Optimized Synthesis of Fmoc/Boc-Protected PNA Monomers and their Assembly into PNA Oligomers.

Article abstract of DOI:10.1002/ejoc.202100278

Continuous advancement of application of peptide nucleic acid (PNA) oligomers encouraged exploration of rapid and efficient synthesis of PNA monomers and oligomers. Among the PNA monomers developed, only a few are commonly used in automated PNA synthesis.

Full text of DOI:10.1002/ejoc.202100278

European Journal of Organic Chemistry
Accepted Article
Accepted Article
Title: Optimized synthesis of Fmoc/Boc-protected PNA monomers and
their assembly into PNA oligomers
Authors: Ashif Yasin Shaikh, Fredrik Fredrik Björkling, Peter Eigil
Nielsen, and Henrik Franzyk
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.202100278
Link to VoR: https://doi.org/10.1002/ejoc.202100278
01/2020

10.1002/ejoc.202100278
European Journal of Organic Chemistry
FULL PAPER
Optimized synthesis of Fmoc/Boc-protected PNA monomers and
their assembly into PNA oligomers
Ashif Y. Shaikh,[a] Fredrik Björkling, [a] Peter E. Nielsen [b] and Henrik Franzyk*[a]
[a]
Dr. Ashif Shaikh, Prof. Fredrik Björkling, Assoc. Prof. Henrik Franzyk
Center for Peptide-based Antibiotics, Department of Drug Design and Pharmacology
Faculty of Heath and Medical Sciences, University of Copenhagen
Jagtvej 162, DK-2100, Copenhagen, Denmark
E-mail: ashif@sund.ku.dk; E-mail: fb@sund.ku.dk; E-mail: henrik.franzyk@sund.ku.dk
Prof. Peter E. Nielsen
[b]
Center for Peptide-based Antibiotics, Department of Cellular and Molecular Medicine,
The Panum Institute, Faculty of Heath and Medical Sciences, University of Copenhagen
Blegdamsvej 3, DK-2200, Copenhagen, Denmark
E-mail: ptrn@sund.ku.dk
Supporting information for this article is given via a link at the end of the document.
Abstract: Continuous advancement of application of peptide nucleic
acid (PNA) oligomers encouraged exploration of rapid and efficient
synthesis of PNA monomers and oligomers. Among the PNA
monomers developed, only a few are commonly used in automated
PNA synthesis. Here, we report short and efficient protocols suitable
for large-scale synthesis of Fmoc/Boc-protected PNA monomers with
advantageous solubility properties; these also facilitate purification
due to the traceless nature of the Boc protecting group. Initially,
several coupling reagents were screened for assembly of a pentamer
containing all four nucleobases, and then the most promising reagents
were tested in the synthesis of a decamer. The Fmoc/Boc-protected
monomers proved compatible with both manual synthesis and
assembly on an automated peptide synthesizer at room temperature
or at 40°C. As compared to the commonly used coupling agent, 1-
[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-
oxide hexafluorophosphate (HATU), both 2-(1H-benzotriazol-1-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and [ethyl
cyano(hydroxyimino)acetato-O2]tri-1-pyrrolidinylphosphonium hexa-
fluorophosphate (PyOxim) proved more favorable, with the latter
being superior. A previously reported side reaction of guanine bases
in the presence of benzotriazol-1-yl-oxytripyrrolidinophosphonium
hexafluorophosphate (PyBOP) was not observed with the
phosphonium salts.
electrostatic repulsion, thus leading to highly efficient PNA–DNA
or PNA–RNA hybridization. Additionally, the peptide-like
backbone possesses stability toward degradation by nucleases
and proteases, thereby emphasizing PNA as a therapeutically
interesting class of biopolymers. Although many derived
subclasses and analogs of PNA have been synthesized in recent
years (e.g., oligomers displaying chiral, cyclic and locked
monomers), the vast majority of biological and application-related
studies still involve the original aeg-based PNA (i.e., oligomers
with aminoethylglycine backbone acylated with nucleobase-
substituted acetic acids) due to its favorable DNA- and RNA-
binding properties as well as high chemical and biological stability.
In order to further promote application of PNA in various fields
facile access to monomers and oligomer assembly via efficient
synthesis protocols are essential.
Solid-phase peptide synthesis (SPPS) has proven
undisputed usefulness as the most convenient approach for
synthesis of peptides and related oligomers in high purity and
good yields. Both Boc- and Fmoc-based chemistry have been
employed for solid-phase synthesis (SPS) of PNA oligomers.
Initially, manual assembly of Boc/Z-protected PNA monomers
was the preferred synthesis method,[1,8-10] but developments
within robotic technologies, compatible with Fmoc chemistry
(including advanced methodologies for constructing combinatorial
libraries), have proved directly transferable to PNA oligomer
synthesis.[11] However, easy access to Fmoc-protected PNA
monomers, suitable for automated synthesis, is still required. At
present, the commercially available Boc/Z-protected monomers
remain incompatible with current automated technology (due to
the risk of corrosion of instrument metal parts), while the Fmoc/
benzhydryloxycarbonyl (Bhoc)-protected monomers are very
expensive, and possess somewhat unfavorable solubility
properties; in addition, crude products often require a work-up
step prior to final purification by preparative HPLC due to
impurities arising from the Bhoc side-chain protecting group.
Several protection schemes have been explored in order to
obtain PNA monomers displaying orthogonal protection of the
backbone amine and the amine functionalities present in the
nucleobases: Boc/Z,[1, 8-10] Fmoc/Z,[12] Fmoc/Bhoc,[13] Fmoc/4,4'-
Introduction
The DNA mimic peptide nucleic acid (PNA) was discovered three
decades ago, and has since inspired researchers from as diverse
fields as molecular and cell biology, drug discovery, medicinal
chemistry, genetic diagnostics, combinatorial chemistry and
nanoscience to utilize PNA oligomers as molecular tools and
reagents.[1-7] Thus, PNA oligomers have been studied extensively
for their therapeutic potential as gene therapeutic drugs as well
as diagnostic and nanotechnology tools. Importantly, PNA is a
biopolymer possessing enhanced sequence-specific binding to
both RNA and DNA. In PNA the phosphodiester backbone is
replaced by an amide backbone, thereby converting the
negatively charged backbone into a neutral backbone devoid of
1
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10.1002/ejoc.202100278
European Journal of Organic Chemistry
FULL PAPER
dichlorobenzhydryloxycarbonyl (Cl-Bhoc),[13] Fmoc/4,4'-difluoro-
benzhydryloxycarbonyl (F-Bhoc),[13] Fmoc/p-methoxybenzyloxy-
carbonyl (4-MeO-Z),[13] Fmoc/Boc,[14-16] and Fmoc/bis(Boc).[17-19]
Besides the traditional use of Boc or Fmoc as temporary
protecting group for the backbone primary amine several
alternative protecting groups have been reported, e.g.,
monomethoxytrityl (Mmt)/(4-tBuBz/isobutyryl),[20] dithiasuccinoyl
(Dts)/Z,[21] 4,4’-dimethoxytrityl (DMT)/p-methoxybenzyl (PMB),[22]
azido/Bhoc,[23] allyloxycarbonyl(Alloc)/Bhoc,[24] nitroveratryloxy-
carbonyl (NVOC)/(anisoyl/isobutyryl),[25] 1-(4,4-dimethyl-2,6-
dioxocyclohex-1-ylidene)ethyl (Dde)/Mmt,[26] 1,3-benzothiazol-2-
yl (Bts)/Boc,[27] monomethyltrityl (Mtt)/Boc,[28] and p-nitrobenzyl-
oxycarbonyl (pNZ)/bis(Boc).[29]
Previously, we have briefly reported on PNA oligomer
synthesis based on the Fmoc/Boc protection scheme,[15-16, 30] but
in the route to the required monomers some critical difficulties
remained to be solved. Thus, further optimization toward more
simple, scalable purification steps was timely in order to provide
robust methods for obtaining PNA monomers in multigram scale
from readily available starting materials.
Initially, 2-aminoethyl glycine was readily obtained in good
yield and purity from the alkylation reaction between chloroacetic
acid and excess ethylenediamine. Conversion into p-toluene-
sulfonic acid (PTSA) salt 1 (storable for a prolonged time until use)
was achieved by treatment with a stoichiometric amount of PTSA
in toluene and subsequent removal of water by (using a Dean-
Stark setup). Here, the carcinogenic benzene solvent was
replaced with the less harmful toluene. Esterification of 1 was
carried out by using a catalytic amount of PTSA in a MeOH–
toluene (1:1 mixture; refluxed overnight). Selective Fmoc
protection of the primary amine in the presence of a secondary
amine was accomplished by using Fmoc-OSu and DIPEA to give
FmocHN(CH2)2NHCH2COOMe (2).[17]
Previously,[14] the direct coupling of the protected
nucleobase-substituted acetic acid to the corresponding acid
derivative (i.e., FmocHN(CH2)2NHCH2COOH) was identified as
the step that impedes a facile purification of the final monomer,
since unreacted amounts of these intermediate carboxylic acids
and the desired products proved surprisingly difficult to separate.
Thus, it was envisioned that consumption of large amounts of
solvent for a time-consuming column purification could be
avoided by instead utilizing the methyl ester derivative 2 in this
amide coupling.
The nucleobase-substituted acetic acid derivatives T-
CH2COOH (3a),[8,32] BocC-CH2COOH (4b),[13] and BocG-CH2COOH
(6a)[13] were synthesized according to reported literature
protocols. Crude BocA-CH2COOBn (5a)[13] was obtained as a
gummy solid, which could be purified by dissolution in a minimum
amount of CH2Cl2 followed by slow crystallization upon keeping
this solution for at least 1 day at room temperature. In contrast,
previous work reported that column chromatography (CC) was
required.[13] Finally, removal of the benzyl group by
hydrogenolysis[13] afforded BocA-CH2COOH (5b), which after
repeated co-evaporation with toluene and subsequent drying in
vacuo could be used directly.
Coupling of 3a[8,32] with compound 2 was carried out by
using O-(N-succinimidyl)-1,1,3,3-tetramethyluronium tetrafluoro-
borate (TSTU) in DMF.[14] The resulting methyl ester (i.e., 7a) was
subjected to saponification with LiOH followed by Fmoc protection
in a one-pot procedure to afford the Fmoc-aeg(T-CH2CO)-OH
(8a).[33]
As comparative systematic studies on the coupling
procedure in SPS of PNA oligomers appeared to be absent in
literature this crucial synthesis step was re-examined in detail. An
overall aim was to enable oligomerization in laboratories with
different types of setups for SPS, e.g., standard equipment with
manual operation as well as advanced automated synthesizers
with a heating option.
Results and Discussion
Currently, a focus in our research is development and synthesis
of
peptide-PNA
conjugates
as
novel
antibacterial
compounds.[14,30] In the present work, we describe synthesis of
Fmoc/Boc-protected PNA monomers and their optimal use in
oligomer synthesis in preparative scale (i.e., >10 mg).
PNA monomer Synthesis
Here, we report improved synthesis protocols for the Fmoc/Boc-
protected PNA monomers (with the A, G and C nucleobase).
These are conveniently obtained by modified routes only involving
a single purification by column chromatography (see Schemes 1
and 2). Monomer synthesis started from the readily available
ethylenediamine, which was alkylated with chloroacetic acid to
obtain the unprotected non-acylated PNA backbone
intermediate.[31] Upon esterification and selective Fmoc protection
of the PNA backbone intermediate (Scheme 1),[17] a direct
coupling with Boc-protected nucleobase-substituted acetic acid
derivatives yielded methyl esters of the desired building blocks.
Subsequent ester hydrolysis and re-introduction of Fmoc (partially
lost during the hydrolysis step) provided the desired Fmoc/Boc-
protected PNA monomers (Scheme 2).
The above nucleobase-substituted acetic acid derivatives
BocC-CH2COOH (4a),[13] BocA-CH2COOH (5b)[13] and BocG-
CH2COOH (6a)[13] were coupled to compound 2 by using TSTU
and DIPEA in DMF.[14] The resulting methyl esters (i.e., 7b-7d)
were purified by vacuum liquid chromatography (VLC), and were
then subjected to ester hydrolysis, followed by Fmoc protection in
situ (as Fmoc was partially lost during the hydrolysis) and work-
up to yield the final monomers Fmoc-aeg(BocC-CH2CO)-OH (8b),
Fmoc-aeg(BocA-CH2CO)-OH (8c), and Fmoc-aeg(BocG-CH2CO)-
OH (8d) - all with a purity sufficient for direct use in PNA
oligomerization. In this modified approach for monomer synthesis
only a single CC purification was required. The NMR data of the
PNA monomers 8b-8d were in agreement with those reported.[30]
2
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10.1002/ejoc.202100278
European Journal of Organic Chemistry
FULL PAPER
O
O
O
H
N
H
N
a,b
+
c,d
NH2
Cl
FmocHN
OMe
H2N
H2N
OH
OH
2 TsOH
1
2
Scheme 1. Synthesis of Fmoc-protected PNA backbone intermediate 2: (a) 4°C → room temp., 12 h, neat (56%);[31] (b) PTSA, toluene, reflux with Dean-Stark setup,
3 h (99%);[17] (c) MeOH–toluene (1:1), reflux at 110 °C, 12 h (94%); (d) Fmoc-OSu, DIPEA, THF, 0 °C → room temp., 3 h (96%).
Screening of Coupling Conditions and Oligomerization
studies
automated synthesis, however, it is required that the coupling
reagent displays sustained solubility (i.e., without a propensity for
precipitation upon standing) in a solvent appropriate for SPS.
Microwave-assisted heating may confer improved yield even with
shorter coupling time as compared to the use of a traditional
heating block. Decomposition of reagents often occurs when
coupling is carried out for a prolonged time in a heating block, and
it may result in some precipitation of byproducts that occasionally
leads to clogging of filters. Hence, we extended our optimization
studies to include testing of promising coupling conditions for
Fmoc/Boc-protected monomers for compatibility with different
SPS options.
Recent developments in assembly of PNA oligomers on solid
support comprise optimization of conditions such as temperature
and reaction time (in order to improve the yield), while
investigations aimed at establishing the optimal coupling reagent
in Fmoc-based SPS of PNA oligomers only include exploration of
a
few commonly used older reagents. Thus,
a more
comprehensive and comparative study was warranted. Once
optimized conditions for manual coupling are obtained these will
be examined with respect to feasibility for direct transfer to
O
O
O
NH
NH
a
NH
ref. [8,32]
N
O
N
O
O
N
O
O
O
H
N
OH
3a
FmocHN
d
OR
: R = Me
7a
8a
: R = H
NHBoc
N
NHBoc
N
NH2
N
a
N
O
ref. [13]
N
O
O
O
O
N
H
O
N
OH
4a
FmocHN
d
OR
: R = Me
: R = H
7b
8b
NHBoc
N
NHBoc
N
NHBoc
N
NH2
N
N
N
N
N
a
N
ref. [13]
ref. [13]
N
N
O
N
N
O
N
N
O
N
H
O
N
OBn
5a
OH
5b
FmocHN
d
OR
c: R = Me
c: R = H
7
8
O
O
N
N
Cl
N
N
HN
BocHN
HN
BocHN
N
a
ref. [13]
N
N
O
N
O
N
H
H2N
N
O
N
OH
6a
FmocHN
d
OR
: R = Me
: R = H
7d
8d
Scheme 2. Synthesis of Fmoc/Boc-protected PNA monomers (a) 2, TSTU, DMF, room temp., 12 h (to give 7a, 91%; 7b, 87%; 7c, 83%; or 7d, 75%); (b) LiOH, and
then Fmoc-OSu, THF–H2O, room temp., 3 h (to give 8a, 90%; 8b, 92%; 8c, 90%; or 8d, 90%).
3
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10.1002/ejoc.202100278
European Journal of Organic Chemistry
FULL PAPER
In a previous report on microwave-assisted synthesis of
peptide-PNA conjugates we performed some preliminary studies
toward an improved coupling protocol.[14] In the present work, we
explore this crucial step in more detail to identify the optimal
coupling conditions for assembly of PNA oligomers, and thus
extends the validation of the general utility of Fmoc/Boc-protected
monomers further.
First, SPS of a short, PNA oligomer (i.e., Fmoc-TGACT-NH2
that contains all four nucleobases) was examined by using
different coupling agents, thereby also validating the general
coupling efficiency of Fmoc/Boc-protected monomers via analysis
In a previously reported comparison of HATU and HBTU as
coupling agents in the synthesis of purine-rich PNA oligomers it
was found that PNA monomer (2.5 equivalents) activated with
HOBt/HBTU performed best when using N-methylmorpholine
(NMM)/pyridine as base.[34]
In our screening experiments standard coupling conditions
were used for assembly of Fmoc-TGACT-NH2: 5 equivalents of
PNA monomer in combination with equal amounts of activating
reagent and base were applied at room temperature with a
coupling time of 30 min. For each coupling reagent, the resulting
crude product was subjected to analytical HPLC. This comparison
(see Table 1) showed that DIC in combination with Oxyma or
HOAt afforded the lowest purity of the crude poduct (50-55%). In
contrast, both aminium and phosphonium coupling reagents
performed better with an HPLC purity of the crude product in the
range 65-80%. Likewise, COMU and HDMA proved better than
DIC with an HPLC purity of 63% and 73%, respectively. Overall,
the most reactive phosphonium salts PyAOP and PyOxim proved
to be superior with 76% and 80% purity, respectively (see Table
1, entries 1-9; for MALDI-TOF MS see SI: S16-S17).
of
the
crude
products
by
matrix-assisted
(MALDI-TOF)
laser
mass
desorption/ionization
time-of-flight
spectrometry (MS). Thus,
a standard Fmoc-Rink amide
polystyrene (PS) resin was used in the initial screening of nine
coupling agents including the most widely used (see Figure 1).
N,N'-Diisopropylcarbodiimide (DIC) in combination with
ethyl cyanohydroxyiminoacetate (Oxyma) or 1-hydroxy-7-
azabenzotriazole (HOAt), aminium salts [i.e., 2-(1H-benzotriazol-
1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU)
and (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]-
O
pyridinium
3-oxide hexafluorophosphate (HATU)],
and
N
phosphonium salts [i.e., (benzotriazol-1-yl-oxy)tripyrrolidino-
phosphonium hexafluorophosphate (PyBOP), (7-azabenzo-
triazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate
N
O
N
C
HO
O
N
N
N
N
O
O
H
ethyl cyano(hydroxyimino)acetato-O2]tri-1-
O
(PyAOP),
and
DIC
PF6
N
pyrrolidinylphosphonium hexafluorophosphate (PyOxim)] were
selected as common coupling reagents together with 1-cyano-2-
Oxyma
COMU
O
ethoxy
2-oxoethylidenaminooxy)dimethylamino-morpholino-
N
N
carbenium hexafluorophosphate (COMU) and 1-[(dimethyl-
amino)(morpholino)methylene]-1H-[1,2,3]triazolo[4,5-b]pyridine-
1-ium-3-oxide hexafluorophosphate (HDMA).
N
N
PF6
N
N
N
N
N
N
HN
PF6
OH
N
X
N
O
N
O
Table 1: Screening of coupling conditions during SPS of FmocHN-TGCAT-NH2
N
HOAt
X = C : HBTU
X = N : HATU
Entry
Activator
Base
HPLC Purity
(260 nm)[a]
HDMA
5.0 eq Monomer, 5.0 eq Activator, 5.0 eq Base, 30 min, RT
1
2
3
4
5
6
7
8
9
DIC/Oxyma
DIC/HOAt
HBTU
Oxyma
HOAt
53.3
50.5
72.9
65.9
64.9
76.0
79.8
63.0
72.7
N
N
N
DIPEA
DIPEA
DIPEA
DIPEA
DIPEA
DIPEA
DIPEA
N
N
O
X
HATU
N
N
P
N
O
PyBOP
PyAOP
PyOxim
COMU
P
N
O
O
PF6
N
PF6
N
HDMA
X = C : PyBOP
X = N : PyAOP
5.0 eq Monomer, 5.0 eq Activator, 5.0 eq Base, 15 min, RT
PyOXim
10
11
12
13
HBTU
HATU
PyOxim
HDMA
DIPEA
DIPEA
DIPEA
DIPEA
74.2
68.6
80.6
69.3
Figure 1.Coupling reagents and auxiliary nucleophile used in oligomerization
Based on these results we selected four of the most
promising coupling agents for further testing. HATU was included
as it is widely used, while PyOxim represented the class of
phosphonium salts. In order to facilitate identification of the best
coupling agent, we reduced the coupling time from 30 min to 15
min under otherwise similar conditions. The purity of the crude
products were estimated by analytical HPLC, which indicated that
PyOxim and HBTU performed best, yielding a purity of 74% and
80%, respectively, while HDMA and HATU only resulted in 69%
purity of the crude product (see Table 1, entries 10-13).
4.0 eq Monomer, 4.0 eq Activator, 4.0 eq Base, 10 min, RT
14
15
16
17
HBTU
HATU
PyOxim
HDMA
DIPEA
DIPEA
DIPEA
DIPEA
72.8
68.6
80.6
70.3
4.0 eq Monomer, 4.0 eq Activator, 4.0 eq Base, 10 min, RT
18
19
20
HATU
PyOxim
PyOxim at 40°C
DIPEA
DIPEA
DIPEA
33.8 (14%)[b]
67.5 (54%)[b]
59.3
[a]
HPLC-based purity of crude PNA products measured at 260 nm (close to
absorption max. of both PNA and Fmoc).
[b] Isolated yield after purification by preparative HPLC.
4
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10.1002/ejoc.202100278
European Journal of Organic Chemistry
FULL PAPER
Figure 2. Analytical HPLC chromatograms showing purityof crude products when using manual synthesis. Coupling conditions: (A) 5.0 eq Monomer, 5.0 eq activator,
5.0 eq base, 30 min, room temperature (RT); (B) 4.0 eq Monomer, 4.0 eq activator, 4.0 eq base, 15 min, RT; (C,D) 4.0 eq Monomer, 4.0 eq activator, 4.0 eq base,
10 min, RT; (E,F) 4.0 eq Monomer, 4.0 eq activator, 4.0 eq base, 10 min, RT or 40°C (automated SPS).
5
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10.1002/ejoc.202100278
European Journal of Organic Chemistry
FULL PAPER
However, it remained quite difficult to clearly distinguish
which reagent was superior for PNA oligomer synthesis at this
stage as no major differences in the HPLC chromatograms were
observed between PyOxim and HBTU (see Figure 2, panel A).
In the next round of experiments, the excess of monomer
was reduced from 5 to 4 equivalents and the coupling time
reduced further to only 10 min to reflect that the ideal is to achieve
satisfactory purity of the PNA oligomer while using the lowest
excess of PNA monomers in the most rapid transformation. By
analysis of the crude products PyOxim was found to be the
optimal coupling reagent with less formation of byproducts (due
to incomplete coupling) as compared to HATU, HBTU and HDMA
(see Figure 2, panels C and D). The main byproducts, formed
during oligomer assembly, were isolated, and found to be a
mixture missing one or two monomers as identified via MALDI-
TOF spectra of the peaks collected by preparative HPLC.
In contrast, HATU-based PNA SPS at room temperature
resulted in only 33% purity of the desired oligomer using manual
synthesis. This relatively lower purity appeared to arise from
formation of N-terminal tetramethylguanidinium byproducts
(Figure 3) also seen in SPPS.[36-37] MALDI-TOF MS analysis of
this isolated fraction by preparative HPLC (see SI: S18-S201) was
in accordance with this side reaction. Since this continuously
occurs during PNA assembly such terminated chains accumulate
resulting in a lower yield of the desired PNA oligomer. To analyse
this further we attempted to separate the constituents of this
byproduct fraction, but this proved unsuccessful.
Instead, we deigned a simple experiment with a shorter
oligomer (i.e., TGACT) to confirm that such byproduct formation
can be observed when using HATU, while it is absent in the
presence of PyOxim. Thus, two portions of resin-bound Fmoc-
TGACT (assembled with PyOxim as coupling reagent) were
Fmoc-deprotected and then separately exposed to HATU and
PyOxim in the absence of monomer building block for 30 min.
After, washing the resins they were subjected to cleavage and the
crude products were analyzed by HPLC and MALDI-TOF. The
latter showed that treatment with HATU resulted in complete
conversion into the N-terminal tetramethylguanidinium derivative,
while PyOxim left the pentamer unchanged (see SI: S21-S22).
In order to further validate the general applicability we also
addressed the previously reported difficulty in SPS of the
pentamer sequence of G monomers seemingly arising from
undesired modification of G residues when using PyBOP.[38]
Nevertheless, this appeared not to occur during incorporation of
neither Fmoc/Boc- or Fmoc/Bhoc-protected G monomers under
the conditions developed here (see SI: S15). Also, in another
report single G deletion sequences were observed,[29] which was
not observed either when performing SPS of Fmoc-GGGGG-
CONH2 oligomer with PyOxim as judged from analytical HPLC
data (Figure 2, panel B, see SI: S15).
Conclusion
Overall, an improved SPS protocol for assembly of PNA
oligomers from Fmoc/Boc-protected monomers was established.
The experiments presented here demonstrate that Fmoc/Boc-
protected PNA monomers can be efficiently synthesized and
applied in oligomer assembly. In addition, Fmoc/Boc-protected
PNA monomers were coupled equally efficiently as compared to
the standard Fmoc/Bhoc-protected PNA monomers, albeit with
the benefit of less byproduct impurities present in the crude due
to Boc being traceless upon TFA cleavage, facilitating purification
by preparative RP-HPLC. Interestingly, the use of PyOxim as
coupling reagent during PNA SPS proved superior to the
commonly used HATU-based protocols. The PyOxim-based
conditions were shown to be compatible with manual SPS at room
temperature and at 40°C. In addition, we did not observe
modification of G-containing residues or deletion of G during
synthesis of a G-based pentamer PNA when using the PyOxim-
based coupling protocol. Finally, we showed compatibility of the
coupling conditions with automated PNA oligomer assembly at
40°C.
Figure 3. Byproduct formation when using HATU for oligomer assembly.
With these promising results at hand, this optimized protocol
was validated in the synthesis of a longer oligomer comparing
PyOxim and HATU. Thus, the efficiency of this protocol was
assessed for assembly of an Fmoc-protected 10-mer antisense
acpPNA sequence[35] (i.e., Fmoc-CTCATACTCT-CONH2).
Moreover, the effect of temperature on coupling efficiency was
examined by comparing SPS at room temperature (∼25°C) with
that at 40°C using automated SPS with PyOxim (see Figure 2,
panels E/F). This relativey low temperature was selected, as we
previously had found that extensive decomposition (mainly loss of
Boc) occurs above 45°C. Assembly of Fmoc-CTCATACTCT-
CONH2 at room temperature using PyOxim or HATU led to a
considerable difference in HPLC-based purity of the crude
products. In case of PyOxim-based SPS using manual as well as
automated synthesis a crude purity of 67% was found for SPS at
room temperature, while manual SPS at 40°C gave rise to 59%
purity.
6
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10.1002/ejoc.202100278
European Journal of Organic Chemistry
FULL PAPER
rinsed with Et2O (100 mL) to give the desired product as an off white solid
(94.2%) that were used directly in the next step.
Experimental Section
General Methods. Starting materials and solvents were purchased from
commercial suppliers (ABCR GmbH, Alfa Aesar, Carbosynth, Fluka, Iris
Biotech, LabScan, Matrix Innovation, Merck, Sigma Aldrich and VWR) and
were used without further purification. Coupling agents were purchased
from Aldrich (PyOxim, COMU), APExBIO (HOAt), Combi-Blocks (PyBOP),
Fluorochem (HATU, PyAOP), Irish (DIC, HBTU, TSTU) and PurePrepTM
(HDMA, Oxyma).
Methyl N-(2-(Fluorenylmethoxycarbonyl)aminoethyl)glycinate[17,39]
Methyl N-(2-aminoethyl)glycinate·2TsOH (from above) and Fmoc-OSu
(7.5 g, 22.2 mmol) were suspended in THF (50 mL), and then the resulting
mixture was cooled to 0°C, and then a solution of DIPEA (11 mL, 63.0
mmol) in THF (15 mL) was added dropwise over 15 min. The mixture was
stirred at 0°C for 1 h, removed from the ice bath, and then stirred for an
additional 15 min. The reaction mixture was poured into a separatory
funnel containing EtOAc (200 mL) and saturated NaHCO3 (50 mL). The
organic layer was washed successively with saturated NaHCO3 (50 mL),
water (50 mL), and brine (2 × 50 mL), dried over Na2SO4, and concentrated
in vacuo. The crude compound 2 gummy solid (8.0 g, 96%) was sufficiently
pure to be used directly in the next step. 1H NMR (400 MHz, Chloroform-
d): δH = 7.78 (dt, J = 7.5 Hz, 2H), 7.63 (d, J = 7.5 Hz, 2H), 7.42 (tt, J = 7.5,
1.0 Hz, 2H), 7.33 (dt, J = 7.5 Hz, 2H), 5.39 (s, 1H), 4.43 (d, J = 7.0 Hz, 2H),
4.24 (t, J = 7.0 Hz, 1H), 3.75 (s, 3H), 3.43 (s, 2H), 3.31 (q, J = 5.7 Hz, 2H),
2.78 (t, J = 5.7 Hz, 2H). 13C NMR (100.6 MHz, Chloroform-d): δC = 172.9,
156.5, 144.0, 141.3, 127.6, 127.0, 125.1, 119.9, 77.4, 77.0, 76.7, 66.6,
51.9, 50.3, 48.7, 47.3, 40.7. Spectroscopic data are in agreement with
those reported in the literature.[39]
Compounds 3a,[8,32] 4a,[13] 5a,[13] 5b,[13] 6a[13] & 7a[13] are synthesized
according to reported literature report.
Deionized water was filtered (0.2 µm) in-house by use of Evoqua Water
Technologies. Column chromatography was performed on silica gel 60
(0.015-0.040 mm) from Merck. TLC analysis was conducted on TLC
aluminium sheets (Merck, silica gel 60, F245). Compounds were visualized
by UV absorption (λ = 254 nm) or by dipping in ninhydrin solution followed
by heating.
1H and 13C NMR spectra were recorded on a Bruker Avance III (400 MHz
and 100.6 MHz, respectively). NMR spectra were recorded in chloroform-
d
or DMSO‑d6 with chemical shifts (δ values) given relative to
tetramethylsilane (δ = 0 ppm). Note: * indicate the presence of peaks
coming from a minor rotamer.
Analytical HPLC was performed on aPhenomenex Luna C18(2) HST
column (100 mm×3 mm; particle size: 2.5 μm; pore size: 100 Å) on a
Shimadzu Prominence and Shimadzu Nexera system using an aqueous
MeCN gradient with 0.1% trifluoroacetic acid (TFA) added (eluent A: 5:95
MeCN-H2O + 0.1% TFA; eluent B: 95:5 MeCN-H2O + 0.1% TFA); a flow
rate of 0.5 mL/min with a linear gradient over 15 min was used.
Preparative reversed-phase high-pressure liquid chromatography (RP-
HPLC) was performed by using a Phenomenex Luna C18(2) column (250
× 21.2 mm, particle size: 5 µm) on a Shimadzu system consisting of a
CBM-20A Prominence communication bus module, two LC-20AP
Prominence pumps, an SPD-M20A Prominence diode array detector, and
an SIL- 20A HT Prominence autosampler. Elution was performed by using
an aqueous MeCN gradient with 0.1% TFA (eluent A: 5:95 MeCN-
H2O:0.1% TFA, eluent B: 95:5 MeCN-H2O:0.1% TFA (using a 0 → 40% B
or a 0 → 60% B gradient).
Modified purification protocol for 5a:[13] Compound 5a was synthesized
according to literature procedure reported in [13] with slight modifications
of the purification protocol. In brief: to benzyl adenine-9-acetate (20 g, 70.5
mmol, 1.0 eq) in DMF (140 mL) was added carbonyldiimidazole (34.3 g,
211 mmol, 3.0 eq), and then the reaction was slowly heated to 105 °C, and
then stirred at that temperature for 2 h. The temperature was reduced to
95 °C, and tert-butanol (20.25 mL, 211, 3.0 eq) was added. The heating
source was removed, and then the mixture was stirred overnight while
cooling to room temperature. The DMF was removed on rotary evaporator,
water (400 mL) was added, and then the mixture was extracted with
CH2Cl2. The organic layer was separated, and then washed with brine. The
organic layer was dried on anhydrous Na2SO4 and evaporated to give a
crude compound, which was crystallized from CH2Cl2 by dissolution in
minimum amount of CH2Cl2 and by keeping the solution at room
temperature for 1 day at room temperature to provide pure 5a (15.0 g,
55%); m.p 159 °C. 1H NMR (400 MHz, DMSO-d6): δH = 10.11 (s, 1H), 8.59
(s, 1H), 8.44 (s, 1H), 7.44 – 7.31 (m, 5H), 5.28 (s, 2H), 5.22 (s, 2H), 1.58
(s, 1H), 1.49 (s, 9H). 13C NMR (101 MHz, DMSO): δC = 168.1, 152.5, 152.1,
151.6, 150.4, 144.8, 135.8, 128.9, 128.7, 128.5, 123.5, 80.6, 67.2, 44.7,
28.3. Spectroscopic data are in agreement with those reported in the
literature.[13]
All intermediates were more than 95% pure as determined by HPLC, and
identity established by MALDI-TOF MS (on a Bruker MicroTOF-Q II
equipped with an electrospray ionization interface operated in positive
ionization mode) and/or HRMS on Solarix XR FTMS Bruker (SI: S23-S30).
N-(2-aminoethyl)glycine ⋅ 2TsOH[17]
A suspension of N-(2-aminoethyl)glycine (19.0 g, 0.16 mol) and PTSA
(68.0 g, 0.36 mol) in toluene (400 mL) was refluxed for 3 h. The solvent
was removed in vacuo, and Et2O (300 mL) was added. After the mixture
was kept at –20 °C for 2 h, the precipitate was collected by filtration, and
then rinsed with more Et2O (100 mL) to give 1 as a white solid (74.0 g,
99%).1H NMR (600 MHz, DMSO-d6): δ = 9.05 (s, 1H), 7.95 (s, 3H), 7.53
(d, J = 8.1 Hz, 4H), 7.15 (d, J = 8.1 Hz, 4H), 3.97 (s, 2H), 3.25 (t, J = 6.9
Hz, 2H), 3.15-3.20 (bs, 2H), 2.30 (s, 6H). 13C NMR (151 MHz, DMSO) δ
168.5, 145.4, 138.60, 128.7, 126.0, 47.7, 44.2, 35.6, 21.3. Spectroscopic
data are in agreement with those reported in the literature.[17]
Coupling of Boc-protected nucleobase-substituted acetic acids to
Methyl N-(2-Fluorenylmethoxycarbonyl-aminoethyl)glycinate
Boc-protected nucleobase-substituted acetic acid (1.5 eq., 3a: 5.8 g, [8,32]
4a: 9.1 g, [13] 5b: 9.9 g, [13] or 6a: 5.9 g[13] was dissolved in DMF (50 mL)
and cooled to 0°C. To this solution TSTU (1.5 eq.) was added, and after 5
min DIPEA (2.0 eq) was added under stirring. After an additional 15 min,
Fmoc-HN(CH2)2NHCH2COOMe (2, 7.4 g, 8.0 g, 8.0 g or 4.5 g) in DMF (25
mL) was added slowly, and then the mixture was stirred for 16 h. DMF was
removed on a rotary evaporator at 45°C by using a high-vacuum pump.
The resulting crude mixture was dissolved in EtOAc, which was washed
with satd brine, satd aq. NaHCO3, and then dried over anhydrous Na2SO4.
After evaporation of the organic layer, the crude product was purified by
VLC (7 × 10 cm, silica gel 60: 15-40 µm) using 0 → 5% MeOH in CH2Cl2
as eluents to give the desired methyl esters as solid (7a: 10.0 g, 91%; 7b:
11.9 g, 87%, 7c: 11.7 g, 83%, or 7d: 6.2 g, 75%).
Methyl N-(2-aminoethyl)glycinate-2TsOH[17]
MeOH (150 mL) was added to a suspension of 1 (11.7 g, 25.0 mmol) and
PTSA (1.0 g, 5.3 mmol) in toluene (150 mL). The resulting suspension was
refluxed (oil bath, 110 °C) for 12 h. The reaction mixture was concentrated
to an oil, and then Et2O (200 mL) was added. The resulting solution was
sonicated for 30 min to initiate precipitation. Then the suspension was kept
at –20 °C for 12 h, after which the precipitate was collected by filtration and
7
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10.1002/ejoc.202100278
European Journal of Organic Chemistry
FULL PAPER
Methyl N-[2-(Fluorenylmethoxycarbonyl)aminoethyl]-N-[(Thymine-1-
yl)acetyl]glycinate (7a)
40.4, 40.2, 39.9*, 39.7, 39.6*, 39.4, 38.4, 28.2*, 28.2*, 28.2. IR ν 3228,
2980, 2970, 1740, 1697, 1675, 1577, 1478, 1355, 1247, 1216, 1150, 1095,
1001, 840, 760, 741 cm-1. HRMS (MALDI-TOF +): m/z calcd for
C32H35N7O8+H-Boc: 646.2625 [M+H-Boc]+; found: 646.2637.
Yield 91%; white solid; m.p 160 °C; 1H NMR (400 MHz, DMSO-d6): δH
=
11.28 (s, 1H)*, 7.89 (d, J = 7.5 Hz, 2H), 7.68 (dd, J = 7.6, 3.5 Hz, 2H), 7.46
– 7.37 (m, 3H), 7.34 (dd, J = 7.5, 1.2 Hz, 2H), 7.32 – 7.21 (m, 2H), 4.66 (s,
1H), 4.47 (d, J = 6.2 Hz, 1H), 4.38 – 4.29 (m, 2H), 4.20 (m, 2H), 4.07 (s,
1H), 3.67 (s, 3H), 3.44 (t, J = 6.8 Hz, 1H), 3.25 (d, J = 6.5 Hz, 1H), 3.16 –
Analytical HPLC chromatograms for compounds 7a, 7b, 7c and 7d: see SI
S14.
3.08 (m, 1H), 1.79 – 1.71 (s, 3H)*. 13C NMR (100.6 MHz, DMSO-d6): δC
=
Hydrolysis of methyl ester intermediates[33]
170.3, 169.9*, 168.1, 167.9*, 164.8, 156.8, 151.4, 144.3, 142.5, 141.2,
128.9, 127.5, 125.6, 120.6, 108.6, 108.5, 65.9, 65.8*, 52.7, 52.2, 48.2,
48.0*, 47.3, 47.2, 40.6, 40.4, 40.2, 40.0, 39.8, 39.6*, 39.4, 38.7. IR ν 3300,
1740, 1697, 1666, 1544, 1475, 1355, 1277,1216, 1155, 1017, 842, 758,
733 cm-1. HRMS (MALDI-TOF +): m/z calcd for C27H28N4O7Na: 543.1856
[M+Na]+; found: 543.1866.
To a solution of the methyl ester of Fmoc/Boc-protected monomer (7a:
10.0 g, 7b: 11.9 g, 7c:11.7 g, 7d: 6.2 g) in dioxane–H2O (2:1; for 7a, 7b
and 7c: 250 mL, and for 7d: 125 mL) at 0 °C was added LiOH·H2O (2.5
eq.). The resulting solution was allowed to warm to room temperature, and
it was then stirred for an additional 30 min. Then 20% citric acid (approx.
8-9 mL) was added to adjust the reaction mixture to pH 7, and then sat.
aq. NaHCO3 (approx. 4.0 mL) was subsequently added to adjust the pH to
8. The resulting mixture was cooled to 0 °C, and then Fmoc-OSu (1.0 eq)
was added. The resulting solution was allowed to warm to room
temperature, and was then stirred at room temperature for 16 h. The
reaction mixture was washed with Et2O (3 × 30 mL). The aqueous layer
was acidified with 20% citric acid to pH 3, and was then extracted with
EtOAc (3 × 50 mL). The combined organic layers were washed with brine,
dried over Na2SO4, filtered, and concentrated under reduced pressure.
The residue was triturated with Et2O (30 mL) to provide a precipitate, which
was filtered and washed with Et2O (3 × 20 mL) to give the desired
compound as a white solid (8a: 8.8 g, 90%; 8b: 10.6 g, 92%; 8c: 11.7 g,
90%; and 8d: 6.2 g, 90%).
Methyl
N-[2-(Fluorenylmethoxycarbonyl)aminoethyl]-N-[(N4-(tert-
butoxycarbonyl)cytosin-1-yl) acetyl]glycinate (7b)
Yield 87%; pale yellow solid; m.p 145 °C;, 1H NMR (400 MHz, DMSO-d6):
δH = 10.29 (s, 1H)*, 7.89 (d, J = 7.4 Hz, 2H), 7.88 – 7.79 (m, 1H), 7.69 (dd,
J = 7.6, 4.0 Hz, 2H), 7.46 – 7.37 (m, 3H), 7.33 (td, J = 7.4, 1.2 Hz, 2H),
6.97 (dd, J = 7.3, 4.7 Hz, 1H), 4.82 (s, 1H), 4.63 (s, 1H), 4.33 (d, J =6.6
Hz, 2H), 4.24 (t, J = 7.2 Hz, 1H), 4.08 (s, 1H), 3.63 (s, 3H), 3.47 (q, J = 5.0,
3.0 Hz, 1H), 3.29 (d, J = 6.4 Hz, 2H), 3.18 – 3.09 (m, 2H), 1.46 (s, 9H). 13
C
NMR (100.6 MHz, DMSO-d6): δC = 169.9, 167.8, 162.8, 156.8, 156.6,
155.5, 152.6, 144.3, 141.2, 128.1, 127.5, 125.6, 120.6, 94.4, 81.4, 65.9,
54.1, 52.7, 52.2, 49.8, 48.3, 47.5, 47.2, 42.3, 40.6, 40.4, 40.2, 40.0, 39.8,
39.6, 39.4, 38.7, 28.2. IR ν 3206, 2980, 2970, 1740, 1671, 1654, 1560,
1503, 1450, 1370, 1227, 1147, 837, 738 cm-1. HRMS (MALDI-TOF +): m/z
calcd for C31H35N5O8+H-Boc: 506.2040 [M+H-Boc]+; found: 506.2049.
N-[2-(Fluorenylmethoxycarbonyl)aminoethyl]-N-[(Thymine-1-
yl)acetyl]glycinate (8a)
Methyl
N-[2-(Fluorenylmethoxycarbonyl)aminoethyl]-N-[(N6-(tert-
Yield 90.2%; 1H NMR (400 MHz, DMSO-d6): δH = 11.24 (br s, 1H), 7.88 (d,
J = 7.5 Hz, 2H), 7.68 (d, J = 7.5 Hz, 2H), 7.62 – 7.49 (m, 1H), 7.41 (t, J =
7.4 Hz, 2H), 7.33 (t, J = 7.4 Hz, 2H), 7.28 – 7.10 (m, 1H), 4.63 (s, 1H), 4.46
(s, 1H), 4.32 (d, J = 7.0 Hz, 1H), 4.22 (q, J = 5.5 Hz, 2H), 3.89 (d, J = 14.2
Hz, 2H), 3.39 (m, 2H), 3.26 (q, J = 6.5 Hz, 1H), 3.14 (q, J = 6.3 Hz, 1H),
1.71 (s, 3H). 13C NMR (100.6 MHz, DMSO-d6): δC = 168.1, 167.3, 164.9,
156.8, 156.5, 151.5, 151.5, 144.4*, 144.3, 142.6, 141.2*, 141.1, 139.9,
134.4*, 134.3, 130.8*, 130.7, 129.4*, 129.4, 128.7, 128.1*, 128.1, 127.7,
127.6, 127.5, 125.8*, 125.7, 125.6, 121.8, 120.6, 120.5*, 120.5, 110.2,
108.4, 66.0, 52.2, 48.2, 47.9, 47.2, 40.6, 40.4, 40.2*, 40.0, 39.8*, 39.6,
39.4, 38.5. HRMS (MALDI-TOF +): m/z calcd for C26H26N4O7+H-Boc:
507.1880 [M+H-Boc]+; found: 507.1895. Spectroscopic data are in
agreement with those reported in the literature.[8]
butoxycarbonyl)adenin9-yl) acetyl]glycinate (7c)
Yield 83%; glassy solid; 1H NMR (400 MHz, DMSO-d6): δH = 10.07 (s, 1H),
8.50 (s, 1H), 8.29 (d, J = 1.4 Hz, 1H), 7.89 (dd, J = 7.5, 2.5 Hz, 2H), 7.68
(t, J = 7.8 Hz, 2H), 7.51 (t, J = 5.8 Hz, 1H), 7.41 (td, J = 7.7, 2.4 Hz, 2H),
7.36 – 7.24 (m, 2H), 5.35 (s, 1H), 5.17 (s, 1H), 4.46 (s, 1H), 4.39 (d, J =
6.8 Hz, 1H), 4.34 – 4.16 (m, 2H), 4.10 (s, 1H), 3.77 (s, 1H), 3.62 (s, 3H),
3.49-3.28(m, 3H), 3.16 (m, 1H), 1.49 (s, 9H). 13C NMR (100.6 MHz,
DMSO-d6): δC = 170.3*, 169.8, 167.6*, 167.2, 156.9, 156.6*, 152.7, 151.9*,
151.6, 150.2, 145.4, 145.3*, 144.3, 141.2, 141.2, 128.9, 127.5, 125.6,
125.5*, 123.4, 123.3, 120.6, 80.5, 65.9, 65.9*, 62.1, 55.3, 52.8, 52.3, 49.5,
48.2, 47.5, 47.4*, 47.2, 47.2*, 44.5, 44.2, 40.6, 40.4, 40.2, 40.0, 39.8*,
39.6, 39.4*, 39.3, 38.4, 28.3. IR ν 2980, 2970, 1735, 1697, 1669, 1450,
1368, 1224, 1145, 1015, 840, 758, 740 cm-1. HRMS (MALDI-TOF +): m/z
calcd for C32H35N7O7+H-Boc: 530.2152 [M+H-Boc]+; found: 530.2162.
N-[2-(Fluorenylmethoxycarbonyl)aminoethyl]-N-[(N4-(tert-
butoxycarbonyl)cytosin-1-yl)acetyl]glycinate (8b)
Methyl
N-[2-(Fluorenylmethoxycarbonyl)aminoethyl]-N-[(N2-(tert-
Yield 92.1%; 1H NMR (400 MHz, DMSO-d6): δH = 10.28 (br s, 1H), 7.91 –
7.87 (m, 2H), 7.87 – 7.79 (m, 1H), 7.68 (dd, J = 7.5, 4.6 Hz, 2H), 7.48 –
7.29 (m, 5H), 6.97 (dd, J = 7.3, 5.3 Hz, 1H), 4.81 (s, 1H), 4.63 (s, 1H), 4.34
(d, J = 6.9 Hz, 1H), 4.31 – 4.17 (m, 2H), 4.05 (s, 1H), 3.97 (s, 1H), 3.45 (t,
J = 7.0 Hz, 1H), 3.33 (t, J = 6.7 Hz, 2H), 3.20 – 3.09 (m, 1H), 1.46 (s, 9H).
13C NMR (100.6 MHz, DMSO-d6): δC = 168.0, 167.4, 163.7, 163.6, 156.6,
155.6, 152.6, 150.9, 144.3, 141.2, 141.2*, 128.1, 127.5, 125.6, 125.6,
120.5, 94.3, 81.4, 66.0, 55.3, 50.0, 49.8, 47.2, 40.6, 40.4, 40.2, 39.9, 39.7,
39.5, 39.3, 28.2. HRMS (MALDI-TOF +): m/z calcd for C30H33N5O8+H-Boc:
492.1883 [M+H-Boc]+; found: 492.1901. Spectroscopic data are in
agreement with those reported in the literature.[13, 14]
butoxycarbonyl)-guanin-9-yl)acetyl)]glycinate (7d)
Yield 75%; white solid; 1H NMR (400 MHz, DMSO-d6): δH = 11.15 (s, 1H),
7.88 (dd, J = 7.5, 5.1 Hz, 2H), 7.79 (d, J = 5.7 Hz, 1H), 7.67 (dd, J = 7.6,
3.5 Hz, 2H), 7.50 – 7.41 (m, 1H), 7.44 – 7.38 (m, 1H), 7.39 (d, J = 3.2 Hz,
1H), 7.35 – 7.21 (m, 2H), 5.08 (s, 1H), 4.92 (s, 1H), 4.43 (s, 1H), 4.38 (d,
J = 6.7 Hz, 1H), 4.31 (d, J = 6.9 Hz, 1H), 4.23 (t, J = 6.7 Hz, 1H), 4.10 (s,
1H), 3.76 (s, 1H), 3.63 (s, 2H), 3.53 (t, J = 6.7 Hz, 1H), 3.35 (d, J = 6.6 Hz,
4H), 1.47 (d, J = 4.9 Hz, 9H). 13C NMR (100.6 MHz, DMSO-d6): δC
=
170.4*, 169.9, 167.6*, 167.2, 156.8*, 155.6, 154.3*, 154.3, 148.1, 144.3,
144.2, 143.0, 141.2, 141.2, 140.7, 139.9, 137.9, 129.4, 128.1, 127.7,
127.5, 127.5, 125.6, 125.5, 124.7, 121.8, 120.6, 120.5, 119.4, 110.2, 82.9,
65.9, 55.4, 52.8*, 52.3, 49.6, 49.1, 48.4, 47.5*, 47.2, 44.4*, 44.2, 40.6*,
N-[2-(Fluorenylmethoxycarbonyl)aminoethyl]-N-[(N6-(tert-
butoxycarbonyl)adenine-9-yl) acetyl]glycinate (8c)
8
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10.1002/ejoc.202100278
European Journal of Organic Chemistry
FULL PAPER
Yield 89.8 %; 1H NMR (400 MHz, DMSO-d6): δH = 10.04 (br s, 1H), 8.53
(s, 1H), 8.27 (d, J = 10.5 Hz, 1H), 7.88 (dt, J = 7.7, 1.0 Hz, 2H), 7.66 (d, J
= 7.7 Hz, 3H), 7.40 (td, J = 7.5, 1.2 Hz, 2H), 7.30 (dtd, J = 8.6, 4.8, 2.5 Hz,
2H), 5.14 (s, 1H), 4.36 (d, J = 7.0 Hz, 1H), 4.30 – 4.17 (m, 3H), 3.97 (s,
1H), 3.89 (s, 1H), 3.56 (s, 1H), 3.43 – 3.32 (m, 2H), 3.18 (s, 1H), 1.48 (s,
9H). 13C NMR (100.6 MHz, DMSO-d6): δC = 173.2, 170.9*, 170.8, 167.5*,
166.9, 156.9*, 156.6, 152.7*, 152.7, 151.8*, 151.6, 150.1*, 150.1, 145.5*,
145.4, 144.3*, 144.3, 141.2*, 141.1, 128.1, 128.0, 127.5, 127.5, 125.6,
125.5, 123.3, 123.3, 120.6, 120.5, 80.5, 80.5, 66.8, 66.0*, 65.9, 50.7, 48.3,
47.7*, 47.4, 47.2*, 47.2, 44.5*, 44.2, 40.6*, 40.4, 40.2, 40.0*, 39.8, 39.6*,
39.3, 38.4, 28.3. HRMS (MALDI-TOF +): m/z calcd for C31H33N7O7+H-Boc:
516.1995 [M+H-Boc]+; found: 516.2015.
remove the Fmoc protecting group on the resin. The deprotection was
performed at room temperature (2 × 10 min with 5 mL) with a CH2Cl2 (5
mL) wash in between. Following the deprotection the resin was washed
successively with DMF, CH2Cl2, and DMF (3 × 3 min each with 5 mL). The
solution of the appropriate Fmoc/Boc PNA monomer (5 equiv) in DMF (1.5
mL) was added to a vial with preweighed coupling reagent (5 equiv)
followed by DIPEA (5 equiv), and then the mixture was left to activate for
10 min prior to addition to the Fmoc-deprotected resin, which was left for
30 min before draining. After coupling of the first monomer, the resin was
washed with DMF, CH2Cl2, and DMF (3 × 3 min each with 5 mL). Then the
next PNA-monomers were coupled successively by using the same
protocol till completion of the desired oligomer. After the final Fmoc
deprotection the resin was washed with DMF and CH2Cl2 (3 × 5 min each
with 5 mL). Finally, cleavage was performed with TFA–CH2Cl2 (95:5). Then
samples were air dried and analytical HPLC was performed (gradient: 0 →
80% B over 15 min for FmocTGACT and 0 → 60% B over 15 min for Fmoc-
acpPNA; flow rate: 0.8 mL/min). Purity of samples was measured at 260
nm and supplemented by MALDI-TOF MS analysis (see SI: S16-S20).
N-[2-(Fluorenylmethoxycarbonyl)aminoethyl]-N-[(N2-(tert-
butoxycarbonyl)-guanin-9-yl)acetyl)]glycinate (8d)
Yield 90.2%; 1H NMR (400 MHz, DMSO-d6): δH = 11.44 ( s, H), 11.04 (s,
1H), 8.02 (d, J = 2.7 Hz, 1H), 7.88 (dd, J = 7.6, 4.4 Hz, 2H), 7.66 (t, J = 6.4
Hz, 2H), 7.43 (dd, 7.8, 4.1 Hz, 2H), 7.36 – 7.22 (m, 3H), 5.11 (s, 1H), 4.95
(s, 1H), 4.41 – 4.27 (m, 3H), 4.03 (s, 2H), 3.57 (s, 1H), 3.51 (t, J = 6.9 Hz,
1H), 3.35 (q, J = 6.7 Hz, 2H), 3.13 (q, J = 6.6 Hz, 1H), 1.47 (s, 9H). 13C
NMR (100.6 MHz, DMSO-d6): δC = 174.9, 171.7, 171.3, 170.8, 167.3,
166.8, 156.9, 155.1, 154.2*, 154.2, 149.9*, 149.8, 148.3, 144.3, 144.3,
141.2, 141.2, 140.8, 128.1, 127.5*, 127.5, 125.6, 125.5, 120.6, 118.2*,
118.1, 83.1*, 83.1, 72.9, 66.8, 65.9, 49.7, 48.4, 47.4*, 47.2, 44.6*, 44.4,
43.1, 40.6*, 40.4, 40.2, 40.0*, 39.8, 39.6*, 39.3, 38.3, 28.2, 25.7. HRMS
(MALDI-TOF +): m/z calcd for C31H33N7O8+H-Boc: 532.1945 [M+H-Boc]+;
found: 532.1970. Spectroscopic data are in agreement with those reported
earlier.[13,14]
Protocol for automated synthesis of Fmoc-CTC-ATA-CCT-TG-NH2
using Biotage® Syro Wave™ at 40 °C with MW-assisted heating:
A sample of 0.05 mmol (94 mg) of a polystyrene Rink Amide resin (Matrix
Innovation; loading 0.53 mol/g, 100-200 mesh) were transferred to the
reaction vessel by using CH2Cl2 and DMF. The resin was allowed to swell
for 5 min, and then automated synthesis was started with an assigned
program on Biotage Microwave synthesizer. Fmoc deprotection was
carried out with 30% and 15 % piperidine in DMF for 3 min and 10 min,
respectively, followed by washing with DMF, CH2Cl2, and DMF (2.6 mL
each). After this, coupling reagents were added: 0.25 mmol/mL PNA
monomer (1.0 mL), 0.24 mmol/mL HATU or PyOxim (1.0 mL) and then
0.50 mmol/mL DIPEA (0.5 mL). Coupling was performed for 20 min at 40
°C MW heating, followed by washing with DMF, CH2Cl2, and DMF (2.6 mL
each). Coupling cycles were repeated until the desired sequence was
assembled. Cleavage was performed with TFA–CH2Cl2 (9:1; 200 µL for 30
min). Then a sample of crude Fmoc-acpPNA was dissolved in MeCN–H2O
(50:50, 0.1% TFA), and then analytical HPLC was performed with a
gradient of 0 → 70% B during 15 min (flow: 0.5 mL/min).
Analytical HPLC data for compounds 8a, 8b, 8c and 8d: see SI S15.
Assembly of PNA monomers on resin:
Experiment design for evaluating coupling conditions (manual SPS):
In the initial screening of different coupling reagents in SPS of Fmoc-
TGACT: 5.0 eq of PNA monomer, DIPEA 5.0 eq and coupling was
performed for 30 min at room temperature for all coupling reagents used
in PNA synthesis.After assembly and cleavage an analysis of the crude
product by analyt. HPLC was performed. Based on these preliminary
results, reduced coupling time to 15 min or 10 min were examined to
observe the effect on coupling efficiency. As reduction in coupling time did
not affect the purity of the desired PNA oligomer the number of equivalents
of PNA monomer was reduced from 5.0 to 4.0 eq which resulted in clear
differences in coupling efficiency of different reagents. Then, in order to
identify the side reaction(s) occurring during each coupling using aminium
reagents (e.g., HATU) a fraction containing the impurities from Fmoc-
acpPNA oligomer synthesis was isolated (see SI: S16-S20), and MALDI-
TOF MS indicated these to result from formation of tetramethylguanidinium
derivatives. Further separation of this fraction into its constituents proved
unsuccessful.
Preparative HPLC of Fmoc-CTC-ATA-CCT-TG-NH2 (Fmoc-acpPNA):
The washed resin was treated with TFA- CH2Cl2; 9:1 (2 × 30 min × 5 mL),
and then the resulting mixture was concentrated. The obtained crude
product was purified immediately. The preparative HPLC was equipped
with a Phenomenex Luna C18(2) column (250 × 21.2 mm; particle size: 5
µm) with detection at 220/260 nm and a flow rate of 20 mL/min for the
gradient 0 → 40% B during 20 min. The crude product was dissolved in
eluent mixture A:B (1:1) to give a concentration of ∼100 mg/mL. In the test
run, a small amount of the sample (300 µL) was injected and major peaks
were collected. The identification of the peak of interest was performed by
MALDI-TOF-MS, and then the peak of interest was collected in all
subsequent runs. Pure fractions were identified by analyt. HPLC (0 → 60%
B during 15 min), and were then combined and lyophilised to give the
product. The amount of desired PNA oligomer, obtained by using HATU or
PyOxim as coupling agent, after preparative HPLC were 33.8 mg (14%)
and 66.7 mg (54%), respectively.
Protocol for manual SPS of PNA oligomers:
Briefly, all Fmoc/Boc-protected PNA monomers were coupled by using a
previously reported methodology[16] The assembly of PNA in the above
screening of coupling conditions was performed manually by using an
Activo PLS synthesizer and PTFE reactors (10 mL, from Activotec)
wherein 0.025 mmol (47 mg) of an Fmoc Rink amide polystyrene resin
(Matrix Innovation; 0.53 mmol/g loading, 100-200 mesh) were transferred
with MeOH and CH2Cl2. The the resin were swelled in DMF (3 × 5 mL) and
drained. Then the resin was treated with 20% piperidine in DMF (5 mL) to
Acknowledgements
We would like to thank Birgitte Simonsen and Uraiwan N.
Adamsen for practical assistance with compound purification and
characterisation. Ashif Y. Shaikh was supported by a postdoctoral
9
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10.1002/ejoc.202100278
European Journal of Organic Chemistry
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grant from the Center for Peptide-Based Antibiotics (CEPAN)
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Keywords: Building blocks • Heating, Automation •
Oligomerization• Peptide Nucleic Acid (PNA) • Solid-phase
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