Synthesis and Characterization of Optically Pure Gamma-PNA Backbones by SIBX-Mediated Reductive Amination

Article abstract of DOI:10.1002/bkcs.12365

Chiral peptide nucleic acid (PNA) is a derivative of regular PNA by introducing a chiral center to its backbone, and is known to bind more strongly to DNA or RNA than regular PNA. In particular, in the case of a γ-backbone, the L isomer stabilizes the PNA/DNA duplex, and the D-isomer has the opposite effect. Therefore, the synthesis of an optically pure γ-backbone is very important. Here, we report a novel synthetic strategy for the suppression of epimerization during the synthesis of the γ-PNA backbone. A stabilized form of 2-iodoxybenzoic acid (SIBX) was used as an oxidative reagent in the key intermediate of the N-Boc-amino acetaldehyde synthesis. This paper reports (1) the synthesis and comparison of three different γ-PNA backbones (lysine, alanine, and glutamate) by three different synthetic routes (SIBX, lithium aluminum hydride, and Red-Al) and (2) the determination of chiral purity from their derivative compounds. The enantiomeric excess purity of SIBX-mediated γ-PNA backbones was determined to be more than 99.4%, as ascertained by the high-performance liquid chromatography (HPLC) chromatogram on a standard RP-C18 column. It is comparatively higher than that of the other methods examined in this work.

Full text of DOI:10.1002/bkcs.12365

Article
DOI: 10.1002/bkcs.12365
BULLETIN OF THE
A. Periyalagan et al.
KOREAN CHEMICAL SOCIETY
Synthesis and Characterization of Optically Pure Gamma-PNA
Backbones by SIBX-Mediated Reductive Amination
Alagarsamy Periyalagan,† Yong-Tae Kim,‡ and In Seok Hong†,
*
†Department of Chemistry, College of Natural Science, Konju National University, Chungnam 32588,
Republic of Korea. *E-mail: ishong@kongju.ac.kr
‡Material Division of Research Institute, SEASUN BIOMATERIALS Inc, Daejeon 34015, Republic of
Korea
Received June 2, 2021, Accepted July 10, 2021
Chiral peptide nucleic acid (PNA) is a derivative of regular PNA by introducing a chiral center to its backbone,
and is known to bind more strongly to DNA or RNA than regular PNA. In particular, in the case of a γ-back-
bone, the L isomer stabilizes the PNA/DNA duplex, and the D-isomer has the opposite effect. Therefore, the
synthesis of an optically pure γ-backbone is very important. Here, we report a novel synthetic strategy for the
suppression of epimerization during the synthesis of the γ-PNA backbone. A stabilized form of
2-iodoxybenzoic acid (SIBX) was used as an oxidative reagent in the key intermediate of the N-Boc-amino
acetaldehyde synthesis. This paper reports (1) the synthesis and comparison of three different γ-PNA back-
bones (lysine, alanine, and glutamate) by three different synthetic routes (SIBX, lithium aluminum hydride,
and Red-Al) and (2) the determination of chiral purity from their derivative compounds. The enantiomeric
excess purity of SIBX-mediated γ-PNA backbones was determined to be more than 99.4%, as ascertained by
the high-performance liquid chromatography (HPLC) chromatogram on a standard RP-C18 column. It is com-
paratively higher than that of the other methods examined in this work.
Keywords: Peptide nucleic acid, γ-Backbone, Optical purity, Stabilized 2-iodoxybenzoic acid,
Epimerization
Introduction
In the case of γ-PNA, the L-isomer PNA in the chiral
center has the property of binding more strongly to comple-
mentary DNA or RNA than regular PNA due to the pre-
organization effect for the α-helical duplex formation. On
the other hand, the D-isomer PNA destabilizes the
PNA/DNA duplex, significantly lowering the melting tem-
perature.29,30 Therefore, it is particularly important to syn-
thesize chiral PNA monomers with a high optical purity for
use in medicinal applications.31
Traditionally, reductive amination is the common method
to synthesize the gamma chiral backbone, using commer-
cially available Boc or Fmoc-protected amino acids.32 That
is, after synthesizing the corresponding aldehyde derivatives
of amino acids, reductive amination between the aldehyde
and glycinate ester provides the gamma chiral backbone.
A critical challenge in the chiral PNA backbone synthe-
sis is the epimerization of chiral carbon during amino alde-
hyde synthesis even under milder conditions.33,34 Amino
aldehyde intermediates have been synthesized either by oxi-
dation of the corresponding alcohols35,36 or by reduction of
the acids and esters.37,38 A typical method to prepare the
optically active amino aldehydes is from N-Boc-protected
Weinreb amide derivatives by reduction with lithium alumi-
num hydride (LAH), and previous studies have shown that
this harsh treatment is prone to racemization in the prepara-
tion of amino aldehyde.39,40
Peptide nucleic acids (PNAs) are synthetic DNA analogs in
which N-(2-aminoethyl) glycine units and nucleobases are
linked via a methylene carbonyl group.1 It has the property of
a strong sequence-selective binding to DNA or RNA.2 Thus,
it has been widely used in the fields of chemistry, biology,
biotechnology, and medicine.3–6 In particular, PNAs have
been studied a lot as therapeutic agents such as antigene or
antisense because of their resistance to the proteolytic and
nucleolytic enzymes in vivo.7 However, its application was
limited due to a remarkably low cell permeability, low solubil-
ity in water, and endosomal escape issues.8
To improve these limitations, there has been much
research on the modification of the PNA structure.9–15 One
of the most distinct modifications has been the insertion of
charged amino acids at the interior of the N-(2-aminoethyl)
glycine backbone16–23 (Figure. 1). Nielsen et al. reported the
first α-chiral PNA, in which the glycine unit of the PNA
backbone was substituted by alanine.24 Subsequently, vari-
ous γ-modified PNA backbones have been reported, includ-
ing Ala, Ser, Lys, PEG, and Cys-based PNAs. Compared
with regular achiral PNA, γ-substituted chiral PNA has
advantages in terms of minimal self-aggregation, good solu-
bility, and stronger PNA-DNA duplex formation.25–28
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Figure 1. Chemical structures of regular PNA and modified PNAs.
Stabilized 2-iodoxybenzoic acid (SIBX) is a non-
explosive formulation of the IBX oxidative reagent that can
be used in a variety of solvents, for instance, refluxing with
ethyl acetate or tetrahydrofuran to safely oxidize organic
alcohol groups into the corresponding aldehydes and
ketones.41,42 The aim of this study was to develop a novel
synthetic method to control the epimerization during the
N-Boc-amino acetaldehyde synthesis. Here, we report that
(1) the synthesis of the gamma-lysine, glutamate, and ala-
nine PNA backbones based on SIBX, LAH, and Red-Al
mediated the corresponding aldehyde synthesis and (2) an
extensive study of these γ-PNA backbones verified their
chiral purity using analytical high-performance liquid chro-
matography (HPLC).
reducing agent than LAH, and the optical purities were
measured and compared with each other. To confirm the
effect of temperature, the γ-backbones were synthesized at
ꢁ
ꢁ
two temperatures (ꢀ20 C and 0 C). In addition, a reagent
that converts an alcohol group to an aldehyde group was
investigated as an alternative to the hydride reduction reac-
tion, which is vulnerable to epimerization. As a result, a
mild oxidation reagent, stabilized IBX reagent, was
selected, and it was reported that there is no change in the
optical purity when the alcohol derivative of an amino acid
is converted to the corresponding aldehyde (Scheme 1(B)).
The next step is to determine the target molecules to
be synthesized. In this study, as shown in Figure 2, the
Boc-Lys(Z)-aeg-OMe, Boc-Glu(cHx)-aeg-OMe, and Boc-
Ala-aeg-OMe γ-backbones were selected as three different
targets that have positive, negative, and neutral charges
respectively in PNA oligomers.
Results and Discussion
As a method of confirming the optical purity of the syn-
thesized γ-backbones, the Ly group introduced a chiral auxil-
iary group containing fluorine atoms into the γ-backbone
that generated a diastereomer structure. Thus, the optical
purity was measured with F-NMR. In addition, Lee’s group
proposed a direct method of separating the enantiomers of
the γ-backbones through a chiral HPLC column and mea-
sured the optical purity without any derivatization process.44
However, in both cases, expensive reagents and equipment
are required, and especially, in the case of chiral columns, it
is necessary to optimize the separation conditions.
Here, the (L)-Fmoc-amino acid as a chiral auxiliary was
attached to the secondary amine of the synthesized γ-backbone
to generate the diastereomers, which were directly analyzed by
C18-RP HPLC to measure the optical purity of the γ-backbone
(Scheme 2).
The typical method of making the corresponding amino acet-
aldehyde from protected amino acids is by reducing the
Weinreb amide with LAH shown in Scheme 1(A). In this
case, epimerization may occur when the α-hydrogen of alde-
hyde derivative is deprotonated as a hydride reduction condi-
tion. In the case of the alanine γ-PNA backbone, the Danith
H. Ly group showed that the LAH reduction of the Weinreb
amide to aldehyde followed by reductive amination could
roughly produce 5% epimerization, which was determined
by fluorine NMR after derivatization to Mosher’s reagent.43
An enantiomeric pure product from a small amount of enan-
tiomeric mixture can be purified by the recrystallization
method; however, it takes a lot of time and labor such as
determining the conditions for recrystallization.
In this study, γ-backbones were synthesized with the tra-
ditional LAH reduction method and with Red Al, a milder
Both the L and D form of the γ-backbone references are
required to determine how much epimerization took place in
the synthesis of the γ-backbone. Thus, each Weinreb amide
from L and D form of the Boc-Lys(Z)-OH, Boc-Glu(cHx)-
OH, and Boc-Ala-OH, respectively, was prepared, and then,
each L and D form of the γ-backbones was synthesized by
the conventional method shown in Scheme 1(A).
(A)
(B)
The (L,L)-5 and (D,L)-5 diastereomers were synthesized
by the coupling reactions of each of the L and D form of
γ-backbones with various (L)-Fmoc-amino acids. Each dia-
stereomer was analyzed with RP-HPLC, and the (L)-Fmoc-
amino acid with the largest difference in the retention time
between the diastereomers was selected. As a result, for
Scheme 1. Synthetic strategies of γ-chiral backbones,
(A) conventional reductive route, (B) SIBX oxidative route;
(a) LiAlH4 or Red-Al, (b) reductive amination with NaHB(OAc)3,
(c) SIBX, DMSO
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Optically pure gamma-PNA backbones
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Figure 2. Target structures of the three different side chain modi-
fied γ-backbones.
each of the Boc-Lys(Z)-aeg-OMe, Boc-Glu(cHx)-aeg-OMe,
and Boc-Ala-aeg-OMe, L forms of the Fmoc-Leu-OH,
Fmoc-Ala-OH, and Fmoc-Phe-OH were optimal as chiral
auxiliary, respectively. As shown in Figure 3(a), each dia-
stereomer reference was fully resolved in this analytical
condition when each diastereomer was coinjected onto an
HPLC column. The (D,L) diastereomers of the three differ-
ent γ-backbones were all eluted about 1 min faster than the
(L,L) diastereomers. Using these chiral auxiliary derivatiza-
tion conditions and HPLC analysis conditions, the degree
of epimerization was measured for the various γ-backbone
synthetic methods. Representative examples are shown in
Figure 3(b), in which the formation of the (D,L) isomer
was almost negligible in the SIBX-mediated oxidative
route. By quantification through integration, all three
γ-backbones showed less than 0.6% epimerization.
Table 1 shows a summary of the analysis results for the
detailed epimerization degree under each synthetic condition.
The Boc-Glu(cHx)-aeg-OMe synthesis of the γ-backbone
through the LAH reduction route failed because the
cyclohexyl ester of the side chain was also reduced by LAH.
From the results, it was observed that an epimerization
of 1.4%–9.7% occurred mostly in the case of the
γ-backbone synthesized by the hydride reduction pathway.
The tendency with temperature seems to improve slightly
in the case of the ꢀ20 ꢁC reaction condition, but it is negli-
gible. However, in the case of the alanine γ-backbone,
Figure 3. HPLC chromatograms of the diastereomer separation;
(a) left, Boc-Lys-(Fmoc-Leu)-aeg-OMe, (D,L)-26.5 min, (L,L)-
27.1 min; middle, Boc-Glu(Fmoc-Ala)-aeg-OMe, (D,L)-26.1 min,
(L,L)-27.0 min; right, Boc-Ala(Fmoc-Phe)-aeg-OMe, (D,L)-
22.7 min, (L,L)-23.4 min. (b) SIBX-mediated γ-backbone; left,
Boc-Lys-(Fmoc-Leu)-aeg-OMe; middle, Boc-Glu(Fmoc-Ala)-aeg-
OMe; right, Boc-Ala(Fmoc-Phe)-aeg-OMe.
epimerization occurred up to almost 10% under the condi-
ꢁ
tion of ꢀ20 C with the Red-Al reduction route. It can be
assumed that this was caused by exposure to an unexpected
basic environment not only under the reaction conditions
but also during the workup and purification in the hydride
reduction pathway. The degree of epimerization in the
Table 1. Optical purities of each γ-backbone synthesized in
different synthetic conditions.
γ-backbones
Route
Temp. (ꢁC)
L-isomera
D-isomera
Lys
LAH
LAH
Red-Al
Red-Al
SIBX
Red-Al
Red-Al
SIBX
LAH
LAH
Red-Al
Red-Al
SIBX
0
ꢀ20
0
ꢀ20
Reflux
0
ꢀ20
Reflux
0
ꢀ20
0
98.0
98.6
97.1
98.1
99.7
98.0
98.1
99.4
96.7
96.2
96.9
90.3
99.4
2.0
1.4
2.9
1.9
0.3
2.0
1.9
0.6
3.3
3.8
3.1
9.7
0.6
Glu
Ala
ꢀ20
Scheme 2. Synthesis of diastereomers of the γ-backbone
using a (L)-Fmoc-amino acid as a chiral auxiliary: (a) HATU,
DIPEA, DMF
Reflux
a Relative % calculated after integration in the HPLC chromatogram.
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LAH reduction pathway of the alanine γ-backbone was
comparable to that observed for the Ly group.
D-amino acids, followed by aldehyde synthesis and reductive
amination, respectively. The main intermediate amino acetal-
dehyde was obtained by three different routes mentioned in
Scheme 1.
The most interesting result is that in the case of the
γ-backbone synthesized by the SIBX oxidative pathway,
the degree of epimerization was significantly reduced for
all three γ-backbones. The degree of epimerization for each
of the Lys, Glu, and Ala-γ-backbones was reduced by 85%,
70%, and 82% on average compared to the hydride reduc-
tion pathway when synthesized by the SIBX oxidative
pathway, respectively. This is a novel method of synthesiz-
ing optically pure γ-backbones that have not yet been
reported as far as we know.
Hydride reagents (LAH, Red-Al, DIBALH, etc.) are par-
ticularly flammable, and when used, the reaction must be
done at low temperature and in an anhydrous environment.
When mass production of target materials is required, spe-
cial equipment is required for the low temperatures and
anhydrous environments, and special attention is required
during the quenching step in the workup. On the other
hand, the SIBX oxidizing agent is a solid reagent and free
from such conditions, and because the reaction conditions
are usually reflux conditions, its use is advantageous
because the SIBX reagents can be easily removed or
regenerated through a filter. Thus, it is particularly suitable
for the mass production of optically pure γ-backbones.
N-(Boc)-Protected Amino Acid Weinreb Amide (1).
N,O-dimethylhydroxylamine hydrochloride, HBTU (1.1 eq.)
and DIEA (3 eq.) were added to a stirred solution of N-(Boc)
protected amino acids in DMF. The resulting mixture was
stirred for 30 min. Upon completion of the reaction, as con-
firmed by the TLC, the solvent was evaporated under reduced
pressure. The resulting crude was dissolved in ethyl acetate,
washed multiple times with 1 N HCl followed by saturated
NaHCO3 and brine solution, and dried over anhydrous
Na2SO4. The concentrated crude mixture was purified by
flash chromatography to give the corresponding Weinreb
amides as a white color solid (1) at a good yield.
N-(Boc)-Protected Amino Alcohol (2). CDI (3 eq.) was
portion-wise added to a solution of N-(Boc) protected amino
acid in anhydrous THF. The solution was stirred for 5 min.
Subsequently, the solution of NaBH4 (3 eq.) in water was
added dropwise for 10 min (effervescence occurred). After
completion of the reaction, a large volume of EtOAc was
added and washed multiple times with 1 N HCl solution,
followed by saturated NaHCO3 solution. The combined
organic layer was dried over anhydrous Na2SO4, and the sol-
vent was evaporated using a rotary evaporator. The resulting
crude was purified by column chromatography to obtain the
target alcohol compound 2.
Experimental Methods
All reagents used in this experiment were purchased from
commercial suppliers and used without further purification
unless otherwise noted. 1H NMR and 13C NMR spectra
were taken with a Bruker 400 MHz NMR spectrometer;
CDCl3 and DMSO-d6 were used as solvents, and tetra-
methylsilane was used as an internal standard. Column
chromatography was performed with CHROMATOREX
GS-40/75 silica gel, and TLC analysis was done with silica
gel 60 F-254 precoated aluminum plate. The HPLC instru-
ment was a Waters binary pump and a Waters 2487 detec-
tor, and the HPLC analytical column used for the optical
purity measurement was INNO C18 (5 μm, 120 Å, 4.6
ꢂ 250 mm). The solvent systems used in the HPLC were
water and MeCN containing 0.1% TFA, respectively.
N-(Boc)-Protected Amino Acetaldehyde (3). Path 1: First
a 2.6 M solution of LAH (1.2 eq.) was added to a chilled
solution of N-(Boc) Weinreb amide (1) in anhydrous THF,
and then the solution was stirred for 30 min. The reaction
was then quenched with 1 N HCl and washed with satu-
rated NaHCO3 followed by saturated brine solution. The
combined organic phase was dried over anhydrous Na2SO4,
and the solvent was concentrated with a rotary evaporator.
This crude mixture 3 was used in the next reductive ami-
nation reaction without further purification.
Path 2: First, a 3.6 M solution of Red-Al (1.5 eq.) was
added to a cold solution of N-(Boc) Weinreb amide (1) in
anhydrous THF, and then the solution was stirred for 30 min.
The reaction was then quenched with 1 N HCl and washed
with saturated NaHCO3 followed by saturated brine solution.
The combined organic phase was dried over anhydrous
Na2SO4, and the solution was concentrated with a rotary
evaporator. This crude mixture 3 was used in the next reduc-
tive amination reaction without further purification.
Path 3: SIBX (5 eq.) was added to a solution of N-(Boc)
amino alcohol in EtOAc in a two-neck round bottom flask,
and then the reaction mixture was refluxed for 5 min. Next,
DMSO (5 eq.) was added to the solution, and the reflux
was continued. After completion of the reaction, the reac-
tion mixture was filtered through a celite pad and washed
with a saturated NaHCO3 solution. The organic phase was
again washed with a saturated brine solution, and the solu-
tion was concentrated with a rotary evaporator after drying
Abbreviations: N, N-dimethylformamide (DMF);
dichloro-methane (DCM); tetrahydrofuran (THF);
dichloroethane (DCE); N,N-diisopropylethylamine (DIEA);
O-(7-azabenzotriazol-1-yl)-N,N,N0N0-tetramethyluronium
hexafluorophosphate (HATU); dimethyl sulfoxide (DMSO);
acetic
acid
(AcOH);
O-(benzotriazol-1-yl)-N,N,N0,N0-
tetramethyluronium hexafluorophosphate (HBTU); 1,10-
carbonyldiimidazole (CDI); stabilized 2-iodoxybenzoic acid (-
SIBX);
triacetoxyborohydride (NaBH(AcO)3; lithium aluminum
hydride (LAH); sodium bis(2-methoxyethoxy)
aluminum hydride (Red-Al).
sodium
borohydride
(NaBH4);
sodium
In general, all γ-modified PNA backbones were synthe-
sized from commercially available N-(Boc)-protected L- and
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over anhydrous Na2SO4. The resulting crude compound (3)
was used in the subsequent reductive amination reaction
without any further purification.
Boc-Glu(cHx)-Weinreb Amide (1b). Yield; 96.7%. 1H
NMR (400 MHz, CDCl3) δ 5.24 (dd, J = 4.8, 4.0 Hz, 1H),
4.67 (td, J = 8.9, 4.2 Hz, 2H), 3.71 (s, 3H), 3.13 (s, 3H),
2.31 (t, J = 7.5 Hz, 2H), 1.98 (qd, J = 7.7, 4.8 Hz, 1H),
1.83–1.71 (m, 3H), 1.68–1.59 (m, 2H), 1.52–1.41 (m,
1H), 1.35 (s, 9H), 1.28 (dd, J = 15.1, 5.8 Hz, 3H), 1.18
(ddd, J = 13.2, 10.9, 3.2 Hz, 2H). 13C NMR (100 MHz,
CDCl3) δ 172.33, 172.00, 155.29, 79.31, 72.47, 61.43,
49.70, 31.88, 31.40, 30.33, 28.12, 27.60, 25.17, 23.53.
N-(Boc)-Protected γ-Backbone (4). Methyl glycinate
hydrochloride (1.5 eq.), DIEA (1.5 eq.), acetic acid
(1.0 eq.), and 1.5 eq. of NaBH(OAc)3 were added to a solu-
ꢁ
tion of aldehyde (3) in DCE at 0 C. Then, the temperature
was raised to ambient temperature, and the reaction was
stirred for 1 hr. Upon the completion of the reaction, an
excess volume of DCM was added, and the organic layer
was washed with saturated NaHCO3 and a brine solution.
The extracted DCM was dried over anhydrous Na2SO4,
and the solvent was evaporated with a rotary evaporator.
The resulting crude was purified by flash column chroma-
tography to yield a colorless syrup compound (4).
Boc-Glu(cHx)-Alcohol (2b). Yield; 82.0%. 1H NMR
(400 MHz, CDCl3) δ 4.86–4.69 (m, 2H), 3.66–3.50 (m,
3H), 2.48–2.41 (m, 1H), 2.40–2.30 (m, 2H), 1.91–1.79
(m, 3H), 1.79–1.74 (m, 1H), 1.74–1.66 (m, 2H), 1.58 (s,
1H), 1.53 (ddd, J = 9.4, 5.2, 3.3 Hz, 1H), 1.42 (s, 9H),
1.38–1.33 (m, 2H), 1.33–1.28 (m, 1H), 1.28–1.18 (m, 1H).
13C NMR (100 MHz, CDCl3) δ 173.27, 156.13, 79.50,
72.95, 64.91, 52.49, 31.63, 31.28, 28.39, 26.07,
25.37, 23.78.
Fmoc-Amino Acid Attached γ-Backbones (5). In a
1.5 mL Eppendorf tube, (L)-Fmoc-amino acid in DMF was
added to Boc-Lys(Z)-aeg-OMe (4) (1.0 eq.), HATU
(1.1 eq.), and DIEA (6.0 eq.). The Fmoc-amino acids used
as a chiral auxiliary were Fmoc-Leu-OH, Fmoc-Ala-OH,
and Fmoc-Phe-OH for the Lys, Glu, and Ala-γ-backbone,
respectively. The solution was left to react for 1 hr, and
then the diastereomer products were monitored by HPLC at
260 nm, flowing from 50% MeCN to 95% MeCN for
30 min at a flow rate of 1 mL/min.
Boc-Glu(cHx)-aeg-OMe γ-Backbone (4b). Yield; obtained
ꢁ
ꢁ
through Red-Al reaction at 0 C; 44.5%, at ꢀ20 C; 62.5%
1
and SIBX route 69.3% respectively. H NMR (400 MHz,
CDCl3) δ 4.75 (td, J = 8.9, 4.0 Hz, 2H), 3.73 (s, 3H), 3.66
(d, J = 2.8 Hz, 1H), 3.42 (q, J = 17.4 Hz, 2H), 2.67 (qd,
J = 12.2, 5.6 Hz, 2H), 2.37 (t, J = 7.6 Hz, 2H), 1.91–1.79
(m, 3H), 1.78–1.68 (m, 3H), 1.64 (s, 1H), 1.55 (ddd,
J = 9.2, 5.1, 3.3 Hz, 1H), 1.47–1.41 (m, 10H), 1.41–1.36
(m, 2H), 1.35–1.30 (m, 1H), 1.30–1.20 (m, 1H). 13C NMR
(100 MHz, CDCl3) δ 172.89, 172.86, 155.78, 79.23, 72.74,
53.06, 51.74, 50.81, 50.27, 31.64, 31.41, 28.38, 28.21,
25.37, 23.76.
The following compounds were prepared, purified, and
characterized using the above general procedure, and their
spectral data are given below.
1
Boc-Lys(Z)-Weinreb Amide (1a). Yield; 93.4%. H NMR
(400 MHz, CDCl3)
δ 7.36–7.19 (m, 5H), 5.30 (d,
J = 8.1 Hz, 1H), 5.10 (d, J = 30.2 Hz, 3H), 4.63 (s, 1H),
3.72 (s, 3H), 3.16 (s, 5H), 1.67 (d, J = 6.4 Hz, 1H), 1.59–
1.44 (m, 3H), 1.40 (s, 11H); 13C NMR (100 MHz, CDCl3)
δ 172.92, 156.32, 155.54, 136.49, 128.30, 127.92, 127.84,
79.37, 66.31, 61.41, 49.90, 40.56, 32.31, 31.89, 29.06,
28.19, 22.29.
Boc-Ala-Weinreb Amide (1c). Yield; 90.0%. 1H NMR
(400 MHz, CDCl3) δ 5.25 (d, J = 7.8 Hz, 1H), 4.73–4.62
(m, 1H), 3.76 (s, 3H), 3.20 (s, 3H), 1.43 (s, 9H), 1.30 (d,
J = 6.9 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 173.62,
155.16, 79.49, 61.61, 46.51, 32.12, 28.36, 18.68.
Boc-Lys(Z)-Alcohol (2a). Yield; 81.1%. 1H NMR
(400 MHz, CDCl3) δ 7.40–7.29 (m, 5H), 5.20–5.05 (m,
2H), 4.90 (d, J = 36.2 Hz, 2H), 3.59 (ddd, J = 14.5, 11.5,
4.1 Hz, 3H), 3.27–3.15 (m, 2H), 2.77 (s, 1H), 1.61–1.48
(m, 3H), 1.45 (s, 9H), 1.42–1.27 (m, 2H); 13C NMR
(100 MHz, CDCl3) δ 156.70, 156.51, 152.64, 136.59,
128.52, 128.11, 79.56, 66.68, 65.48, 52.48, 40.41, 30.78,
29.77, 28.40, 22.77.
1
Boc-Ala-Alcohol (2c). Yield; 85.9%. H NMR (400 MHz,
CDCl3) δ 4.77 (d, J = 6.5 Hz, 1H), 3.73 (d, J = 4.2 Hz,
1H), 3.60 (ddd, J = 10.1, 5.8, 4.0 Hz, 1H), 3.48 (dt,
J = 10.9, 5.5 Hz, 1H), 3.01 (s, 1H), 1.42 (s, 9H), 1.12 (d,
J = 6.8 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 156.34,
79.63, 67.19, 48.52, 28.36, 17.29.
Boc-Ala-aeg-OMe γ-Backbone (4c). Yield; obtained
ꢁ
ꢁ
Boc-Lys(Z)-aeg-OMe γ-Baꢁckbone (4a). Yield; obtained
through LAH reaction at 0 C; 81.4%, at ꢀ20 C; 83.7%;
Red-Al reaction at 0 ꢁC; 79.1%, at ꢀ20 ꢁC; 71.1% and
through SIBX reaction; 66.7%, respectively. 1H NMR
(400 MHz, CDCl3) δ 5.00 (s, 1H), 3.52 (s, 4H), 3.23 (q,
J = 17.4 Hz, 2H), 2.44 (d, J = 5.9 Hz, 2H), 1.62 (s, 1H),
1.24 (s, 9H), 0.95 (d, J = 6.7 Hz, 3H). 13C NMR
(100 MHz, CDCl3) δ 172.72, 155.46, 78.60, 54.24, 51.46,
50.35, 45.90, 28.20, 18.78.
ꢁ
through LAH reaction at 0 C; 75.0%, at ꢀ20 C; 70.0%;
Red-Al reaction at 0 ꢁC; 97.3%, at ꢀ20 ꢁC; 99.5% and
SIBX reaction; 57.1% respectively. 1H NMR (400 MHz,
CDCl3) δ 7.39–7.29 (m, 5H), 5.09 (s, 2H), 4.97 (s, 1H),
4.77 (s, 1H), 3.72 (s, 3H), 3.63 (s, 1H), 3.41 (q,
J = 17.4 Hz, 2H), 3.19 (dd, J = 12.6, 6.4 Hz, 2H), 2.71–
2.56 (m, 2H), 1.67 (s, 1H), 1.60–1.29 (m, 15H). 13C NMR
(100 MHz, CDCl3) δ 172.92, 156.45, 155.91, 136.66,
128.44, 128.06, 127.99, 79.14, 66.51, 52.94, 51.71, 50.72,
50.09, 40.70, 32.74, 29.56, 28.36, 22.88.
1H NMR and 13C NMR data of the various intermediates
and γ-backbones synthesized in this study are included in
the Supporting Information.
Bull. Korean Chem. Soc. 2021
© 2021 Korean Chemical Society and Wiley-VCH GmbH.
www.bkcs.wiley-vch.de
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BULLETIN OF THE
Article
Optically pure gamma-PNA backbones
KOREAN CHEMICAL SOCIETY
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Conclusion
Three types of γ-modified PNA backbones were synthe-
sized from the relatively inexpensive N-protected L-amino
acid. N-protected amino acetaldehyde derivatives, which
are key intermediates of the γ-backbone synthesis, were
synthesized through the LAH, Red-Al reduction, and SIBX
oxidative routes, and the final γ-backbone was synthesized
by the conventional reductive amination between the Boc-
amino acetaldehyde and glycinate ester. The chiral purity
of each γ-backbone was determined by the diastereomer
formation of the corresponding γ-backbones with the suit-
able (L)-Fmoc-protected amino acids as chiral derivatizing
reagents. In the SIBX-mediated route, γ-backbones with an
optical purity of over 99.4% were obtained from all three
types of γ-backbones. Finally, this methodology is robust
and can be applied to the preparation of large-scale γ-PNA
backbones without any concerns over loss of optical purity.
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Acknowledgment. This research was supported by research
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Bull. Korean Chem. Soc. 2021
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www.bkcs.wiley-vch.de
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