Synthesis of 7-benzylguanosine cap-analogue conjugates for eIF4E targeted degradation

Article abstract of DOI:10.1016/j.ejmech.2019.01.080

Eukaryotic translation initiation factor 4E (eIF4E) is a key player in the initiation of cap-dependent translation through recognition of the m⁷GpppX cap at the 5’ terminus of coding mRNAs. As eIF4E overexpression has been observed in a number of human diseases, most notably cancer, targeting this oncogenic translation initiation factor has emerged as a promising strategy for the development of novel anti-cancer therapeutics. Toward this end, in the present study, we have rationally designed a series of Bn⁷GxP-based PROTACs for the targeted degradation of eIF4E. Herein we describe our synthetic efforts, in addition to biochemical and cellular characterization of these compounds.

Full text of DOI:10.1016/j.ejmech.2019.01.080

Accepted Manuscript
Synthesis of 7-benzylguanosine cap-analogue conjugates for eIF4E targeted
degradation
Tanpreet Kaur, Arya Menon, Amanda L. Garner
PII:
S0223-5234(19)30100-X
DOI:
https://doi.org/10.1016/j.ejmech.2019.01.080
EJMECH 11087
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 December 2018
Revised Date: 29 January 2019
Accepted Date: 29 January 2019
Please cite this article as: T. Kaur, A. Menon, A.L. Garner, Synthesis of 7-benzylguanosine cap-
analogue conjugates for eIF4E targeted degradation, European Journal of Medicinal Chemistry (2019),
doi: https://doi.org/10.1016/j.ejmech.2019.01.080.
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ACCEPTED MANUSCRIPT

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Synthesis of 7-Benzylguanosine Cap Analogue Conjugates for eIF4E Targeted
Degradation
Tanpreet Kaur, Arya Menon, and Amanda L. Garner*
Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, 1600 Huron
Parkway, Ann Arbor, Michigan 48109, USA
*algarner@umich.edu
KEYWORDS: eIF4E, PROTACs, m7G cap, cancer, proteasomal degradation
ABSTRACT:
Eukaryotic translation initiation factor 4E (eIF4E) is a key player in the initiation of cap-
dependent translation through recognition of the m7GpppX cap at the 5’ terminus of coding
mRNAs. As eIF4E overexpression has been observed in a number of human diseases, most
notably cancer, targeting this oncogenic translation initiation factor has emerged as a promising
strategy for the development of novel anti-cancer therapeutics. Toward this end, in the present
study, we have rationally designed a series of Bn7GxP-based PROTACs for the targeted
degradation of eIF4E. Herein we describe our synthetic efforts, in addition to biochemical and
cellular characterization of these compounds.

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1. INTRODUCTION
Dysregulation of cap-dependent translation is a hallmark of many cancers [1,2,3]. Initiation of
cap-dependent translation is dependent upon recognition of the m7GpppX cap (Figure 1A) at the
5’-terminus of a mRNA by eukaryotic translation initiation factor 4E (eIF4E) [4]. Binding of
eIF4E to the mRNA cap results in recruitment of the translation machinery, which then initiates
protein synthesis at the transcript’s start codon. eIF4E plays an essential role in controlling
cellular growth and proliferation [5], and overexpression of this translation initiation factor is
observed in many malignant cell lines and primary tumors, including breast [6], head and neck
[7], and non-Hodgkin’s lymphomas [8]. In previous literature reports, it has been shown that
inhibition of eIF4E function slows tumor growth and induces apoptosis [9]. Thus, development
of selective eIF4E inhibitors or degradation-inducing molecules could be a promising approach
for the treatment of many cancers, in addition to providing valuable chemical tools for studying
cap-dependent translation.
OH OH
A
B
O
O
N
H2N
HN
N
O
O
N
N
N
N
NH
NH2
O P OH
O
3
Me
O
O
OH
mRNA
m7GpppX cap
Figure 1. eIF4E biological activity and structure. (A) Structure of the m7GpppX cap. (B) Crystal
structure of eIF4E bound to the m7GpppX cap (PDB: 1EJ1). The key tryptophan residues for cap
binding are highlighted, in addition to lysines close to the cap-binding pocket.

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Molecular recognition of the m7GpppX cap by eIF4E involves cation-π interactions, in which
Trp56 and Trp102 of eIF4E sandwich the positively-charged methylated guanine nucleobase of
the cap (Figure 1B) [10]. This interaction is of utmost significance, as the binding affinity of
m7GTP has been shown to be ~100-fold tighter than that of GTP itself (Kds of 0.15 and 14 µM,
respectively) [10]. Based on the demonstrated importance of eIF4E in cellular and disease
biology [3,11], many structure-activity relationship (SAR) campaigns have been carried out to
rationally design m7G-based competitive inhibitors of eIF4E binding to capped mRNA (Figure
2) [12,13,14,15] These studies have revealed that analogues containing benzyl or substituted
benzyl groups at the 7-position (~10-fold enhancement in binding affinity) and at least one
phosphate group are optimal inhibitors of eIF4E-cap binding [16,17,18]. In addition, efforts have
been made to improve the cellular permeability of cap analogues through various pro-drug
strategies (4Ei-1,2,3 in Figure 2) [19,20,21].
HN
CO2Me
O
O
N
R
NH
R
O
N
N
n
NH
NH2
OH
HO P O
O
NH
HO P O
O
HO P O
O
N
N
N
NH
NH2
NH
NH2
N
N
N
O
O
O
OH OH
OH OH
R = Me, Bn, substituted Bn
OH OH
4Ei-1
4Ei-2; R = Bn
4Ei-3; R = Me
Figure 2. Structures of previously reported m7G cap analogues [21].
Recently, PROteolysis-TArgeting Chimeras (PROTACs) [22,23] have emerged as a
powerful alternative to traditional small molecule-based drug discovery approaches in that these
bifunctional molecules can trigger intracellular protein degradation by marking proteins for
proteosomal digestion [24,25,26,27,28]. In short, a small molecule ligand for the protein-of-
interest is conjugated to an E3 ubiquitin ligase ligand, such as lenalidomide or VHL peptide,
generating a novel two warhead probe [29] to enable the directed ubiquitination, and subsequent
degradation, of the target protein [30]. To date, this strategy has been successfully extended to
BET family [25,31,32,33], Sirtuin 2 [34], CDK9 [35], Skp1-cullin-F-box [36], BCR-ABL [37]
and ERRα proteins [38]. Thus, based on the previous successes of m7G cap analogues as
inhibitors of eIF4E, we became interested in developing PROTACs based upon this scaffold for

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the targeted proteasomal degradation of eIF4E [14,39,40]. To this end, 4Ei-1 has been previously
shown to induce eIF4E proteasomal degradation in a dose-dependent manner; however, high
concentrations (100-500 µM) of the compound were required to see an effect [41]. By using a
PROTAC strategy, our goal was to achieve this biological effect with lower concentrations due
to the catalytic nature of these conjugates, thereby, providing in-roads toward the development of
these molecules for therapeutic applications. Herein we disclose our synthetic efforts toward cap-
competitive eIF4E-targeted PROTACs and their preliminary in vitro and cellular
characterization.
2. RESULTS AND DISCUSSION
2.1. Assessing eIF4E as a Candidate for PROTAC Development
Little is known about the endogenous turnover of the eIF4E protein [42,43,44]. To date, only the
C-terminus of Hsc-70 interacting protein (CHIP) and cellular inhibitor of apoptosis protein-1
(cIAP1) E3 ligases have been implicated in eIF4E ubiquitination and degradation under
conditions of cellular stress [43,44]. eIF4E contains 16 total lysine residues, 13 of which have
been shown to be solvent accessible via X-ray crystallography (Lys21, 36, 49, 52, 54, 65, 106,
108, 119, 159, 162, 192, 212) [45] for possible ubiquitination. Notably, several of these residues
are within 15 Å of the cap-binding pocket (Figure 1B) [45], which is an essential requirement for
the use of PROTAC technology [46]. Thus, we were encouraged that eIF4E degradation could be
induced with appropriate Bn7GxP-based PROTAC design.
2.2 Design and Synthesis of GMP-based PROTACs
As an initial PROTAC design strategy, we generated a small library of guanosine
monophosphate (GMP) derivatives conjugated to lenalidomide (1), a ligand for the well-known
E3 ligase, cereblon [47,48]. GMP was chosen as the Bn7GxP scaffold based on its successful use
in previously reported inhibitors of eIF4E (Figure 2) [21,39,40,49]. The synthetic route is shown
in Scheme 1 and began with HATU-mediated coupling of 1 with Boc-6-aminohexanoic acid to
form Boc-protected amide 2 in 89% yield, followed by Boc deprotection in quantitative yield.
The unmasked free amine 3 was then subjected to coupling with GMP disodium salt to provide
GMP-lenalidomide conjugate 4 in 31% yield [18,50]. The low reaction yield for this step was
due to solvent incompatibility between the hydrophobic lenalidomide derivative 3 and

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hydrophilic GMP. Alkylation at the 7-position with benzyl bromide or 4-fluorobenzyl bromide
[18,51] yielded Bn7GMP PROTACs 5a and 5b in 4% and 29% yields, respectively. 4-
Fluorobenzyl substitution was also examined, as this modification was previously shown to
further enhance binding affinity to eIF4E [10]. Again, yields were low due to poor solubility.
BocHN
H2N
O
NH2
NHBoc
5
5
NH
O
HO
O
NH
O
NH
50% TFA/DCM
5
O
O
N
O
NH
NH
HATU, DIPEA
100%
N
O
N
O
89%
O
O
O
1
2
3
O
N
ONa
NaO P O
O
N
N
NH
NH2
EDCI.HCl, pH 7.2
31%
O
OH OH
O
O
N
O
N
O
NH
NH
O
O
HN
O
HN
O
R-Br
O
O
N
R
N
5
5
NH
NH
N
N
NH
NH2
NH
NH2
HO P O
O
HO P O
O
N
N
O
O
OH OH
OH OH
4
5a: R = Bn, 4%
5b: R = 4-FBn, 29%
Scheme 1. Synthesis of Bn7GMP-based lenalidomide conjugates.
2.3 Design and Synthesis of Bioisoteres of GMP-based PROTACs
Aryl sulfamates have previously been reported as bioisosteres of phosphate groups [51]. Thus,
we were also interested in the synthesis of such non-hydrolyzable Bn7GMP-based PROTACs.
The synthesis, which is shown in Scheme 2, began with TBS protection of guanosine to yield 6
in 70% yield. Sulfamoylation at the 5’ position was then carried out using sulfamoyl chloride
[51] generated in situ from formic acid and chlorosulfonyl isocyanate [51] to yield 7 in 95%
yield. Coupling with N-hydroxysuccinic (NHS) ester 8 in the presence of DBU, followed by
deprotection of the tert-butyl protecting group resulted in 10. Interestingly, conjugation of the
free acid of this compound with lenalidomide (1) under HATU- or PyBrOP-mediated coupling

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conditions did not yield the desired product. Since we speculated that this may be due to the
weak basicity of 1, we utilized the lenalidomide derivative 3, which resulted in the formation of
11 in 40% yield. Subsequent deprotection of the TBS groups with TBAF and alkylation at the 7-
position produced the target PROTACs 13a and 13b in 45% and 55% yields, respectively. Yields
for the alkylation of sulfamate derivative 12 were better than that of the corresponding GMP due
to its enhanced solubility in DMSO.
O
N
O
N
O
N
NH2
N
N
N
N
N
N
1. TBSCl, DMF
2. MeOH, 50 ºC
NH2SO2Cl
DMA
NH
NH2
NH
NH2
NH
NH2
O
S
O
HO
O
HO
70%
95%
O
O
O
OH OH
TBSO
OTBS
6
TBSO
OTBS
7
O
O
O
DBU, DMF
42%
N
O
O
2
O
8
O
N
O
NH
OH
O
2
O
HN
O
3, EDCI.HCl,
NMM, DMSO, H2O,
pH 6.0
O
2
NH
O
O
2
O
N
NH
O
5
N
N
DCM:TFA:TES
O
NH
NH
NH2
O S O
O
NH
NH2
O
40%
O S O
O
90%
O
N
N
N
O
NH
O
N
N
O
NH
NH2
O S O
O
TBSO OTBS
10
N
TBSO OTBS
9
O
TBSO OTBS
11
TBAF/THF
71%
O
O
N
O
N
O
NH
NH
O
2
O
2
HN
O
HN
O
NH
NH
5
5
R-Br
O
O
O
N
O
N
R
O
NH
O
NH
N
N
N
NH
NH2
NH
NH2
O S O
O
O S O
O
N
O
O
OH OH
12
OH OH
13a: R = Bn, 45%
13b: R = 4-FBn, 55%
Scheme 2. Synthesis of sulfamate-containing Bn7GMP-based lenalidomide conjugates.

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2.4 Biochemical Characterization of GMP-based PROTACs
To test the ability of the Bn7GMP-based PROTACs to bind to eIF4E and compete with the
m7GpppX cap, we developed an in vitro cap competition assay (Figure 3A). In this assay, cell
lysate is first incubated with m7GxP (GMP or GDP) agarose resin [52] to enable the affinity
purification of eIF4E. The preparation of this resin is shown in Figure 3B and uses aniline-
catalyzed reductive amination chemistry for chemical conjugation. Resin-immobilized eIF4E is
then incubated with compounds, such as m7GxP or a PROTAC. If the compound is active, eIF4E
will be competed off the resin; however, if there is no inhibitory activity, eIF4E will be retained
and detected by Western blot analysis.

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Figure 3. Cap-competition assay. (A) Assay overview. (B) Synthesis of m7GxP agarose resin.
Each of the synthesized Bn7GMP-based PROTACs (5a, 5b, 13a, 13b), in addition to the
GMP-lenalidomide conjugates 4 and 12, were tested in the m7GMP cap competition assay using
m7GMP as a positive control. As shown in Figure 4, upon treatment of eIF4E immobilized from
HEK293 cell lysate, negligible inhibition was observed with either the Bn7GMP- or sulfamate-
based PROTACs in comparison to the control. No inhibition with the GMP derivatives was
observed as expected. We hypothesize that this is due to the decreased or absent negative charge

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on the phosphoramide and sulfamate moieties, respectively, as a similar effect has been observed
with other GxP-binding proteins [53].
Figure 4. Western blot analysis of eIF4E following the m7GMP cap competition assay.
Immobilized eIF4E was treated with 500 µM of m7GMP or a GMP- or Bn7GMP-based
PROTAC. DMSO was used as a negative control.
2.5 Design and Synthesis of GDP-based PROTACs
As m7GDP has been shown to exhibit 50-fold greater inhibitory activity against eIF4E-cap
binding as compared to m7GMP [17], we next synthesized Bn7GDP-based PROTACs with hopes
that they would exhibit activity in the cap competition assay. In this case, we chose to
incorporate both lenalidomide and VHL ligand conjugates as part of our PROTAC design, in
addition to varying linker lengths, which has been shown to play a crucial role in the success of
this approach [26],[54].
For the lenalidomide conjugates, we followed a similar synthetic route to those used for the
GMP compounds (Scheme 3). The synthesis began with HATU-mediated coupling of
lenalidomide (1) with alkyl and PEG linkers to form Boc-protected amides 14−16 in 18-89%
yields. In case of the PEG7 analogue 16, we suspect that the observed low yield was due to its
aggregation propensity. Following deprotection, the unmasked free amines 17−19 were subjected
to EDCI-mediated coupling with the sodium salt of GDP to yield conjugates 20−22 in 32-35%
yields [18,50]. Alkylation was performed with benzyl or 4-fluorobenzyl bromide [18,51], and
resulted in Bn7GDP-based PROTACs 23−25 in 9-20% yields. Low yields were observed due to
both poor solubility and minor hydrolysis of GDP.

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O
NH2
O
LINKER
NHBoc
NH
HO
50% TFA/DCM
N
O
HATU, DIPEA
O
NH
O
O
O
NH
O
NH
NH
N
O
1
N
O
O
O
O
14: alkyl, 89%
15: PEG3, 56%
16: PEG7, 18%
17: alkyl, 100%
18: PEG3, 100%
19: PEG7, 100%
NHBoc
HO
5
O
O
=
NHBoc
3
LINKER
NHBoc
HO
HO
O
HO
O
ONa
N
N
O
NH
NH2
HO P O
O
NHBoc
7
EDCI.HCl, pH 7.2
O
2
N
O
OH OH
O
O
LINKER
HO P
LINKER
NH
NH
HO P
HN
HN
O
O
O
O
R
O
O
N
HN
HN
O
HO P O
O
O
HO P O
O
O
N
O
N
R-Br
N
N
N
NH
NH2
NH
NH2
O
O
N
N
O
O
OH OH
OH OH
23a: alkyl, R = Bn, 16%
20: alkyl, 35%
21: PEG3, 32%
22: PEG7, 35%
23b: alkyl, R = 4-FBn, 11%
24a: PEG3, R = Bn, 20%
24b: PEG3, R = 4-FBn, 11%
25a: PEG7, R = Bn, 13%
25b: PEG7, R = 4-FBn, 9%
Scheme 3. Synthesis of Bn7GDP-based lenalidomide conjugates.
The VHL ligand (26) was conjugated to GDP through a similar synthetic route (Scheme 4).
In this case, however, only the alkyl linker was used. HATU-mediated coupling of 26 with Boc-
6-aminohexanoic acid to yield Boc-protected amide 27 in 75% yield. After deprotection, amine
28 was conjugated to GDP via known EDC coupling [21], which produced GDP-VHL conjugate
29 in 32% yield. Subsequent alkylation [18,51] then generated the target PROTACs 30a and 30b
in 11% and 41% yields, respectively. Similar challenges with solubility and stability were
encountered.

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N
N
S
N
S
S
O
NHBoc
HO
5
50% TFA/DCM
OH
OH
NH
OH
NH
O
NH
O
HATU, DIPEA
90%
N
N
N
O
75%
H
H
O
O
O
NH2
N
N
5
5
NHBoc
NH2
O
O
26
27
28
O
ONa
N
N
NH
HO P O
O
EDCI.HCl,
NMM, pH 6.0
32%
2
N
NH2
O
OH OH
N
N
S
S
OH
OH
NH
NH
N
N
O
O
H
H
R-Br
O
O
N
N
5
5
NH
NH
O
O
HO P
O
O
HO P
O
O
O
O
R
N
N
NH
NH2
NH
HO P O
O
HO P O
O
N
N
N
N
NH2
O
O
OH OH
OH OH
30a: R = Bn, 11%
30b: R = 4-FBn, 41%
29
Scheme 4. Synthesis of Bn7GDP-based VHL conjugates.
2.6 Biochemical Characterization of GDP-based PROTACs:
Each of the synthesized GDP- (20−22 and 29) and Bn7GDP-based PROTACs (23−25 and 30)
was tested for eIF4E inhibitory activity in the m7GDP cap competition assay (Figure 3A), in this
case, using m7GDP as a positive control. To our delight, each of the Bn7GDP conjugates was
found to be active (Figure 5A), while the GDP derivatives showed no activity as expected.
Within the alkyl linker series (Figure 5A, top), no observable inhibitory activity differences were
observed between the lenalidomide and VHL conjugates. Interestingly, however, the PEG3
lenalidomide (21, 24a-b) PROTAC showed enhanced activity in comparison the PEG7
analogues (22, 25a-b) (Figure 5A, bottom). Inhibition of eIF4E-cap binding was also found to be
dose-dependent for each of the PROTACs, and representative data for 23a is shown in Figure
5B.

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A
B
Figure 5. Western blot analysis of eIF4E following the m7GDP cap competition assay in
HEK293 cell lysate. (A) Immobilized eIF4E was treated with 500 µM of m7GDP or a GDP- or
Bn7GDP-based PROTAC. DMSO was used as a negative control. (B) Dose-dependent inhibition
by 23a.
2.7 Cellular Characterization of GDP-based PROTACs:
Encouraged by the promising biochemical data obtained with the Bn7GDP-based PROTACs, we
proceeded to cellular assays to determine if these compounds were able to induce the
proteasomal degradation of eIF4E. MDA-MB-231 breast cancer cells were treated with varying
concentrations (25−200 µM) of PROTACs for 24−72 h. Unfortunately, decreased eIF4E protein
levels were not detected with any of the conjugates (data not shown). Because we suspected that
low cellular permeability may be a problem, we next tested the PROTACs in K562 cells, which
have been found to more readily uptake purine nucleotides [55]. Across a similar set of
conditions, again we saw no intracellular degradation of eIF4E (representative data in Figure 6).
Thus, we hypothesize that the permeability of these compounds is still low, as has been observed

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with other cap analogues[14,18,21] or that ternary complex formation between eIF4E and the E3
ligase is not possible with the designed molecules. Future efforts should be focused on
examining additional linkers, as this has been shown to play a crucial role in successful
PROTAC design, in addition to the exploration of additional E3 ligase ligands, such as those for
cIAP and MDM2 [26, 54].
Figure 6. Western blot of total eIF4E following the treatment of K562 cells with Bn7GDP-based
PROTACs. Actin was used as a loading control.
3. CONCLUSION
In conclusion, we have synthesized a library of Bn7GxP-based PROTACs for the targeted
degradation of eIF4E, the m7GpppX cap-binding translation initiation factor. The targeting of
aberrant eIF4E activity with m7G cap analogues is rich with history [2,11,13], yet continues to be
plagued with issues of cellular permeability due the necessity for nucleotide-based scaffolds
[18,39,40]. Unfortunately, our efforts described herein were similarly unsuccessful in translating
to cellular assays. While pro-drugs have been developed (Figure 2), the requirement for high
micromolar concentrations in cells dampens enthusiasm for clinical development [19,20, 41].
Thus, future efforts in targeting eIF4E should focus on the discovery of non-nucleotide small
molecule competitive antagonists of cap binding or inhibiting its protein-protein interactions,
which are also crucial for its activity in initiating cap-dependent translation [1,3].
4. EXPERIMENTAL
4.1. General Materials and Methods: Lenalidomide (1) was purchased from AstaTech and used
as received. VHL ligand (26) was purchased from Abosyn and used as received. GMP and GDP
were purchased from ChemImpex and used as received. All solvents and reagents were used

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without further purification. Yields refer to chromatographically and spectroscopically (1H
NMR) homogeneous materials, unless otherwise stated. Reactions were monitored by thin-layer
chromatography (TLC) carried out on 0.25-mm SiliCycle silica gel plates (60F-254) using UV-
light (254 nm). Flash chromatography was performed using SiliaFlash P60 silica gel. Analytical
RP-HPLC was performed using an Agilent 1260 Infinity HPLC equipped with a ZORBAX
Eclipse XDB-C18 column (4.6 × 150 mm; 5 µm) at a flow rate of 1 mL/min, with detection at
214 and 254 nm. Semi-preparative HPLC was carried out on Agilent 1260 Infinity HPLC
equipped with a PrepHT XDB-C18 column (21.2 x 150 mm; 5 µm) at a flow rate of 18 mL/min
using 95% water as a mobile phase and detection at 254 nm and 214 nm. NMR spectra were
performed on a 300 MHz Bruker instrument and calibrated using a solvent peak as an internal
reference. Spectra were processed using MestReNova software. Mass spectrometry (HRMS) was
performed using an Agilent 6520 Accurate-Mass Q-TOF LC/MS spectrometer using ESI
ionization with less than 5-ppm error for all HRMS analyses.
4.2 General procedure A: Boc-protected acids (1.1 mmol, 1.1 equiv) were added to a stirring
solution of lenalidomide/VHL ligand (1.0 equiv), HATU (1.2 equiv) and DIPEA (2.0 equiv) in
dry DMF (0.22 M) at 25 °C. The reaction mixture was allowed to stir at 25 °C for 12 h. Upon
completion of the reaction as determined via TLC, the mixture was acidified with dilute HCl
(5%, 5 mL) and extracted with EtOAc (3×). The organic layers were dried over anhydrous
sodium sulfate and concentrated under reduced pressure in vacuo. The crude reaction mixture
was purified by silica gel chromatography using EtOAc in hexanes (30−100% EtOAc) to yield
the desired products as a white solid in 18−56% yields.
4.3 General procedure B: To an ice-cold stirring solution of Boc-protected derivatives (1 equiv)
in DCM (5 mL) was added trifluoroacetic acid (TFA) (5 mL). The reaction mixture was warmed
to 25 °C and stirred for an additional 2 h. Upon completion of the reaction, the solvent was
evaporated under reduced pressure in vacuo. The crude product was dried further under high
vacuum for 4 h and was used directly for the next step.
4.4 General procedure C: To a stirring solution of GMP or GDP sodium salt (1.0 equiv) in water
at pH 7.2 (0.06 M) was added N-methyl morpholine (10 equiv), EDCI. HCl (5 equiv), and free

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amine derivatives (1.0 equiv) in DMSO (0.06 M). The reaction mixture was stirred overnight at
25 °C. Upon completion, the mixture was diluted with water and purified by semi-preparative
HPLC (2−20% acetonitrile in water). Product-containing fractions were pooled and lyophilized
to dryness to yield the desired products as white solids.
4.5 General procedure D: To a stirring solution of guanosine analogues (1.0 equiv) in dry
DMSO (0.006 M) was added alkyl bromide (10 equiv). The reaction mixture was stirred
overnight at 25 °C. Upon completion, the mixture was diluted with water and extracted with
diethyl ether (3×). The combined water layers were lyophilized and purified by semi-preparative
HPLC (2−40% acetonitrile in water). Product containing fractions were pooled and lyophilized
to dryness to yield the Bn7GxP analogues as white solids.
4.6 Compound characterization:
tert-Butyl(6-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)-6-oxohexyl)
1
carbamate (2). General procedure A, 89% yield. Rf 0.40 (EtOAc). H NMR (500 MHz,
DMSO-d6) δ 11.01 (s, 1H), 9.78 (s, 1H), 7.81 (d, J = 7.2 Hz, 1H), 7.47 (d, J = 11.7 Hz, 2H), 6.75
(d, J = 5.4 Hz, 1H), 5.13 (d, J = 18.4 Hz, 1H), 4.43–4.27 (m, 2H), 2.89 (d, J = 24.4 Hz, 4H), 2.35
(s, 3H), 2.01 (dd, J = 9.7, 4.5 Hz, 1H), 1.58 (d, J = 14.8 Hz, 2H), 1.42–1.36 (m, 2H), 1.34 (s,
13
9H), 1.27 (p, J = 7.5, 6.9 Hz, 2H). C NMR (126 MHz, DMSO-d6) δ 173.28, 171.78, 171.49,
168.29, 162.74, 156.03, 134.26, 134.12, 133.09, 129.02, 125.67, 119.39, 77.75, 52.00, 46.97,
40.44, 40.22, 36.22, 31.66, 29.76, 28.71, 26.45, 25.30, 23.09. HRMS (ESI-TOF) m/z calcd for
C24H32N4O6 [M + H]+ 473.2400, found 473.2421.
6-Amino-N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)hexanamide
(3).
General
procedure B, 100% yield. Rf 0.2 (EtOAc). HRMS (ESI-TOF) m/z calcd for C19H24N4O4 [M +
H]+ 373.1876, found 373.1882.
((2S,3R,4S,5S)-5-(2-Amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydro-
furan-2-yl)methylhydrogen(6-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)-6-
oxohexyl)phosphoramidate (4). General procedure C, 31% yield. 1H NMR (500 MHz, D2O) δ
7.94 (t, J = 3.5 Hz, 1H), 7.57–7.52 (m, 1H), 7.46–7.34 (m, 2H), 5.73–5.63 (m, 1H), 5.06–4.96

ACCEPTED MANUSCRIPT
(m, 1H), 4.57–4.49 (m, 1H), 4.35–4.29 (m, 1H), 4.25–4.13 (m, 3H), 3.93–3.82 (m, 2H), 3.01 (d,
J = 50.7 Hz, 5H), 2.87–2.75 (m, 2H), 2.61 (d, J = 3.2 Hz, 2H), 2.44–2.34 (m, 1H), 2.26–2.16 (m,
3H), 1.75 (d, J = 6.9 Hz, 2H), 1.41 (d, J = 5.4 Hz, 2H), 1.30 (d, J = 3.2 Hz, 2H), 1.19–1.00 (m,
3H), 0.93 (d, J = 17.5 Hz, 2H). 31P NMR (202 MHz, D2O) δ 9.18 (d, J = 6.1 Hz). HRMS (ESI-
TOF) m/z calcd for C29H35N9O11P [M - H]- 716.2194, found 716.2234.
2-Amino-7-benzyl-9-((2S,3S,4R,5S)-5-(((((6-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-
yl)amino)-6-oxohexyl)amino)(hydroxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydro-
furan-2-yl)-6-oxo-6,9-dihydro-1H-purin-7-ium (5a). General procedure D, 4% yield. HRMS
(ESI-TOF) m/z calcd for C36H41N9O11P [M - H]- 806.2658, found 806.2683.
2-Amino-9-((2S,3S,4R,5S)-5-(((((6-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-
yl)amino)-6-oxohexyl)amino)(hydroxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydro-
furan-2-yl)-7-(4-fluorobenzyl)-6-oxo-6,9-dihydro-1H-purin-7-ium (5b). General procedure D,
29% yield. HRMS (ESI-TOF) m/z calcd for C36H40FN9O11P [M - H]- 824.2563, found
824.2593.
2-Amino-9-((2R,3R,4R,5R)-3,4-bis((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetra
hydrofuran-2-yl)-1,9-dihydro-6H-purin-6-one (6). To a stirring solution of guanosine (10 g,
35.3 mmol, 1.0 equiv) and imidazole (19 g, 283 mmol, 8 equiv) in dry DMF (70 mL) was added
TBSCl (21.2 g, 142 mmol, 4 equiv) under nitrogen atmosphere. The resulting solution was
stirred overnight at 25 °C. The reaction mixture was diluted with water (200 mL) and extracted
with DCM (3 × 50 mL). The combined DCM layers were washed with sat. aq. NH4Cl (1 × 25
mL), water (1 ×50 mL), dried over sodium sulfate and concentrated under vacuum. The resultant
white solid was suspended in 80% acetic acid (300 mL) and the reaction mixture was heated at
60 ºC for 12 h. Upon completion, the solvent was evaporated under vacuum and the crude
product was purified by silica gel column chromatography using DCM/methanol (0−20%
1
methanol) to yield 6 as a white solid (12.7 g, 70% yield). H NMR (500 MHz, DMSO-d6) δ
10.62 (s, 1H), 7.98 (s, 1H), 6.44 (s, 3H), 5.73 (d, J = 7.3 Hz, 1H), 5.29–5.19 (m, 2H), 4.69 (dd, J
= 7.3, 4.5 Hz, 1H), 4.27–4.18 (m, 1H), 3.92 (ddd, J = 5.0, 3.5, 1.3 Hz, 1H), 3.67 (dt, J = 11.9, 5.2
Hz, 1H), 3.56 (ddd, J = 11.9, 5.9, 3.5 Hz, 1H), 0.91 (s, 10H), 0.73 (s, 9H), 0.11 (d, J = 8.3 Hz,

ACCEPTED MANUSCRIPT
6H), -0.09 (s, 3H), -0.31 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 157.10, 154.09, 151.87,
136.14, 117.18, 87.17, 86.16, 75.43, 73.57, 61.71, 40.48, 40.31, 40.14, 39.97, 39.81, 39.64,
39.47, 26.26, 26.20, 25.96, 18.26, 18.02, -2.74, -4.21, -4.30, -5.09. HRMS (ESI-TOF) m/z calcd
for C22H42N5O5Si2 [M + H]+ 512.2724, found 512.2724.
Sulfamoyl chloride [51]. To an ice-cold stirring solution of chlorosulfonyl isocyanate (600 μL,
6.85 mmole, 1 equiv.), formic acid (285.5 μL, 6.85 mmole, 1 equiv.) was added drop-wise. After
10 min, the reaction mixture was warmed to 25 °C and stirred for 1 h. It was used directly for
the synthesis of 7.
((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-bis((tert-butyldimethyl-
silyl)oxy)tetrahydrofuran-2-yl)methyl sulfamate (7). To an ice-cold stirring solution of 6 (1.5
g, 3.0 mmol, 1.0 equiv) in dry DMF (10 mL) was added sulfamoyl chloride (345 mg, 3.3 mmol,
1.1 equiv) under nitrogen atmosphere. The resulting solution was warmed to 25 °C and stirred
for 1 h. Triethyl amine (3 mL) was then added and the reaction was stirred for 10 min. The
reaction mixture was cooled to 0 ºC, quenched with methanol (5 mL) and concentrated under
vacuum. The reaction mixture was dissolved in ethyl acetate (50 mL), washed with brine and
water (200 mL), dried over sodium sulfate and concentrated under vacuum. The crude reaction
mixture was purified by silica gel column chromatography using DCM/methanol (0−20%
1
methanol with 0.1% TEA) to yield 7 as a white solid (1.64 g, 95% yield). H NMR (500 MHz,
DMSO-d6) δ 10.66 (d, J = 6.0 Hz, 1H), 7.89 (s, 1H), 7.63 (s, 2H), 6.43 (s, 2H), 5.74 (d, J = 7.4
Hz, 1H), 4.76 (dd, J = 7.3, 4.4 Hz, 1H), 4.29 (dd, J = 11.3, 5.2 Hz, 1H), 4.26–4.19 (m, 2H), 4.13
(t, J = 5.2 Hz, 1H), 0.90 (s, 9H), 0.71 (s, 9H), 0.11 (d, J = 10.5 Hz, 6H), -0.08 (s, 3H), -0.30 (s,
3H). 13C NMR (126 MHz, DMSO-d6) δ 157.14, 154.13, 151.98, 136.09, 117.25, 86.26, 83.39,
74.49, 73.12, 68.50, 40.46, 40.30, 40.13, 39.96, 39.80, 39.63, 39.46, 26.16, 25.93, 18.19, 17.97, -
4.28, -4.30, -4.40, -5.09. HRMS (ESI-TOF) m/z calcd for C22H43N6O7SSi2 [M + H]+ 591.2452
found 591.2460.
tert-butyl-4-(((((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-bis((tert-
butyldimethylsilyl)oxy)tetrahydrofuran-2-yl)methoxy)sulfonyl)amino)-4-oxobutanoate (9).
To a stirring solution of 4-(tert-butoxy)-4-oxobutanoic acid (693 mg, 3.0 mmol, 1 equiv) in

ACCEPTED MANUSCRIPT
DCM (5.0 mL), DIPEA (1.04 mL, 6.0 mmol, 2 equiv), and N-hydroxysuccinimide (379.5 mg,
3.3 mmol, 1.1 equiv) was added HATU (1.25 g, 3.3 mmol, 1.1 equiv). The resulting solution was
stirred under argon at 25 °C for 4 h. Upon completion of reaction, sat. brine (20 mL) was added
and the aqueous layer was extracted with DCM. The combined DCM layers were dried over
anhydrous sodium sulfate and concentrated under vacuum. The flask was left under high vacuum
for 2 h and crude 8 was used without further purification. 8 (1.7 g, 3.0 mmol, 1.5 equiv) and
compound 7 (1.8 g, 2.0 mmol, 1.0 equiv) was re-dissolved in dry DMF (5 mL) and was cooled at
0 ºC. DBU (492 µL, 3.3 mmol, 1.1 equiv) was added and the reaction mixture was stirred at 25
°C overnight. Next, the reaction mixture was dried under high vacuum and purified by silica gel
column chromatography using DCM/methanol (0−20% methanol) to yield 9 as a white solid
1
(939 mg, 42% yield). H NMR (500 MHz, DMSO-d6) δ 7.92 (d, J = 9.5 Hz, 1H), 7.76 (s, 1H),
6.49 (s, 2H), 5.75 (d, J = 7.4 Hz, 1H), 4.77 (dd, J = 7.2, 4.7 Hz, 1H), 4.54–4.42 (m, 2H), 4.40–
4.32 (m, 1H), 4.21 (dd, J = 21.1, 4.7 Hz, 2H), 4.13 (s, 1H), 2.49–2.40 (m, 3H), 1.38 (d, J = 5.8
Hz, 9H), 0.91 (s, 9H), 0.72 (s, 9H), 0.11 (s, 7H), -0.07 (s, 3H), -0.30 (s, 3H). 13C NMR (126
MHz, DMSO-d6) δ 171.87, 171.56, 157.19, 154.13, 152.00, 136.10, 117.17, 92.79, 86.18, 83.29,
82.85, 80.28, 77.40, 74.47, 73.20, 72.14, 70.95, 53.56, 40.48, 40.31, 40.15, 39.98, 39.81, 39.65,
39.48, 31.39, 29.78, 28.22, 28.14, 26.21, 26.15, 25.92, 25.67, 18.30, 18.19, 18.11, 17.95, -4.00, -
4.23, -4.32, -4.41, -4.45, -4.51, -5.17. HRMS (ESI-TOF) m/z calcd for C30H54N6O10SSi2 [M +
H]+ 747.3239 found 747.3232.
4-(((((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-bis((tert-butyl-
dimethylsilyl)oxy)tetrahydrofuran-2-yl)methoxy)sulfonyl)amino)-4-oxobutanoic acid (10).
Compound 9 (185 mg, 0.26 mmol) was dissolved in DCM/TFA/Et3SiH (2:1:0.1, 2 mL) and
stirred for 2 h. The solution was concentrated to dryness and left on high vacuum for 2 h. It was
used directly for next reaction (90% yield). HRMS (ESI-TOF) m/z calcd for C26H47N6O10SSi2
[M + H]+ 691.2613 found 691.2610.
((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-bis((tert-butyldimethyl-
silyl)oxy)tetrahydrofuran-2-yl)methyl(4-((6-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-
yl)amino)-6-oxohexyl)amino)-4-oxobutanoyl)sulfamate (11). To a stirring solution of 10 (100
mg, 0.26 mmol, 1 equiv), 3 (185 mg, 0.26 mmol, 1 equiv) and NMM (289 µL, 2.6 mmol, 10

ACCEPTED MANUSCRIPT
equiv) in DMSO/water at pH 7.0 (6 mL, 1:1) was added EDC.HCl (248.3 mg, 1.3 mmol, 5
equiv). The resultant solution was stirred overnight at 25 °C. The reaction mixture was diluted
with water and purified by semi-preparative HPLC (20−80% acetonitrile in water) to yield 11 as
1
a white solid (112 mg, 40%). H NMR (500 MHz, DMSO-d6) δ 10.99 (s, 1H), 10.67 (s, 1H),
9.73 (s, 1H), 7.89 (s, 1H), 7.85 (q, J = 5.6 Hz, 1H), 7.81–7.78 (m, 1H), 7.51–7.41 (m, 2H), 6.43
(s, 2H), 5.73 (d, J = 7.5 Hz, 1H), 5.12 (dd, J = 13.3, 5.1 Hz, 1H), 4.73 (dd, J = 7.4, 4.5 Hz, 1H),
4.50 (dd, J = 10.9, 5.3 Hz, 1H), 4.40 (dd, J = 10.7, 4.7 Hz, 2H), 4.37–4.27 (m, 2H), 4.21 (d, J =
4.7 Hz, 1H), 4.11 (t, J = 5.2 Hz, 1H), 3.03 (d, J = 6.6 Hz, 2H), 2.91 (dd, J = 13.5, 5.3 Hz, 2H),
2.58 (d, J = 16.8 Hz, 1H), 2.50–2.46 (m, 3H), 2.33 (q, J = 7.1, 6.6 Hz, 5H), 2.03–1.96 (m, 1H),
1.57 (p, J = 7.5 Hz, 2H), 1.43–1.35 (m, 2H), 1.32–1.24 (m, 2H), 0.88 (s, 9H), 0.69 (s, 9H), 0.09
(d, J = 9.9 Hz, 6H), -0.09 (s, 3H), -0.32 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 173.28,
171.72, 171.56, 171.49, 170.84, 168.27, 157.14, 154.15, 152.00, 135.99, 134.25, 134.08, 133.10,
129.02, 125.64, 119.39, 117.18, 86.12, 83.12, 74.46, 73.11, 71.35, 51.98, 46.93, 40.87, 40.46,
40.29, 40.13, 39.96, 39.79, 39.62, 39.46, 38.99, 36.20, 31.66, 31.36, 29.64, 29.36, 26.59, 26.20,
26.14, 25.90, 25.28, 23.09, 18.16, 17.94, -4.32, -4.35, -4.46, -5.19. HRMS (ESI-TOF) m/z calcd
for C45H68N10O13SSi2 [M + H]+ 1045.4305 found 1045.4295.
((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydro-
furan-2-yl)methyl(4-((6-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)-6-oxo
hexyl)amino)-4-oxobutanoyl)sulfamate (12). To an ice-cold stirring solution of 11 (60 mg,
0.06 mmol, 1 equiv) in THF (3.0 mL) was added 1M TBAF (171 µL, 0.2 mmol, 3 equiv). The
resultant solution was warmed to 25 °C and stirring was continued for 5 min. The reaction was
diluted with methanol and concentrated under vacuum. The crude residue was purified by silica
gel chromatography (20% methanol in DCM) followed by further purification via semi-
1
preparative HPLC (20−80% acetonitrile in water) to yield 12 as white solid (35 mg, 71%). H
NMR (500 MHz, DMSO-d6) δ 11.00 (s, 1H), 9.82 (s, 2H), 8.36 (s, 2H), 7.90 (s, 1H), 7.86–7.75
(m, 3H), 7.46 (d, J = 6.8 Hz, 2H), 6.82 (s, 2H), 5.68 (d, J = 6.5 Hz, 1H), 5.14–5.06 (m, 1H),
4.50–4.29 (m, 5H), 4.12–3.90 (m, 5H), 3.01 (d, J = 6.5 Hz, 3H), 2.93–2.83 (m, 2H), 2.64–2.56
(m, 1H), 2.48 (s, 2H), 2.36 (dt, J = 22.2, 5.9 Hz, 4H), 2.04–1.96 (m, 1H), 1.55 (s, 9H), 1.52 (d, J
13
= 7.7 Hz, 8H), 1.44–1.35 (m, 2H). C NMR (126 MHz, DMSO-d6) δ 177.14, 173.28, 172.49,
171.90, 171.46, 168.29, 165.79, 157.42, 154.49, 151.97, 135.87, 134.35, 134.11, 133.09, 128.96,

ACCEPTED MANUSCRIPT
125.60, 119.24, 116.92, 86.69, 83.14, 73.87, 71.40, 67.52, 57.99, 52.06, 47.07, 38.79, 36.22,
35.32, 32.64, 31.66, 29.36, 26.53, 25.32, 23.05. HRMS (ESI-TOF) m/z calcd for C33H41N10O13S
[M + H]+ 817.2575 found 817.2558.
2-Amino-7-benzyl-9-((2R,3R,4S,5R)-5-(((N-(4-((6-((2-(2,6-dioxopiperidin-3-yl)-1-oxoiso
indolin-4-yl)amino)-6-oxohexyl)amino)-4-oxobutanoyl)sulfamoyl)oxy)methyl)-3,4-di-
hydroxytetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-7-ium (13a). General procedure
1
D, 45%. H NMR (500 MHz, DMSO-d6) δ 10.99 (s, 1H), 10.12 (d, J = 66.0 Hz, 1H), 9.73 (s,
1H), 7.85–7.21 (m, 9H), 5.85 (d, J = 25.5 Hz, 1H), 5.61 (t, J = 13.1 Hz, 3H), 5.47 (s, 1H), 5.11
(d, J = 12.5 Hz, 2H), 4.64–3.75 (m, 11H), 3.08–2.83 (m, 2H), 2.59 (d, J = 16.3 Hz, 1H), 2.41–
2.19 (m, 6H), 2.04–1.89 (m, 1H), 1.63–1.19 (m, 7H). HRMS (ESI-TOF) m/z calcd for
C40H45N10O13S [M - H]- 905.2883 found 905.2874.
2-Amino-9-((2R,3R,4S,5R)-5-(((N-(4-((6-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-
yl)amino)-6-oxohexyl)amino)-4-oxobutanoyl)sulfamoyl)oxy)methyl)-3,4-dihydroxytetra-
hydrofuran-2-yl)-7-(4-fluorobenzyl)-6-oxo-6,9-dihydro-1H-purin-7-ium (13b). General
procedure D, 55%. 1H NMR (500 MHz, DMSO-d6) δ 11.01 (s, 1H), 10.18 (s, 1H), 9.75 (s, 2H),
8.16 (s, 1H), 7.84–7.80 (m, 1H), 7.78–7.69 (m, 4H), 7.50 (d, J = 6.4 Hz, 2H), 7.17 (t, J = 8.8 Hz,
3H), 5.83 (s, 1H), 5.71–5.52 (m, 3H), 5.13 (d, J = 18.4 Hz, 2H), 4.43–4.30 (m, 4H), 4.21 (s, 1H),
4.12 (d, J = 17.4 Hz, 2H), 4.02 (d, J = 10.9 Hz, 1H), 3.19–3.12 (m, 2H), 2.94 (d, J = 35.4 Hz,
3H), 2.61 (d, J = 18.0 Hz, 2H), 2.41–2.20 (m, 9H), 2.10–1.98 (m, 2H). 19F NMR (470 MHz,
DMSO-d6) δ -148.28, -148.33. HRMS (ESI-TOF) m/z calcd for C40H46FN10O13S [M + H]+
925.2945 found 925.2951.
tert-Butyl(2-(2-(2-(3-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)-3-oxo-
propoxy)ethoxy)ethoxy)ethyl)carbamate (15). General procedure A, 56% yield. 1H NMR (500
MHz, DMSO-d6) δ 11.01 (s, 1H), 9.82 (s, 1H), 7.81 (d, J = 8.6 Hz, 1H), 7.50 (d, J = 10.3 Hz,
2H), 6.72 (t, J = 5.3 Hz, 1H), 5.15–5.08 (m, 1H), 4.44–4.27 (m, 2H), 3.70 (t, J = 6.3 Hz, 2H),
3.53–3.46 (m, 6H), 3.46–3.42 (m, 2H), 3.34 (s, 6H), 3.03 (q, J = 5.9 Hz, 2H), 2.59 (d, J = 6.3 Hz,
2H), 1.35 (s, 9H). 13C NMR (126 MHz, DMSO-d6) δ 173.28, 171.49, 169.78, 168.25, 156.01,
134.09, 133.11, 129.09, 125.63, 119.53, 78.03, 70.15, 70.08, 69.93, 69.59, 67.04, 51.97, 46.88,

ACCEPTED MANUSCRIPT
40.46, 40.30, 40.30, 40.13, 39.96, 39.80, 39.63, 39.46, 37.04, 31.65, 28.67, 23.12. HRMS (ESI-
TOF) m/z calcd for C27H39N4O9 [M + H]+ 563.2717, found 563.2708.
tert-Butyl(24-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)-24-oxo-3,6,9,12,15,
1
18,21-heptaoxatetracosyl)carbamate (16). General procedure A, 18% yield. H NMR (500
MHz, D2O) δ 7.61 (t, J = 8.5 Hz, 1H), 7.50 (dt, J = 15.2, 7.4 Hz, 2H), 5.05 (dd, J = 13.3, 5.3 Hz,
1H), 4.44–4.28 (m, 2H), 3.75 (t, J = 5.8 Hz, 2H), 3.65–3.35 (m, 26H), 3.10 (t, J = 5.3 Hz, 2H),
2.90–2.70 (m, 3H), 2.62 (t, J = 5.8 Hz, 2H), 2.48–2.32 (m, 2H), 2.24–2.09 (m, 2H), 1.27 (s, 9H).
13C NMR (126 MHz, D2O) δ 175.70, 172.89, 172.22, 170.72, 158.07, 136.24, 131.84, 131.64,
129.52, 128.42, 121.59, 69.51, 69.46, 69.32, 66.57, 52.39, 47.58, 39.57, 36.11, 30.72, 27.60,
22.13. HRMS (ESI-TOF) m/z calcd for C35H54N4O13 [M + K]+ 777.3354, found 777.3316.
3-(2-(2-(2Aaminoethoxy)ethoxy)ethoxy)-N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-
yl)propenamide (18). General procedure B, 100%. HRMS (ESI-TOF) m/z calcd for
C22H31N4O7 [M + H]+ 463.2193, found 463.2177.
1-Amino-N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)-3,6,9,12,15,18,21-heptaoxa
tetracosan-24-amide (19). General procedure B, 100%. HRMS (ESI-TOF) m/z calcd for
C30H46N4O11 [M + H]+ 639.3241, found 639.3239.
6-(((((((2S,3R,4S,5S)-5-(2-Amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetra-
hydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(l1-oxidaneyl)phosphoryl)amino)-N-
(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)hexanamide (20). General procedure C, 35%
yield. 1H NMR (500 MHz, DMSO-d6) δ 11.00 (s, 1H), 10.85 (s, 1H), 10.01 (s, 1H), 7.95 (s, 2H),
7.82 (d, J = 6.5 Hz, 1H), 7.50–7.41 (m, 2H), 5.69 (d, J = 5.8 Hz, 1H), 5.10 (dd, J = 18.8, 6.4 Hz,
1H), 4.54–4.47 (m, 1H), 4.43 (s, 1H), 4.41–4.38 (m, 1H), 4.35 (s, 1H), 4.31 (s, 1H), 4.19 (s, 1H),
4.01 (d, J = 16.1 Hz, 3H), 3.02 (s, 2H), 3.00–2.92 (m, 5H), 2.41–2.29 (m, 3H), 2.03–1.94 (m,
31
1H), 1.60–1.53 (m, 3H), 1.43 (s, 1H), 1.36–1.24 (m, 3H). P NMR (202 MHz, DMSO-d6) δ -
2.48 (d, J = 20.7 Hz), -10.98 (d, J = 20.8 Hz). HRMS (ESI-TOF) m/z calcd for C29H36N9O14P2
[M - H]- 796.1857, found 796.1867.

ACCEPTED MANUSCRIPT
3-(2-(2-(2-(((((((2S,3R,4S,5S)-5-(2-Amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-
dihydroxytetrahydrofuran-2-yl)methoxy)(l1-oxidaneyl)phosphoryl)oxy)(hydroxy)
phosphoryl)amino)ethoxy)ethoxy)ethoxy)-N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-
yl)propenamide (21). General procedure C, 32% yield. 1H NMR (500 MHz, D2O) δ 9.03 (d, J =
10.8 Hz, 1H), 7.67–7.50 (m, 3H), 5.97 (d, J = 31.4 Hz, 2H), 5.12 (d, J = 13.3 Hz, 2H), 4.61 (d, J
= 10.6 Hz, 1H), 4.45 (d, J = 17.1 Hz, 2H), 4.37 (d, J = 17.4 Hz, 3H), 4.23 (d, J = 10.8 Hz, 1H),
3.84 (t, J = 5.7 Hz, 2H), 3.73–3.57 (m, 10H), 3.15 (t, J = 6.4 Hz, 4H), 3.12–3.06 (m, 5H), 2.72 (t,
J = 5.7 Hz, 2H), 1.89–1.77 (m, 4H). 13C NMR (126 MHz, D2O) δ 175.67, 172.24, 170.62,
162.49, 160.43, 155.22, 154.75, 135.97, 131.75, 129.39, 128.14, 121.36, 119.64, 117.32, 115.00,
89.82, 83.67, 74.67, 74.50, 69.36, 66.52, 66.22, 55.24, 52.36, 47.49, 42.65, 38.95, 36.34, 34.88,
31
30.69, 24.88, 22.07; P NMR (202 MHz, D2O) δ -0.15, -0.19, -11.11, -11.21, -11.77, -23.59, -
23.65. HRMS (ESI-TOF) m/z calcd for C32H42N9O17P2 [M + NH4]+ 886.2174, found 886.2175.
1-(((((((2S,3R,4S,5S)-5-(2-Amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetra
hydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(l1-oxidaneyl)phosphoryl)amino)-N-
(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)-3,6,9,12,15,18,21-heptaoxatetracosan-24-
1
amide (22). General procedure C, 35% yield. H NMR (500 MHz, DMSO-d6) δ 11.01 (s, 1H),
10.56 (s, 2H), 10.06 (s, 1H), 9.89 (s, 1H), 7.95–7.86 (m, 2H), 7.83 (d, J = 7.2 Hz, 1H), 7.76 (s,
1H), 7.45 (dd, J = 18.2, 5.7 Hz, 2H), 7.21 (s, 1H), 6.61 (d, J = 45.8 Hz, 4H), 5.96 (s, 3H), 5.65
(d, J = 5.4 Hz, 1H), 5.12 (dd, J = 13.3, 5.0 Hz, 1H), 4.78–4.69 (m, 1H), 4.65–4.56 (m, 2H), 4.39
(ddd, J = 45.1, 25.5, 10.9 Hz, 4H), 4.24 (s, 1H), 4.02–3.89 (m, 3H), 3.73–3.54 (m, 5H), 3.13 (s,
2H), 3.03–2.93 (m, 3H), 2.91 (s, 1H), 2.87 (s, 2H), 2.71 (s, 1H), 2.62 (d, J = 5.0 Hz, 3H), 2.52 (s,
2H), 2.32 (d, J = 25.4 Hz, 5H), 2.06 (d, J = 6.2 Hz, 2H), 1.86 (s, 2H), 1.59–1.50 (m, 2H), 1.09 (d,
J = 42.2 Hz, 3H). 31P NMR (202 MHz, DMSO-d6) δ -1.69, -10.05. HRMS (ESI-TOF) m/z calcd
for C40H58N9O21P2 [M - H]- 1062.3222, found 1062.3217.
2-Amino-7-benzyl-9-((2S,3S,4R,5S)-5-(((((((6-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-
4-yl)amino)-6-oxohexyl)amino)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)
methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-7-ium
(23a).
General procedure D, 16% yield. HRMS (ESI-TOF) m/z calcd for C36H42N9O14P2 [M - H]-
886.2321, found 886.2361.

ACCEPTED MANUSCRIPT
2-Amino-9-((2S,3S,4R,5S)-5-(((((((6-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)
amino)-6-oxohexyl)amino)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-3,4-
dihydroxytetrahydrofuran-2-yl)-7-(4-fluorobenzyl)-6-oxo-6,9-dihydro-1H-purin-7-ium
(23b). General procedure D, 11% yield. HRMS (ESI-TOF) m/z calcd for C36H41FN9O14P2 [M -
H]- 904.2227, found 904.2212.
2-Amino-7-benzyl-9-((2S,3S,4R,5S)-5-(((((((2-(2-(2-(3-((2-(2,6-dioxopiperidin-3-yl)-1-oxo
isoindolin-4-yl)amino)-3-oxopropoxy)ethoxy)ethoxy)ethyl)amino)(hydroxy)phosphoryl)
oxy)(hydroxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-6-oxo-6,9-
dihydro-1H-purin-7-ium (24a). General procedure D, 20% yield. HRMS (ESI-TOF) m/z calcd
for C39H48N9O17P2 [M - H]- 976.2638, found 976.2677.
2-Amino-9-((2S,3S,4R,5S)-5-(((((((2-(2-(2-(3-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-
yl)amino)-3-oxopropoxy)ethoxy)ethoxy)ethyl)amino)(hydroxy)phosphoryl)oxy)(hydroxy)
phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-7-(4-fluorobenzyl)-6-oxo-6,9-
dihydro-1H-purin-7-ium (24b). General procedure D, 11% yield). HRMS (ESI-TOF) m/z
calcd for C39H47FN9O17P2 [M - H]- 994.2544, found 994.2543.
2-Amino-7-benzyl-9-((2R,3R,4S,5R)-5-(((((((24-((2-(2,6-dioxopiperidin-3-yl)-1-oxo
isoindolin-4-yl)amino)-24-oxo-3,6,9,12,15,18,21-heptaoxatetracosyl)amino)(hydroxy)
phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-6-
oxo-6,9-dihydro-1H-purin-7-ium (25a). General procedure D, 13% yield. HRMS (ESI-TOF)
m/z calcd for C47H64N9O21P2 [M - H]- 1152.3686, found 1152.3695.
2-Amino-9-((2R,3R,4S,5R)-5-(((((((24-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)
amino)-24-oxo-3,6,9,12,15,18,21-heptaoxatetracosyl)amino)(hydroxy)phosphoryl)oxy)
(hydroxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-7-(4-fluorobenzyl)-
6-oxo-6,9-dihydro-1H-purin-7-ium (25b). General procedure D, 9% yield. HRMS (ESI-TOF)
m/z calcd for C47H63FN9O21P2 [M - H]- 1170.3592, found 1170.3599.

ACCEPTED MANUSCRIPT
tert-Butyl(6-(((2S)-1-((4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)
pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-6-oxohexyl)carbamate (27). General
1
procedure A, 75% yield. H NMR (500 MHz, Chloroform-d) δ 8.60 (s, 1H), 7.88 (d, J = 36.1
Hz, 1H), 7.17 (s, 1H), 6.57–6.43 (m, 1H), 4.69–4.58 (m, 2H), 4.52–4.39 (m, 3H), 4.24 (dd, J =
15.1, 5.2 Hz, 2H), 4.03 (d, J = 11.1 Hz, 1H), 3.62–3.48 (m, 1H), 3.01–2.91 (m, 2H), 2.86 (s, 1H),
2.79 (s, 1H), 2.42 (s, 3H), 2.39–2.20 (m, 2H), 2.10 (dd, J = 13.6, 7.3 Hz, 3H), 1.56–1.42 (m, 2H),
13
1.31 (s, 9H), 1.17 (d, J = 11.7 Hz, 3H), 0.85 (s, 9H). C NMR (126 MHz, Chloroform-d) δ
174.67, 174.11, 171.92, 170.91, 162.69, 156.06, 150.43, 148.27, 138.45, 138.12, 131.70, 130.79,
129.45, 128.02, 79.16, 77.27, 77.01, 76.76, 70.00, 58.89, 57.52, 56.97, 43.17, 40.33, 36.10,
34.94, 31.49, 29.57, 28.40, 26.39, 26.10, 25.16, 20.81, 15.91. HRMS (ESI-TOF) m/z calcd for
C33H49N5O6S [M + H]+ 644.3482, found 644.3476.
(4R)-1-((S)-2-(6-Aminohexanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methyl-
1
thiazol-5-yl)benzyl) pyrrolidine-2-carboxamide (28). General procedure B, 90%. H NMR
(500 MHz, DMSO-d6) δ 8.97 (s, 1H), 8.61 (s, 1H), 8.44 (s, 1H), 7.89 (d, J = 9.2 Hz, 1H), 7.45–
7.32 (m, 5H), 4.53 (d, J = 9.3 Hz, 1H), 4.48–4.38 (m, 3H), 4.34 (s, 2H), 4.21 (dd, J = 15.8, 5.2
Hz, 2H), 3.65 (q, J = 12.2, 11.5 Hz, 3H), 2.72 (t, J = 7.4 Hz, 3H), 2.25 (dd, J = 14.1, 7.3 Hz, 2H),
2.12 (dt, J = 14.2, 7.1 Hz, 2H), 2.07–1.99 (m, 1H), 1.97–1.81 (m, 2H), 1.49 (dd, J = 17.0, 7.7 Hz,
6H), 1.27 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 172.41, 170.13, 166.85, 151.88, 148.14,
139.95, 131.61, 130.07, 129.30, 129.07, 128.52, 127.85, 69.31, 59.15, 56.81, 56.78, 42.10, 40.43,
40.35, 40.26, 40.09, 39.93, 39.76, 39.59, 39.43, 39.18, 38.42, 35.66, 35.27, 35.11, 27.60, 27.59,
26.83, 26.03, 25.90, 25.52, 25.39, 16.38. HRMS (ESI-TOF) m/z calcd for C28H41N5O4 [M + H]+
544.2958, found 544.2939.
(4S)-1-((2S)-2-(6-(((((((2S,3R,4S,5S)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-
dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(λ1-oxidaneyl)
phosphoryl)amino)hexanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-
1
yl)benzyl)pyrrolidine-2-carboxamide (29). General procedure C, 32% yield. H NMR (500
MHz, DMSO-d6) δ 9.10 (s, 1H), 8.97 (d, J = 4.3 Hz, 2H), 8.59–8.53 (m, 1H), 7.87 (d, J = 9.2 Hz,
2H), 7.48–7.29 (m, 7H), 6.61 (d, J = 52.9 Hz, 2H), 5.68 (d, J = 4.8 Hz, 1H), 4.58–4.15 (m, 9H),
3.69–3.57 (m, 3H), 3.49–3.23 (m, 8H), 2.43 (d, J = 3.0 Hz, 4H), 2.32–2.17 (m, 2H), 2.17–2.07

ACCEPTED MANUSCRIPT
(m, 1H), 2.05 (s, 1H), 1.95–1.83 (m, 2H), 1.59–1.41 (m, 4H), 1.32–1.20 (m, 2H). 13C NMR (126
MHz, DMSO-d6) δ 172.38, 170.13, 169.28, 152.02, 151.89, 148.33, 148.15, 139.95, 138.97,
131.60, 131.42, 130.63, 130.07, 129.40, 129.07, 128.25, 127.86, 69.87, 69.31, 59.15, 58.69,
56.79, 54.11, 42.51, 42.09, 40.46, 40.29, 40.12, 39.96, 39.79, 39.62, 39.46, 38.42, 35.68, 35.10,
27.24, 26.84, 26.00, 25.36, 16.40. HRMS (ESI-TOF) m/z calcd for C38H53N10O14P2S [M+H]+
969.3095, found 969.3085.
2-Amino-7-benzyl-9-((2S,3S,4R,5S)-3,4-dihydroxy-5-(((hydroxy((hydroxy((6-(((2S)-1-((4S)-
4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-
oxobutan-2-yl)amino)-6-oxohexyl)amino)phosphoryl)oxy)phosphoryl)oxy)methyl)tetra-
hydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-7-ium (30a). General procedure D, 11% yield.
HRMS (ESI-TOF) m/z calcd for C45H59N10O14P2S [M - H]- 1057.3403, found 1057.3411
2-Amino-9-((2S,3S,4R,5S)-3,4-dihydroxy-5-(((hydroxy((hydroxy((6-(((2S)-1-((4S)-4-
hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-
oxobutan-2-yl)amino)-6-oxohexyl)amino)phosphoryl)oxy)phosphoryl)oxy)methyl)tetra-
hydrofuran-2-yl)-7-(4-fluorobenzyl)-6-oxo-6,9-dihydro-1H-purin-7-ium (30b). General
procedure D, 41% yield. HRMS (ESI-TOF) m/z calcd for C45H59FN10O14P2S [M - H]-
1075.3308, found 1075.3315.
((2R,3S,4R,5R)-5-(2-Amino-7-methyl-6-oxo-1,6-dihydro-9H-purin-7-ium-9-yl)-3,4-
dihydroxytetrahydrofuran-2-yl)methyl phosphate (m7GMP). General procedure D, 75%
1
yield. H NMR (500 MHz, D2O) δ 9.12 (s, 1H), 6.06 (d, J = 3.4 Hz, 1H), 4.67–4.63 (m, 1H),
4.45 (t, J = 5.2 Hz, 1H), 4.41–4.37 (m, 1H), 4.28–4.21 (m, 1H), 4.14–4.11 (m, 1H), 4.10 (s, 3H).
13C NMR (126 MHz, D2O) δ 155.49, 154.99, 149.47, 136.43, 108.39, 89.67, 83.99, 74.67, 69.14,
63.28, 35.87. 31P NMR (202 MHz, D2O) δ 0.12. HRMS (ESI-TOF) m/z calcd for C11H15N5O8P
[M - H]- 376.0664, found 376.0669.
4.7 Preparation of m7GxP Agarose Resin
The synthesis was adapted from that of Edery et al. [52]. m7GMP (synthesized; see below) or
m7GDP (Sigma) sodium salts was dissolved in water (10 mM final), and a solution of sodium

ACCEPTED MANUSCRIPT
periodate (1.0 equiv) in sodium acetate buffer (0.1 M, pH 6; 51 mM final) was added. The
resulting mixture was agitated at 25 °C for 30 min protected from light. Adipic acid dihydrazide
agarose (1 mL packed; Sigma) was washed with water (1× 20 mL) followed by sodium acetate
buffer (1× 20mL), and then resuspended in sodium acetate buffer (2 mL). To this slurry was
added aniline (20 equiv) and the oxidized m7GxP solution. The resin mixture was then shaken at
25 °C for 45 min before adding sodium cyanoborohydride (16 equiv). The mixture was agitated
overnight at 4 °C. The resin was then washed with NaCl (1M; 5× 5 mL), equilibrated in buffer A
(5 mL; 50 mM HEPES, pH 7, 200 mM KCl), and stored at 4 °C.
4.8 Cell Culture
HEK293 cells were grown in DMEM supplemented with 10% FBS and 1 mM Glutamine. MDA-
MB-231 cells were grown in RPMI-1640 supplemented with 10% FBS and 1 mM Glutamine.
K562 cells were grown in Iscove's Modified Dulbecco's Medium supplemented with 10% FBS
and 1 mM Glutamine. Cells were grown at 37 °C with 5% CO2 in a humidified incubator. All
cell lines were authenticated by STR profiling and regularly tested for mycoplasma
contamination
4.9 m7G Cap Competition Assay and Western Blot
The cap competition assay was carried out as previously described (Yanagiya et al., 2012).
Briefly, HEK293 cells were lysed in cap pull-down buffer (50 mM HEPES-KOH pH 7.5, 150
mM KCl, 1 mM EDTA, 2 mM DTT and 0.1% Tween 20) containing protease inhibitors and
freeze-thawed thrice. Cell lysate was centrifuged at 20,000 × g for 25 min. Total protein in the
supernatant was measured using the BCA reagent (Thermo Scientific). 350 µg of total protein
from the cell lysate was then incubated for 2 h at 4 °C with m7GMP or m7GDP agarose resin.
The resin was washed 3× with cap pull-down buffer followed by 1× Tris Buffered Saline (TBS)
and 1× Buffer A (50 mM HEPES-KOH pH 7.5, 150 mM KCl). The resin was then incubated
with compounds (in Buffer A) overnight. The resin was again washed 1× with cap pull down
buffer followed by 1×TBS and 1× water. Proteins were then eluted from the resin by boiling in
2× LDS sample buffer (10 min at 70 °C) resolved on a 4−12% Bis-Tris gel, and transferred to
PVDF membrane in Towbin’s Buffer. The membrane was blocked in 5% milk for 1 h at 25 °C,

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and then incubated with a primary antibody (overnight at 4 °C) and secondary antibody (1 h at
25 °C). Proteins were visualized by autoradiography. Anti-eIF4E antibody was purchased from
Cell Signaling Technology (cat# 9742). Figures were formatted in Adobe Illustrator.
ACKNOWLEDGMENTS:
We would like to thank Sydney Rosenblum for giving creative inputs while preparing the
manuscript. This work was supported by the NIH (R01 CA202018 to A.L.G).
COMPETING INTERESTS:
The authors declare no competing interests.
SUPPORTING INFORMATION:
Spectra for all synthesized compounds.
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