Continuous-Flow Synthesis of Cationic Lipid SST-01 via Safe and Scalable Aerobic Oxidation and Reductive Amination

Article abstract of DOI:10.1021/acs.oprd.0c00084

The synthesis of the cationic lipid SST-01, a key component of Kyowa Kirin's siRNA-lipid nanoparticles, through a two-step homogeneous continuous-flow process is described. Safe and scalable aerobic oxidation with a catalytic CuI/TEMPO system and diluted oxygen (5% O2 in N2) was utilized in the first step to provide a lipid aldehyde. Subsequent reductive amination of the first-step eluent with methylamine using tetramethylammonium triacetoxyborohydride provided the target SST-01 in an overall yield of 95%.

Full text of DOI:10.1021/acs.oprd.0c00084

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Full Paper
Continuous-flow Synthesis of Cationic Lipid SST-01 via Safe
and Scalable Aerobic Oxidation and Reductive Amination
Katsuaki Fujiwara, Haruro Ishitani, and Shu Kobayashi
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.0c00084 • Publication Date (Web): 22 Jul 2020
Downloaded from pubs.acs.org on July 22, 2020
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Continuous-flow Synthesis of Cationic Lipid SST-01 via Safe and
Scalable Aerobic Oxidation and Reductive Amination
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Authors
Katsuaki Fujiwara,*†‡ Haruro Ishitani,§ and Shū Kobayashi*‡§
Affiliation
†Fuji Research Park, Research Functions Unit, R&D Division, Kyowa Kirin, Co., Ltd.,
1188, Shimotogari, Nagaizumi-cho, Sunto-gun, Shizuoka 411-8731, Japan
‡Department of Chemistry, School of Science, and §Green & Sustainable Chemistry Social
Cooperation Laboratory, Graduate School of Science, The University of Tokyo, Hongo, Bunkyo-ku,
Tokyo 113-0033, Japan
Abstract Graphic
Two-step Continuous flow
1) Aerobic Oxidation
N
OH
2) Reductive Amination
SST-01
Linoleyl Alcohol
(Cationic lipids for siRNA-Lipid Nanoparticle)
Yield: 95% (2steps)
Productivity: 2.2 g/h
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Abstract
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The synthesis of a cationic lipid SST-01, a key component of Kyowa Kirin’s siRNA-lipid
nanoparticle (LNP), through a two-step homogeneous continuous-flow process is described. Safe
and scalable aerobic oxidation with a catalytic CuI/TEMPO system and a diluted oxygen (5% O2 in
N2) was utilized in the first step to provide a lipid aldehyde. Subsequent reductive amination of the
first step eluent with methylamine using tetramethylammonium triacetoxyborohydride provided the
target SST-01 with an overall yield of 95%.
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Keywords
Continuous-flow chemistry, Catalytic oxidation, Reductive amination, Cationic lipid, Lipid
nanoparticle
INTRODUCTION
Continuous-flow synthesis1 is a technology that has attracted a great deal of attention from
industry as well as academia, because it provides safe, compact, efficient and environmentally
benign chemical manufacturing processes, including those for manufacturing fine and specialty
chemicals like drugs.2–5 In comparison to conventional batch methods, continuous-flow methods
offer large benefits to pharmaceutical industries, such as accurate quality control, efficient inventory
management, easy automation, and faster scale-up applications;6–8 these allow compounds from the
laboratory to be brought to markets faster and cheaper.
Lipid nanoparticle (LNP) is one of the most successful lipid-mediated delivery vehicle systems for
short interfering RNA (siRNA) and is now considered a reliable therapeutic modality after the U.S.
Food and Drug Administration (FDA) approved Alnylam Pharmaceuticals’ PATISIRAN9. LNPs are
approximately 100 nm in size and consist of siRNA, neutral lipids, PEG-lipids, and cationic lipids.
Cationic lipids, key components of siRNA-LNP, are protonated in acidic endosomes, increase the
electric charge on the surface of nanoparticles, and induce the migration of siRNA into cytosol.
Medicinal chemists have shown that sophisticated structural modification of cationic lipids
influences the in vivo performance of siRNA-LNPs. In a previous study10, 11 at Kyowa Kirin Co.,
Ltd., we conducted a medicinal chemistry campaign and found that cationic lipid SST-01 (1; Figure
1), which possesses a relatively simple chemical structure of two long alkyl chains connected to a
nitrogen atom, exerts prominent siRNA delivery activity, especially for solid tumors.
N
SST-01 1
( )
Figure 1. Structure of cationic lipid SST-01 (1)
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We have been conducting a batch process to supply SST-01 (1) for use in a preclinical study
(Scheme 1).12 However, the manufacturing cost of the present batch method, which consists of
dialkylation with lipid mesylate 2, was inadmissibly high due to the use of expensive raw material 2.
In addition, the formation of a byproduct, the corresponding quaternary ammonium salt (3),
decreased the yield of the desired 1. In order to reduce the manufacturing cost, the present batch
method with conventional N-alkylation should be improved. We therefore predict that the utilization
of continuous-flow methods with a novel synthetic route using inexpensive raw material would
achieve such an improvement. In this paper, we report the details of our improvement in preparing 1
with sequential continuous-flow reactions.
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Scheme 1. Structure of cationic lipid SST-01 (1) and the first-generation synthetic route in batch
OMs
2
40% MeNH2 in H2O
61%
90 oC, 3 h
N
SST-01 1
( )
N
MsO
3
RESULTS AND DISCUSSION
Prior to investigations into continuous-flow methods, we conducted comprehensive research on the
synthesis of 1 and envisioned a new two-step protocol starting with less-expensive linoleyl alcohol
(4). For the new protocol, oxidation of an alcohol and reductive double amination would provide
target 1 without the formation of an undesired quaternary ammonium salt. However, preliminary
studies revealed that a known process shown in Scheme 2 gave the corresponding aldehyde in
relatively low yield, and a co-oxidant, PhI(OAc)2, was hard to dissolve into the reaction medium. In
addition, a reductant in the amination step, NaBH(OAc)3, has no solubility in THF, producing a
heterogeneous system. We expected that these drawbacks would account for troubles in
continuous-flow; thus, we re-investigated both steps individually.
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Scheme 2. New synthetic route of SST-01 (1)
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OH
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TEMPO (0.1 eq.)
PhI(OAc)2 (1.1 eq.)
EtOAc (3 v/w)
73%
25 oC, 4 h
CHO
5
2 M MeNH2 (1.0 eq.) in THF
NaBH(OAc)3 (1.4 eq.)
AcOH (1.0 eq.)
85%
THF(10 v/w)
25 oC, 5 h
N
SST-01 1
( )
Since the oxidation of primary alcohols to aldehydes using the flow method has been reported 13-17
,
some of these conditions were examined in the batch method (Table S1). As a result, it was found
that aerobic oxidation conditions with a CuI/TEMPO catalyst system developed by Stahl et al.18 gave
the desired aldehyde in a satisfactory 81% isolated yield (Table S1, entry 6). Although this Stahl
oxidation was already applied into a homogeneous flow reaction,19 it was necessary to adjust the
original conditions with our substrates to maintain the reduced cost. Several conditions, such as
solvents, concentrations of substrates, reaction temperature, oxygen amounts, and Cu sources20 and
ligands21 were widely screened (Table S2-S6), and it was found that the combination of CuI and
4,4’-dimethoxy-2,2’-bipyridine showed sufficient activity in NMP at 60°C. We also examined an
oxygen concentration by considering the limiting oxygen concentration (LOC) of NMP (7.6% at
200°C, 20 bar)22 for the safety aspect in continuous-flow conditions, and decided to use 5% oxygen
diluted with nitrogen instead of air. Under these optimized conditions, a complete homogeneous
system could be realized and a 92% yield of the desired aldehyde was obtained (Scheme 3).
Scheme 3. Optimized conditions of the oxidation step in batch
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Heterogeneous
TEMPO (0.1 eq.), PhI(OAc)2 (1.1 eq.)
EtOAc (3v/w), 25 oC, 4 h
73%
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Condition screening &
Optimization
CHO
OH
4
5
Homogeneous
TEMPO (0.1eq.), CuI (0.1 eq.)
4,4'-dimethoxy-2,2'-bipyridine (0.1 eq.)
N-methylimidazole (0.2 eq.)
5% O2 in N2, NMP (20 v/w), 60 oC, 4.5 h
4
92% (3% of was remained)
To apply the abovementioned optimized conditions to a homogeneous continuous-flow system, the
flow set-up shown in Figure 2 was assembled. An NMP solution of substrate 4 (Solution A in
Reservoir A) was flowed using Pump A, and an NMP solution of a catalyst system (Solution B in
Reservoir B), consisting of CuI, the ligand, TEMPO, and N-methylimidazole (NMI), was fed using
Pump B at the same flow rate of Solution A. These streams were mixed at a T-shaped mixer (M1),
and then 5% oxygen in nitrogen that was regulated with a mass flow controller (MFC) at another
T-shaped mixer (M2) was combined. Finally, the whole mixture was fed into a 30 m SUS tube
reactor (i.d. 1.0 mm). The tube reactor was heated in an aluminum heating block and a back pressure
regulator (BPR) was attached at the outlet.
Pump A
SUS tube reactor
OH
(1.58 mm o.d., 1.0 mm i.d., 30 m)
4
M1
0.38 M in NMP under N2
(Solution A in Reservoir A)
Pump B
CuI, Ligand, TEMPO, 0.038 M in NMP
NMI, 0.075 M in NMP under N2
(Solution B in Reservoir B)
M2
BPR
Heater
MFC
trap&valve
CHO
5
5% O2 in N2
Figure 2. Set-up of the continuous-flow oxidation step.
The results of representative optimization studies using this flow system are shown in Table 1.
High yield and high selectivity were obtained under slow feed rate conditions (0.04 mL/min, entry 1).
In addition, the reaction time was shown to be markedly shorter than with the batch method (Table 1,
entry 1 vs. Scheme 3). The flow regime was an annular mist flow. We considered a tube reactor in
flow had a larger interfacial area to volume ratio than that of a stirred tank reactor in batch, and the
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reaction solution and diluted oxygen gas were sufficiently mixed. We assumed this was why the
reaction time was significantly shortened. On the other hand, the stability of the catalyst was
maintained for up to 21 hours, which was monitored in Reservoir A (Table S7, entry 3). A decrease
in yield at 143 hours raised the suspicion of catalyst deterioration; however, the yield recovered to
92% yield (HPLC area) when the reaction temperature was elevated to 80°C (Table S7, entries 4 and
5).
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Table 1. Optimization of reaction conditions of the oxidation step in flow
TEMPO (0.1 eq.)
CuI (0.1 eq.)
4,4'-dimethoxy-2,2'-bipyridine (0.1 eq.)
N-methylimidazole (0.2 eq.)
CHO
OH
5% O2 in N2
4
5
NMP
Residence
time
Flow rate (mL/min)
HPLC area (%)
Pressure Temp. Oxygen
Entry
(bar)
(oC)
(eq.)
Solution A, B a
5% O2 in N2
4
2
5
(min) b
1
2
0.040, 0.040
0.10, 0.10
0.10, 0.10
0.10, 0.10
0.10, 0.10
0.10, 0.10
0.20, 0.20
0.20, 0.20
0.20, 0.20
0.40, 0.40
0.40, 0.40
0.40, 0.40
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15
38
15
20
15
15
15
30
50
50
50
0
0
0
5
5
5
5
5
5
8
8
8
60
60
60
60
60
70
70
80
80
80
90
100
1.8
0.7
1.8
4.3
5.7
4.2
2.1
2.0
4.0
5.1
4.9
4.8
1.6
1.6
0.6
1.6
1.2
1.6
1.5
1.5
0.8
0.5
0.5
0.5
97
64
85
91
92
98
79
80
96
91
94
95
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6
1
7
20
19
2
8
9
10
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12
8
5
3
aSolution A: 4 in NMP (0.38 M). Solution B: CuI, Ligand, TEMPO in NMP (0.038 M), NMI in NMP (0.075 M).
bThe residence time was calculated based on the assumption that the total flow rate was the sum of the flow rate of
the gas and liquid components.
To improve productivity, the flow rates of Solutions A and B were increased from 0.04 mL/min to
0.40 mL/min in a stepwise manner. The decreased conversion of substrate 4 was essentially
observed by increasing the rate; however, this could be overcome by increasing the oxygen feed rate,
back pressure, and reaction temperature (entries 2-9). Final optimization at 0.40 mL/min was started
with an oxygen feed rate of 50 mL/min, 8 bar back pressure, and a reaction temperature of 80°C, and
a 91% yield (HPLC area) was obtained with the co-existence of 8% substrate 4. Elevating the
reaction temperature to 100°C improved the conversion of the desired 5 and 95% yield (HPLC area)
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was obtained (entry 12). Furthermore, continuous operation for 20 hours under optimized conditions
produced 40 g of the target aldehyde (5) (95% yield). Under these conditions, the flow regime was a
slug flow (Figure S2). In comparison to the previously reported continuous-flow CuI/TEMPO
oxidation of aliphatic alcohol, the usage of inexpensive CuI, low pressure of oxygen gas, and a short
residence time were successfully achieved.
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Next, a preliminary investigation of the reductive amination process was conducted in batch. To
avoid the use of an insoluble metal hydride reductant, such as NaBH(OAc)3, screening of other
conditions was investigated (Table S8-S12). Sodium bis(2-methoxyethoxy)aluminum hydride
(Red-Al) and catalytic hydrogenation conditions were both invalid; however, switching the cation of
the acetoxyborohydride from Na+ to Me4N+ made borohydride species soluble and a reasonable yield
of the desired SST-01 (1) was attained (Table S12, entry 2). Under these conditions, the one-pot
two-step reaction was confirmed to be possible using a crude reaction mixture from the first
oxidation step (Scheme 4).
Scheme 4. Optimized conditions of the reductive amination step in batch
Heterogeneous
2 M MeNH2 (1.0 eq.) in THF
NaBH(OAc)3 (1.4 eq.), AcOH (1.0 eq.)
THF(10 v/w), 25 oC, 5 h
85%
Condition Screening &
Optimization
CHO
N
5
SST-01 1
( )
Homogeneous
2M MeNH2 (0.55 eq.) in MeOH,
Me4NBH(OAc)3 (2.5 eq.)
NMP(10 v/w), 70 oC, 1.5 h
4
51% (from alcohol
)
To apply the above system in a flow process, other set-ups consisting of the following apparatuses
were assembled (Figure 3). A 0.19 M NMP solution of aldehyde 5 (Solution C) and an unpurified
crude mixture from the oxidation step were flowed from Reservoir C using Pump C, and this stream
was mixed with a MeOH solution of methylamine (Solution D) fed from Pump D at a T-shaped
mixer (M3). The combined substrate solutions were then forgathered with a NMP solution of
Me4NBH(OAc)3 from Pump E (Solution E) at a T-shaped mixer (M4), and were carried into a 20 m
SUS tube reactor (i.d. 1.0 mm), which was heated by an aluminum heating block.
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CHO
Pump C
5
SUS tube reactor
0.19 M in NMP under N2 (Solution C in Reservoir C)
(Reaction mixture from oxidation step without any purification)
(1.58 mm o.d., 1.0 mm i.d., 20 m)
M3
9
Pump D
Pump E
MeNH2
M4
0.20 M or 0.078 M in MeOH under N2
(Solution D in Reservoir D)
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Heater
Me4NBH(OAc)3
0.45 M in NMP under N2
(Solution E in Reservoir E)
N
SST-01
1
( )
Figure 3. Set-up of the continuous-flow reductive amination step.
The conditions optimized in batch were first applied to the flow reaction. The conversion of
aldehyde 5 was not full and a moderate 61% yield of the dialkylated amine 1 was obtained along
with 6% of monoalkylated amine 6 (Table 2, entry 1). In this case, precipitation in the resulting
solution could be found, possibly due to the evaporation of MeOH in the reactor. We assumed that
the precipitation was a derivative of Me4NBH(OAc)3 since MeOH was able to dissolve the reductant.
To increase the relative MeOH amount and suppress the evaporation of MeOH, we diluted the
solution D to 0.078 M and the reaction temperature was reduced to 25°C. These modifications were
effective for homogenization of the reaction medium, and a better yield of the desired 1 was
obtained when the reaction was performed at 40°C (entries 2 and 3). Increasing the flow rate of
Solution C did not affect the conversion or yield; thus, productivity of 0.15 mmol/min could be
achieved under the conditions shown in entry 5. The stability of the present flow system was also
checked under the conditions of entry 4, and no clogging and no change in the outcome could be
confirmed during the 11-hour operation.
Table 2. Optimization of the reaction condition of the reductive amination step in flow
MeNH2 (0.5-0.53 eq.) in MeOH
Me4NBH(OAc)3 (2.5-3.0 eq.)
CHO
N
+
N
H
NMP, Temp.
5
6
SST-01
1
)
(
Residence
Time
Two Steps
Yield
Flow rate (mL/min)
HPLC area (%)
Temp.
(oC)
Entry
Solution C a Solution D b Solution E c
5
SST-01
6
6
8
7
8
7
(min)
(%) g
1
2
3
4
5
0.20
0.20
0.15
0.30
0.80
0.10 d
0.24
0.21 f
0.21 f
0.19
0.38
1.00
70
25
40
40
40
31
24
30
15
6
19
12
0
61
64
82
83
81
0.19 e
0.38 e
0.96
70
71
75
0
2
a 5 in NMP (0.19 M), reaction mixture from the oxidation step without any purification. b MeNH2 in MeOH (0.078 M,
0.5 eq.). c Me4NBH(OAc)3 in NMP (0.45 M, 3.0 eq.). d MeNH2 in MeOH (0.20 M, 0.5 eq.). e MeNH2 in MeOH
(0.078 M, 0.53 eq.). f Me4NBH(OAc)3 in NMP (0.45 M, 2.5 eq.). g Yield from alcohol 4. Yield based on HPLC assay
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using biphenyl as an internal standard.
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Using a combined flow set-up, as illustrated in Figure 4, we next tried to connect the homogeneous
CuI/TEMPO-catalyzed oxidation with the reductive amination. To avoid any contamination of
oxygen into the reduction step in the first step, a gas-liquid separation flask (Reservoir C) was placed
between the two units, and Reservoir C was purged by nitrogen gas to keep the oxygen
concentration at ≤5% (considering LOC of NMP).
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Pump A
OH
SUS tube reactor
4
(1.58 mm o.d., 1.0 mm i.d., 30 m)
0.38 M in NMP under N2
(Solution A in Reservoir A)
M1
0.40 mL/min
Pump B
N2 purge
CuI, Ligand, TEMPO, 0.038 M in NMP
NMI, 0.075 M in NMP under N2
(Solution B in Reservoir B)
M2
BPR
0.40 mL/min
Heater, 100 o
8 bar
C
trap&valve
MFC
50 mL/min
5% O2 in N2
CHO
5
0.19 M in NMP (Solution C in Reservoir C)
Pump C
SUS tube reactor
(1.58 mm o.d., 1.0 mm i.d., 20 m)
M3
0.80 mL/min
Pump D
M4
MeNH2
0.078 M in MeOH under N2
(Solution D in Reservoir D)
Clogged
0.86 mL/min
Pump E
Heater, 40 o
C
Me4NBH(OAc)3
0.45 M in NMP under N2
(Solution E in Reservoir E)
1.0 mL/min
N
SST-01
1
( )
4
The equivalent of reagents for : CuI (0.1 eq.), Ligand (0.1 eq.), TEMPO (0.1 eq.), NMI (0.2 eq.), methylamine (0.45 eq.), Me4NBH(OAc)3 (3.0 eq.)
Figure 4. Set-up of the continuous-flow oxidation and reductive amination steps.
The two-step flow reaction proceeded smoothly for 30 minutes; however, clogging occurred at the
T-shaped mixer (M4) on the inlet part of the second reduction reactor due to the deposition of a
reddish-brown solid. We suspected that the solid was a derivative of Cu species due to its very low
solubility in any solvent. Although one potential problem of this system was compatibility of copper
species supplied in the oxidation step for the following reductive amination step, the previous single
reductive amination investigation under flow conditions proved that there was no deposition from
the copper-included substrate solution (Solution C) in the reductive amination step.
To clarify the causes of the generation of the insoluble solid, we carefully checked the precipitation
process among Solutions C, D, and E, especially for a solid growth process from a seed. i) Methanol
and an insoluble solid seed were added to Solution C, resulting in the immediate generation of a
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considerable amount of solid (Table 3, entry 1). ii) A methanol solution of methylamine and
insoluble solid seed were added to Solution C, resulting in the immediate generation of a
considerable amount of solid (entry 2). iii) A methanol solution of methylamine and a NMP solution
of Me4NBH(OAc)3 were added to Solution C at the same time in the presence of the solid seed,
resulting no further growth of the solid (entry 3). These careful observations suggested that the solid
growth occurred in the intersection of Solutions C and methanol. We assume that methanol
generated the solid by working as an anti-solvent. Given the results of entry 3, solution E was
considered important for dissolving the solid, and we suspected that acidic sources from
Me4NBH(OAc)3 would be effective for dissolving the solid.
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Table 3. Investigation of the causes of the generation of the insoluble solid
Insoluble
solid
Entry
Condition
1
2
3
Solution Ca +MeOH + Seed of the insoluble solid
Solution Ca +MeNH2 in MeOH + Seed of the insoluble solid
Yes
Yes
No
Solution Ca +MeNH2 in MeOH + Me4NBH(OAc)3 in NMP + Seed of the insoluble solid
a 5 in NMP (0.19 M), reaction mixture from oxidation step without any purification.
Therefore, we changed the method to mix them all together using a cross-shaped mixer (M5, Figure
5). As a result, no insoluble solid was observed, and continuous operation without clogging became
possible. The overall yield of SST-01 (1) from the oxidation step was determined to be 95% (HPLC
assay) from samples collected between 4.5 and 5 hours, and the target compound was obtained with
an isolated yield of 76%. Productivity of 2.2 g/h was achieved.
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Pump A
OH
SUS tube reactor
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4
(1.58 mm o.d., 1.0 mm i.d., 30 m)
M1
0.38 M in NMP under N2
(Solution A in Reservoir A)
0.40 mL/min
N2 gas flow
Pump B
CuI, Ligand, TEMPO, 0.038 M in NMP
NMI, 0.075 M in NMP under N2
(Solution B in Reservoir B)
M2
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BPR
0.40 mL/min
Heater, 100 o
8 bar
C
trap&valve
MFC
Residence time = 0.5 min
50 mL/min
5% O2 in N2
CHO
5
0.19 M in NMP (Solution C in Reservoir C)
Pump C
SUS tube reactor
(1.58 mm o.d., 1.0 mm i.d., 20 m)
0.80 mL/min
Pump E
M5
Me4NBH(OAc)3
0.45 M in NMP under N2
(Solution E in Reservoir E) 1.0 mL/min
Heater, 40 o
Residence time = 6 min
C
4
Yield = 95% from (HPLC assay)
Isolated yield= 76% from
Productivity:2.2 g/h
4
Pump D
MeNH2
0.078 M in MeOH under N2
(Solution D in Reservoir D)
N
0.92 mL/min
SST-01
1
( )
4
The equivalent of reagents for : CuI (0.1 eq.), Ligand (0.1 eq.), TEMPO (0.1 eq.), NMI (0.2 eq.), methylamine (0.48 eq.), Me4NBH(OAc)3 (3.0 eq.)
Figure 5. Modified set-up of the continuous-flow oxidation and reductive amination steps
Finally, the comparison of the batch and flow methods examined in the present report is
summarized in Table 4. Compared to the original batch method (entry 1), the final optimized flow
method (entry 4) showed a reduction in the raw material cost, shortened reaction time and improved
total yield.
Table 4. The comparison with the synthetic route and method of SST-01 (1)
Total
Total Isolated
Synthetic route and method
Cost of
Starting Material
Number of
Steps
Reaction
Time
(h)
Entry
Yield
(%)
of SST-01 (1)
Shown in Scheme 1,
Batch method
Shown in Scheme 2,
Batch method
1
2
3
4
High
Low
Low
Low
1
3
9
61
62
51
76
2
Shown in Scheme 3 and 4,
Batch method
2 (1) a
2 (1) a
6
Shown in Figure 5,
Flow method
0.1b
aThe reaction mixture from the oxidation step without any purification was directly used for the reductive amination
step. b The residence time of the oxidation step was calculated based on the assumption that the total flow rate was the
sum of the flow rate of gas and liquid components.
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CONCLUSION
A two-step continuous-flow process for the cationic lipid SST-01 (1), which is an important
component of Kyowa Kirin’s siRNA-lipid nanoparticle (LNP), was developed. Safe, scalable
catalytic oxidation with 5% oxygen and subsequent homogeneous reductive amination were utilized
in this process. To maintain homogeneity during the two-step operation, the solvents, reagents, and
mixing order were finely optimized and the target lipid amine SST-01 (1) could be synthesized in
95% yield (HPLC assay, 76% isolated yield) and with 2.2 g/h productivity from inexpensive linoleyl
alcohol (4).
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EXPERIMENTAL SECTION
General Information. All reagents and solvents were procured from commercial sources and used
as received. Amino silica gel (CHROMATOREX NH-DM1020) was purchased from Fuji Silysia
Chemical Ltd. 1H and 13C NMR spectra were recorded with JEOL ECA-500 spectrometers in CDCl3
(7.24 and 77.0 ppm). HPLC analyses were performed on a Hitachi Chromaster ® system. HPLC
analyses were performed with the following conditions: Agilent ZORBAX SB-CN (5 μm, 4.6 × 150
mm); column temperature, 60°C; flow rate, 1.0 mL/min; photodiode array detection (206 nm), and
mobile phase gradient, A/B=60/40 (0-10 min), A/B=0/100 (30 min), A/B=0/100 (45 min), mobile
phase A, 2-PrOH/10 mM aq.NH4OAc=15/85; mobile phase B, 2-PrOH/10 mM aq.NH4OAc=85/15.
The HPLC yield was calculated by an HPLC assay using biphenyl as an internal standard and with
purified standard compounds. The HPLC area % was calculated without reagent and solvent peaks.
High-resolution mass spectrometry was carried out with a Synapt G2 HDMS (Waters). As
apparatuses for the flow systems, an HPLC pump (SHIMADZU LC 20AD x 2), plunger pump
(FLOM, Intelligent Pump Model UI-22-110 x 1, FLOM, Dual Pump KP-22 x 2) were used.
Aluminum block heaters SynFlex (EYELA, RCH-1000, BBS-230RB, Aluminum beads) were used
for flow reactions. PTFE tubes (1.0 mm i.d., 1.58 mm o.d.) were used to connect the pumps with the
SUS tube reactor (1.0 mm i.d., 1.58 mm o.d., 20 m or 30 m). 5 % O2 in N2 gas was regulated by a
mass-flow controller (HORIBA STEC, SEC-E40MK3, 10-50mL/min, 10 bar) connected with
control unit (HORIBA STEC, CU-2130). An SUS cross (GL Sciences, U-430, Cat. No. 6010-72358)
and PCTFE fitting 3 neck joint (GL Sciences, Cat. No. 6010-47210) were used as a mixer. The
pressure was regulated by a back pressure regulator (DFC, FC-BPV-500) and monitored at the
HPLC pump display.
Preparation of starting material and reagent solution
Continuous-flow oxidation: Linoleyl alcohol (4) (20.0 g, 75.1 mmol) was added to a 200 mL
volumetric flask and dissolved in NMP (0.375 M). Then, the solution was transferred to a recovery
flask (Reservoir A) and purged with N2 (Solution A). CuI (3.57 g, 18.8 mmol), 4,4’-dimethoxy-2,
2’-bipyridine (4.06 g, 18.8 mmol), TEMPO (2.93 g, 18.8 mmol) and NMI (2.99 mL, 37.5 mmol)
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were mixed in a 500 mL volumetric flask and dissolved in NMP (Cu concentration: 0.0375 M). Then,
the solution was transferred to a recovery flask (Reservoir B) and purged with N2 (Solution B).
Solution A (0.400 mL/min) and Solution B (0.400 mL/min) were flowed by HPLC pumps
(SHIMADZU LC 20AD x 2) and mixed in a T-shape mixer (M1, GL Sciences, PCTFE fitting 3
neck joint). The resulting stream and 5% O2 diluted by N2 (50 mL/min) were introduced into an SUS
tube reactor (1.0 mm i.d., 1.58 mm o.d., 30 m) through a T-shape mixer (M2, GL Sciences, PCTFE
fitting 3 neck joint). The tube reactor was heated to 100°C and the pressure of the reactor was
adjusted to 8 bar by a back pressure regulator (DFC, FC-BPV-500). The resulting solution (Solution
C) was collected in a flask (Reservoir C) purging by N2. To analyze the resulting Solution C, an
appropriate volume of Solution C was picked up and analyzed by HPLC.
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Two-step continuous-flow: 9.8 M MeNH2 in methanol (4.00 mL, 39.2 mmol) was added to a 500
mL volumetric flask and diluted with methanol to 0.0784 M. Then, the solution was transferred to a
recovery flask (Reservoir D) and purged with N2 (Solution D). Me4NBH(OAc)3 (59.3 g, 225 mmol)
was added to a 500 mL volumetric flask and dissolved in NMP (0.450 M). Then, the solution was
transferred to a recovery flask (Reservoir E) and purged with N2 (Solution E). Solution C was
pumped out from Reservoir C by a plunger pump (FLOM Intelligent Pump, UI-22-110) and
combined with streams of Solutions D and E, fed from plunger pumps, in a cross-shaped mixer (M5,
GL Sciences, SUS cross). The combined reaction solution was introduced into a SUS tube reactor
(1.0 mm i.d., 1.58 mm o.d., 20 m) heated to 40°C. The outlet solution was collected for 35 min (94.9
mL) in a recovery flask and analyzed to determine the yields by an HPLC assay using biphenyl as an
internal standard (HPLC yield of SST-01=94.5% from 4). For the isolation of the product, the
recovered reaction mixture (94.9 mL) was diluted with heptane (200 mL), washed successively with
1 M aq. NaOH solution (100 mL) and saturated aq. NaCl solution (200 mL), and dried over
anhydrous magnesium sulfate. The resultant was concentrated under reduced pressure, and was
purified by amino silica gel column chromatography (heptane only) to give SST-01 (1) (1.02 g,
isolated yield=75.9 %, HPLC area =93.6%).
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI: .
Optimization studies for the oxidation and reductive amination in batch; Evaluation of Stability of
catalyst solution in flow; Characterization.
AUTHOR INFORMATION
Corresponding Author
*E-mail: katsuaki.fujiwara.wj@kyowakirin.com
*E-mail: shu_kobayashi@chem.s.u-tokyo.ac.jp.
ORCID
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Shu
̅
Kobayashi: 0000-0002-8235-4368
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
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We thank Koji Hagihara, Masashi Taga for giving us helpful advice on this research subject. We
thank for Takayuki Atago for developing the original batch method. We thank Dr. Hideyuki
Onodera, Akira Ogawa, and Dr. Takeshi Kuboyama for supporting the editing of this manuscript.
ABBREVIATIONS
TEMPO, 2,2,6,6-tetramethylpiperidine 1-oxyl;
NMP, N-methylpyrrolidone; NMI, N-Methylimidazole
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