Catalytic Synthesis of PEGylated EGCG Conjugates that Disaggregate Alzheimer's Tau

Article abstract of DOI:10.1055/a-1509-5904

The naturally occurring flavonoid ( )-epigallocatechin gallate (EGCG) is a potent disaggregant of tau fibrils. Guided by the recent cryo-electron microscopy (cryoEM) structure of EGCG bound to fibrils of tau derived from an Alzheimer s brain donor, methods to site-specifically modify the EGCG D-ring with aminoPEGylated linkers are reported. The resultant molecules inhibit tau fibril seeding by Alzheimer s brain extracts. Formulations of aminoPEGylated EGCG conjugated to the (quasi)-brain-penetrant nanoparticle Ferumoxytol inhibit seeding by AD-tau with linker length affecting activity. The protecting groupfree catalytic cycloaddition of amino azides to mono-propargylated EGCG described here provides a blueprint for access to stable nanoparticulate forms of EGCG potentially useful as therapeutics to eliminate Alzheimer s-related tau tangles.

Full text of DOI:10.1055/a-1509-5904

SYNTHESIS
0
0
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9
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7
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8
1
1
4
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7
-
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1
0
X
Georg Thieme Verlag KG Rüdigerstraße 14, 70469 Stuttgart
2
021, 53, A–I
special topic
A
Special Issue dedicated to Prof. Sarah Reis-
Synthesis
A. El Khoury et al.
Special Topic
Catalytic Synthesis of PEGylated EGCG Conjugates that Disaggregate
Alzheimer’s Tau
Anton El Khoury
Paul M. Seidler1
N
N
cat.
Bn
N
N
N
Re-assigned Structure
N
Bn
OH
David S. Eisenberg*
Patrick G. Harran*
N
N
OH
OH
OH
0
0
0
0
-
0
0
0
1
-
8
7
4
8
-
5
1
6
7
OH
OH
HO
O
HO
O
N
N
Department of Chemistry and Biochemistry, Molecular
Biology Institute, Howard Hughes Medical Institute,
University of California–Los Angeles, 607 Charles E.
Young Drive East, Los Angeles, CA 90095-1569, USA
harran@chem.ucla.edu
Bn
O
O
sodium ascorbate
cat. CuSO4·5H2O
DMSO/H2O
25 °C, 1 h
OH
OH
OH
O
OH
O
salt formation
or conjugation site
O
O
OH
OH
NH2
Published as part of the
Special Issue dedicated to Prof. Sarah Reisman, recipient of
the 2019 Dr. Margaret Faul Women in Chemistry Award
Protecting-group-free
N
O
N
n
N
2
steps, scalable syntheses
N3
NH2
O
PDYe Maoea tuvpar naiidCclrgkothSmDr.Ga r Eree. irinvHsas petna ono@rE nrfbacadeCshnitrhne,g emLg mo A. sui uscAtl nrha yg.oe eardslnue ds , BC i Ao c
9
h
0e 0m9 i5s -t 1r y5 ,6 M9 ,o
U
le
n
ci ut el ad rS Bt ia ot leos gy Institute, Howard Hughes Medical Institute, University of California–Los Angeles, 607 Charles E.
n
Received: 16.04.2021
Accepted after revision: 17.05.2021
Published online: 17.05.2021
play a decisive role in cognitive decline. Recent advances in
imaging showed tau tangles to be the best predictors of
4
DOI: 10.1055/a-1509-5904; Art ID: ss-2021-m0212-st
Alzheimer’s progression as well as the species responsible
for driving brain atrophy. Oligomeric and fibrillar tau ap-
pear to be promising targets for therapeutics.
Abstract The naturally occurring flavonoid (–)-epigallocatechin gal-
late (EGCG) is a potent disaggregant of tau fibrils. Guided by the recent
cryo-electron microscopy (cryoEM) structure of EGCG bound to fibrils
of tau derived from an Alzheimer’s brain donor, methods to site-specifi-
cally modify the EGCG D-ring with aminoPEGylated linkers are report-
ed. The resultant molecules inhibit tau fibril seeding by Alzheimer’s
brain extracts. Formulations of aminoPEGylated EGCG conjugated to
the (quasi)-brain-penetrant nanoparticle Ferumoxytol inhibit seeding
by AD-tau with linker length affecting activity. The protecting group-
free catalytic cycloaddition of amino azides to mono-propargylated
EGCG described here provides a blueprint for access to stable nanopar-
ticulate forms of EGCG potentially useful as therapeutics to eliminate
Alzheimer’s-related tau tangles.
The polyphenolic flavanoid (–)-epigallocatechin gallate
(
EGCG) inhibits aggregation of proteins involved in neuro-
generative amyloidoses including huntingtin, amyloid-,
5
6
and -synuclein. Wobst et al. reported EGCG blocks the
fibrillization of tau by sequestering unfolded protein mono-
mers. Recently, cryoEM was used to determine the binding
site for EGCG on fibrils of tau deriving from the brain tissue
7
of a donor with AD. Relative to the apo AD-tau fibril, the
bound form contains EGCG wedged into an interfacial cleft
(
Figure 1).
Key words (–)-epigallocatechin gallate, Alzheimer’s disease, tau dis-
aggregation, nanoparticle conjugation, click reaction, TBTA
Alzheimer’s disease (AD) is the 6th leading cause of
death in the United States and 7th in the world. Personal
and economic burdens associated with this most common
type of dementia are enormous. Approximately 6.8 million
Americans currently suffer from the disease. By 2050, its
annual costs to the healthcare system are anticipated to
2
reach $1.1 trillion. Despite decades of research and numer-
ous attempts at treatment, much is still unknown about the
etiology of Alzheimer’s. Two main markers have been iden-
tified: plaques of aggregated -amyloid and neurofibrillary
tangles of tau. However, the precise cause of cognitive de-
cline and effective drug targets have been elusive. Early fo-
cus on -amyloid led to clinical trials of multiple therapeu-
tic candidates with limited success.3 Those failed trials
called into question the hypothesis that amyloid plaques
Figure 1 CryoEM structure of non-liganded AD-tau fibrils (A, PDB
6
HRE) and fibrils bound to disaggregant EGCG (B), from reference 7a.
EGCG is rendered green with oxygens shown in red. Residues from the
Tau protein are rendered grey with oxygens red, nitrogens blue, and
sulfur gold. The surface on EGCG that remains solvent accessible in the
fibril-bound pose is labeled. C: Chemical structure of EGCG showing the
nomenclature of ring systems. Density map of EGCG-tau binding cleft
(
green: EGCG, blue/grey: tau fibril).
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2021. Thieme. All rights reserved. Synthesis 2021, 53, A–I
Georg Thieme Verlag KG, Rüdigerstraße 14, 70469 Stuttgart, Germany

B
Synthesis
A. El Khoury et al.
Special Topic
The structure of EGCG bound to AD tau indicated posi-
tions on the small molecule that might serve as anchor
points for nanoparticle conjugation, wherein the ability to
bind tau fibrils would be retained. Despite numerous re-
ports of therapeutic potential for EGCG, the compound is
prone to auto-oxidation, has poor pharmacokinetics, and is
largely excluded from the brain when administered system-
ically. Stable conjugation to brain penetrant nanoparticles
exhibited correlations to C6-H and the C4 methylene pro-
tons, but not to the propargyl group or D ring aryl protons.
These data indicated the proper structure assignment
should be the ether 3, wherein alkylation had occurred at
the p-OH of the gallate ester. This phenol is presumably the
most acidic in EGCG.
A second product 3′ isolated from the reaction was dou-
bly etherified (see SI for structure) and showed HMBC cor-
relations between a second propargylic methylene (OCH2,
4.67 ppm) and C4′ (137.0 ppm), and between C4′ and C2′-H
(6.53 ppm). Data indicated the second propargyl ether
formed on ring C. The phenols on ring A appeared to be the
least susceptible to alkylation under basic conditions.
With the structure of 3 confirmed, conditions were
screened to optimize its formation while avoiding the use
of NaH in DMF – a potentially explosive combination, par-
8
was seen as potential means to offset those limitations. We
selected Ferumoxytol as a nanoparticle carrier. Ferumoxy-
tol exhibits moderate brain penetration, with penetration
increasing coincident with pathologies that alter the neuro-
vascular unit.
As best modeled (Figure 1B), the tau bound form of
EGCG oriented its A-ring C5 phenol and a major portion of
the gallate D-ring towards solvent. We sought to selectively
derivatize the natural product ($ 17/g, Oakwood Chemicals)
at one position along this periphery (see Scheme 1) with
end-functionalized ethylene glycol chains of varying length.
1
0
ticularly when heated. It was eventually found that treat-
ing EGCG with 1 equivalent of propargyl bromide and 0.5
equivalent of powdered K CO in DMF at room temperature
2
3
afforded 3 in 45% isolated yield – versus the 33% yield ob-
tained using the NaH/DMF procedure.
OH
EGCG (1)
4
' OH
2
'
We next synthesized a set of glycol based -amino
azides 4a–e with chain lengths varying from 5 to 17 atoms
(see Table 2 and experimental section for details) in order
to produce EGCG conjugates with incrementally increasing
chain lengths. Cycloaddition reaction conditions were first
HO
O
4
OH
6
O
5
2
''
OH
OH
O
4''
OH
1
1
optimized with 4c using copper catalysis (Table 1). Stan-
OH
solvent-exposed
periphery when bound to
fibrillar tau
1
2
dard conditions using catalytic Cu(II) and sodium ascor-
bate in aqueous THF (Table 1, entries 1, 2), failed to cycloadd
4c to 3 due to substrate insolubility. When THF was re-
placed with t-BuOH, desired triazole 5c was detected, but
only in trace quantities (entry 3). An attempt to replace so-
NaH (or K2CO3)
DMF
Br
OH
OH
1
3
dium ascorbate with Cu(0) was unsuccessful (entry 4), as
was the use of stoichiometric Cu(I) (entry 6). Notably, when
OH
OH
OH
OH
HO
O
HO
O
stoichiometric amounts of CuSO were employed (entry 5),
4
O
O
starting materials were consumed and a highly insoluble
O
OH
OH
OH
OH
O
O
precipitate formed. The use of H O/DMSO co-solvent mix-
2
ture resulted in the formation of the product in 12% yield
(entry 7), which remained unchanged even in the presence
of excess amino azide partner (entry 8). We suspected this
material was a copper/product complex and hypothesized
that earlier attempts at catalysis may have been poisoned
by 5c. To fortify the copper catalyst against possible product
sequestration, we turned to polydentate ligands reported
O
HMBC
3 (45%)
Corrected Structure (this work)
OH
OH
2
Previous Assignment (ref. 9)
Scheme 1 Properly assigning the structure of mono-propargylated
EGCG
14
Wang and co-workers had reported that a sodium salt of
EGCG reacted with propargyl bromide in DMF at 80 °C to
afford predominantly A-ring mono-ether 2, along with less-
by Sharpless. Gratifyingly, in the presence tris[(1-benzyl-
4-triazolyl)methyl]amine (TBTA), 20 mol% of CuSO rapidly
4
catalyzed the cycloaddition of 4c to 3 in H O/DMSO at room
2
9
er amounts of further propargylated compounds. We re-
temperature to afford triazole adduct 5c in 52% isolated
yield (entry 9). When the catalyst load was decreased from
20 to 5 mol%, isolated yield decreased, and conversion pla-
teaued at roughly 80%. The reactions required no workup
and product was easily purified as its TFA salt via prepara-
tive reverse-phase HPLC (see experimental section for de-
tails).
peated this reaction and found spectroscopic data for the
major etherification product was inconsistent with struc-
ture 2. HMBC spectra showed a correlation between the
propargylic methylene protons (OCH , 4.76 ppm) and C-4′′
2
(
137.0 ppm). C4′′ exhibited coupling with C2′′-H (6.89
ppm), and C2′′-H also correlated to the carbonyl carbon
165.7 ppm). C5 (95.2 ppm), the linkage site assigned in 2,
(
©
2021. Thieme. All rights reserved. Synthesis 2021, 53, A–I

C
Synthesis
A. El Khoury et al.
Special Topic
Table 1 Protecting Group-Free, Copper-Catalyzed Huisgen Cycloaddition to Form AminoPEGylated EGCGa
OH
OH
OH
OH
HO
O
OH
OH
sodium ascorbate
HO
O
O
[
Cu], additive
OH
OH
N3
O
NH2
O
O
O
O
solvent
OH
OH
O
4c
O
OH
O
NH2
O
N
O
O
3
N
N
OH
5
c
Entry
[Cu]
Cu(OAc)
Additive
Solvent (4:1)
Yield (%)b
1
2
(0.2 equiv)
–
H
H
H
H
H
H
H
H
H
2
2
2
2
2
2
2
2
O/THF
O/THF
0
0
2
CuSO
CuSO
CuSO
CuSO
4
4
4
4
·5H
·5H
·5H
·5H
2
2
2
2
O (0.2 equiv)
O (0.2 equiv)
O (0.2 equiv)
O (1 equiv)
–
3
–
O/t-BuOH
O/t-BuOH
O/t-BuOH
O/DMSO
O/DMSO
O/DMSO
trace
0
4c
5
–
–
–d
6e
7
CuBr (1 equiv)
–
–f
CuSO
CuSO
4
4
·5H
2
2
O (0.5 equiv)
O (0.5 equiv)
–
12
12
52
8g
·5H
–
9
CuSO
4
·5H
2
O (0.2 equiv)
TBTAh
2
O/DMSO
a
Reaction conditions: 3 (1 mmol), 4c (1 mmol), [Cu], additive (50 mol%), sodium ascorbate (2.0 equiv), solvent (0.1 M), 1 h.
Isolated yield.
Cu (powder) was used as a reductant instead of sodium ascorbate.
Reaction resulted in the formation of insoluble precipitate.
No sodium ascorbate was added.
b
c
d
e
f
Complex mixture.
g
h
2
mmol of 4c were used.
TBTA: Tris((1-benzyl-4-triazolyl)methyl)amine.
Table 2 A Set of EGCG Derivatives Having Varied Linker Lengthsa
OH
OH
OH
OH
OH
sodium ascorbate
CuSO4·5H2O
TBTA
HO
O
HO
O
OH
O
DMSO/H2O
O
OH
OH
OH
OH
O
O
O
O
3
OH
OH
a–e (n = 1–5)
NH2
N
O
N
n
N
5
N3
NH2
O
n
4a–e (n = 1–5)
Compound
n
Yield (%)b
5
5
5
5
5
a
b
c
1
2
3
4
5
68
58
52
46
45
d
e
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2021. Thieme. All rights reserved. Synthesis 2021, 53, A–I

D
Synthesis
A. El Khoury et al.
Special Topic
Using the conditions shown in Table 1, entry 9, a set of
EGCG conjugates having increasing chain lengths were syn-
thesized (Table 2). As the number of glycol units increased,
the yields of triazole products 5 decreased slightly. But in all
cases, analytically pure product was isolated readily using
preparative reversed phase HPLC.
Intracellular tau aggregates are seen as bright green
puncta in cells that were seeded with crude AD brain ex-
tract in the absence of inhibitor. The number of puncta in
inhibitor-treated cells are a proxy used to assess the disag-
gregating activity of EGCG-linked nanoparticles. To our de-
light, all of the EGCG-linker conjugates inhibited seeding by
AD brain extracts by at least 90% with 5c displaying potency
nearly on par with EGCG itself. As a comparison with other
analogues of EGCG, we tested ECG, which lacks the meta-
OH group of the C ring. Consistent with the structure of
EGCG bound to tau, which shows no contact with the meta-
OH, ECG was seen to inhibit seeding as well as the linker-
conjugated analogues and nearly as well as the parent natu-
ral product, EGCG. These data demonstrate that D ring deri-
vatizations are well tolerated, consistent with our observa-
tion that the D ring remains largely solvent-exposed in the
binding cleft of tangled tau filaments from AD brain.
1
Interestingly, when analyzing amine salts 5 by H NMR
in protic solvents (i.e., CD OD or D O), the aryl protons in
3
2
the A-ring (5.93 ppm) quickly disappeared. Their integra-
tion (relative to stable resonances) decreased ~75% in 3
hours. Overnight storage of the NMR samples saw complete
disappearance of both signals. HRMS identified the prod-
ucts as [M + 1] + 2 ions, indicating C–H bonds in the A ring
had been replaced by C–D bonds. Notably, C–H bonds in the
C and D rings showed no exchange, even after prolonged
storage. Deuteration of flavonoids has been observed in the
gas phase by mass spectrometry.15 Jordheim and co-work-
ers reported anthocyanidin natural products are deuterated
EGCG is subject to off-target binding and rapid metabo-
lism, which restricts its therapeutic potential. We reason
that covalent conjugation of EGCG to nanoparticles may re-
1
6
in 15 vol% TFA in CD OD over a period of days. Rapid deu-
3
teration of compounds 5 at room temperature may derive
from the acidity of their amine salt appendages, wherein
deuteration of the A-ring was presumably occurring via a
dearomatized species of type i (Scheme 2). The A-ring ap-
peared to be considerably more basic than the C and D
rings. Along those lines, we observed that EGCG itself would
react with N-iodosuccinimide to rapidly and selectively io-
dinate the A-ring (data not shown).
OH
i
OH
OH
H
O
X
C
O
HO
X
O
H
A
O
D
OH
OH
OH
O
D
O
F3CCO2
O
OH
5
6
a–e X = H
a–e X = D
NH3
CD3OD
N
N
n
N
no detectable exchange
in C and D rings
Scheme 2 Rapid and selective A-ring deuteration of EGCG conjugates
We next tested if the D-ring site of triazole-linked ami-
noPEGylation would interfere with tau fibril disaggregation
observed for EGCG. AD crude brain extracts have been
shown to seed aggregation of fluorescently labeled tau in
HEK293 recipient biosensor cells expressing an aggrega-
Figure 2 Linker conjugated EGCG analogues retain inhibitory activity
towards AD crude brain extracts. A: Seeding by crude AD brain extract
pre-treated with EGCG or experimental linker-conjugated analogues, as
indicated. Inhibitor activity is read-out by measuring seeding in tau bio-
sensor cells. Seeding is taken as a proxy for the fibril load that is con-
tained within the AD crude brain extracts. Reduction in fibril load
following treatment with experimental linker-conjugated analogues of
EGCG reduces prion-like seeding by AD-tau nearly as effectively as
EGCG itself. B: Representative fluorescence images of tau biosensor
cells experiments from A. Intracellular aggregates seeded by crude AD
brain extracts are identified as puncta (green dots in the ‘No inhibitor’
treated sample, left fluorescence micrograph). Inhibitor treatment re-
duces the number of puncta (right fluorescence micrograph). The num-
ber of puncta as a function of inhibitor pre-treatment is plotted in A.
1
7
tion-prone fragment of tau called K18, and seeding is in-
7
a
hibited by EGCG. We compared inhibition of seeding by
EGCG and D-ring analogues 5a–c as a preliminary proof-of-
concept. Crude extract of autopsied brain tissue of a donor
with AD (prepared as described in the SI) were pre-incubat-
ed with inhibitors (10 M final concentration on cells) for
1
6–18 hours and resulting homogenates were added to the
cells for imaging 3 days later. The data obtained are shown
in Figure 2.
©
2021. Thieme. All rights reserved. Synthesis 2021, 53, A–I

E
Synthesis
A. El Khoury et al.
Special Topic
duce binding to metabolic and off-target proteins, which
accommodate EGCG inside of buried active sites of globular
proteins that are sterically inaccessible to nanoparticle-
bound molecules of EGCG. Thus, we sought to synthesize a
series of EGCG-nanoparticle conjugates that varied by
linker length to identify a minimal linker that retains inter-
action of EGCG with the solvent exposed binding cleft of
fibrillar tau.
Ferumoxytol is an FDA approved carbohydrate-coated
iron nanoparticle with widespread use in the clinic with
applications ranging from anemia treatment to off-label MR
imaging of neurovasculature.8a,b,d,18 We conjugated an ex-
panded series of EGCG bearing linkers of incrementally in-
creasing length, 5a–e, to Ferumoxytol nanoparticles using
standard amidation conditions (sulfo-NHS, EDC, 2 h, rt).
Unlike previous inclusion-based, labile EGCG nanoparticle
formulations that release EGCG at sites of action,19 we load-
ed the small molecule via covalent attachment. Covalent
conjugation is likely to reduce off-target binding and has
added potential to improve potency by exploiting the mul-
tivalency of the nanoparticle (each nanoparticle displays
~
50 potential linking sites).
We tested the activity of Ferumoxytol conjugated EGCG
analogues using the biosensor cell assay described above,
except we omitted the pre-incubation step such that our as-
say more closely resembled the scenario of therapeutic in-
tervention, for which there is no pre-incubation period.
Nanoparticle conjugated EGCG derivative was mixed with
crude AD brain extract and immediately transfected into
tau biosensor cells. We find that all the analogues except for
the compound with the shortest linker 5a exhibited desired
activity inhibiting seeding by at least 50% (Figure 3A). Over-
all, our data demonstrate that nanoparticle conjugates re-
tain the inhibitory properties of the parent compound, and
underscores that functional EGCG nanoparticles can be suc-
cessfully designed based on information that is gleaned
from the cryoEM structure.
Figure 3 Nanoparticle-conjugated EGCG retains inhibitor activity and
clusters with fibrils of AD-tau. A: Seeding by crude AD brain extract
measured in tau biosensor cells that were co-transfected with nanopar-
ticles coupled to EGCG by linkers of varying length. B, C: Negative-stain
electron micrographs of EGCG-conjugated and non-conjugated nano-
particles. Nanoparticle coupled with EGCG analogue 5c (B) cluster with
fibrils of AD-tau. No clustering is seen between non-conjugated
nanoparticles and AD-tau fibrils (C).
refinements to the EGCG molecule and optimized nanopar-
ticulate formulations could provide means to deliver a po-
tent tau fibril disaggregant to the brains of Alzheimer’s pa-
tients.
As added evidence of its inhibitory action, we also ob-
served an interesting effect of EGCG nanoparticle incuba-
tion with tau paired helical filaments purified from AD
brain by negative-stain electron microscopy. Nanoparticles
loaded with 5c form dense clouds that engulf AD-tau fibrils,
in some cases apparently unwinding the paired helical fila-
ment (Figure 3B). Non-conjugated control nanoparticles ex-
hibited no apparent interaction with AD-tau fibrils (Figure
All reagents were purchased from commercial suppliers (Sigma-
Aldrich, Combi-Blocks, or Oakwood Chemicals) and were used with-
out further purification. When necessary, reaction solvents were
dried using an activated alumina solvent drying system. TLC was per-
formed on pre-coated plates Sorbent Technologies, silica gel 60 PF254
(
0.25 mm). TLC plates were visualized with UV light (254 nm) or
stained using KMnO4 or ninhydrin. Flash chromatography was per-
formed on silica gel 60 (240–400 mesh). Purification of final products
was performed using an Agilent 1200 HPLC system equipped with
G1361A preparative pumps, and a Waters Sunfire C18 column (5 m,
3
C).
In summary, we have optimized a monoetherification of
naturally occurring EGCG and properly assigned the re-
giochemistry of the reaction. We have established a proce-
dure to directly catalyze cycloaddition of glycol based -
amino azide chains to this molecule. The resultant amino
polyphenolic conjugates retain the ability to disaggregate
AD brain-derived tau – both as isolated species and when
loaded onto Ferumoxytol nanoparticles. These promising
results provide a blueprint for future work wherein further
1
9 mm × 250 mm). Analytical HPLC was performed using the same
system, but with a G1312A binary pump. NMR spectra were recorded
on a Bruker Avance (500 MHz) spectrometer using CDCl or CD OD as
3
3
solvents and referenced relative to residual CHCl ( = 7.26) or CD OD
3
3
(
 = 3.31). Chemical shifts are reported in ppm and coupling con-
1
3
stants J in hertz (Hz). C NMR spectra were recorded on the same in-
struments (125 MHz) with total proton decoupling referenced rela-
tive to residual CHCl ( = 77.16) or CD OD ( = 49.00). IR spectra were
3
3
©
2021. Thieme. All rights reserved. Synthesis 2021, 53, A–I

F
Synthesis
A. El Khoury et al.
Special Topic
1
3
obtained on Jasco FT/IR-4100 equipped with a universal ATR sam-
pling accessory. High-resolution mass spectra were recorded on Wa-
ters LCT Premier. Optical rotations were measured on a Rudolph Au-
topol III Automatic Polarimeter and are quoted in units of degree.
C NMR (125 MHz, CDCl ):  = 73.1, 70.71, 70.66, 70.6, 70.3, 70.1,
3
50.7, 41.7.
4-Azido-3,6,9,12-tetraoxatetradecan-1-amine (4d)23
1
Experimental procedures for biological studies are provided in the
Supporting Information.
Compound was prepared according to GP1; starting with 3.39 g (14.2
mmol) of pentaethylene glycol; yield: 2.7 g (73%); yellow oil.
1
H NMR (500 MHz, CDCl ):  = 3.72–3.60 (m, 14 H, 3-H, 4-H, 5-H, 6-H,
3
Amino Azides 4; General Procedure 1 (GP1)
7
-H, 8-H, 9-H), 3.55 (t, J = 3.0 Hz, 2 H, 2-H), 3.38 (t, J = 4.7 Hz, 2 H, 10-
The corresponding diol (1.0 equiv) was dissolved in DCM (0.6 M), fol-
lowed by the addition of TsCl (2.1 equiv). The reaction was cooled to 0
H), 2.90 (t, J = 4.7 Hz, 2 H, 1-H).
13
C NMR (125 MHz, CDCl ):  = 71.9, 70.7–70.0 (wide peak), 50.7,
3
°
C. KOH (8.0 equiv) was then added in one portion, the reaction mix-
4
1.4.
ture was warmed to rt and stirred for 4 h. The mixture was then dilut-
ed with H O (100 mL) and extracted with DCM (3 × 100 mL). The
2
7-Azido-3,6,9,12,15-pentaoxaheptadecan-1-amine (4e)24
1
combined organic layers were dried (MgSO ), filtered, and concen-
4
Compound was prepared according to GP1; starting with 3.0 g (10.6
mmol) of hexaethylene glycol; yield: 2.27 g (70%); yellow oil.
trated in vacuo. The crude bis-tosylate was obtained as a white solid
and used directly in the next step.
1
H NMR (500 MHz, CDCl ):  = 3.96 (t, J = 3.2 Hz, 2 H, 11-H), 3.62–3.80
To the crude bis-tosylate (1.0 equiv) in DMF (0.6 M) under argon was
3
(
m, 18 H, 2-H, 3-H, 4-H, 5-H, 6-H, 7-H, 8-H, 9-H, 10-H), 3.50 (t, J = 4.90
Hz, 2 H, 12-H), 3.15 (t, J = 4.90 Hz, 2 H, 1-H).
added NaN (4.0 equiv). The reaction was stirred overnight at 80 °C.
3
The mixture was then cooled to rt, diluted with H O (100 mL), and ex-
2
1
3
tracted with EtOAc (3 × 100 mL). Combined organic layers were
washed with H O (2 × 50 mL) and brine (2 × 50 mL), dried (MgSO ),
C NMR (125 MHz, CDCl ):  = 70.6–69.8 (wide peak), 66.9, 50.7,
3
40.6.
2
4
filtered, and concentrated in vacuo. The resulting bis-azide was used
directly in the next step without purification.
(
2R,3R)-5,7-Dihydroxy-2-(3,4,5-trihydroxyphenyl)chroman-3-yl
Crude bis-azide from the previous step (1.0 equiv) was dissolved in
THF/Et O/H O (5:1:5, 0.6 M). PPh (1.0 equiv) in Et O (0.7 M) was then
added over 1 h using a syringe pump. The resulting solution was
stirred at rt overnight at which time precipitate formation was ob-
3,5-Dihydroxy-4-(prop-2-yn-1-yloxy)benzoate (3) and (2R,3R)-2-
[3,5-Dihydroxy-4-(prop-2-yn-1-yloxy)phenyl]-5,7-dihydroxychro-
man-3-yl 3,5-Dihydroxy-4-(prop-2-yn-1-yloxy)benzoate (3′)
2
2
3
2
Using NaH/DMF: To NaH (15.7 mg, 0.65 mmol, 1.5 equiv) at 0 °C was
added a solution of EGCG (200 mg, 0.44 mmol, 1.0 equiv) in anhyd
DMF (1.45 mL, 0.3 M). The resulting mixture was stirred at rt for 30
min. Propargyl bromide (53 L, 0.48 mmol, 1.1 equiv, 80% w/w) was
then added and the reaction mixture was heated to 80 °C and stirred
overnight. Upon cooling to rt, the mixture was concentrated in vacuo
and subjected to flash column chromatography (silica gel, CHCl3/
MeOH 100:0 to 15:1 to 13:1 to 11:1). Desired mono-propargylated
product (TLC: CHCl /MeOH 8:2, R = 0.3) was obtained in 33% yield
served. The reaction mixture was diluted with H O (100 mL) and
2
washed with Et O (3 × 100 mL). The aqueous layer was then basified
2
via the addition of solid NaOH to pH 11 and extracted with DCM (3 ×
1
00 mL). Combined organic layers were dried (MgSO ), filtered, and
4
concentrated in vacuo. The crude product (TLC: DCM/MeOH 8:2 R =
f
0
.2) was purified via flash column chromatography (silica gel,
DCM/MeOH/Et N 100:0:0 to 90:10:0 to 80:10:10) to give the desired
3
amino azide.
3
f
(
72 mg) along with 15% (35 mg) of bispropargylated product (R = 0.6)
f
2
-(2-Azidoethoxy)ethan-1-amine (4a)20
as white solids.
Compound was prepared according to GP1; starting with 849 mg (8.0
mmol) of diethylene glycol: yield: 936 mg (90%); yellow oil.
Monopropargylated Product 3
Mp, decomposed at >120 °C; [] 21 –162.0 (c = 0.1, MeOH).
1
H NMR (500 MHz, CDCl ):  = 3.65 (t, J = 5.0 Hz, 2 H, 4-H), 3.54 (t, J =
D
3
5
.1 Hz, 2 H, 2-H), 3.38 (t, J = 5.0 Hz, 2 H, 5-H), 2.90 (t, J = 5.1 Hz, 2 H, 1-
FT-IR (neat): 3358, 3290, 2124, 1697, 1606, 1522, 1454, 1371, 1347,
1242, 1196, 1147, 1056, 1039, 1017, 826, 769, 640 cm–1
.
H), 2.40 (s, 2 H, NH2).
1
3
1
C NMR (125 MHz, CDCl ):  = 72.7, 70.0, 50.7, 41.6.
H NMR (500 MHz, CD OD):  = 6.89 (s, 2 H, Gal H-2, H-6), 6.47 (s, 2 H,
3
3
H-2′, H-6′), 5.93 (s, 2 H, H-6, H-8), 5.52 (m, 1 H, H-3), 4.95 (s, 1 H, H-
2
-[2-(2-Azidoethoxy)ethoxy]ethan-1-amine (4b)21
2), 4.76 (d, J = 2.4 Hz, 2 H, OCH R), 2.99–2.94 (dd, J = 17.3, 4.5 Hz, 1 H,
2
H-4), 2.85–2.80 (dd, J = 17.3, 2.3 Hz, 1 H, H-4), 2.77 (t, J = 2.4 Hz, 1
H, ≡CH).
Compound was prepared according to GP1; starting with 2.3 g (15.0
mmol) of triethylene glycol; yield: 2.35 g (90%); yellow oil.
13C NMR (125 MHz, CD
OD):  = 165.7, 156.5, 156.4, 155.8, 150.5,
1
3
H NMR (500 MHz, CDCl ):  = 3.68–3.63 (m, 6 H, 4-H, 5-H, 8-H), 3.53
3
1
7
45.3, 137.0, 132.4, 129.3, 125.7, 108.7, 105.4, 97.9, 95.2, 94.5, 78.6,
7.1, 75.3, 68.9, 58.6, 25.4.
(t, J = 5.0 Hz, 2 H, 2-H), 3.39 (t, J = 5.2 Hz, 2 H, 6-H), 2.88 (t, J = 5.0 Hz,
2
H, 1-H), 2.17 (s, 2 H, NH2).
+
1
3
HRMS (ESI): m/z calcd for C25
97.1102.
H
20
O
11 [M + H] : 497.1039; found:
C NMR (125 MHz, CDCl ):  = 73.0, 70.7, 70.3, 70.1, 50.7, 41.6.
3
4
2
-{2-[2-(2-Azidoethoxy)ethoxy]ethoxy}ethan-1-amine (4c)22
Bispropargylated Product 3′
Mp, decomposed at >120 °C; [] 21 –133.0 (c = 0.1, MeOH).
Compound was prepared according to GP1; starting with 2.62 g (13.5
mmol) of tetraethylene glycol; yield: 2.5 g (85%); yellow oil.
D
1
FT-IR (neat): 3359, 3282, 2926, 2858, 2362, 2124, 1695, 1601, 1519,
H NMR (500 MHz, CDCl ):  = 3.68–3.60 (m, 10 H, 3-H, 4-H, 5-H, 6-H,
-H), 3.50 (t, J = 5.1 Hz, 2 H, 2-H), 3.39 (t, J = 5.1 Hz, 2 H, 8-H), 2.86 (t,
3
1
451, 1363, 1235, 1174, 1142, 1049, 1014, 982, 754, 736, 711, 632
7
–
1
cm
.
J = 5.1 Hz, 2 H, 1-H), 1.89 (s, 2 H, NH2)
©
2021. Thieme. All rights reserved. Synthesis 2021, 53, A–I

G
Synthesis
A. El Khoury et al.
Special Topic
1
1
H NMR (500 MHz, CD OD):  = 6.88 (s, 2 H, Gal H-2, H-6), 6.50 (s, 2 H,
H NMR (500 MHz, CD OD):  = 7.90 (s, 1 H, triazole-H), 6.88 (s, 2 H,
3
3
H-2′, H-6′), 5.94 (s, 2 H, H-6, H-8), 5.55 (m, 1 H, H-3), 4.99 (s, 1 H, H-
Gal H-2, H-6), 6.50 (s, 2 H, H-2′, H-6′), 5.95 (s, 2 H, H-6. H-8), 5.53–
2
), 4.76 (d, J = 2.4 Hz, 2 H, OCH R), 4.66 (d, J = 2.4 Hz, 2 H, OCH R′),
5.52 (m, 1 H, H-3), 5.26 (s, 2 H, OCH R), 4.97 (s, 1 H, H-2), 4.52–4.50 (t,
2
2
2
3.01–2.95 (dd, J = 17.4, 4.6 Hz, 1 H, H-4), 2.86–2.81 (dd, J = 17.3, 2.2
J = 4.6 Hz, 2 H, NCH CH OCH CH OCH CH NH ), 3.82–3.73 (m, 2 H,
2
2
2
2
2
2
2
Hz, 1 H, H-4), 2.77 (t, J = 2.4 Hz, 1 H, ≡CH), 2.71 (t, J = 2.4 Hz, 1 H,
NCH CH OCH CH OCH CH NH ),
3.50–3.48
3.41
3.02–2.96
(m,
(m,
(m,
2
H,
H,
H,
2
2
2
2
2
2
2
≡
CH′).
NCH CH OCH CH OCH CH NH ),
4
2
2
2
2
2
2
2
1
3
NCH
2
CH
2
OCH
2
CH
2
OCH
2
CH
2
NH
2
),
3
C NMR (125 MHz, CD OD):  = 165.6, 156.6, 156.5, 155.6, 150.5,
3
NCH CH OCH CH OCH CH NH and H-4), 2.86–2.82 (dd, J = 18.1, 1.9
150.4, 137.0, 134.8, 132.3, 125.6, 108.7, 105.5, 97.9, 95.2, 94.5, 79.0,
2
2
2
2
2
2
2
Hz, 1 H, H-4).
13C NMR (125 MHz, CD
78.6, 78.1, 75.3, 75.0, 68.8, 58.8, 58.6, 26.4.
+
3
OD):  = 165.6, 156.54, 156.46, 155.8, 150.4,
45.3, 137.1, 132.3, 129.4, 125.6, 108.9, 105.3, 97.8, 95.1, 94.5, 77.0,
HRMS (ESI): m/z calcd for C28H23O11 [M + H] : 535.1240; found:
1
7
535.1252.
0.1, 69.8, 68.98, 68.95, 66.3, 63.8, 53.7, 50.1, 39.3, 25.5.
Using K CO /DMF: To EGCG (1 g, 2.18 mmol, 1.0 equiv) in DMF (11 mL,
2
3
+
HRMS (ESI): m/z calcd for C31H35N O [M + H] : 671.2201; found:
0
.2 M) at 0 °C was added K CO (166 mg, 1.2 mmol, 0.5 equiv) in one
4
13
2
3
6
71.2170.
portion. The reaction was stirred at rt for 1 h. Propargyl bromide
0.24 mL, 2.18 mmol, 1.1 equiv, 80% w/w) was then added and the
(
mixture was stirred at the same temperature overnight. The mixture
was then concentrated in vacuo and purified as above furnishing the
product in 45% yield (491 mg) along with 10% of bispropargylated
side product. All the analytical data matched the one obtained using
the above alternative procedure.
(2R,3R)-5,7-Dihydroxy-2-(3,4,5-trihydroxyphenyl)chroman-3-yl
4-{[1-(2-{2-[2-(2-Aminoethoxy)ethoxy]ethoxy}ethyl)-1H-1,2,3-
triazol-4-yl]methoxy}-3,5-dihydroxybenzoate (5c)
Compound was prepared according to GP2; yield: 30 mg (52%); white
solid; mp, decomposed at >120 °C; []D21 –62 (c = 0.1, MeOH).
FT-IR (neat): 3203, 2970, 1674, 1602, 1523, 1437, 1368, 1200, 1143,
Click Reaction with PEGylated Linkers; General Procedure 2 (GP2)
–1
1
060, 981, 831, 718, 647, 622, 610 cm
.
To a flame-dried microwave vial was added 3 (40 mg, 0.081 mmol, 1.0
equiv) and the corresponding azide 4 (0.081 mmol, 1.0 equiv). In a
separate vial, a solution of CuSO ·5H O (4 mg, 0.016 mmol, 0.2 equiv),
1
H NMR (500 MHz, CD OD):  = 7.87 (s, 1 H, triazole-H), 6.89 (s, 2 H,
3
Gal H-2, H-6), 6.50 (s, 2 H, H-2′, H-6′), 5.94 (s, 2 H, H-6. H-8), 5.54–
5.53 (m, 1 H, H-3), 5.25 (s, 2 H, OCH R), 4.97 (s, 1 H, H-2), 4.52 [t, J =
4
4
2
2
sodium ascorbate (34 mg, 0.17 mmol, 2.0 equiv), and TBTA (21 mg,
.04 mmol, 0.5 equiv) in DMSO/H O (4:1, 0.81 mL, 0.1 M) was pre-
.9 Hz, 2 H, NCH CH (OCH CH ) OCH CH NH ], 3.81–3.73 [m, 2 H,
2 2 2 2 2 2 2 2
0
2
NCH CH (OCH CH ) OCH CH NH ],
3.59–3.57
[m,
2
H,
H, N-
H,
NCH CH (OCH CH ) OCH CH NH ], 3.07–3.05 [t, J = 5.5 Hz, 2 H,
2
2
2
2
2
2
2
2
pared and added to the first vial. The reaction was stirred at rt for 1 h.
The crude mixture was purified by preparative reverse-phase HPLC
NCH CH (OCH CH ) OCH CH NH ], 3.54–3.47 [m,
4
2
2
2
2
2
2
2
2
CH CH (OCH CH ) OCH CH NH ],
3.45–3.42
[m,
4
2
2
2
2
2
2
2
2
(
33–60% MeCN/H O + 0.1% (v/v) TFA in 8.5 min] to give the desired
2
2
2
2
2
2
2
2
2
triazole adduct 5 (t = 5.2 min).
R
NCH CH (OCH CH ) OCH CH NH ], 3.01–2.96 (dd, J = 17.3, 4.5 Hz, 1
2 2 2 2 2 2 2 2
H, H-4), 2.86–2.82 (dd, J = 17.3, 2.3 Hz, 1 H, H-4).
(
4
3
2R,3R)-5,7-Dihydroxy-2-(3,4,5-trihydroxyphenyl)chroman-3-yl
-({1-[2-(2-Aminoethoxy)ethyl]-1H-1,2,3-triazol-4-yl}methoxy)-
,5-dihydroxybenzoate (5a)
13
C NMR (125 MHz, CD OD):  = 165.6, 156.54, 156.48, 155.8, 150.4,
3
1
7
45.3, 137.0, 132.3, 129.4, 125.6, 124.9, 108.9, 105.3, 97.8, 95.1, 94.5,
7.0, 70.0, 69.9, 69.6, 69.0, 66.3, 63.9, 50.1, 39.3, 25.5.
Compound was prepared according to GP2; yield: 35 mg (68%); white
solid; mp, decomposed at >120 °C; []D –81 (c = 0.1, MeOH).
+
HRMS (ESI): m/z calcd for C33H38N O Na [M + Na] : 737.2282; found:
2
1
4
14
7
37.2308.
FT-IR (neat): 3374, 2951, 2934, 1676, 1626, 1523, 1448, 1370, 1196,
1
–
1
146, 1061, 1015, 770, 724, 650, 612 cm .
(
2R,3R)-5,7-Dihydroxy-2-(3,4,5-trihydroxyphenyl)chroman-3-yl
1
H NMR (500 MHz, CD OD):  = 7.88 (s, 1 H, triazole-H), 6.87 (s, 2 H,
4-{[1-(14-Amino-3,6,9,12-tetraoxatetradecyl)-1H-1,2,3-triazol-4-
3
Gal H-2, H-6), 6.50 (s, 2 H, H-2′, H-6′), 5.95 (s, 2 H, H-6, H-8), 5.54 (m,
yl]methoxy}-3,5-dihydroxybenzoate (5d)
1
H, H-3), 5.25 (s, 2 H, OCH R), 4.97 (s, 1 H, H-2), 4.56 (t, J = 4.8 Hz, 2 H,
2
Compound was prepared according to GP2; yield: 28 mg (46%); white
solid; mp, decomposed at >120 °C; []D –69 (c = 0.1, MeOH).
NCH CH OCH CH NH ), 3.83–3.75 (m, 2 H, NCH CH OCH CH NH ),
21
2
2
2
2
2
2
2
2
2
2
3.50–3.48 (m,
2
H, NCH CH OCH CH NH ), 3.01–2.97 (m,
3
H,
2
2
2
2
2
FT-IR (neat): 3179, 2926, 1681, 1627, 1523, 1451, 1372, 1349, 1203,
NCH CH OCH CH NH , and H-4), 2.86–2.82 (dd, J = 17.3, 2.2 Hz, 1 H,
H-4).
2
2
2
2
2
1
146, 1061, 1038, 831, 726, 641, 618 cm–1
.
1H NMR (500 MHz, CD
OD):  = 7.90 (s, 1 H, triazole-H), 6.87 (s, 2 H,
1
3
3
C NMR (125 MHz, CD OD):  = 165.6, 156.5, 156.5, 155.8, 150.4,
3
Gal H-2, H-6), 6.48 (s, 2 H, H-2′, H-6′), 5.93 (s, 2 H, H-6, H-8), 5.52–
.53 (m, 1 H, H-3), 5.23 (s, 2 H, OCH R), 4.95 (s, 1 H, H-2), 4.51–4.48 [t,
145.3, 137.1, 132.3, 129.4, 125.7, 124.9, 108.8, 105.4, 97.8, 95.1, 94.5,
5
2
77.0, 69.1, 69.0, 66.3, 63.9, 49.9, 39.0, 25.4.
J = 4.7 Hz, 2 H, NCH CH (OCH CH ) OCH CH NH ], 3.78–3.70 [m, 2 H,
2
2
2
2
3
2
2
2
+
HRMS (ESI): m/z calcd for C29H32N O [M + H] : 627.1938; found:
4
12
NCH CH (OCH CH ) OCH CH NH ], 3.61–3.58 [m,
2
6
6
H,
H,
H,
2
2
2
2
3
2
2
2
627.1954.
NCH CH (OCH CH ) OCH CH NH ), 3.55–3.50 [m,
2
2
2
2
3
2
2
2
NCH CH (OCH CH ) OCH CH NH ], 3.46–3.40 [m,
2
2
2
2
3
2
2
2
(
4
2R,3R)-5,7-Dihydroxy-2-(3,4,5-trihydroxyphenyl)chroman-3-yl
-[(1-{2-[2-(2-Aminoethoxy)ethoxy]ethyl}-1H-1,2,3-triazol-4-
NCH CH (OCH CH ) OCH CH NH ], 3.05–3.03 [t, J = 5.0 Hz, 2 H,
NCH CH (OCH CH ) OCH CH NH ], 3.00–2.94 (dd, J = 17.5, 4.6 Hz, 1
2 2 2 2 3 2 2 2
2 2 2 2 3 2 2 2
yl)methoxy]-3,5-dihydroxybenzoate (5b)
H, H-4), 2.85–2.80 (dd, J = 17.5, 1.8 Hz, 1 H, H-4).
Compound was prepared according to GP2; yield: 32 mg (58%); white
solid; mp, decomposed at >120 °C; []D –73 (c = 0.1, MeOH).
13
C NMR (125 MHz, CD OD):  = 165.6, 156.50, 156.45, 155.8, 150.4,
3
2
1
145.3, 137.2, 132.3, 129.4, 125.6, 125.0, 108.9, 105.3, 97.8, 95.1, 94.5,
77.0, 70.05, 69.94, 69.88, 69.82, 69.78, 69.5, 69.0, 68.9, 66.3, 64.0,
50.1, 39.2, 25.5.
FT-IR (neat): 3170, 2964, 2952, 1678, 1627, 1609, 1523, 1450, 1376,
–
1
1
347, 1201, 1146, 1058, 1039, 969, 836, 720, 677, 602 cm .
©
2021. Thieme. All rights reserved. Synthesis 2021, 53, A–I

H
Synthesis
A. El Khoury et al.
Special Topic
+
HRMS (ESI): m/z calcd for C35H42N O Na [M + Na] : 781.2544; found:
(7) (a) Seidler, P. M.; Boyer, D. R.; Sawaya, M. R.; Ge, P.; Shin, W. S.;
4
15
781.2525.
DeTure, M. A.; Dickson, D. W.; Jiang, L.; Eisenberg, D. S. bioRxiv
2
020, preprint DOI: 10.1101/2020.05.29.124537. (b) Sonawane,
S. K.; Chidambaram, H.; Boral, D.; Gorantla, N. V.; Balmik, A. A.;
Dangi, A.; Ramasamy, S.; Marelli, U. K.; Chinnathambi, S. Sci.
Rep. 2020, 10, 12579.
(
4
2R,3R)-5,7-Dihydroxy-2-(3,4,5-trihydroxyphenyl)chroman-3-yl
-{[1-(17-Amino-3,6,9,12,15-pentaoxaheptadecyl)-1H-1,2,3-tri-
azol-4-yl]methoxy}-3,5-dihydroxybenzoate (5e)
(
8) (a) Lu, M.; Cohen, M. H.; Rieves, D.; Pazdur, R. Am. J. Hematol.
2010, 85, 315. (b) Muldoon, L. L.; Sàndor, M.; Pinkston, K. E.;
Neuwelt, E. A. Neurosurgery 2005, 57, 785. (c) Neubert, J.;
Wagner, S.; Kiwit, J.; Bräuer, A. U.; Glumm, J. Int. J. Nanomed.
Compound was prepared according to GP2; yield: 29 mg (45%); white
solid; mp, decomposed at >120 °C; []D –79 (c = 0.1, MeOH).
2
1
FT-IR (neat): 3307, 2908, 1678, 1625, 1521, 1449, 1374, 1349, 1238,
1
–
1
201, 1147, 1096, 845, 772, 722, 652, 633 cm .
2
015, 10, 2033. (d) Nguyen, K. L.; Yoshida, T.; Kathuria-Prakash,
1
H NMR (500 MHz, CD OD):  = 7.90 (s, 1 H, triazole-H), 6.87 (s, 2 H,
N.; Zaki, I. H.; Varallyay, C. G.; Semple, S. I.; Saouaf, R.; Rigsby, C.
K.; Stoumpos, S.; Whitehead, K. K.; Griffin, L. M.; Saloner, D.;
Hope, M. D.; Prince, M. R.; Fogel, M. A.; Schiebler, M. L.; Roditi,
G. H.; Radjenovic, A.; Newby, D. E.; Neuwelt, E. A.; Bashir, M. R.;
Hu, P.; Paul Finn, J. Radiology 2019, 293, 554.
3
Gal H-2, H-6), 6.49 (s, 2 H, H-2′, H-6′), 5.93 (s, 2 H, H-6. H-8), 5.52–
5
J = 4.7 Hz, 2 H, NCH CH (OCH CH ) OCH CH NH ], 3.82–3.71 [m, 2 H,
NCH CH (OCH CH ) OCH CH NH ], 3.63–3.60 [m,
.53 (m, 1 H, H-3), 5.24 (s, 2 H, OCH R), 4.95 (s, 1 H, H-2), 4.52–4.50 [t,
2
2
2
2
2
4
2
2
2
2
H,
2
2
2
2
4
2
2
2
NCH CH (OCH CH ) OCH CH NH ], 3.57–3.38 [m, 16 H,
(9) Wang, J.; Sun, P.; Wang, Q.; Zhang, P.; Wang, Y.; Zi, C.; Wang, X.;
Sheng, J. Cancer Cell Int. 2019, 19, 266.
2
2
2
2
4
2
2
2
NCH CH (OCH CH ) OCH CH NH ], 3.01–2.94 [m,
3
H,
2
2
2
2
4
2
2
2
NCH CH (OCH CH ) OCH CH NH , H-4], 2.85–2.80 (dd, J = 17.5, 1.8
Hz, 1 H, H-4).
(10) Yang, Q.; Sheng, M.; Henkelis, J. J.; Tu, S.; Wiensch, E.; Zhang, H.;
Zhang, Y.; Tucker, C.; Ejeh, D. E. Org. Process Res. Dev. 2019, 23,
2210.
2
2
2
2
4
2
2
2
1
3
C NMR (125 MHz, CD OD):  = 165.6, 156.56, 156.49, 155.8, 150.4,
3
(
11) (a) Aufort, M.; Herscovici, J.; Bouhours, P.; Moreau, N.; Girard, C.
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1
9
6
45.3, 143.6, 137.2, 132.3, 129.4, 125.6, 124.9, 108.9, 105.3, 97.8, 95.1,
4.5, 77.0, 69.94, 69.87, 69.84, 69.80, 69.76, 69.70, 69.68, 69.43, 69.0,
8.9, 66.3, 63.9, 50.0, 39.2, 25.5.
+
HRMS (ESI): m/z calcd for C37H47N O [M + H] : 803.2987; found:
4
16
803.2997.
Conflict of Interest
(e) Sun, P.; Chen, J.; Liu, J.; Zhang, K. Macromolecules 2017, 50,
1463.
The authors declare no conflict of interest.
(
12) Himo, F.; Lovell, T.; Hilgraf, R.; Rostovtsev, V. V.; Noodleman, L.;
Sharpless, K. B.; Fokin, V. V. J. Am. Chem. Soc. 2005, 127, 210.
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(
Funding Information
Funding provided by the Donald J. and Jane M. Cram Endowment (to
(14) Chan, T. R.; Hilgraf, R.; Sharpless, K. B.; Fokin, V. V. Org. Lett.
2004, 6, 2853.
P.H.), the NIH (R56 AG070895 to D.E.), and the HHMI (D.E.).
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. Cram
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15) Madhusudanan, K. P.; Sachdev, K.; Harrison, D. A.; Kulshreshtha,
D. K. Int. J. Mass Spectrom. Ion Processes 1984, 62, 289.
Supporting Information
(16) Jordheim, M.; Fossen, T.; Songstad, J.; Andersen, Ø. M. J. Agric.
Food Chem. 2007, 55, 8261.
Supporting information for this article is available online at
https://doi.org/10.1055/a-1509-5904.
(17) (a) Sanders, D. W.; Kaufmann, S. K.; DeVos, S. L.; Sharma, A. M.;
Mirbaha, H.; Li, A.; Barker, S. J.; Foley, A. C.; Thorpe, J. R.; Serpell,
L. C.; Miller, T. M.; Grinberg, L. T.; Tolnay, M.; Seeley, W. W.;
Diamond, M. I. Neuron 2014, 82, 1271. (b) Seidler, P. M.; Boyer,
D. R.; Murray, K. A.; Yang, T. P.; Bentzel, M.; Sawaya, M. R.;
Rosenberg, G.; Cascio, D.; Williams, C. K.; Newell, K. L.; Ghetti,
B.; DeTure, M. A.; Dickson, D. W.; Vinters, H. V.; Eisenberg, D. S.
J. Biol. Chem. 2019, 294, 16451.
S
u
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p
orting InformationS
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p
p
orting Informatio
n
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