2.10 (m, 2H), 1.47 – 1.39 (m,

2

H), 1.33 – 1.22 (m, 8H), 0.88 (t, J = 7.0 Hz, 3H).

13

C NMR (151 MHz, CDCl

3

): d 173.0, 147.7, 146.4, 128.4, 118.4, 33.2, 31.9, 29.3, 29.2, 28.8, 22.8, 14.2.

–

HRMS (ESI-TOF): calculated for C12

19 2

H O ([M–H] ) 195.1391, found 195.1392

(

(2E,4E)-dodeca-2,4-dienoyl)-L-threonine (S32). tert-butyl O-(tert-butyl)-N-((2E,4E)-dodeca-2,4-dienoyl)-L-threoninate S31 was

ꢀ

prepared in a similar fashion to S8. S31 (100 mg, 0.244 mmol, 1 eq) was dissolved in 2.4 mL triﬂuoroacetic acid at 0 C, then stirred

at room temperature for 30 minutes. The solution was then diluted with 8 mL toluene and concentrated in vacuo to dryness, followed

by two additional evaporations from 8 mL toluene to provide pure S32 as a colorless resin (92 mg, quantitative).

1

3

H NMR (400 MHz, CDCl ): d 7.25 – 7.15 (m, 1H), 7.08 (d, J = 8.4 Hz, 1H), 6.21 – 6.03 (m, 2H), 5.92 (d, J = 15.0 Hz, 1H), 4.65 (d, J =

8

.0 Hz, 1H), 4.55 – 4.37 (m, 1H), 2.20 – 2.07 (m, 2H), 1.45 – 1.35 (m, 2H), 1.33 – 1.24 (m, 8H), 1.21 (d, J = 6.3 Hz, 3H), 0.88 (t, J =

.9 Hz, 3H).

6

13

3

C NMR (101 MHz, CDCl ): d 173.8, 168.4, 145.1, 143.5, 128.3, 120.4, 67.9, 57.8, 33.3, 31.9, 29.4, 29.3, 29.0, 22.8, 19.4, 14.2.

2

D

3

½

aꢂ = 13.5 (c 0.720, MeOH)

–

4

HRMS (ESI-TOF): calculated for C16H26NO ([M–H] ) 296.1867, found 296.1870.

(

2E,4E)-N-((2S,3R)-3-hydroxy-1-(((5S,8S,10S,E)-10-hydroxy-5-methyl-2,7-dioxo-1,6-diazacyclododec-3-en-8-yl)amino)-1-oxobu-

tan-2-yl)dodeca-2,4-dienamide (2). Acid S32 (60 mg, 0.223 mmol, 2 eq) and crude glidobamine 13 (129 mg, ꢁ0.111 mmol as judged

1

by H NMR analysis, 1 eq) were dissolved in 5.6 mL anhydrous DMF and cooled to 0 C. To the mixture was added diisopropylethyl-

ꢀ

amine (116 mL, 0.668 mmol, 6 eq), followed by solid DEPBT (67 mg, 0.223 mmol, 2 eq). The reaction was warmed to room temperature

ꢀ

and stirred overnight, and reaction progress was monitored by LCMS. At 12 hours, the reaction was cooled to 0 C and quenched

ꢀ

with 1 mL water, then concentrated in vacuo to dryness. The residue was precipitated into 50 mL diethyl ether at 0 C from 0.75 mL

ꢀ

methanol, and the resulting solids were collected by centrifugation at 4200 RPM at 4 C for 15 minutes. The solids were dissolved in

methanol, ﬁltered through a 0.2 mm PTFE membrane ﬁlter, then puriﬁed by reversed-phase HPLC (32% acetonitrile in water) to pro-

vide glidobactin A (2) as a ﬂuffy white powder after lyophilization (25 mg, 0.0480 mmol, 43%, 25% from 12).

1

3 2

H NMR (600 MHz, (CD ) SO): d 8.79 – 8.51 (m, 1H), 7.91 (d, J = 8.8 Hz, 1H), 7.74 (d, J = 7.9 Hz, 1H), 7.41 (t, J = 6.5 Hz, 1H), 7.00 (dd,

J = 15.1, 10.8 Hz, 1H), 6.40 (d, J = 13.9 Hz, 1H), 6.23 – 6.05 (m, 4H), 4.88 (s, 1H), 4.67 (s, 1H), 4.46 – 4.30 (m, 2H), 4.28 (dd, J = 8.8,

4

.1 Hz, 1H), 4.02 – 3.88 (m, 1H), 3.64 – 3.46 (m, 1H), 3.10 – 2.90 (m, 2H), 2.18 – 2.07 (m, 2H), 1.92 – 1.77 (m, 1H), 1.58 (d, J = 13.3 Hz,

H), 1.50 – 1.41 (m, 1H), 1.41 – 1.33 (m, 3H), 1.30 – 1.22 (m, 8H), 1.22 – 1.16 (m, 2H), 1.00 (d, J = 6.3 Hz, 3H), 0.86 (t, J = 6.8 Hz, 3H).

1

13

C NMR (151 MHz, (CD

2.5, 32.2, 31.2, 28.5, 28.5, 28.4, 22.1, 19.9, 18.5, 14.0.

3 2

) SO): d 171.0, 169.4, 167.6, 165.4, 143.1, 142.1, 139.7, 128.6, 123.3, 123.1, 66.9, 66.7, 58.1, 51.2, 44.7,

4

2

½aꢂ = –92.0 (c = 0.460, MeOH)

D

–

43 4 6

HRMS (ESI-TOF): calculated for C27H N O ([M–H] ) 519.3188, found 519.3180.

Cell Toxicity Assays

RPMI 8226 (20,000 cells/well) or MM1.R (20,000 cells/well) cells were seeded in a 96-well plate in cell culture media supplemented

with indicated concentrations of either 1 or bortezomib (BTZ) (1,000x stock in DMSO) and cultured for either 24 or 48 hours. Toxicity

was determined using the WST-1 assay (Roche) according to the protocol of the manufacturer. EC50 values were calculated with

GraphPad Prism (V7.02).

Cell Chemical Biology 27, 1–9.e1–e18, October 15, 2020 e16

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Brief Communication

Sample Preparation for SDS-PAGE Experiments

Proteins in full cell lysate (2 mg/mL, 25 mL) were treated with indicated concentrations of 20 (1 mL of 25x stock in DMSO) or DMSO for

one hour at r.t. Click chemistry was then initiated by the addition of TAMRA azide (Click Chemistry Tools, 25x stock in DMSO), tris(2-

carboxyethyl)phosphine hydrochloride (TCEP, Alfa Aesar, 1 mM, fresh 50x stock in water), tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]

amine (TBTA, Sigma-Aldrich, 100 mM, 16x stock in DMSO:tBuOH 1:4), and copper(II) sulfate (1 mM, 50x stock in water) to the lysate

and incubated in the dark for one hour at r.t. SDS-PAGE reducing loading buffer (4x) was added and proteins were separated using an

11% SDS-PAGE gel. Gels were visualized using a Sapphire Biomolecular Imager (Azure Biosystems), then stained using Coomassie

Brilliant Blue (CBB). Images were quantiﬁed with ImageJ.

Gel-Based In Situ Competitive Experiments

RPMI 8226 cells were seeded in a 6-well plate (1,000,000 cells/well) in cell culture media supplemented with indicated concentrations

of 1 (1,000x stock in DMSO) or DMSO and cultured for 6 hours. Cells were then collected and lysed as described above. Lysates

(2 mg/mL, 25 mL) were treated with 20 (10 mM; 1 mL of 25x stock in DMSO) for one hour at r.t. Click chemistry, reducing SDS-

PAGE and visualization were performed as described above.

In Situ 20 Competitive Experiment for MS

RPMI 8226 cells were seeded in 14 cm Petri dishes and cultured as previously described until they reached conﬂuence. Cell culture

media was then removed and the cells were incubated in fresh media with either 1 (100 nM; 25 mL of 1000x stock in DMSO) or DMSO

for 6 hours. Cells were washed two times with PBS and harvested by centrifugation, resuspended in PBS and lysed as described

above. The protein concentration of the lysate was determined using the Bradford assay. Lysates (2.32 mg/mL, 645 mL) were treated

with 20 (10 mM; 6.51 mL of 100x stock in DMSO) for one hour at r.t. and subjected to click chemistry using biotin azide (30 mM, 50x

stock in DMSO), tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (1 mM, 50x fresh stock in water), tris[(1-benzyl-1H-1,2,3-triazol-

4

Protein was precipitated by adding methanol/chloroform/water (4:1:3 v/v) to the reaction mixture and the turbid mixture was centri-

-yl)methyl]amine (TBTA) (100 mM, 16x stock in DMSO:tBuOH 1:4), and copper(II) sulfate (1 mM, 50x stock in water) for one hour at r.t.

ꢀ

fuged for ﬁve minutes at 14,000 x g at 4 C yielding a protein layer between the aqueous and organic layers. The protein layer was

isolated, dried and solubilized in 2 % SDS in PBS via sonication. The proteome was centrifuged at 4,700 x g for ﬁve minutes and the

supernatant was transferred to a new tube. PBS was added to give a ﬁnal SDS concentration of 0.2 %. Streptavidin agarose beads

(

150 mL, ProteoChem) were added and the mixture was rotated overnight at r.t. Beads were washed with 1 % SDS in PBS (1x 10 mL),

PBS (3x 10 mL), and water (3x 10 mL). Beads were resuspended in 6 M urea in PBS (500 mL), reduced with 10 mM neutralized TCEP

200 mM fresh stock in water) for 30 minutes at r.t. and alkylated with 25 mM iodoacetamide (400 mM fresh stock in water) for 30 mi-

nutes at r.t. in the dark. Beads were pelleted by centrifugation (1’400 x g, 2 minutes) and resuspended in 150 mL of 2 M urea in 50 mM

(

NH

4

HCO

3

, 1 mM CaCl

ꢀ

2

(100x stock in water) and trypsin (Thermo Scientiﬁc, 1.5 mL of 0.5 mg/mL). The digestion was performed for 12

hours at 37 C. Samples were acidiﬁed to a ﬁnal concentration of 5 % acetic acid, desalted over a self-packed C18 spin column and

dried. Samples were analyzed by LC-MS/MS (described below) and the MS data was processed with MaxQuant (described below).

LC-MS/MS Analysis

Peptides were resuspended in water with 0.1 % formic acid (FA) and analyzed using EASY-nLC 1200 nano-UHPLC coupled to a Q

Exactive HF-X Quadrupole-Orbitrap mass spectrometer (Thermo Scientiﬁc). The chromatography column consisted of a 50 cm long,

7

5 mm i.d. microcapillary capped by a 5 mm tip and packed with ReproSil-Pur 120 C18-AQ 2.4 mm beads (Dr. Maisch GmbH). LC

solvents were 0.1 % FA in H

2

O (Buffer A) and 0.1 % FA in 90 % MeCN: 10 % H

a ﬂow rate of 300 nL/min. over a 240 min. linear gradient (5-35 % Buffer B) at 65 C. Data was acquired in data-dependent mode

2

O (Buffer B). Peptides were eluted into the MS at

ꢀ

(top-20, NCE 28, R = 7,500) after full MS scan (R = 60,000, m/z 400-1,300). Dynamic exclusion was set to 10 s, peptide match to

prefer and isotope exclusion was enabled.

MaxQuant Analysis

The MS data was analyzed with MaxQuant (V1.6.1.0) (Cox and Mann, 2008) and searched against the human proteome (Uniprot) and

a common list of contaminants (included in MaxQuant). The ﬁrst peptide search tolerance was set at 20 ppm, 10 ppm was used for

the main peptide search and fragment mass tolerance was set to 0.02 Da. The false discovery rate for peptides, proteins and sites

identiﬁcation was set to 1%. The minimum peptide length was set to 6 amino acids and peptide re-quantiﬁcation and label-free quan-

tiﬁcation (MaxLFQ) were enabled. The ‘match between runs’ function was activated for the global changes in protein expression

experiment. The minimal number of peptides per protein was set to two. Methionine oxidation was searched as a variable modiﬁca-

tion and carbamidomethylation of cysteines was searched as a ﬁxed modiﬁcation.

Transfection and Western Blot

HEK293T cells were seeded (6,000,000 cells in 20 mL DMEM 10% FCS without Pen/Strep) in a 10 cm Petri dish and left overnight to

attach. Cells were transfected using a mixture of 96 mg PEI MAX 40000 (Polysciences Inc.) and 24 mg DNA in 2 mL opti-MEM. After

2

4 hours, cells were harvested by scraping, washed two times with PBS and lysates were prepared and treated with 30 mM 20

as described above. Click chemistry, reducing SDS-PAGE and visualization were performed as described above. Proteins were

transferred to a PVDF membrane using a Trans-Blot Turbo transfer system (Bio-Rad). Membrane was blocked with 5% milk in

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Brief Communication

Tris-Buffered Saline with 0.1 % Tween 20 (TBST) for one hour. Rabbit polyclonal antibody against DYKDDDDK (FLAG) (CST, #2368S,

ꢀ

1

/1,000 in 5% milk in TBST) and a-tubulin (CST, #2144S, 1/1,000 in 5% milk in TBST) were added and incubated overnight at 4 C.

Membrane was washed three times for ﬁve minutes with TBST and incubated with Alexa Fluor 647-AfﬁniPure Donkey Anti-Rabbit IgG

H+L) (Jackson ImmunoResearch Inc., 711-605-152, 1/500 in 5 % milk in TBST) for one hour at r.t. Membrane was washed four times

for ﬁve minutes with TBST and visualized with a Sapphire Biomolecular Imager (Azure Biosystems).

(

In Situ b5 Proteasome Activity Assay

RPMI 8226 cells were seeded in a 6-well plate (1,000,000 cells/well) in cell culture media supplemented with indicated concentrations

of 1, 2, 21 or 22 (1,000x stock in DMSO) or DMSO and cultured for 6 hours. Cells were then collected and lysed as described above.

2

Lysates (1 mg/mL, 25 mL) were dispensed into 100 mL assay buffer (PBS (pH 7.4), MgCl (1.5 mM), EDTA (1 mM), EGTA (1 mM), su-

crose (250 mM) and freshly added DTT (5 mM), ATP (2 mM) and proteasome substrate Suc-LLVY-AMC (Enzo Life Sciences, 100 mM,

ꢀ

1

mL of 100x stock in DMSO)) in the wells of a black / clear bottom 96-well plate. The plate was incubated at 37 C for two hours and

ﬂuorescence was read at A360ex/A460em using a Synergy H1 hybrid multi-mode microplate reader (Biotek). The protocol was modi-

ﬁed from Maher (2014). IC50 values were calculated with GraphPad Prism (V7.02).

Western Blot for Mono-/Poly-Ub and PARP-1

RPMI 8226 cells were seeded in a 6-well plate (1,000,000 cells/well) in cell culture media supplemented with indicated concentrations

of 1 (1,000x stock in DMSO) or DMSO and cultured for 6 hours. Cells were then quickly washed twice in PBS and lysed using RIPA

buffer (20 mM Tris, 150 mM NaCl, 2 mM EDTA, 0.1 % SDS, 1 % NP-40, 0.25 % deoxycholate, 1 mM PMSF) plus protease inhibitor

cocktail (100x, RPI Corp., Cat.No. P50700-1) for ﬁve minutes on ice. Cells were quickly sonicated using a needle sonicator, the lysate

ꢀ

was spun down in a tabletop centrifuge at 17000 x g for 10 minutes at 4 C and the cleared lysate was transferred into a new micro-

tube. Protein concentration was measured using the BCA protein assay (Thermo Fisher, Cat No. 23225). SDS-PAGE was performed

as previously described and wet transfer to a PVDF membrane was done at 350 mA for one hour. Membrane was blocked with 5 %

milk in TBST for one hour. Primary antibodies against respective proteins: PARP-1 (CST, #9532, 1:1,000 in 5 % BSA in TBST), mono- /

polyubiquitinated conjugate antibody (Enzo Life Sciences, Prod. No. BML-PW8805, 1:500 in 5 % milk in TBST), and a-tubulin anti-

ꢀ

body (CST, #2144S, 1:1,000 in 5 % milk in TBST)), were added and incubated overnight at 4 C. Membrane was washed three times

for ﬁve minutes with TBST and incubated for one hour at r.t. with Alexa Fluor 647-AfﬁniPure Donkey Anti-Rabbit IgG (Jackson Im-

munoResearch Inc., 711-605-152) or Alexa Fluor 488-AfﬁniPure Donkey Anti-Mouse IgG (Jackson ImmunoResearch Inc., 715-

545-150). Membrane was washed four times for ﬁve minutes with TBST and visualized with a Sapphire Biomolecular Imager (Azure

Biosystems).

Global MS Proteomics Proﬁling of 1 and BTZ

RPMI 8226 cells were seeded in a 6-well plate (1,000,000 cells/well) in cell culture media supplemented with 1 (100 nM, 1,000x stock

in DMSO), bortezomib (BTZ, 2.5 nM, 1,000x stock in DMSO) or DMSO and cultured for 6 hours. Cells were then collected and lysed in

PBS as described earlier and protein concentration was measured using the Bradford assay. Proteins (30 mg) were then denatured in

6

M urea in 50 mM NH

4

HCO

3

, reduced with 10 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) for 30 minutes and alkylated

HCO , and digested with

(1003 stock in water). The digestion was performed for

with 25 mM iodoacetamide for 30 minutes in the dark. Samples were diluted to 2 M urea with 50 mM NH

4

3

trypsin (Thermo Scientiﬁc, 1.5 mL of 0.5 mg/mL) in the presence of 1 mM CaCl

2

ꢀ

12 hours at 37 C. Samples were acidiﬁed to a ﬁnal concentration of 5% acetic acid, desalted over a self-packed C18 spin column,

and dried. Samples were analyzed by LC-MS/MS (see above), and the MS data were processed with MaxQuant (see above).

QUANTIFICATION AND STATISTICAL ANALYSIS

Statistical analysis was performed with GraphPad Prism unless noted otherwise. Statistical values including the number of times the

data was replicated (‘‘n’’) and statistical signiﬁcance are reported in the Figure legends. Data are represented as mean ± SD or ± SEM

and p-values calculated using an unpaired Student’s t-test. LC-MS/MS proteomics experiments were analyzed with MaxQuant using

high conﬁdence parameters indicated in the corresponding section. Statistical analysis of the global proteomics dataset was per-

formed with Perseus using indicated parameters in the Figure legend S2B.

Cell Chemical Biology 27, 1–9.e1–e18, October 15, 2020 e18

Article Doi

Concise Chemoenzymatic Total Synthesis and Identification of Cellular Targets of Cepafungin I

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