Synthesis of (R)- and (S)-Fmoc-Protected Diethylene Glycol Gamma PNA Monomers with High Optical Purity

Article abstract of DOI:10.1021/acs.joc.8b02714

A robust synthetic route has been developed for preparing optically pure, Fmoc-protected diethylene glycol-containing (R)- and (S)-γPNA monomers. The strategy involves the application of 9-(4-bromophenyl)-9-fluorenyl as a temporary, safety-catch protecting group for the suppression of epimerization in the O-alkylation and reductive amination steps. The optical purities of the final monomers were determined to be greater than 99.5% ee, as assessed by ¹⁹F-NMR and HPLC. The new synthetic methodology is well-suited for large-scale monomer production, with most synthetic steps providing excellent chemical yields without the need for chromatographic purification other than a simple workup and precipitation.

Full text of DOI:10.1021/acs.joc.8b02714

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Article
Synthesis of (R)- and (S)-Fmoc-Protected Diethylene
Glycol Gamma PNA Monomers with High Optical Purity
Wei-Che Hsieh, Ashif Yasin Shaikh, J. Dinithi R. Perera, Shivaji Ashokrao Thadke, and Danith H. Ly
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b02714 • Publication Date (Web): 04 Jan 2019
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Synthesis of (R)- and (S)-Fmoc-Protected Diethylene Glycol
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Gamma PNA Monomers with High Optical Purity
Wei-Che Hsieh, Ashif Y. Shaikh, J. Dinithi R. Perera, Shivaji A. Thadke, and Danith H. Ly*
Institute for Biomolecular Design and Discovery (IBD) and Department of Chemistry,
Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213 (U.S.A).
D.H.L: 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213; 412-268-4010;
dly@andrew.cmu.edu
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Abstract
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A robust synthetic route has been developed for preparing optically-pure, Fmoc-protected
diethylene glycol-containing (R)- and (S)-PNA monomers. The strategy involves the application
of 9-(4-bromophenyl)-9-fluorenyl as a temporary, safety-catch protecting-group for the
suppression of epimerization in the O-alkylation and reductive amination steps. The optical
purities of the final monomers were determined to be greater than 99.5% ee, as assessed by
19F-NMR and HPLC. The new synthetic methodology is well-suited for large-scale monomer
production, with most synthetic steps providing excellent chemical yields without the need for
chromatographic purification other than a simple workup and precipitation.
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Introduction
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Synthetic oligonucleotides are valuable as molecular tools and reagents for biology,
biotechnology, and medicine, as well as for molecular engineering and computation.1-4
Paramount to the success of many of these applications is a reliance on their ability to
recognize and bind the designated DNA or RNA targets, or their obligatory partners with high
affinity and sequence-specificity. Other desirable biophysical attributes, e.g. cellular
transduction, biodistribution, metabolism, and clearance may be necessary for intracellular and
in vivo applications; however, they are not required for in vitro experiments. It is therefore
essential to be able to modify the structures and chemical functionalities of these synthetic
oligonucleotide molecules with ease, so that they can be tailor-made to meet the specific
requirements in hand, since, in general, there is no one particular nucleic acid system or
chemical outfit that can fulfill all the application requirements.
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A particular class of nucleic acid mimic that is endowed with synthetic flexibility is peptide
nucleic acid (PNA). Developed by Nielsen and coworkers5 in the early 1990’s, PNA comprises
an entirely different backbone skeleton from that of DNA or RNA, made up of N-(2-aminoethyl)
glycine instead of the usual sugar phosphodiester units. PNA has several appealing features,
including tight and sequence-specific binding of DNA or RNA, and resistance to proteolytic and
nucleolytic degradation.6 Furthermore, the acyclic and achiral backbone provides a convenient
means for installing new chemical groups or for making structural modifications. However, PNA
is not without shortcomings. The charge-neutral polyamide backbone that confers PNA with
many of its attractive features renders it less soluble in water than the natural counterparts. As a
consequence, PNA has a tendency to aggregate and adhere to surfaces and other
macromolecules in a non-specific manner.7,8 While this issue has been addressed to some
degree,9-11 further improvements in this and other areas, including synthetic methodology and
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recognition property, are warranted in order to expand the scope and utility of PNA in the
biological and biomedical arenas.
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A promising approach involves installation of a chiral center at the gamma backbone.12-20 The
presence of a chiral center at this position has been shown to transform PNA from a random-
fold into either a right-handed (RH) or a left-handed (LH) helical motif depending on the
stereochemistry.21,22 The LH conformer is unable to hybridize to RH, or to DNA or RNA, due to
conformational mismatch. In contrast, due to the complementarity in conformation, the RH
conformer hybridizes to DNA or RNA with exquisite affinity and sequence-specificity, and is the
only class of oligonucleotide molecules developed to date that has been shown to be capable of
invading double helical B-form DNA (B-DNA) without sequence limitation.23 Although locked
nucleic acid (LNA) has been reported to be able to invade DNA, strand invasion requires
simultaneous binding of both strands of DNA double helix. Such a demand has practical
limitations.24 Furthermore, by incorporating a diethylene glycol (commonly referred to as
miniPEG, or MP) moiety at the gamma backbone, the water solubility and biocompatibility of
PNA can be significantly improved without the need to introduce charged groups,25 which can
compromise the binding affinity and/or specificity of the oligonucleotide molecule. These newly
endowed properties of PNA have been exploited in a number of applications including
biosensing,26-28 gene editing,29,30 and molecular engineering.22 The challenge with this
undertaking, however, is in the production of chemical building blocks in large scale and with
high optical purity, and ones that are compatible with automated solid-phase peptide synthesis
(SPPS). The former is essential, since the presence of even a trace amount of enantiomeric
impurity could have profound adverse effects on the conformation and hybridization property of
oligomer. The previously reported Boc-chemistry is not ideally-suited for automated SPPS due
to the strong acid (TFA) typically employed to remove Boc group, which is conducive to acid
corrosion and rapid deterioration of the instrument.25 Here we report the development of a
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robust method for scaling up the production of enantiomerically-pure Fmoc-protected (R)- and
(S)-MPPNA monomers that are suitable for automated synthesis from relatively inexpensive
starting materials (Chart 1).
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O
O
O
B
O
B
O
O
O
O
O
O
(R)
(S)
OH
FmocHN
N
N

FmocHN
OH


1a-d (RH)
2a-d (LH)
O
NHBoc
NHBoc
O
N
N
NH
N
N
NH
B =
T
C
N
A
N
G
N
N
O
O
N
N
NHBoc
a
b
d
c
Chart 1
Results and Discussion
Rationale. To date most chiral PNA monomers were prepared through the reductive amination
route, starting with Boc- or Fmoc-protected amino acids.31 Such a coupling reaction, however, is
known to produce configurationally unstable products due to the propensity of Boc- and Fmoc-
aminoaldehyde intermediates to undergo epimerization even under mild experimental
conditions.32-35 Previously, we were able to minimize the extent of epimerization at gamma()-
carbon by carrying out O-alkylation of the serine sidechain under a strict reaction condition and
by employing Mitsunobu reaction in the preparation of backbone (Scheme 1).25 We managed to
produce small quantities of Boc-protected backbone and the corresponding Boc-MPPNA
monomers for the initial proof-of-concept studies (Scheme 1a). However, it is daunting to scale
up monomer production via this synthetic route without compromising optical purity. One reason
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is that the alkylation step is performed at a relatively low temperature and for a short reaction
time to minimize the degree of epimerization at the expense of chemical yield, and another
reason is the difficulty in removing triphenylphosphine oxide byproduct in the Mitsunobu reaction
from the desired backbone (Scheme 1b). To circumvent the shortcomings of scalability and
optical purity, and the harsh reaction conditions employed in Boc-chemistry, we examined
several amino-protecting groups including trityl (Trt),36,37 fluorenylmethyloxycarbonyl (Fmoc),38
dibenzyl,39 9-phenylfluorenyl (PhF),40 and 9-(4-bromophenyl)-9-fluorenyl (BrPhF),41 along with
numerous synthetic routes. Among them we found BrPhF to be the most robust temporary
protecting group, and the synthetic route outlined in Scheme 2 and 4 to be the most facile in the
production of Fmoc-MPPNA monomers 1a-d and 2a-d with high optical purity (Chart 1). The
other protecting groups were not as successful due to several setbacks as outlined in Scheme
S1 and S2 (Supplemental Information).
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B (T, CZ, AZ, GZ)
(a) Previous work
RO
O
BocHN
HO
CO2H
6 steps
N
CO2H
BocHN
R = (CH2CH2O)2Me
(b) Critical reaction steps
Alkylation
BocHN
HO
CO2H
BocHN
CO2H
NaH, DMF,
Br(CH2CH2O)2Me,
0 oC, 1 h, 50-60%
RO
RO
Mitsunobu
BocHN
OH
N
CO2Et
NO2
DIAD, TPP, THF,
BocHN
S
o,p-diNBS-Gly-OEt
RO
O
O
58%
NO2
Scheme 1
Choice of PhF as a temporary protecting group. Previously we have shown that PhF, an
amino-protecting group originally developed by Lubell and Rapoport42 in the early 1980’s, was
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effective in shielding the α-center of amino acids against epimerization under highly basic
conditions.43 The protective capacity of PhF has been largely attributed to its sterically imposing
size and preference as a leaving group. The demanding size forces the α-proton of ester to be
in an unfavorable orientation for deprotonation. Under a harsh deprotonation condition, such as
treatment with base under refluxing condition, the reprotonation of α-anion is significantly slower
than the elimination of PhF. This preserves the stereochemistry of the intact PhF-protected
ester, whereas the eliminated byproduct can be readily removed with aqueous workup.42
However, we found PhF to be difficult to remove in the subsequent steps, requiring either a
strong acid or a prolonged hydrogenolysis. The former is incompatible with other protecting
groups, while the latter could lead to partial reduction of the cytosine nucleobase.44 To obviate
this challenge we adopted the halogenated derivative, namely BrPhF, as reported by
Surprenant and Lubell,45 which retains the full protection of PhF but that can be selectively
removed under a mild acidic condition following the insertion of an electron-donating group,
such as morpholine.
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The strategy for incorporating BrPhF is outlined in Scheme 2. The sidechain of L-serine 3 was
initially protected with tetrahydropyranyl acetal (THP) group, followed by N-alkylation with
BrPhF. We found THP protection,46 instead of the reported in situ trimethylsilylation,40 to be
necessary in providing high yield in the preparation of BrPhF-L-Ser(THP)-OMe 5. The
conversion of 5 to Weinreb amide 8 was performed in three steps: hydrolysis, amidation, and
THP removal, with 95% overall yield. Such a reaction sequence appears lengthy and could
potentially be replaced by a single transformation step by employing a Lewis acid, such as
Me2AlCl, and N,O-dimethylhydroxylamine hydrochloride;47 however, we found such a reaction to
be low yielding and not reproducible, making it difficult to scale up in comparison to those shown
in Scheme 2. O-alkylation of 8 with MP-sidechain followed by Buchwald-Hartwig amination
yielded the acid-labile MPhF-protected Weinreb amide 10. For optimization purpose, we
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screened several reagents and reaction conditions (Table S1, Supplemental Information), and
found Cs2CO3 and XPhos to be the best combination. Overall, compound 10 was prepared in
seven steps with greater than 56% overall yield, with several reaction steps carried out in nearly
quantitative yields without purification.
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O
O
1. DHP, PTSA,
DCM; 83% [4]
H N
2
BrPhFHN
HCl
OMe
OMe
2. [13], Pb(NO3)2
K3PO4, ACN; 96%
OH
OTHP
3
5
1. LiOH, THF/H2O; [6]
2. CH3ONHCH3 HCl,
DCC, DhBtOH,
= BrPhF
O
DMF, DCM; [7]
BrPhFHN
O
O
N
3. PTSA, MeOH
95% (3 steps)
OH
Br
8
O
= MPhF
1. [17], NaH, TBAI,
DMF; 77% [9]
MPhFHN
N
2. Pd(OAc)2, XPhos,
Cs2CO3, morpholine,
toluene; 96%
OR
10
N
O
Scheme 2
Preparation of BrPhF and MP-sidechain. The required BrPhFBr precursor 13 and tosylated
MP-sidechain 17 were prepared as shown in Scheme 3A and B, respectively. Treatment of 1,4-
dibromobenzene with 9-fluorenone in the presence of n-BuLi gave BrPhF-OH 12 in good yield.
Bromination of this compound with acetyl bromide in toluene provided 13 with quantitative
yield.48 We have also attempted the HBr aqueous condition as reported by Christie and
Rapoport,49 but we found it to be relatively inefficient due to the rapid solvolysis of the target
compound 13. The MP-sidechain precursor 17 was prepared from a commercially available
benzyl diethylene glycol in three steps: conversion of alcohol to t-butyl ether, removal of Bn
group, and tosylation. The first step was carried out in accordance with the protocol reported by
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Bartoli and Sambri,50 involving a simple treatment with magnesium perchlorate and Boc-
anhydride in DCM. Hydrogenation of 15 followed by tosylation yielded the desired MP-sidechain
precursor 17. This route is much simpler to install t-butyl ether than the conventional method,
which requires harsh conditions employing isobutylene in the presence of a strong acid
catalyst.51
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Br
(A)
1. i. n-BuLi, ether
ii. 9-Fluorenone;
87% [12]
Br
Br
2. AcBr, toluene,
Quant. yield
Br
11
13
(B)
(Boc)2O, Mg(ClO4)2, DCM; 71%
HO
O
O
O
BnO
BnO
14
15
1. H2, Pd/C, MeOH; [16]
O
2. TsCl, Et3N, DMAP, DCM
90% (2 steps)
O
TsO
17
Scheme 3
Synthesis of backbones and monomers. The backbone intermediates, both (R)- and (S)-
configuration, were prepared in the same way starting from L- and D-serine, respectively.
However, only preparation of the L-configuration is shown (Scheme 4). Conversion of Weinreb
amide 10 to an aldehyde followed by standard reductive amination52 yielded the desired
backbone 19 with high optical purity, as shown in the section below. Coupling the backbone with
Boc-protected carboxymethylnucleobases (T, CBoc, ABoc, and GBoc 44,53
yielded the respective
)
monomer esters 20a-d. Removal of MPhF was accomplished by treatment with citric acid
without affecting Boc, t-butyl ether, or allyl protecting group, as assessed by ESI-MS (Figure
S28 and S29). We have also assessed the dichloroacetic acid/triethylsilane condition as
reported by Surprenant and Lubell,45 but found that such a reaction condition led to the removal
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of both MPhF and Boc protecting groups. In situ Fmoc protection, followed by removal of the
allyl group under a neutral condition afforded the right-handed (R)-MPPNA monomers 1a-d.
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RO
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BCH2CO2H, HBTU,
DMF; 70-96%
1. LiAH, THF; [18]
MPhFHN
10
NH
O
2. PTSA NH2CH2CO2Allyl,
DIEA, NaBH(OAc)3, DCE;
82% (2 steps)
19
O
B
B
RO
MPhFHN
O
RO
O
1. HOC(CO2H)3,
ACN/H2O; [21a-d]
N
O
N
FmocHN
2. Fmoc-Cl,
NaHCO3, THF;
82-91% (2 steps)
O
O
O
20a-d
22a-d
B
t
RO
FmocHN
O
Pd(PPh3)4, PhSiH3,
DCM; 88-96%
R = (CH2CH2O)2 Bu
O
a, B = T; b, B = ABoc
;
N
c, B = CBoc; d, B = GBoc
OH
1a-d
Scheme 4
Assessment of optical purity. The optical purities of monomers were determined by 19F-NMR
and HPLC following the conversion of monomers 1a (RH) and 2a (LH) to the respective Mosher
derivatives 23 and 24. It is interesting to note that in this case 19F-NMR spectra showed a single
peak for compound 23 and a doublet for 24 (Figure 1). The extra peak in 24 is not due to the
presence of a racemate, but rather due to the compound existing in two rotameric forms, as
documented in the literature.25 The difference in the 19F-NMR patterns is attributed to the
difference in the local chemical environments of the two compounds. Taking the difference in
the mole fraction of the two enantiomers, as determined by the peak areas, yielded an
enantiomeric excess (ee) greater than 99.5% for both monomers 1a and 2a.54 This finding was
further substantiated by HPLC analysis (Figure 2), which showed no trace evidence of the
racemate present in these monomers, with an ee value determined to be at least 99.5%. The
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broader shoulders observed in each trace could be due to the presence of chemical purities
since the samples were not column-purified; they were injected into HPLC as crude mixtures.
Collectively, these results demonstrate the effectiveness of BrPhF and MPhF in suppressing
epimerization in the two critical reaction steps, O-alkylation of serine sidechain and reductive
amination, in the preparation of Fmoc-MPPNA backbones and the corresponding monomers—
even under a relatively harsh condition, treatment with base at an elevated temperature, carried
out in the alkylation step to drive the reaction to completion.
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Figure 1. 19F-NMR spectra of compounds 23 and 24. The doublet pattern observed with
compound 23 is the result of the formation of rotamers in DMSO-d6.
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Figure 2. HPLC chromatograms of compounds 23 and 24. Overlapping peaks, an indication of
the presence of racemate, were not observed. HPLC condition: C18 column (dimensions 4.6
mm X 250 mm), 1 mL/min flow rate, 60 oC oven temperature, 25 to 75% ACN/H2O with 0.1%
TFA in 30 min.
Conclusion
In summary, we have shown that optically-pure (R)- and (S)-Fmoc-MPPNA monomers could
be prepared in large scales from relatively inexpensive L- and D-serine, respectively. The new
methodology employed the safety-catch BrPhF-protecting group and its morpholine derivative,
namely MPhF, in shielding the α-carbon from undergoing epimerization in the O-alkylation and
reductive amination steps. The ee values of monomers produced by this synthetic route were
determined to be greater than 99.5%, as assessed by 19F-NMR and HPLC. Although the
synthetic route is relatively lengthy in comparison to the published report,25 many of these
reaction steps provided excellent chemical yields and were used in the subsequent steps
without purification. Upon the insertion of morpholine, the resulting MPhF-protecting group can
be removed under a mild acidic condition without affecting Boc, t-butyl ether, or allyl protecting
group. The resulting amine can be readily re-protected with Fmoc or any other functional groups
as desired. This is in direct contrast to all the other synthetic routes that have been
examined,25,43 which were prone to epimerization and required a strenuous chromatographic
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purification in every step. The reported methodology is general and robust, and could be applied
to the preparation of PNA monomers with other chemical functionalities at the gamma
backbone without the concern for erosion of optical purity, even under basic conditions at an
elevated temperature. It should be pointed out that such a synthetic route may not be practical
for the synthesis of monomers for basic research consumption due to its potentially high cost.
However, it will be essential for the production of GMP-grade materials for clinical testing—for
instance, in gene regulation and editing—where obtaining a single enantiomeric product in large
scale is of paramount.
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Experimental Section
All commercial reagents were used without further purification. Solvents were dried by standard
methods and distilled freshly prior to use. 1H-, 13C- and 19F-NMR spectra were recorded on a
Bruker 500.13 MHz Avance NMR spectrometer using standard Bruker software. The 1H NMR
spectra were referenced to DMSO-d6 (2.50 ppm) or TMS in CDCl3 (0.00 ppm). The 13C{1H}
NMR spectra were referenced to CDCl3 (77.23 ppm) or DMSO-d6 (39.52 ppm). The 19F NMR
spectra were referenced to TFA in DMSO-d6 (-76.55 ppm). Flash chromatography was
performed using standard silica gel (60Å, 63-200 µm) or NH2 silica gel (100 Å, 40-75
µm). TLC was generally performed with silica gel 60 F-254 precoated plates or NH2
HPTLC plate. High Resolution Mass Spectrometry (HRMS) was performed with a Thermo
Scientific Exactive Plus EMR Orbitrap ESI mass spectrometer. GC-MS analysis was performed
on a Hewlett-Packard Agilent 6890-5973 GC-MS workstation with a Hewlett-Packard fused
silica capillary column cross-linked with 5% phenylmethylsiloxane and Helium as the carrier gas.
The following conditions were used for all GC-MS analyses: injector temperature, 250 °C; initial
temperature, 70 °C; temperature ramp, 10 °C/min; final temperature, 280 °C. High-performance
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liquid chromatography was performed on a Shimadzu UFLC system with a C18 column
(dimensions 4.6 mm X 250 mm) and 1 mL/min flow rate. Gradients and temperature were
indicated in the figure captions.
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O3-(Tetrahydropyran-2-yl)-L-serine methyl ester hydrochloride (4).46 To a suspension of L-serine
methyl ester hydrochloride 3 (19.5 g, 127.3 mmol) in anhydrous CH2Cl2 (100 mL) was added
3,4-dihydro-1-H-pyran (17.1 mL, 187.8 mmol) and p-toluenesulfonic acid monohydrate (0.476 g,
2.5 mmol). The reaction mixture was allowed to stir at room temperature for 20 h. The resulting
mixture was chilled in an ice bath, filtered and washed with chilled CH2Cl2 to afford white
powder without further purification (25.7 g, 83%). C9H18ClNO4; white solid; TLC (MeOH/CH2Cl2,
10:90) Rf= 0.6; diastereomeric ratio: 83:17; 1H NMR (500.13 MHz, DMSO-d6) δ 8.77 (3H, s),
4.63‒4.57 (1H, m), 4.33‒4.28 (1H, m), 4.04‒3.99 (1H, m), 3.82‒3.75 (4H, m), 3.63‒3.58 (1H,
m), 3.46‒3.44 (1H, m), 1.79‒1.41 (6H, m); 13C{1H} (125.77 MHz, DMSO-d6) δ 168.1, 98.3/97.7,
64.6/64.4, 61.3/61.0, 52.8, 52.3/52.2, 29.7/28.5, 24.8, 18.5. HRMS (ESI) calcd for C9H18NO4:
204.1236; found: m/z 204.1223 [M – HCl + H]+.
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N-(9-(4-Bromophenyl)-9-fluorenyl)-O3-(tetrahydropyran-2-yl)-serine methyl ester (5). To a
solution of serine ester 4 (8.00 g, 33.4 mmol) in anhydrous ACN (110.0 mL) was added
Pb(NO3)2 (55.32 g, 167.0 mmol) and K3PO4 (35.5 g, 167.0 mmol) and stirred for 30 min. To the
suspension was transferred bromide compound 13 in CH2Cl2 (44 mL) and allowed to stir at
room temperature for 24 h. The reaction was quenched with MeOH (10 mL). The mixture was
then filtered by Celite pad. The filtrate was concentrated in vacuo and purified by flash
chromatography on silica gel (EtOAc/hexane, 8:92 to 15:85) to afford yellow syrup as product.
(16.7 g, 96% from serine ester 4). C28H28BrNO4; TLC (EtOAc/hexane, 15:85) Rf= 0.4;
diastereomeric ratio: 83:17; 1H NMR (500.13 MHz, DMSO-d6) δ 7.85 (2 H, t, J = 8.0 Hz), 7.43–
7.35 (4 H, m), 7.32–7.22 (5 H, m), 7.18 (1 H, d, J = 7.5 Hz), 4.42 (0.17H, m), 4.32 (0.83H, t, J =
3.4 Hz), 3.55–3.51 (3H, m), 3.35–3.30 (2H, m), 3.17 (2.49H, s), 3.15 (0.51H, s), 2.76–2.71 (1 H,
m), 1.57–1.28 (6 H, m) ; 13C{1H} (125.77 MHz, DMSO-d6) δ 173.8/173.6, 148.5/148.4,
148.2/148.1, 144.1, 140.0, 139.9, 131.0, 128.6/128.5, 128.1, 128.0, 127.6, 125.5, 124.7, 120.3,
120.1, 98.0/96.8, 72.0, 68.4/67.7, 61.2/60.5, 55.9/55.6, 51.1/51.0, 29.9/29.7, 24.8, 18.8/18.4;
HRMS (ESI) calcd for C28H2981BrNO4: 524.1259.; found: m/z 524.1252 [M + H]+.
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2-N-(9-(4-Bromophenyl)-9-fluorenyl)-3-hydroxyl-N-methoxy-N-methylpropanamide (8). To a
chilled solution of methyl ester 5 (10.0 g, 19.1 mmol) in a co-solvent of ethanol and water (400
mL, 1:1) was added LiOH-H2O (3.21 g, 76.6 mmol) portion-wise at 0 oC in an ice bath. The
reaction mixture was allowed to gradually warm to and stir at ambient temperature for 12 h. The
mixture was neutralized with Dowex resin and filtered. The filtrate was concentrated in vacuo
and co-evaporated with toluene (3x) to afford compound 6 as white solid. The crude mixture
was used in the next step without further purification.
To the above compound 6 was added anhydrous DMF and CH2Cl2 (200 mL, 3:1), followed by
DCC (5.93 g, 28.7 mmol) and DhBtOH (4.68 g, 28.7 mmol) at 0 oC in an ice bath. After 1 h of
stirring, a mixture of N,O-dimethylhydroxylamine (2.80 g, 28.7 mmol), DIEA (5.00 mL, 28.7
mmol) in anhydrous DMF and CH2Cl2 (200 mL, 3:1) was transferred to the above reaction
mixture at 0 oC. The resulting solution was stirred for 2 h at ambient temperature. The reaction
was then quenched with methanol and concentrated in vacuo. The resulting syrup was diluted
with EtOAc (50 mL). The organic layer was washed with sat. NaHCO3 (3x) and brine (3x), dried
over Na2SO4, and concentrated in vacuo to afford Weinreb amide 7 as yellowish foaming solid.
The crude product was used in the next step without further purification.
To the above compound 7 was added methanol (95.7 mL) and p-toluenesulfonic acid
monohydrate (3.64 g, 19.1 mmol) at 0 oC and stirred at ambient temperature for 18 h. The
reaction mixture was neutralized with sat. NaHCO3 and concentrated in vacuo. The resulting
solid was diluted with EtOAc and H2O. The aqueous layer was washed with EtOAc (2x). The
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combined organic layer was washed with brine, dried over Na2SO4, and concentrated in vacuo.
The residue was then purified by column chromatography (EtOAc: hexane= 40:60 to 85:15) to
afford alcohol 8 (8.50 g, 95% in three steps). Colorless syrup; C24H23BrN2O3; TLC
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(EtOAc/hexane, 65:35) Rf= 0.4; 1H NMR (500.13 MHz, DMSO-d6) δ 7.83 (2 H, d, J = 8.1 Hz),
7.43–7.35 (5 H, m), 7.30–7.24 (4 H, m), 7.10 (1 H, d, J = 7.6 Hz), 4.63 (1H, t, J = 5.9 Hz), 3.33–
3.24 (3H, m), 2.95 (4H, br), 2.72 (3H, br); 13C{1H} NMR (125.77 MHz, DMSO-d6) δ 173.8, 149.0,
148.2, 144.5, 140.5, 139.5, 131.1, 128.4, 128.1, 127.9, 127.5, 125.7, 125.7, 120.1, 72.1, 64.0,
59.9, 53.8, 31.5; HRMS (ESI) calcd for C24H24BrN2O3: 467.0970; found: m/z 467.0954 [M + H]+.
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2-N-(9-(4-Bromophenyl)-9-fluorenyl)-3-(2-(2-(tert-butoxy)ethoxy)ethoxy)-N-methoxy-N-
methylpropanamide (9). To a chilled solution of alcohol 8 (7.73g, 16.5 mmol eq) in DMF (0.2 M)
in an ice bath was added 60% NaH (1.00 g, 25.0 mmol) and stirred at the same temperature.
After 30 min, tetrabutylammonium iodide (0.10 g, 0.17 mmol) and tosylate 17 (10.5 g, 19.9
mmol) were added to the above suspension. After 12 h, the reaction was quenched with sat.
NH4Cl. The mixture was concentrated in vacuo, followed by stirring with EtOAc and water. The
aqueous layer was washed with EtOAc (2x). The combined organic layer was then washed with
brine, dried over Na2SO4, and concentrated in vacuo. The residue was purified by column
chromatography on silica gel (EtOAc/hexane, 22:78 to 53:47) to afford colorless syrup (7.80 g,
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77%). C32H39BrN2O5; TLC (EtOAc/hexane, 50:50) Rf= 0.4; 1H NMR (500.13 MHz, DMSO-d6) δ
7.83 (2 H, d, J = 6.3 Hz), 7.42–7.34 (5 H, m), 7.29–7.23 (4 H, m), 7.10 (1 H, d, J = 7.5 Hz),
3.40–3.27 (11H, m), 3.17 (1H, br), 2.99 (3H, br), 2.71 (3 H, br), 1.09 (9 H, s); 13C{1H} NMR
(125.77 MHz, DMSO-d6) δ 173.5, 148.8, 148.2, 140.4, 139.6, 131.1, 128.5, 128.0, 127.9, 127.5,
125.6, 124.9, 120.2, 73.1, 72.2, 72.0, 70.4, 70.0, 69.6, 60.6, 60.0, 51.4, 31.5, 27.2; HRMS (ESI)
calcd for C32H4081BrN2O5: 613.2100; found: m/z 613.2085 [M + H]+.
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3-(2-(2-(tert-butoxy)ethoxy)ethoxy)-N-methoxy-N-methyl-2-N-(9-(4-morpholinophenyl)-9-
fluorenyl)-propanamide (10). To a solution of bromide 9 (7.50 g, 12.2 mmol) in degassed
toluene (24 mL) was added Pd(OAc)2 (137.7 mg, 0.61 mmol), XPhos (292.31 mg, 0.61 mmol),
cesium carbonate (20.0 g, 61.3 mmol) and morpholine (1.29 mL, 14.7 mmol). The resulting
mixture was stirred at 80 oC for 2 h. After gradual cooling to ambient temperature, the reaction
mixture was filtered through Celite pad. The filtrate was concentrated in vacuo and purified by
flash column chromatography on NH2 silica gel (EtOAc/hexane, 18:72 to 63:37) to afford 10 as
colorless syrup (7.27 g, 96%). C36H47N3O6; TLC (EtOAc/hexane, 50:50) Rf= 0.2; 1H NMR
(300.13 MHz, DMSO-d6) δ 7.82–7.78 (2 H, m), 7.38–7.07 (8 H, m), 6.78 (2H, d, J = 9.0 Hz),
3.68 (4H, t, J = 4.7 Hz), 3.38–3.22 (11 H, m), 3.11 (1H, br), 3.03–2.97 (7 H, m), 2.72 (3H, br),
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1.10 (9H, s); 13C{1H} NMR (125.77 MHz, DMSO-d6) δ 173.7, 150.0, 149.7, 148.8, 140.3, 139.4,
135.0, 128.1, 127.7, 127.2, 126.3, 125.6, 124.9, 120.0, 1144.7, 73.3, 72.2, 72.0, 70.4, 70.0,
69.6, 66.0, 60.6, 59.3, 51.5, 48.3, 31.5, 27.2; HRMS (ESI) calcd for C36H47N3NaO6: 640.3363;
found: m/z 640.3338 [M + Na]+.
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Br
OH
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9-(4-Bromophenyl)-9-fluorenol (12).45 To a solution of 1,4-dibromobenzene (72.1 g, 305.5 mmol)
in 1L of ether at – 50 oC in an ACN-dry ice bath was added n-butyllithium (1M, 25 mL) over a
period of 1 h and stirred for another 30 min. To this suspension was transferred fluorenone
(38.5 g, 213.8 mmol) in 400 mL of ether under N2 atmosphere and stirred at the same
temperature for another 1 h. The reaction mixture was gradually warmed to ambient
temperature and stirred for 2 h. The mixture was cooled to 0 oC and quenched with water (200
mL). The aqueous layer was washed with ether (300 mL 2x). The combined organic layer was
washed with brine (300mL x 2), dried over MgSO4, and concentrated in vacuo to afford the
crude product as yellow syrup. The residue was purified by flash column chromatography on
silica gel (EtOAc/hexane, 1:49 to 1:9) to afford compound 12 (62.6 g, 87% yield from
fluorenone). C19H13BrO, white solid; TLC (EtOAc/hexane, 15:85) Rf= 0.4; 1H NMR (500.13 MHz,
CDCl3) δ 7.65 (2 H, d, J = 7.8 Hz), 7.38‒7.35 (4H, m), 7.28‒7.22 (6H, m), 2.41 (1H, br); 13C{1H}
NMR (125.77 MHz, CDCl3) δ 150.0, 142.3, 139.5, 131.3, 129.3, 128.6, 127.3, 124.7, 121.2,
120.2, 83.3.
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9-(4-bromophenyl)-9-fluorenyl bromide (13): To an anhydrous solution of 9-(4-bromophenyl)-9-
fluorenol 1245 (14.1 g, 41.8 mmol) in toluene (208.0 mL) was added acetyl bromide (61.9 mL,
835.1 mmol) at 0 oC and refluxed at 120 oC for 2 h. Upon the completion of the reaction as
confirmed by GC-MS, the reaction mixture was concentrated under reduced pressure and co-
evaporated with toluene three more times. The residue was dried in vacuo for 12 h to afford the
bromide 13 as yellowish syrup. This compound was used in the next step without further
purification. For analytical sample: 1H NMR (500.13 MHz, CDCl3) δ 7.66 (2H, d, J = 7.7 Hz),
7.47 (2H, d, J = 7.4 Hz), 7.41 (2H, d, J = 8.6 Hz), 7.37‒7.34 (4H, m), 7.29‒7.26 (2H, m); 13C{1H}
NMR (125.77 MHz, CDCl3) δ 149.1, 140.4, 138.0, 131.4, 129.2 (2x), 128.6, 125.9, 122.2, 120.4,
66.5. GC-MS tR= 17.8 min.
((2-(2-(tert-butoxy)ethoxy)ethoxy)methyl)benzene (15). To a chilled solution of ethylene glycol
monobenzyl ether (157.0 g, 0.80 mol) in CH2Cl2 (1.0 L) at -50 oC in an ACN/dry ice bath was
added Boc anhydride (401.6 g, 1.84 mol) and Mg(ClO4)2 (17.9 g, 0.80 mol). The reaction
mixture was allowed to gradually warm to room temperature and refluxed for 36 h. The reaction
was quenched by adding sat. NaHCO3 (200 mL). The aqueous layer was washed with CH2Cl2
(200 mL 2x). The combined organic layer was washed with brine, dried over Na2SO4, and
concentrated in vacuo. The crude mixture was purified by flash column chromatography on
silica gel (EtOAc/hexane, 1:99 to 16:84) to afford compound 15 (144.3 g, 71%). C15H24NO3;
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colorless syrup; TLC (EtOAc/hexane, 15:85) Rf= 0.6; 1H NMR (500.13 MHz, CDCl3) δ 7.35–7.26
(5 H, m), 4.57 (2 H, s), 3.70–3.68 (2 H, m), 3.64–3.60 (4H, m), 3.53 (2H, t, J= 5.4 Hz), 1.20 (9H,
s); 13C{1H} NMR (125.77 MHz, CDCl3) δ 138.3, 128.3, 127.7, 127.5, 73.2, 73.0, 71.2, 70.7, 69.4,
61.1, 27.5; HRMS (ESI) calcd for C15H24NaO3: 275.1623; found: m/z 275.1606 [M + Na]+.
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2-(2-(tert-butoxy)ethoxy)ethyl-4-methylbenzenesulfonate (17). To a chilled solution of tert-butyl
ether 15 (144.3 g, 0.57 mol) in MeOH (50 mL) at 0 oC was added Pd/C (608.5 mg, 0.57 mmol).
The reaction was stirred at 100 psi of H2 gas for 12 h at ambient temperature. The reaction
mixture was then cooled to 0 oC and filtered through Celite. The filtrate was concentrated in
vacuo to afford alcohol 16, which was used in the next step without purification.
To a chilled solution of alcohol 16 in CH2Cl2 (1.0 L) at 0 oC was added DMAP (41.9 g, 0.34 mol),
Et3N (79.7 mL, 9.57 mol) and tosyl chloride (130.8 g, 0.69 mol). The reaction mixture was stirred
at ambient temperature for 12 h before quenching with 10% citric acid (200 mL). The organic
layer was further washed with 10% citric acid (200 mL) and brine, dried over Na2SO4 and
concentrated in vacuo. The crude mixture was purified by flash column chromatography on
silica gel (EtOAc/hexane, 1:99 to 35:65) to afford tosylate 17 (163.1 g, 90% in two steps) as
colorless syrup. C15H24O5S; colorless syrup; TLC (EtOAc/hexane, 22:78) Rf= 0.4; 1H NMR
(500.13 MHz, CDCl3) δ 7.80 (2H, t, J = 8.2 Hz), 7.34 (2H, t, J = 8.2 Hz), 4.16 (2H, t, J = 9.8 Hz),
3.70 (2H, t, J = 9.8 Hz), 3.53 (2H, t, J = 10.3 Hz), 3.44 (2H, t, J = 10.3 Hz) 2.44 (3H, s), 1.17
(9H, s); 13C{1H} NMR (125.77 MHz, CDCl3) δ 144.7, 133.1, 129.8, 128.0, 73.0, 71.4, 69.3, 68.7,
61.2, 27.4, 21.6; HRMS (ESI) calcd for C15H24NaO5S: 339.1242; found: m/z 339.1228 [M + Na]+.
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[CH2N]Gly-O-allyl (19). To a chilled solution of amide 10 (423.9 mg, 0.69 mmol) in anhydrous
THF (7 mL) at 0 oC was added lithium aluminum hydride (26.0 mg, 0.69 mmol). The reaction
mixture was stirred for 1 h before quenching with sat. Na2SO4 and further diluted with ether. The
aqueous layer was then extracted with ether (2x). The combined organic layer was washed with
brine, dried over Na2SO4 and concentrated in vacuo to afford aldehyde 18. The residue was
used in the next step without purification.
To a chilled solution of aldehyde 18 in anhydrous dichloroethane (7 mL) at 0 oC was added
glycine allyl ester toluenesulfonic acid salt55 (197.2 mg, 0.69 mmol), N,N-diisopropylethylamie
(239.0 µL, 1.37 mmol) and sodium triacetoxyborohydride (218.1 mg, 1.03 mmol). The reaction
mixture was stirred at ambient temperature for 20 h before quenching with sat. NaHCO3. The
mixture was then diluted with ether. The aqueous layer was extracted with ether (2x). The
combined organic layer was washed with brine, dried over Na2SO4 and concentrated in vacuo.
The residue was purified by flash column chromatography on silica gel (EtOAc/hexane, 24:76 to
99:1) to afford backbone 19 as colorless syrup (370.1 mg, 82% in two steps). C39H51N3O6; TLC
(EtOAc/hexane, 80:20) Rf= 0.4; 1H NMR (500.13 MHz, DMSO-d6) δ 7.81–7.79 (2 H, m), 7.37–
7.20 (6 H, m), 7.17 (2H, d, J = 9.0 Hz), 6.77 (2H, d, J = 9.0 Hz), 5.95–5.87 (1 H, m), 5.30 (1H,
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dq, J = 17.3, 1.6 Hz), 5.22 (1H, dq, J = 10.5, 1.4 Hz), 4.56 (2H, d, J = 4.8 Hz), 3.68 (4H, t, J =
4.8 Hz), 3.37–3.30 (7 H, m), 3.13 (2H, t, J = 4.8 Hz), 3.09 (1H, d, J = 17.2 Hz), 3.03–2.92 (7 H,
m), 2.26 (1H, dd, J = 11.4, 5.3 Hz), 2.16–2.08 (2 H, m), 2.02 (1H, br), 1.08 (9H, s); 13C{1H} NMR
(125.77 MHz, DMSO-d6) δ 171.8, 150.6, 150.1, 150.0, 139.7(2x), 136.1, 132.6, 128.0(2x),
127.5(2x), 126.4, 125.3, 125.0, 120.0(2x), 117.8, 114.7, 72.2, 72.0(2x), 70.4, 69.5, 69.4, 66.0,
64.3, 60.6, 51.4, 50.8, 50.2, 48.4, 27.3; HRMS (ESI) calcd for C39H52N3O6: 658.3856; found: m/z
658.3826 [M + H]+.
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A general procedure for nucleobase coupling
To a solution of amine (1.0 eq) in anhydrous DMF (0.1 M) was added N,N-diisopropylethylamine
(2.0 eq), nucleobase (2.0 eq) and HBTU (1.8 eq). The reaction was stirred for 12 h before
quenching with MeOH. The reaction mixture was concentrated in vacuo. The residue was
diluted with EtOAc, washed with sat. NaHCO3 and brine, dried over Na2SO4 and concentrated in
vacuo. The crude was purified by flash column chromatography on NH2 silica gel
(MeOH/EtOAc, 1:99 to 15:85).
3-(2-(2-(Tert-butoxy)ethoxy)ethoxy)-2-N-(9-(4-morpholinophenyl)-9-fluorenyl)-L-serine thymine
allyl ester (20a). This compound was prepared using the aforementioned general procedure
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starting with amine 19 (62.5 mg) and thymine acetic acid (34.99 mg). Yield: (75 mg, 96%);
C46H57N5O9; colorless syrup; TLC (MeOH/EtOAc, 5:95) Rf= 0.5 with NH2 Silica gel; rotamer
ratio=4:1; 1H NMR (500.13 MHz, DMSO-d6) δ 11.27–11.22 (1H, m), 7.84–7.78 (2H, m), 7.44–
7.11 (9H, m), 6.79–6.75 (2H, m), 5.97–5.79 (1H, m), 5.37–5.20 (2H, m), 4.60–4.36 (4H, m),
3.69–3.67 (4H, m), 3.60 (1H, d, J= 17.1 Hz), 3.49–3.23 (9H, m), 3.15–2.74 (9H, m), 2.21–2.07
(1H, m), 1.74–1.71 (3H, m), 1.07–1.06 (9H, m); 13C{1H} NMR (125.77 MHz, DMSO-d6) δ
168.7/168.2, 167.8/167.4, 164.4/164.3, 150.9/150.8, 150.6/150.3, 150.0/149.9, 149.8/149.5,
142.1/141.9, 140.1/139.9, 139.6/139.5, 135.6/135.2, 132.2, 128.3, 128.2/128.1, 127.8,
127.7/127.6, 126.6/126.4, 125.5/125.4, 125.3/125.2, 120.2, 120.1/120.0, 118.4/118.0, 114.8,
108.2/108.0, 72.2(2x), 71.2, 70.4/70.3, 69.9/69.6, 69.5/69.4, 66.0, 65.5/64.8, 60.6/60.5,
51.0/50.6, 50.5/50.3, 48.4, 47.8/47.6, 47.5, 27.3/27.2, 11.9; HRMS (ESI) calcd for C46H58N5O9:
824.4234; found: 824.4204 m/z [M + H]+.
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3-(2-(2-(Tert-butoxy)ethoxy)ethoxy)-2-N-(9-(4-morpholinophenyl)-9-fluorenyl)-L-serine
adenine(Boc) allyl ester (20b). This compound was prepared using the aforementioned general
procedure starting with amine 19 (71.1 mg) and adenine (Boc) acetic acid (63.40 mg). Yield: (70
mg, 70%); C51H64N8O9; colorless syrup; TLC (MeOH/CHCl3, 5:95) Rf= 0.4; rotamer ratio=1:3; 1H
NMR (500.13 MHz, DMSO-d6) δ 10.05–10.02 (1H, m), 8.50–8.47 (1H, m), 8.22–8.20 (1H, m),
7.83–7.73 (2H, m), 7.48–7.07 (8H, m), 6.80–6.69 (2H, m), 6.01–5.78 (1H, m), 5.40–5.10 (4.75H,
m), 4.66–4.46 (2H, m), 4.17 (0.25H, m), 3.69–3.68 (6H, m), 3.43–2.75 (16H, m), 2.20 (1H, br),
1.48 (9H, s), 1.07 (2.25H, s), 1.01 (6.75H, s); 13C{1H} NMR (125.77 MHz, DMSO-d6) δ
168.8/168.2, 167.3/166.8, 152.2, 151.6/151.3, 151.4/151.1, 150.6/150.3, 150.1/149.9,
149.8/149.6, 149.7/149.5, 144.8, 140.2/139.9, 139.6/139.5, 135.6/135.2, 132.2/132.1, 128.3,
128.1, 127.8, 127.7/127.6, 126.6/126.4, 125.5/125.4, 125.3, 123.0/122.9, 120.2, 120.1,
118.4/118.0, 114.8/114.7, 80.1/80.0, 72.3/72.2, 72.2/71.9, 71.1, 70.4/70.3, 70.1/69.7, 69.5/69.4,
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66.0, 65.6/64.8, 60.6/60.4, 51.0/50.7, 50.3, 48.4, 47.6, 44.0/43.5, 27.9, 27.2; HRMS (ESI) calcd
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3-(2-(2-(Tert-butoxy)ethoxy)ethoxy)-2-N-(9-(4-morpholinophenyl)-9-fluorenyl)-L-serine
cytosine(Boc) allyl ester (20c). This compound was prepared using the aforementioned general
procedure starting with amine 19 (74.8 mg) and cytosine (Boc) acetic acid (61.23 mg). Yield:
(85.4 mg, 83%); C50H64N6O10; colorless syrup; TLC (MeOH/CHCl3, 15:85) Rf= 0.5; rotamer
ratio=1:4; 1H NMR (500.13 MHz, DMSO-d6) δ 10.26–10.23 (1H, m), 7.83–7.71 (3H, m), 7.45–
7.18 (8H, m), 7.12–6.75 (4H, m), 5.98–5.50 (1H, m), 5.37–5.20 (2H, m), 4.71–4.46 (4.8H, m),
4.05–4.01 (0.2H, m), 3.69–3.67 (4.2H, m), 3.58 (0.8H, d, J= 17.1 Hz), 3.47–2.75 (16H, m),
2.23–2.08 (1H, m), 1.46–1.45 (9H, m), 1.07–1.05 (9H, m); 13C{1H} NMR (125.77 MHz, DMSO-
d6) δ 168.8/168.2, 167.9/167.3, 163.2, 154.9, 152.1, 150.6/150.4, 150.2/150.1, 149.9/149.7,
149.5, 140.1/140.0, 139.6/139.5, 135.6/135.2, 132.2, 128.2, 128.1, 127.8, 127.7, 126.6/126.4,
125.4, 125.2, 120.2/120.0, 120.1/119.9, 118.3/118.0, 114.8/114.7, 93.9/93.8, 80.9, 72.3, 72.2,
71.1, 70.4/70.3, 69.9/69.6, 69.5/69.4, 66.0, 65.5/64.8, 60.6/60.4, 51.0/50.7, 50.5/50.2, 49.4/48.8,
48.4, 47.6, 27.8, 27.3/27.2; HRMS (ESI) calcd for C50H64N6NaO10: 931.4552; found: m/z
931.4542 [M + Na]+.
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3-(2-(2-(tert-butoxy)ethoxy)ethoxy)-2-N-(9-(4-morpholinophenyl)-9-fluorenyl)-L-serine
guanine(Boc) allyl ester (20d). This compound was prepared using the aforementioned general
procedure starting with amine 19 (71.1 mg) and guanine (Boc) acetic acid (66.85 mg). Yield:
(76.8 mg, 75%); C51H64N8O10; colorless syrup; TLC (MeOH/CHCl3, 10:90) Rf= 0.2 with NH2
Silica gel; rotamer ratio=1:3; 1H NMR (500.13 MHz, DMSO-d6) δ 11.3 (1H, br), 11.0–10.9 (1H,
br), 7.84–7.77 (2H, m), 7.69–7.67 (1H, m), 7.47–7.08 (8H, m), 6.80–6.73 (2H, m), 6.01–5.80
(1H, m), 5.40–5.20 (2H, m), 4.89–4.80 (2.5H, m), 4.64–4.63 (0.5H, m), 4.48–4.47 (1.5H, m),
4.20–4.02 (0.5H, m), 3.70–3.68 (4H, m), 3.54–2.76 (18H, m), 2.22–2.12 (1H, m), 1.49–1.48 (9H,
m), 1.07 (2.25H, s), 1.01 (6.75H, s); 13C{1H} NMR (125.77 MHz, DMSO-d6) δ 168.9/168.1,
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167.2/166.7, 155.1, 153.6, 150.5/150.2, 150.1/149.9, 149.7/149.4, 149.5/149.4, 147.3, 140.2,
140.1/139.9, 139.7/139.5, 135.6/135.1, 132.2, 128.3, 128.2, 128.1, 127.9, 127.7, 126.5/126.3,
125.4, 125.3, 125.2, 120.2/120.1, 119.0/118.9, 114.8/114.7, 82.5/82.4, 72.3/72.2, 72.2/72.0,
71.0, 70.4/70.2, 70.0/69.7, 69.5/69.4, 66.0, 65.6/64.9, 60.6/60.3, 51.1/50.6, 50.4, 48.4, 47.5,
43.7/43.4, 27.8, 27.3/27.1; HRMS (ESI) calcd for C51H65N8O10: 949.4823; found: m/z 949.4807
[M + H]+.
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A general method for converting MPhF- to Fmoc-protected allyl ester 22a-d
To a chilled solution of MPhF-protected allyl ester 20 (1 eq) in ACN (0.1M) at 0 oC was added
1M citric acid (3.6 eq). The reaction mixture was allowed to gradually warm to and stir at
ambient temperature for 12 h. The reaction progress was monitored by HRMS (See Figure S28
and S29). Upon completion, the reaction mixture was chilled in an ice bath for 10 min, followed
by the addition of NaHCO3 (14 eq) and Fmoc-Cl (2.5 eq). After 3 h, the reaction mixture was
mixed with silica gel and concentrated in vacuo and directly purified by column chromatography
(EtOH/CHCl3= 1:99 to 10:90) to afford the final Fmoc-MPPNA allyl ester 22.
Fmoc-3-(2-(2-(tert-butoxy)ethoxy)ethoxy)-L-serine thymine monomer allyl ester (22a). This
compound was prepared using the aforementioned general procedure starting with ally ester
20a (59.50 mg). Yield: (45 mg, 86%); C38H48N4NaO10; colorless syrup; TLC (MeOH/CHCl3, 5:95)
Rf= 0.5; rotamer ratio= 2:3; 1H NMR (500.13 MHz, DMSO-d6) δ 11.27–11.26 (1H, m), 7.89 (2H,
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d, J= 7.5 Hz), 7.70 (2H, d, J= 7.4 Hz), 7.43–7.22 (5H, m), 5.99–5.85 (1H, m), 5.40–5.19 (2H, m),
4.74–4.04 (10H, m), 3.90–3.80 (1H, m), 3.59–3.08 (12H, m), 1.73–1.72 (3H, m), 1.09–1.08 (9H,
m); 13C{1H} NMR (125.77 MHz, DMSO-d6) δ 169.0/168.5, 168.0/167.7, 164.3, 155.8/155.7,
150.9, 143.9/143.8, 142.0/141.9, 140.7, 132.2, 127.6, 127.0, 125.2/125.1, 120.1, 118.2/117.8,
108.2/108.0, 72.2, 70.5, 70.4, 69.9/69.8, 69.6/69.5, 65.5, 64.8, 60.6/60.5, 49.9/49.3, 48.4,
48.2/48.0, 47.8/47.7, 46.7, 27.3/27.2, 11.9; HRMS (ESI) calcd for c 743.3268; found: m/z
743.3244 [M + Na]+.
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Fmoc-3-(2-(2-(tert-butoxy)ethoxy)ethoxy)-L-serine adenine(Boc) monomer allyl ester (22b). This
compound was prepared using the aforementioned general procedure starting with allyl ester
20b (43.50 mg). Yield: (33 mg, 85%); C43H55N7O10; colorless syrup; TLC (MeOH/CHCl3, 5:95)
Rf= 0.5; rotamer ratio= 2:3; 1H NMR (500.13 MHz, DMSO-d6) δ 10.06 (1H, br), 8.55–8.53 (1H,
m), 8.34–8.29 (1H, m), 7.92–7.90 (2H, m), 7.74–7.71 (2H, m), 7.53–7.32 (4H, m), 6.00–5.85
(1H, m), 5.46–5.19 (4H, m), 4.73–3.15 (21H, m), 1.51 (9H, m), 1.11 (9H, m); 13C{1H} NMR
(125.77 MHz, DMSO-d6) δ 169.0/168.4, 167.4/167.1, 155.9/155.8, 152.2, 151.4, 151.1, 149.7,
144.8, 143.9/143.8, 140.7, 132.2, 127.6, 127.0, 125.2, 123.0/122.9, 120.1, 118.2/117.8,
80.0/79.1, 72.2, 70.5, 70.4, 70.1/69.8, 69.6, 65.6/65.5, 64.8, 60.6, 50.2/49.9, 48.5, 48.2, 46.7,
27.8, 27.3/27.2; HRMS (ESI) calcd for C43H56N7O10: 830.4088; found: m/z 830.4069 [M + H]+.
Fmoc-3-(2-(2-(tert-butoxy)ethoxy)ethoxy)-L-serine cytosine(Boc) monomer allyl ester (22c). This
compound was prepared using the aforementioned general procedure starting with allyl ester
20c (59.80 mg). Yield: (48 mg, 91%); C42H55N5O11; colorless syrup; TLC (MeOH/CHCl3, 5:95)
Rf= 0.3; rotamer ratio=1:1; 1H NMR (500.13 MHz, DMSO-d6) δ 10.27 (1H, br), 7.89–6.96 (10H,
m), 5.98–5.84 (1H, m), 5.37–5.18 (2H, m), 4.88–4.79 (1H, m), 4.65–4.55 (3H, m), 4.41–4.21
(4H, m), 4.15–4.04 (1H, m), 3.91–3.80 (1H, m), 3.57–3.10 (13H, m), 1.45 (9H, s), 1.09–1.08
(9H, m); 13C{1H} NMR (125.77 MHz, DMSO-d6) δ 169.0/168.5, 168.0/167.7, 163.3, 155.9/155.7,
155.0, 152.1, 150.4, 143.9/143.8, 140.7, 132.3/132.2, 127.6, 127.0, 125.1, 120.8, 118.2/117.8,
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49.3, 48.3/48.2, 46.7, 27.8, 27.3/27.2; HRMS (ESI) calcd for C42H56N5O11: 806.3976; found: m/z
806.3930 [M + H]+.
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Fmoc-3-(2-(2-(tert-butoxy)ethoxy)ethoxy)-L-serine guanine(Boc) monomer allyl ester (22d). This
compound was prepared using the aforementioned general procedure starting with allyl ester
20d (53.20 mg) as the starting material. Yield: (39 mg, 82%); C43H55N7O11; colorless syrup; TLC
(MeOH/CHCl3, 5:95) Rf= 0.1; rotamer ratio=1:1; 1H NMR (500.13 MHz, DMSO-d6) δ 11.31–
10.69 (2H, br), 7.89–7.22 (9H, m), 6.01–5.83 (1H, m), 5.37–5.19 (2H, m), 5.18–3.35 (23H, m),
1.45–1.39 (9H, m), 1.08–1.05 (9H, m); 13C{1H} NMR (125.77 MHz, DMSO-d6) δ 169.1/168.5,
167.4/167.1, 156.1/155.8, 155.1/155.0, 153.7/153.6, 149.6/149.4, 147.4, 143.8, 143.6, 140.7,
140.3/140.1, 132.2/132.1, 127.6, 127.5/127.3, 127.0, 125.1/125.0, 121.3, 120.2/120.1, 118.9,
118.3/117.8, 82.5/82.4, 72.2, 70.6, 70.4, 70.0/69.9, 69.8/69.6, 65.6/65.5, 65.4/64.9, 60.6/60.5,
50.5, 49.6/49.4, 48.4/48.2, 46.7, 43.8/43.6, 27.7/27.6, 27.2; HRMS (ESI) calcd for C43H56N7O11:
846.4038; found: m/z 846.3983 [M + H]+.
A general method for converting Fmoc-protected allyl esters 22a-d to the final monomers
1a-d
To a solution of allyl ester 22 (1 eq) in DCM (0.1M) was added Pd[PPh3]4 (0.2 eq) and PhSiH3 (2
eq). The reaction was stirred at ambient temperature for 12 h. The reaction mixture was then
concentrated in vacuo with silica gel. The crude was then purified by column chromatography
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(A= EtOAc, B= ACN/MeOH/H2O= 2:1:1, A:B= 97:3 to 66:34) to afford the final Fmoc-MPPNA
monomers 1.
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Fmoc-3-(2-(2-(tert-butoxy)ethoxy)ethoxy)-L-serine thymine monomer (1a). 96% yield;
C35H44N4O10; white powder; TLC (EtOAc/ACN/MeOH/H2O, 6:1:1:1) Rf= 0.5; rotamer ratio=2:3;
1H NMR (500.13 MHz, DMSO-d6) δ 12.9 (1 H, br), 11.3–11.2 (1 H, m), 7.88 (2 H, J= 7.6 Hz, d),
7.69 (2 H, J= 7.4 Hz, d), 7.43–7.22 (5 H, m), 4.72–4.64 (1 H, m), 4.46 (1 H, s), 4.35–4.28 (2 H,
m), 4.23–4.22 (1 H, m), 4.11 (1 H, m), 4.02–3.80 (2 H, m), 3.60–3.07 (13 H, m), 1.72 (3 H, m),
1.09–1.08 (9 H, m); 13C{1H} NMR (125.77 MHz, DMSO-d6) δ 170.9/170.4, 168.0/167.5, 164.4,
155.9/155.8, 151.0/150.9, 143.9/143.8, 142.1/142.0, 140.7, 127.6, 127.0, 125.2, 120.1,
108.1/108.0, 72.2, 70.5, 70.4, 69.9/69.8, 69.6, 65.6/65.4, 60.6/60.5, 49.8/49.4, 48.5, 48.1/47.7,
47.8, 46.7, 27.3/27.2, 11.9; HRMS (ESI) calcd for C35H44N4NaO10: 703.2955; found: m/z
703.2927 [M + H]+.
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Fmoc-3-(2-(2-(tert-butoxy)ethoxy)ethoxy)-L-serine adenine(Boc) monomer (1b). 90% yield;
C40H51N7O10; white powder; TLC (EtOAc/ACN/MeOH/H2O, 6:1:1:1) Rf= 0.4; rotamer ratio=2:3;
1H NMR (500.13 MHz, DMSO-d6) δ 12.8 (1 H, br), 10.0 (1 H, s), 8.51–8.50 (1 H, m), 8.27 (1 H,
s), 7.88 (2 H, J= 7.3 Hz, d), 7.72–7.69 (2 H, m), 7.51–7.14 (4 H, m), 5.42–5.10 (2 H, m), 4.38–
4.21 (4 H, m), 4.06–3.81 (2 H, m), 3.70–3.12 (13 H, m), 1.48 (9 H, m), 1.08 (3.6 H, s), 1.04 (5.4
H, s); 13C{1H} NMR (125.77 MHz, DMSO-d6) δ 170.7/170.1, 167.4/166.9, 155.9/155.8, 152.2,
151.4, 151.1, 149.7, 144.9/144.8, 143.9/143.8, 140.7, 128.9/127.6, 128.2/127.0, 125.3/125.2,
122.9, 120.1, 80.0, 72.2, 70.5, 70.4, 70.0, 69.7, 69.6, 65.6/65.4, 60.6/60.5, 49.8/49.3, 48.5, 48.0,
46.7 , 27.8, 27.2; HRMS (ESI) calcd for C40H52N7O10: 790.3775; found: m/z 790.3749 [M + H]+.
Fmoc-3-(2-(2-(tert-butoxy)ethoxy)ethoxy)-L-serine cytosine(Boc) monomer (1c). 92% yield;
C39H51N5O11; white powder; TLC (EtOAc/ACN/MeOH/H2O, 6:1:1:1) Rf= 0.5; rotamer ratio=1:1;
1H NMR (500.13 MHz, DMSO-d6) δ 10.26 (1 H, br), 7.88 (2 H, J= 7.6 Hz, d), 7.84–7.79 (1 H,
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