α-Chymotrypsin and l-acylase aided synthesis of 5-hydroxypipecolic acid via Jacobsen's hydrolytic kinetic resolution of epoxy amino acids

Article abstract of DOI:10.1039/c5ra09207h

Diethyl malonate derivatives were used to synthesize racemic 2-amino-5-hexenoic acid. These racemic 2-amino-5-hexenoic acid (homoallylyglycine) derivatives were efficiently resolved aided by α-chymotrypsin or l-acylase, giving rise to l- and d-enantiomers

Full text of DOI:10.1039/c5ra09207h

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Page 1 of 12
RSC Advances

-Chymotrypsin and
L
-acylase aided synthesis of 5-hydroxypipecolic acid via Jacobsen's
hydrolytic kinetic resolution of epoxy amino acids.
View Article Online
Suvratha Krishnamurthy,†* Jalli Venkataprasad,† Tarun Chand Vagvala,‡ TetsujiDMOI:o1r0.i1g0u39c/hC5i,R†A09207H
and Akihiko Tsuge.†
†Department of Applied Chemistry, Kyushu Institute of Technology, Kitakyushu 804-8550, Japan.
‡Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, Kitakyushu 808-
0196, Japan.
e-mail : suvrathak@gmail.com
Table of Contents/Abstract Graphic
O
HO
H
N
CO2Et
N
CO2Et
OH
O
Boc
Boc
(RR)-Co-salen
HO
H
HO
N
CO2Et
Boc
N
CO2Et
H
N
CO2Et
Boc
Boc
5-hydroxypipecolic acid synthesis from intramolecular reaction of epoxy amino acids; enatiomers
separated by hydrolase and diastereomeric epoxide separated by Jacobsen's hydrolytic kinetic
resolution.
ABSTRACT: Diethyl malonate derivatives were used to synthesize racemic 2-amino-5-hexenoic acid. This
racemic 2-amino-5-hexenoic acid (homoallylyglycine) derivatives were efficiently resolved aided by -
chymotrypsin or L-acylase, giving rise to L- and D- enatiomers. These isolated enatiomerically pure amino acids
with tert-butoxycarbonyl (Boc) protection were oxidised with 3-chloroperbenzoic acid. The oxidation gave rise
to an inseparable diastereomeric epoxides due to newly generated chiral center at C5 carbon. The isolation of
one of the diastereomeric epoxide was possible by selectively converting the remaining diastereomer into a
dihydroxyl compound catalysed by Jacobsen's hydrolytic kinetic resolution (HKR).The isolated epoxide was
regioselectively attacked by LiBr to give vicinal halohydrin, with bromide attacking on terminal C6 carbon.
Upon (Boc) deprotection of the halohydrin, lead to intramolecular cyclization by attack of free amine at C6
carbon, generating a single isomer of 5-hydroxypipecolic acid which was effortlessly recovered in good yield
after re-protection of the amine with (Boc) group. Similarly the dihydroxyl compound isolated earlier was
converted to an halohydrin with iodine at the C6 carbon. This was feasible by efficient regioselective mono-
tosylation with catalytic (Bu2SnO) followed by iodine substitution. This was utilized to synthesize the 5-
hydroxypipecolic acid derivative in the same described sequence consisting of Boc removal by acid treatment,
cyclization, reprotection and purification. Finally the same sequence was repeated with D-isomer and two
diastereomers were isolated.
1

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Page 2 of 12
1. Introduction
5-Hydroxypipecolic acid (5-hydroxypiperidine-2-carboxylic acid) (1, Fig.1), a vital non-proteinogenic
View Article Online
cyclic -hydroxy--amino acid found in plants1,2 and animals3 occurs as a metabolic by-product.DDOeI:r1iv0.a1t0i3v9e/sC5oRfA09207H
5-hydroxypipecolic acid are constituents of a tumor necrosis-converting enzyme inhibitor, TNF- (3, Fig.1).4
Recently cis-5-hydroxypipecolic acid (2, Fig.1) was utilized as a precursor for the synthesis of β-lactamases
inhibitor MK-76555 (4, Fig.1), which combined with Merck’s Primaxin® is in phase II clinical trials for the
treatment of gram-negative bacterial infections.6 It is also employed in the synthesis of 5-guanidino pipecolates,
as constrained arginine mimetic and exhibited weak to moderate inhibition of nitric oxide synthase (NOS).7 cis-
5-hydroxypipecolic acid demonstrates inhibition of Aspergillus spp responsible for spoilage of stored grains.8 It
is also reported to display potent inhibitory effects on human platelet aggregation induced by serotonin.9
Due to its above mentioned biological relevance and ability to induce rigidity owing to its cyclic
structure, several groups have attempted the synthesis of 5-hydroxypipecolic acid.7,10-14 In this context glycine
amino acid residues functionalized with epoxide side chains are synthetically valuable precursors for generation
of cyclic amino acids. We recently synthesized 4-hydroxyproline isomers from epoxide derived from 2-amino-
4-pentenoic acid (allyl glycine), by selective intramolecular reaction of amine on C5 carbon.15 Similarly the
synthesis of 5-hydroxypipecolic acid can be achieved by selective attack of amine group on C6 carbon of 2-
amino-5-hexenoic acid epoxide (homoallyl glycine). Several reports exists on the synthesis of 5-
hydroxypipecolic acid derivatives via intramolecular reactions of epoxy amino acid.1,16 However these synthetic
procedures yield an unresolved diastereomeric mixture of desired cis- and trans-5-hydroxypipecolic acids, 1,16,17
along with the undesirable 5-hydroxymethylprolines.1,17
A straightforward method generating 5-
hydroxypipecolic acid derivatives bypassing the laborious need to isolate the cis- and trans- diastereomers in the
final stages combined with elimination of the formation of regioisomeric proline is desired. In this paper we
would like to present the synthetic scheme circumventing the drawbacks imposed by the established synthetic
procedures involving generation of 5-hydroxypipecolic acid by intramolecular reaction of epoxy amino acid. To
the best of our knowledge this is the first work reporting the synthesis of 5-hydroxy pipecolic acid wherein the
2S (L-) and 2R (D-) isomers were efficiently separated by enzymatic kinetic resolution. Whereas the epoxide
precursors that give rise to cis- and trans- isomers of 5-hydroxypipecolic acid were separated via Jacobsen's Co
catalyzed HKR.
2

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2. Results and Discussion
-acylase and -Chymotrypsin catalyzed enzymatic resolution of 2-amino-5-hexenoic acid
derivatives.
L

View Article Online
DOI: 10.1039/C5RA09207H
Enatiomerically pure 2-amino-5-hexenoic acid derivatives were synthesized by enzymatic resolution of acetyl
amino acid and tert-Boc amino acid ester, with L-aminoacylase and -chymotrypsin respectively (Scheme 1).
CO2Et
L-aminoacylase
AcHN
CO2Et
AcHN
CO2H
H2N
(S)-7
CO2H
AcHN
CO2H
5
(RS)-6
(R)-6
CO2Et
CO2Et
chymotrypsin
BocHN
BocHN
CO2H
BocHN
(RS)-9
Scheme 1. Enzymatic resolution of 2-amino-5-hexenoic acid derivatives.
CO2Et
BocHN
CO2Et
(R)-9
8
(S)-10
Diethyl acetamidomalonate (5) was reacted with excess of 4-bromo-1-butene in the presence of sodium ethoxide
in absolute ethanol to obtain the alkylated diester. The diester was half saponified and decarboxylated
simultaneously, by treating with solid NaOH in EtOH:H2O mixture (1:1, v/v) and refluxing for 24 h. This gave
the desired racemic acetyl amino acid (RS)-6, (47 % overall yield based on 5) suitable for the L-acylase
catalyzed enzymatic resolution.
An aqueous solution of (RS)-
treated with
-aminoacylase from Aspergillus genus.18 After 24 h, the solution was concentrated and
acidified with 1M HCl to pH 3 and extracted with EtOAc to remove the unreacted (R)- . The remaining
acidic solution was passed through ion exchange resin (DOWEX 50WX8) to separate (S)-
, which was
6, maintained at 38 C, with pH 7.5 adjusted with LiOH (aq) was
L
6
7
selectively eluted with NH4OH (1M). Due to high water solubility of the acetyl amino acid the yields
were less, but pure D- and L-2-amino-5-hexenoic acid were obtained with good chemical and
enatiomeric purity ascertained by 1H NMR and chiral HPLC.
Subsequently diethyl (2-Boc-amino)malonate (8), was treated with excess 4-bromo-1-butene in
presence of sodium ethoxide to obtain alkylated diester. The later was selectively saponified with solid
LiOH to obtain the half acid half ester, which was successfully decarboxylated in refluxing toluene to
obtain (RS)-
Enzymatic resolution was carried by suspending the racemic (RS)-
mmol) at 38 C and treating with -chymotrypsin. To avoid slight deviance of pH caused by formation
9 (59 % overall yield with respect to 8).
9
in phosphate buffer (pH 8, 0.1


of (S)-10, minimal amount of NH4OH (1M) was added and maintained at pH 8. After 24 h, the mixture
was cooled to room temperature and alkalinity was enhanced by addition of (4% NaHCO3) followed by
extraction with EtOAc to recover unreacted (R)-
9 (51 %). The remaining alkaline aqueous solution was
subsequently acidified with solid citric acid to pH 3, extracted with EtOAc to obtain (S)-10 (46%).
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Page 4 of 12
O
O
*
5
5
*
View Article Online
DOI: 10.1039/C5RA09207H
m-CPBA
2
2
HN
Boc
CO2Et
HN
Boc
CO2R
HN
Boc
CO2Et
-R = H; S-10
-R = Et;S-9
(2S,5R)-11
(2S,5S)-11
Scheme 2. m-CPBA oxidation of the alkene side chain of (S)-9.
m
-CPBA oxidation.(Scheme 2)
Kinetically resolved Boc-amino acid (S-10) was esterified with ethyl bromide in the presence of Et3N to
obtain (S)- in 84% yield. This fully protected Boc-amino acid ester with unsaturated side chain was
epoxidized with excess 3-chloroperbenzoic acid (m-CPBA) by treating the solution of ( -9 in DCM
with excess m-CPBA at 0 C, followed by 24 h stirring at room temperature. Subsequently excess m-
CPBA was cautiously reduced with 10% Na2SO3 at 0 C. The DCM layer was separated and washed
9
S
)


(4% NaHCO3 and brine), concentrated and chromatographed (SiO2) to obtain 11 (90%, yield).
The resulting epoxide was a diastereomeric mixture (2S,5R)-11 and (2S,5S)-11 due to the newly
generated chiral centre at C5 carbon. However the diastereomers moved as a single spot with same Rf
value on TLC and could not be separated.1H NMR analysis in (CDCl3) did not show any set of
distinguishable peaks for the individual diastereomers. In this context m-CPBA oxidation of similar
epoxides is believed to produce 1:1 mixture of both diastereomers, nonetheless it was reported that
these diastereomers were inseparable.16,19
OH
O
HO
O
(RR)-Co-Salen
HN
Boc
CO2Et
HN
Boc
CO2Et
HN
Boc
CO2Et
(2S,5R)-11
(2S,5S)-12
11
47%
46%
Scheme 3. Jacobsen's hydrolytic kinetic resolution of (2S,5S)-11 and
(2S,5R)-11
Hydrolytic kinetic resolution (HKR) of epoxy amino acids.20,21 (Scheme 3)
To aid the separation of the diastereomeric epoxide, 11 was treated with (0.55 eq) of H2O in the
presence of (0.5 mol%) of (RR)-Co-Salen and AcOH, at room temperature in THF. This produced the
chiral diol (2S,5S)-12 and chiral epoxide (2S,5R)-11 which had a considerable Rf difference. In fact the
chiral epoxide (2S,5R)-11 and chiral diol (2S,5S)-12, were easily separated by silica gel column
chromatography in good yield, 47% and 46%, respectively.
tert-butoxycarbonyl (Boc) removal and intramolecular cyclization.(Scheme 4)
Next, the chiral epoxide (2S,5R)-11 was treated with LiBr in the presence of AcOH in THF, which
attacked the C6 carbon selectively giving rise to bromohydrin 13 22
. This bromo amino acid was isolated
in good yield (92%) after (SiO2) purification, which was suitable for synthesis of 5-hydroxypipecolic
acid. To aid the intramolecular cyclization, Boc group of 13 was removed by treating it with TFA. After
4

Page 5 of 12
RSC Advances
1h TFA was removed to obtain, 14.TFA in quantitative yield which was not purified owing to its
hydrophilic character, instead it was used for next step directly.
View Article Online
14.TFA, was dissolved in THF at room temperature and treated with DIEA (2 eq) and DstOirI:r1e0d.1.0D39I/CE5ARA09207H
treatment leads to the neutralization of TFA and simultaneously gives rise to highly nucleophilic and
free amine. This amine will attack intramolecularly the C6 carbon, thereby producing the 5-
hydroxypipecolic acid. Due to the formation of HBr after intramolecular cyclization, another equivalent
of DIEA was required to neutralize this and complete the cyclization. TLC analysis at the end of 4 h,
(butanol/pyridine/acetic acid/H2O, 4:1:1:2, v/v/v/v) by staining with ninhydrin showed the absence of
the reactant and appearance of a yellow spot, which is characteristic of the cyclic amino acids. This
confirmed that the nitrogen intramolecularly attacked the side chain to produce the piperidine ring, i.e.,
(2S,5R)-15. To aid the purification process the nitrogen of the piperidine ring was again reprotected
with Boc group by treatment with Boc2O in presence of DIEA to generate the Boc-5-hydroxypipecolic
acid derivatives. 5-hydroxypipecolic acid, (2S,5R)-16 was separated as an oil (80%) by silica gel
column chromatography (50% EtOAc in hexane).
Subsequently the oil (2S,5R)-16 was converted to 1.HCl. The spectral data, melting point and
specific rotation of 1.HCl, were in good agreement with the reported values.23
Br
Br
O
HO
HO
HO
DIEA
TFA
LiBr
THF
Br
AcOH,THF
CO2Et
HN
Boc
(2S,5R)-11
CO2Et
HN
Boc
CO2Et
H2N
TFAH2N
CO2Et
(2S,5R)-13
(2S,5R)-14.TFA
HO
HO
HO
Boc2O
Ref 11
N
H
CO2H
DIEA,THF
N
Boc
CO2Et
N
H
CO2Et
(2S,5R)-15
(2S,5R)-16
1
Scheme 4. Formation of (2S,5R)-hydroxypipecolic acid by intramolecular cyclization
Regioselective halogenation,
N-Boc removal and intramolecular cyclization.
In order to transform the chiral diol 12 into 5-hydroxypipecolic acid, it was necessary to transform 12
into amino acid with a good leaving group at C6 carbon similar to 13. This was achieved by selective
tosylation of diol by treating with TsCl in the presence of catalytic Bu2SnO (30 mol%), DMAP (cat.)
and triethylamine at -10
temperature was maintained between -10

C, yielding the mono tosylated product in almost quantitative yield.24-26 The

C to 0 C in order to retain the acid labile Boc group. The

tosyl group in 17 was replaced with iodo group by refluxing it in the presence of NaI (6 eq) in acetone.
After 8 h, acetone was replaced with EtOAc washed with (sat.Na2S2O3), brine and chromatographed on
(SiO2) resulting (2S,5S)-18 (95%) used for synthesis of 5-hydroxypipecolic acid (Scheme 5).
5

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Page 6 of 12
OH
OTs
I
HO
HO
HO
HO
HO
Ref 11
NaI
i-iii
TsCl
View Article Online
Bu2SnO
DMAP, DCM
DOI: 10.1039/C5RA09207H
acetone
reflux
N
CO2Et
N
H
CO2H
Boc
BocHN
CO2Et
BocHN
CO2Et
BocHN
CO2Et
(2S,5S)-12
(2S,5S)-17
(2S,5S)-18
(2S,5S)-19
2
Scheme 5. Formation of (2S,5S)-hydroxypipecolic acid by intramolecular cyclization i.TFA; ii. DIEA, THF; iii. Boc2O, DIEA,THF.
To aid the intramolecular cyclization Boc group of 18 was removed by treating with TFA at
room temperature. After 1h the solution was concentrated to obtain Boc- deprotected amino acid salt of
TFA in quantitative yield, which was used for subsequent step directly.
After dissolving in THF at room temperature and treating it with DIEA (2 eq) free amine was
generated. The free amine attacked the C6 carbon intramolecularly generating 5-hydroxypipecolic acid.
After 3 h, to aid the purification process the nitrogen of the amino acid was again reprotected with Boc
group by treating with Boc2O in presence of DIEA to generate the Boc-5-hydroxypipecolic acid
derivatives. The fully protected 5-hydroxypipecolic acid (2S,5S)-19 was easily separated as an oil by
silica gel column chromatography (50% EtOAc in hexane) in 76 % yield. Subsequently the oil (2S,5S)-
19 was converted to 2.HCl. The spectral data, melting point and specific rotation of 2.HCl, were in
good agreement with the reported values.27,28
Synthesis of (2R,5R)-20 and (2R,5S)-21. Finally the synthesis of the (2R,5R) and (2R,5S)- isomers of the 5-
hydroxypiecolic acid was easily achieved starting from (2R)-9 (Scheme 6), [ESI-S2].
HO
HO
N
CO2Et
N
CO2Et
HN
Boc
(R)-9
CO2R
Boc
Boc
(2R,5R)-20
(2R,5S)-21
Scheme 6. Formation of (2R,5S) and(2R,5R) isomers by intramolecular
cyclization
3. Experimental section
General: Reagents, dry solvents, and enzymes were from commercial sources. DOWEX 50WX8 (50–100) was
used as an ion exchange resin, after washing with 1M HCl. (R,R)-(−)-N,N′-Bis(3,5-di-tert-butylsalicylidene)-
1,2-cyclohexanediaminocobalt(II) was purchased from Sigma-Aldrich®. TLC was visualized by one of the
following, UV (F254), I2, H3(PMo12O4) or ninhydrin. Chiral HPLC analysis were performed on Column I:
CHIRALPAK IA column (4.6 × 250 mm, Diacel Chemical Industries, Ltd.), n-hexane:2-propanol:TFA
(97:3:0.1, v/v/v) with a flow rate of 1mL/min, detection at 220 nm; Column II: CHIRALCEL OD column (4.6 ×
250 mm, Diacel Chemical Industries, Ltd.), n-hexane:2-propanol:TFA (90:10:0.1) with a flow rate of 1mL/min,
1
detection at 220 nm. H were measured in CDCl3, CD3OD or D2O at 500 or 400 MHz and 13C NMR were
measured in CDCl3, CD3OD or D2O at 125 MHz. Normal- and high resolution-FAB mass were measured
routinely. Optical rotations were measured using Jasco Polarimeter (P-1010). Melting point (m.p.) were
recorded by As One melting point instrument (Model Number ATM-01).
Kinetic resolutions by -acylase from Aspergillus genus.
L
6

Page 7 of 12
RSC Advances
(
RS)-2-(acetamido)-5-hexenoic acid (RS)-6
.
Sodium metal (5.10 g, 222.0 mmol) was added to a flask
. After complete dissolution of sodium, 5 (4V3ie.w5Agrt,icle Online
containing 195 mL of absolute ethanol maintained at 0

C
200 mmol) was added in one portion and the resulting solution was refluxed. After 3h to this reflDuOxiI:n1g0.1s0o3l9u/tCio5RnA09207H
4-bromo-1-butene (25.0 g, 185 mmol) was added and continued to reflux. After 24 h the mixture was
concentrated and the residue dissolved in EtOAc (600 mL) and washed with 10% citric acid, 4% NaHCO3, brine,
dried (MgSO4), filtered and evaporated to get the alkylated diester.
This diester was dissolved in 1:1 (v/v), mixture of ethanol and water (230 mL) at 0 C, and to this solution
solid NaOH (7.0 g, 175 mmol) was added, this solution was then refluxed. After 24h the ethanol was removed
and the resulting solution was dissolved in 4% NaHCO3 and extracted with diethyl ether to remove the
unreacted starting material. Then the remaining alkaline aqueous solution was acidified to pH 3 with 1M HCl
and extracted with EtOAc (20 mL × 20). The combined organic layer was washed with brine, dried (MgSO4),
filtered and evaporated to give (RS)-6 as a white powder (15 g, 47 % w.r.t. 5). mp 119-121 C [lit.29 106-108
1
C]; H NMR (400 MHz, CD3OD) 5.89-5.79 (m, 1H), 5.10-5.00 (m, 2H), 4.37 (m, 1H), 2.14 (m, 2H), 1.99 (s,
3H), 1.96-1.71 (m, 2H); 13C NMR (125 MHz, CD3OD) 175.6, 173.5, 138.5, 116.2, 53.3, 32.1, 31.2, 22.5;
LRMS-FAB (m/z) 343 (67), 194 (58), 172 (100), 126 (68), 83 (68); HRMS-FAB (m/z) calcd for C8H14N1O3
([M+H]+)172.0974 found 172.0909.
(S)-2-amino-5-hexenoic acid (S)-7 and (R)-2-(acetamido)-5-hexenoic acid (R)-6. (RS)-6 (3.00 g, 17.5
mmol) and CoCl2•6H2O (14 mg, 50 μmol) was dissolved in 60 mL of H2O at 38 C, while maintaining pH at
7.5 with aqueous LiOH.
pH was monitored at 7.5 with small amount of NH4OH (1M). After 24 h, the reaction mixture was
concentrated, acidified to pH 2-3 with 1M HCl. The unreacted (R)- was extracted with EtOAc, washed
with brine, dried (MgSO4), filtered and evaporated to give (R)- (1.23 g, 41%). (Column II. ee 88%).
L
-aminoacylase from Aspergillus genus (0.8 g) was then added and stirred. The
6
6
The remaining acidic aqueous phase was applied to a column of ion exchange resin (DOWEX
50WX8). After sample charging and elution with 1M HCl, followed by H2O, finally the compound (S)-
7
was eluted with 1M NH4OH. Evaporation of this eluant afforded (S)-7 as white solid (0.72g, 32 %).
1
(after N acetylation, Column II. ee > 99%) H NMR (D2O, 500 MHz) 5.84-5.76 (m, 1H), 5.12-5.03 (m,
2H), 3.72-3.69 (m, 1H), 2.15-2.11 (m, 2H), 1.99-1.85 (m, 2H); 13C NMR (D2O, 125 MHz) 174.7, 137.0,
115.9, 54.3, 29.7, 28.6; LRMS-FAB (m/z) 130 (100).
Kinetic resolutions by
RS)-ethyl 2- -(tert-butoxycarbonyl)-2-amino-5-hexenoate (RS)-9. Metallic sodium (0.92 g, 39.9
mmol) was added to EtOH (17.0 mL) at 0 C and stirred until complete dissolution of sodium. (10.0 g,
-chymotrypsin.
(
N

8
36.3 mmol) was then added at the same temperature and stirred for 30 min. Finally 4-bromo-1-butene
(5.6 mL, 54.8 mmol) was added in one portion and the resulting yellow solution was refluxed for 24 h.
After 24 h, the solution was partitioned between H2O and EtOAc, extracting the desired product into
EtOAc. The EtOAc was then washed with 10% citric acid, brine, dried (MgSO4), filtered and
evaporated to obtain an oil (9.51 g), which was suspended in EtOH:H2O (18:8, v/v) and cooled to 0
C.
This solution was treated with solid LiOH.H2O (1.33 g, 31.8 mmol) and stirred overnight at 0 C. The

solution was then mixed with 4% NaHCO3 to make the solution alkaline and extracted with diethyl ether
to remove any unreacted starting material. The remaining alkaline solution was then acidified to pH 3
with solid citric acid and extracted with EtOAc. The EtOAc was then washed with brine, dried
(MgSO4), filtered and evaporated to give the mono ester mono acid (8.30 g). This was dissolved in
7

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Page 8 of 12
toluene (25 mL) and refluxed. After 18 h the solvent was evaporated at 40-50 C to give a crude
mixture which directly purified by silica gel column chromatography (hexane:EtOAc, 80:20, v/Vvie)wtAorticle Online
DOI: 10.1039/C5RA09207H
1
give (RS)-
9
(5.5 g, 59%, after 3 steps). H NMR (500 MHz, CDCl3) 5.84-5.76 (m, 1H), 5.07-5.00 (m,
2H), 4.31-4.30 (m, 1H), 4.21 (m, 2H), 2.15-2.09 (m, 2H), 1.95-1.88 (m, 1H), 1,75- 1.68 (m,1H), 1.45 (s,
9H) 1.28 (m, 3H); 13C NMR (125 MHz, CDCl3) 172.8, 155.3, 137.1, 115.6, 79.8, 61.3, 53.1, 32.1, 29.5,
28.3, 14.2; LRMS-FAB (m/z) 258 (50), 230 (21), 202 (100), 158 (93) and 128(18); HRMS-FAB (m/z)
calcd for C13H24 N1O4 ([M+H]+) 258.1705, found 258.1707.
(R)-ethyl
2-N-(tert-butoxycarbonyl)-2-amino-5-hexenoate
(R)-9
and
(S)
2-N-(tert-
butoxycarbonyl)-2-amino-5-hexenoic acid (S)-10. (RS)-9 (4.80 g, 18.6 mmol) was suspended in phosphate
buffer ( 360 mL, 0.1 M; pH 8.00), warmed to 38 C and α-chymotrypsin (28 mg, 40 units/mg) was added in one
portion. The resulting mixture was stirred at 38 C for 24 h while monitoring the pH 8 with little amount of (1M
NH4OH). After 24 h the above solution was made more alkaline by addition of 4% NaHCO3 and was extracted
with EtOAc (20 ml × 10). The combined EtOAc layer (200 mL) was washed with brine, dried (MgSO4),
filtered and evaporated to give (R)-9 ( 2.42 g, 51 %). (Column I. ee > 64%); [α]D25 = - 9.30 (c = 2.44, CH2Cl2).
The remaining alkaline solution was then acidified to pH 3, with solid citric acid and extracted with EtOAc (30
ml × 10). The combined EtOAc layer (300 mL) was washed with brine, dried (MgSO4), filtered and evaporated
to give (S)-10 (1.95 g, 46 %). (Column I. ee > 99%); [α]D20 = - 1.18 (c = 1.30, CH3OH). {lit.30[α]D20 = - 1.1 (c
= 1.30, CH3OH)}; 1H NMR (500 MHz, CD3OD) 5.81-5.73 (m, 1H), 5.01-4.98 (m, 3H), 4.04-4.02 (m, 1H), 2.18-
2.08 (m, 2H), 1.91-1.85 (m, 1H), 1.75-1.68 (m, 1H), 1.44 (s, 9H); 13C NMR (125 MHz, CD3OD) 175.0, 156.7,
137.1, 114.6, 79.1, 52.9, 30.8, 29.7, 27.3; LRMS-FAB (m/z) 275 (35), 252 (16), 230 (17), 174 (100) 130 (65);
HRMS-FAB (m/z) calcd for C11H20N1O4 ([M+H]+) 230.1392, found 230.1336.
m-CPBA oxidation.
(S)-ethyl 2-N-(tert-butoxycarbonyl)-2-amino-5-hexenoate (S)-9. (S)-10 (1.31 g, 5.72 mmol) was dissolved in
DMF (15 mL) and cooled with an ice-water bath. This solution was treated with triethylamine (1.20 mL, 8.58
mmol) and finally ethyl bromide (2.6 mL, 35 mmol). The resulting solution was then allowed to attain room
temperature and continued to stir. After 24 h the solution was concentrated to get a crude mixture as an oil. This
crude mixture was purified by silica gel column chromatography (hexane:EtOAc, 80:20, v/v) to give (S)-9 (1.24
g, 84 %). [α]D25 = + 8.53 (c = 2.46, CH2Cl2). [1H NMR same as (RS)-9].
ethyl 2-[(tert-butoxycarbonyl)amino]-4-(2-oxiranyl)butanoate, (2S,5R)-11 and (2S,5S)-11. (S)-9
(1.2 g, 4.67 mmol) was dissolved in DCM 20.0 mL and cooled to 0 C with ice-water bath. To this solution m-
CPBA (1.57 g, 7.0 mmol) was added in one portion and stirred. The ice-water bath was removed once the m-
CPBA completely dissolved and allowed to stir at room temperature. After 24 h the crude reaction mixture was
again cooled with in an ice-water bath, treated with 10% aqueous Na2SO3 (30 mL), then the solution was stirred
vigorously to reduce the excess m-CPBA. After 2h the organic phase was separated and washed with 4%
NaHCO3, brine, dried (MgSO4), filtered and evaporated to get a clear oil. This was purified by silica gel column
1
chromatography (hexane:EtOAc, 70:30, v/v) to get pure 11 as a clear oil (1.15 g, 90 %). H NMR (500 MHz,
CDCl3) 5.10-5.05 ( m, 1H), 4.32 (m, 1H) 4.20 (q, 2H), 2.93 (m, 1H), 2.76 (m, 1H), 2.49 (m, 1H,) 1.99 (m, 1H)
1.8 (m, 1H), 1.68 (m, 1H) 1.51 (m, 1H), 1.45 (s, 9H), 1.29 (t, 3H); 13C NMR (125 MHz, CDCl3) 172.5 (172.4),
155.4, 79.9, 61.5 (61.4), 53.2 (53.0), 51.6 (51.5), 47.1 (47.0), 29.2, 28.4, 28.3, 14.2(14.1); LRMS-FAB (m/z)
274 (42), 218 (100), 174 (49); HRMS-FAB (m/z) calcd for C13H24N1O5 ([M+H]+) 274.1654, found 274.2659.
Jacobsen’s Hydrolytic Kinetic Resolution (HKR).
8

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RSC Advances
ethyl 2-[(tert-butoxycarbonyl) amino]-4-(2-oxiranyl)butanoate (2S,5R)-11 and ethyl 2-[(tert-
butoxycarbonyl) amino]-5,6-dihydroxyhexanoate, (2S,5S)-12. 11 (1 g, 3.65 mmol) was taken in a vial
View Article Online
equipped with a stir bar, to this was added (RR)-Co-Salen (0.5 mol %, 12 mg), followed by acetiDcOaIc: 1id0.1(03309/μCL5R)A09207H
and THF (0.3 mL). Finally H2O (2 mmol, 36 μL) was added in one portion at room temperature and stirred.
After 48 h, the solution was directly purified by silica gel column chromatography, eluting the column by
(EtOAc:hexane, 30:70, v/v) gave (2S,5R)-11 (0.47 g, 47 %) as a clear oil. Further elution with
1
(EtOAc:haxane:methanol, 60:40:2, v/v/v) gave (2S,5S)-12 (0.49 g, 46 % ). H (500 MHz, CDCl3) 5.25-5.14 (m,
0.66H), 4.36 (m, 0.73H), 4.21 (q, 2H), 3.75 (br s, 0.91H), 3.64 (br s, 0.96H), 3.46 (m, 0.98H), 2.03-1.93(m,
1.90H), 1.84-1.68 (m, 0.82H), 1.60-1.48 (m, 5H), 1.45, 1.44 (both s, total 9H) 1.29 (t, 3H); 13C NMR (125 MHz,
CDCl3) 173.0 (172.8), 155.8, 80.0 (79.9), 71.7 (71.5), 66.6, (66.5), 61.4, 53.4, 29.0, 28.7, 28.3, 14.1; LRMS-
FAB (m/z) 356 (33), 354 (35), 300 (53), 298 (55), 256 (98), 254 (100); HRMS-FAB (m/z) calcd for C13H26N1O6
([M+H]+) 292.1760, found 292.1767.
Synthesis of (2S,5R)-5-hydroxypipecolic acid derivatives. (Scheme 4)
ethyl 2-[(tert-butoxycarbonyl) amino]-5-hydroxy-6-bromohexanoate (2S,5R)-13. 11 (0.46 g, 1.71 mmol)
was dissolved in THF (15 mL) and cooled to 0 C. To this solution AcOH (0.30 mL, 5.25 mmol) was added
followed by LiBr (0.24 g, 2.76 mmol). The solution was then warmed to room temperature and stirred. After
16h, when the TLC analysis showed the complete disappearance of the epoxide, the solution was concentrated
in vacuum and the residue taken in EtOAc. Washed with 4% NaHCO3, brine, dried( MgSO4), filtered and
evaporated to get crude product which was purified by silica gel column chromatography eluting with
1
(EtOAc:hexane, 40:60 v/v) to get 13 (0.55 g, 92 %). H NMR (500 MHz, CDCl3) 5.21 (bd, 1H), 4.37 (m, 1H),
4.21 (m, 2H), 3.82 (br s, 1H), 3.50-3.38 (m, 2H), 2.87 (bd, 1H), 2.05-1.91 (m, 1H), 1.86-1.54 (m, 3H), 1.45 (s,
9H), 1.29 (t, 3H); 13C NMR (125 MHz, CDCl3), 172.5, 155.7, 80.2, 70.8, 61.5, 53.1, 40.1, 30.6, 29.7, 28.3,
14.2; LRMS-FAB (m/z) 356 (33), 354 (35), 300 (53), 298 (55), 256 (98), 254 (100); HRMS-FAB (m/z) calcd for
C13H25N1O5Br1 ([M+H]+) 354.0916, found 354.0881.
1-tert-butyl 2-ethyl 5-hydroxypiperidine-1,2-dicarboxylate (2S,5R)-16. 13 (0.26 g, 0.73 mmol) was
dissolved in TFA (5 ml) and allowed to stand at room temperature. After 1 h when the reaction was complete
(TLC) the excess TFA was evaporated. The crude product was redissolved in diethyl ether and evaporated to
remove excess TFA, to get 14.TFA as a white sticky oil.
14.TFA was dissolved in THF (5 mL) and to this DIEA (0.25 mL, 1.46 mmol) was added, and the
resulting solution was stirred at room temperature. After 4h the solution was treated with additional DIEA (0.13
mL, 0.73 mmol) and Boc2O (0.32 g, 1.46 mmol). The resulting solution stirred at room temperature. After 12 h
the solution was concentrated and the residue was dissolved in ethyl acetate and washed with 4% NaHCO3, 10%
citric acid, brine, dried (MgSO4), filtered and evaporated to get an oil purified by silica gel column
1
chromatography (EtOAc:hexane, 50:50, v/v) to get 16 as an oil (0.16 g, 80%). H (500 MHz, CDCl3) 4.91, 4.74
(both br s, 1H), 4.20 (m, 2H), 4.08-3.95 (m, 2H), 3.24-3.10 (m, 1H), 2.17-2.00 (m, 2H), 1.89-1.76 (m, 2H), 1.47,
1.45 ( both s, total 9H); 13C (125 MHz, CDCl3) 171.7 (171.5), 156.5 (156.2), 80.4, 61.1, 54.7 (53.5), 48.1 (47.1),
28.2, 27.2 (26.9), 20.3, 14.3; LRMS-FAB (m/z) 274 (57), 218 (100), 174 (100), 144 (50); HRMS-FAB (m/z)
calcd. for C13H24 N1O5 ([M+H]+) 274.1654, found 274.1656.
9

RSC Advances
Page 10 of 12
Synthesis of (2S,5S) 5-hydroxypipecolic acid derivatives. (Scheme 5)
ethyl 2-[(tert-butoxycarbonyl)amino]-5-hydroxy-6-tosylhexanoate (2S,5S)-17. 12 ( 0.45 g, 1.53 mmolV)ieww aArsticle Online
DOI: 10.1039/C5RA09207H
dissolved in DCM (25.0 mL) and cooled to -10 C, to this stirred solution triethylamine (0.25 mL, 1.8 mmol)
followed by Bu2SnO (0.11 g, 0.46 mmol, 30 mol%) and DMAP (10 mg, cat.) were added. The solution was then
stirred for 15 min at -10 C finally TsCl (0.32 g, 1.68 mmol) was added portion wise over a period of 1 h. The
resulting solution was stirred for an additional 1 h. At the end of 2h, the reaction was quenched with 4%
NaHCO3 and DCM was replaced with EtOAc. The EtOAc layer was washed with brine, dried(MgSO4), filtered
and evaporated to give a crude mixture purified by silica gel column chromatography (EtOAc:hexane, 30:70 to
50:50, v/v) to give 17 (0.67 g, 97 %). 1H (500 MHz, CDCl3) 7.80 (d, J = 8.3 Hz, 2H), 7.36 (d, J = 8.1 Hz, 2H),
5.10 (br d, 1H), 4.27 (m, 1H), 4.19 (m, 2H), 4.12 (m, 1H), 4.00 (m, 1H), 3.88 (m, 2H), 2.46 (s, 3H), 1.91-1.77
(m, 2H), 1.55-1.46 (m, 2H), 1.43 (s, 9H); 13C (125 MHz, CDCl3) 172.5, 155.5, 145.1, 132.6, 130.0, 128.0, 80.0,
73.7, 68.8, 61.5, 53.0, 28.7, 28.3, 21.7, 14.2; LRMS-FAB (m/z) 446 (15), 390 (42), 347 (37), 346 (100);
HRMS-FAB (m/z) calcd for C20H32 N1O8S1 ([M+H]+) 446.1849, found 446.1864.
ethyl 2-[(tert-butoxycarbonyl)amino]-5-hydroxy-6-iodohexanoate (2S,5S)-18. 17 (0.55 g, 1.23
mmol) was dissloved in acetone (30 mL) and to this solution NaI (1 g, 6.67 mmol) was added and the resulting
solution was refluxed for 8 h. After 8 h, the acetone was replaced with EtOAc and washed with sat.Na2S2O3,
brine, dried (MgSO4), filtered and evaporated to give an oil purified by silica gel chromatography
1
(EtOAc:hexane, 50:50, v/v) to give 18 (0.47 g, 95 %). H (500 MHz, CDCl3) 5.11 (bd, 1H), 4.32 (m, 1H), 4.21
(m, 2H), 3.58 (m, 1H), 3.36 (m, 1H), 3.22 (m, 1H), 2.16 (bd, 1H), 1.93-1.92 (m, 1H), 1.82-1.80 (m, 1H), 1.70-
1.66 (m,1H), 1.60-1.53 (m, 1H), 1.45 (s, 9H), 1.28 (m, 3H); 13C NMR (125 MHz, CDCl3) 172.5, 155.5, 80.1,
70.3, 61.5, 53.1, 32.1, 29,1, 28.3, 15.8, 14.2; LRMS-FAB (m/z) 402 (35), 346 (69), 302 (100), 284 (37); HRMS-
FAB (m/z) calcd for C13H25N1O5I1 ([M+H]+) 402.0777, found 402.0794.
1-tert-butyl 2-ethyl 5-hydroxypiperidine-1,2-dicarboxylate (2S,5S)-19. 18 (0.262 g, 0.65 mmol) was
dissolved in TFA (4 ml) and allowed to stand at room temperature. After 1 h when the reaction was complete
(TLC) the excess TFA was evaporated, the crude product was redissolved in diethyl ether and evaporated to
remove excess TFA, to obtain Boc deprotected product as a salt of TFA, which was dissolved in THF (5 mL)
and to this DIEA (0.23 mL, 1.30 mmol) was added, and the resulting solution was stirred at room temperature.
After 4h when the reaction was complete which was confirmed by TLC, the solution was treated with additional
DIEA (0.12 mL, 0.65 mmol) and Boc2O (0.28 g, 1.30 mmol) and the resulting solution stirred at room
temperature. After 12 h the solution was concentrated and the residue was dissolved in ethyl acetate and washed
with 4% NaHCO3, 10% citric acid, brine, dried (MgSO4), filtered and evaporated to get an oil purified by silica
gel column chromatography (EtOAc:hexane, 50:50, v/v) to get 19 as an oil (0.13 g, 76 %). 1H NMR (500 MHz,
CDCl3) 4.87-4.58 (m, 1H), 4.28-4.04 (m, 3H), 3.71-3.58 (m, 1H), 2.82-2.64 (m, 1H), 2.35-2.19 (m, 1H) 2.04-
1.94 (m, 1H), 1.78-1.67 (m, 1H), 1.62-1.53 (m, 1H), 1.47, 1.44 (both s, total 9H); 13C NMR (125 MHz, CDCl3)
171.5 (171.4), 155.5 (155.2), 80.5, 66.8 (66.7), 61.3, 54.0 (52.8), 48.5 (47.6), 30.5 (30.0), 28.3, 25.0 (24.8),
14.2; LRMS-FAB (m/z) 274 (70), 218 (60), 174 (100), 144 (45); HRMS-FAB (m/z) calcd for C13H24 N1O5
([M+H]+) 274.1654, found 274.1658.
4. Conclusion
Starting from a common intermediate all the four isomers of 5-hydroxypipecolic acid (2S,5R)-, (2S,5S)-,
(2R,5S)- and (2R,5R) were synthesized from commercially available malonate derivatives.
Enatiomerically pure 2-amino-5-hexenoic acids were obtained by enzymatic resolution of the racemic
10

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RSC Advances
amino acid ester (

-chymotrypsin) or acetyl aminoacid (
L
-acylase). Oxidation of the unsaturated side
chain of the enatiomerically pure
L
-2-amino-5-hexenoic acid with m-CPBA, generatedViewthAerticle Online
DOI: 10.1039/C5RA09207H
diastereomeric, inseparable epoxides. Co catalysed hydrolytic kinetic resolution converted the
diastereomeric epoxide into separable components, each of them were correspondingly transformed into
C6 halo amino acids. Followed by, intramolecular nucleophilic attack of the amino -NH2 group at C6
carbon generating the respective 5-hydroxypipecolic acid diastereomers. Similarly starting from -2-
D
amino-5-hexenoic acid, two diastereomers of 5-hydroxypipecolic acid were synthesized. Ultimately
synthesis of a single diastereomer was successfully achieved surpassing the difficulty of isolation of the
cis- and trans- diastereomers, in a facile fashion. Application of these compound to synthesize
biologically important cyclic tetra peptides are being investigated which will be reported in future.
■ Associated content
Supporting Information
Synthesis of (2R) isomers, 1H NMR and 13C NMR spectra.
■ Author information
Corresponding Author
*suvrathak@gmail.com
■ Acknowledgements
We are grateful to the Center for Instrumental Analysis, Kyushu Institute of Technology (KITCIA) for 1H NMR
and 13C NMR spectra. We also like to thank Dr. Tamaki Kato for helping us in chiral HPLC analysis.
■ References
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