Preparation of α-hydroxy-β-Fmoc amino acids from N-Boc amino acids

Article abstract of DOI:10.1055/s-0031-1289586

A general method for the conversion of N-Boc amino acids into their homologated α-hydroxy-β-Fmoc amino acids is described. The protocol involved preparation of the amino aldehyde by reduction of the corresponding Weinreb amides, hydrocyanation, and hydrol

Full text of DOI:10.1055/s-0031-1289586

PAPER
4023
Preparation of a-Hydroxy-b-Fmoc Amino Acids from N-Boc Amino Acids
Erik P. Johnson,a M. Patricia Hubieki,a Andrew P. Combs,b,1 Christopher A. Teleha*b,2
a
J-STAR Research Inc., 3001 Hadley Road, Unit 1–3, South Plainfield, NJ 07080, USA
Bristol-Myers Squibb Pharmaceutical Co., Pharmaceutical Research Institute, Discovery Chemistry, P.O. Box 80336, Wilmington,
b
DE 19880, USA
Fax +1(215)5404611; E-mail: cteleha@its.jnj.com
Received 6 September 2011; revised 7 October 2011
developing methodology that preserved the integrity of
Abstract: A general method for the conversion of N-Boc amino
the chiral center along the route. Commercially available
acids into their homologated a-hydroxy-b-Fmoc amino acids is de-
N-Boc-D-amino acids 4 were converted by the action of
scribed. The protocol involved preparation of the amino aldehyde
by reduction of the corresponding Weinreb amides, hydrocyana-
CDI/MeNH(OMe)·HCl into their corresponding Weinreb
tion, and hydrolysis of the nitrile group, followed by reprotection of amides 55–8 in 85–92% yield. The amides were smoothly
reduced to the aldehydes 65,6,9,10 with LiAlH4 in 92–99%
the amino acid as the Fmoc derivative.
yield. Compound 6c was prepared by a slightly different
route: commercially available N-Boc-phenylglycinol was
oxidized using Dess–Martin periodinane in quantitative
yield.11,12
Key words: N-Boc amino acid, reduction, hydrocyanation, N-
Fmoc amino acid
The development of combinatorial and parallel synthesis
techniques in drug discovery has greatly increased the de-
mand for reagents and scaffolds for the rapid assembly of
new molecules for biological screening. Although these
assays require small quantities of final compound, any
solid-phase endeavor requires the proper choice of resins,
loading requirements, and a ready supply of reagents.
R1
R
R1
AA
CONHR2
AA
CO2H
AA
CONHR2
N
N
N
H
H
H
O
1
OH
OH
2
3a R = Me 3d R = Bn
3b R = Et 3e R = i-Pr
3c R = Ph 3f R = i-Bu
α-keto amide
statin
Figure 1 Retrosynthesis of aspartyl (statin) and serine (a-keto
In support of the preparation of libraries directed towards
the discovery of novel statin-based 2 and a-keto amide in-
hibitors 1 of aspartyl and serines proteases,3 respectively,
we required the facile and scaleable synthesis of a series
of Fmoc-protected b-amino acids 3 as intermediates in li-
brary formation (Figure 1). We developed a general meth-
od that maintained good control of the R-configuration of
the b-amino acid, was robust enough to handle a range of
R groups, and was scaleable to multi-gram quantities
needed for combinatorial library development. There was
no desire to control the hydroxy group stereochemistry in
3 since both R- and S-enantiomers of the hydroxy group
of the statin can bind aspartyl proteases and would be ox-
idized to a carbonyl group after incorporation into the pro-
tease library. At this early juncture, there was no
prerequisite on route selection to account for orthogonal
protecting groups on R if other amino acid groups were to
be considered. A method to prepare 3a,e,f with a stereo-
defined hydroxy group using enantioselective addition of
HCN catalyzed by an enzyme has been reported.4 Consid-
ering our need to support library development with larger
quantities, we chose to investigate an independent route
with special attention to methods that were scaleable.
amide) protease libraries
With these aldehydes 6 in hand, we decided to convert
them into their cyanohydrins 7,5,13–15 and Myers’ method16
(KCN, AcOH, EtOAc, MeOH) worked extremely well for
the examples under investigation. As mentioned earlier,
the formation of diastereomeric mixtures at the hydroxy
center was not a concern because this group would be later
transformed to the a-carbonyl group. It is worthwhile not-
ing that for the easily epimerizable 6c, a mixture of TFA
and AcOH was used to effect conversion to 7c
(Scheme 2).
The hydrolysis of the cyanide 7 to the carboxylic acid 84,17
was accomplished with concentrated HCl in 1,4-dioxane.
It is important to note that anisole was beneficial to the re-
action, presumably by acting as a cation scavenger, and
improved the quality of the isolated product.18 Running
the reaction in aqueous 6 M HCl alone provided the prod-
uct, but isolation in pure form or further reaction was
problematic. However, 8 could be passed through an ion-
exchange column for the purpose of purifying the material
to analytical quality.
The general method used for the preparation of 3 is shown
in Scheme 1. The amino acid pool was seen as an excel-
lent source of chirality for our work. Thus, we focused on
Finally, conversion of 8 into their Fmoc derivatives 3 was
smoothly accomplished using Fmoc-OSuc under aqueous
acetone conditions. The products 3 were isolated in suffi-
cient purity by simple partitioning between water and eth-
yl acetate.
SYNTHESIS 2011, No. 24, pp 4023–4026
x
x.
x
x
.
2
0
1
1
Advanced online publication: 03.11.2011
DOI: 10.1055/s-0031-1289586; Art ID: M87311SS
© Georg Thieme Verlag Stuttgart · New York

4024
E. P. Johnson et al.
PAPER
Me
Me
MeNH(OMe)⋅HCl
KCN, AcOH
R
R
R
R
O
N
CDI, i-Pr2NEt, DMF
LAH, THF
92–99%
EtOAc, MeOH
CN
H
BocHN
BocHN
CO2H
BocHN
BocHN
82–95%
92–97%
OH
O
O
4
5
6
7
O
R
R
concd HCl
1,4-dioxane
anisole
Fmoc-OSu, NaHCO3
acetone, H2O
CO2H
OH
CO2H
a R = Me d R = Bn
b R = Et e R = i-Pr
c R = Ph f R = i-Bu
O
N
HCl⋅H2N
H
OH
57–79% from 7
8
3
Scheme 1 General synthesis route
KCN, TFA, AcOH
EtOAc, MeOH
Dess–Martin
quant.
H
CN
BocHN
OH
BocHN
92–97%
BocHN
O
6c
OH
7c
4
Scheme 2 Specific preparation of 6c by Dess–Martin oxidation, and formation of 7c
mL, 833 mmol, 2.78 equiv). The separated aqueous layer (pH 1–2)
was extracted with EtOAc (200 mL) and the combined organic
phases were washed successively with H2O (100 mL), sat. aq
NaHCO3 (200 mL), and H2O (100 mL). Concentration and azeotro-
pic drying with fresh EtOAc under reduced pressure afforded a syr-
upy product (255–276 mmol, 85–92%), suitable for use in the next
step. These materials solidified after all traces of solvent had been
removed.
In summary, we have described a general method for the
multi-gram preparation of epimeric Fmoc-protected a-
amino-b-hydroxy acids 3 from commercially available
Boc-amino acids 4. Our method relies on high yielding
formation of Weinreb amides 5, chiral aldehydes 6, cy-
anohydrins 7 and the key hydrolysis to acid 8, and repro-
tection of the amino group. Although our method provides
a mixture of hydroxy diastereomers, a potential benefit of
this feature in library design would allow for more exten-
sive coverage of the diversity space when screening for
inhibitors in other proteases.19 Use of reagents 3 in con-
junction with combinatorial library development will be
reported elsewhere.
(R)-tert-Butyl 1-[Methoxy(methyl)amino]-1-oxopropan-2-yl-
carbamate (5a)
Mp 148.5–149.5 °C; [a]D20 +28.5 (c = 1.0, MeOH).
IR (Nujol): 3290, 2900, 1710, 1650, 1300, 1250, 1065, 980 cm–1.
1H NMR (CDCl3): d = 5.33 (d, J = 8.0 Hz, 1 H), 4.68 (m, 1 H), 3.78
(s, 3 H), 3.21 (s, 3 H), 1.44 (s, 9 H), 1.31 (d, J = 6.9 Hz, 3 H).
13C NMR (CDCl3): d = 173.5, 155.0, 79.2, 61.4, 46.4, 32.0, 28.1,
Starting materials were used as supplied without further treatment
unless otherwise noted. Work-ups requiring concentrations were
done under reduced pressure. Optical rotations were measured on a
Perkin-Elmer 241 polarimeter. Nuclear magnetic resonance spectra
(1H, 13C) were obtained on a Varian VXR300S multinuclear spec-
trometer at 300 MHz and 75 MHz, respectively, and are referenced
to Me4Si (d = 0.0 ppm). Infrared (IR) spectra were recorded as Nujol
mulls (unless otherwise noted) on a Perkin-Elmer 783 infrared spec-
trometer and are reported in reciprocal wave numbers (cm–1). Ele-
mental analyses were performed by Robertson Microlit Labs, Inc.,
Madison, NJ. Melting points are uncorrected.
18.4.
HRMS: m/z calcd for C10H20N2O4: 233.1502; found: 233.1508
[M + H].
Anal. Calcd for C10H20N2O4: C, 51.71; H, 8.68; N, 12.06. Found: C,
51.75; H, 8.57; N, 11.94.
N-Boc-(R)-2-amino Aldehydes 6; General Procedure
A slurry of LiAIH4 (250 mmol, 1.10 equiv) in anhyd THF (250 mL)
agitated with an overhead stirrer was chilled to –15 °C. A solution
of N-Boc-(R)-Weinreb amide (227 mmol, 1.00 equiv) in anhyd THF
(250 mL) was added with cooling so as to keep the reaction temper-
ature <5 °C. The reaction mixture was stirred for 45 min, quenched
by slow addition of EtOAc (500 mL), keeping the internal temper-
ature <5 °C, followed by treatment with aq 2 M NaHSO4 (600 mL),
keeping the internal temperature <5 °C. During the quench, the re-
action mass gelled but later thinned to a slurry. The layers were sep-
arated and the gray aqueous phase was extracted with EtOAc (500
mL). The combined organic layers were washed successively with
aq 1 M NaHSO4 (100 mL), sat. aq NaHCO3 (100 mL) and brine
(100 mL). After drying (MgSO4), filtration, and concentration, the
product aldehyde (209–225 mmol, 92–99%) was obtained as an oil,
which was used directly in the next step. Analytically pure material
was not obtained unless the aldehyde crystallized or solvent was
rigorously removed.
Experimental procedures are given for the preparation of 5a, 6a, 6c,
7a, 7c, 8a, and 3a.
N-Boc-(R)-2-amino Weinreb Amides 5; General Procedure
The appropriate N-Boc-(R)-amino acid 4 (300 mmol, 1.00 equiv)
was dissolved in anhyd THF (400 mL), and N,N¢-carbonyldiimida-
zole (360 mmol, 1.20 equiv) was added portionwise over 1 h so as
to control foaming. The mixture was stirred at r.t. for 1 h. A solution
of N,O-dimethylhydroxylamine hydrochloride (330 mmol, 1.10
equiv) and i-Pr2NEt (315 mmol, 1.05 equiv) in anhyd DMF (185
mL) was introduced over 0.5 h (with cooling as necessary to main-
tain the reaction temperature <30 °C). This mixture was stirred at
r.t. for 18 h. Most of the solvent was removed, and the residue was
partitioned between EtOAc (500 mL) and 1.67 M aq NaHSO4 (500
Synthesis 2011, No. 24, 4023–4026 © Thieme Stuttgart · New York

PAPER
Preparation of a-Hydroxy-b-Fmoc Amino Acids
4025
(R)-tert-Butyl 1-Oxopropan-2-ylcarbamate (6a)
Mp 89–89.5 °C; [a]D20 +35.0 (c = 1.1, MeOH).
IR (Nujol): 3320, 2900, 1700, 1680, 1560, 1370, 1250, 1160 cm–1.
1H NMR (CDCl3): d = 9.56 (s, 1 H), 5.21 (m, 1 H), 4.21 (m, 1 H),
1.46 (s, 9 H), 1.33 (d, J = 7.3 Hz, 3 H).
13C NMR (CDCl3): d = 199.7, 155.3, 79.9, 55.4, 28.2, 14.7.
(R)-tert-Butyl 2-Cyano-2-hydroxy-1-phenylethylcarbamate (7c)
Crude N-Boc-(R)-phenylglycinal (22.10 g, 94.0 mmol) dissolved in
EtOAc (150 mL), was treated with a solution of KCN (6.43 g, 98.7
mmol), TFA (9.64 g, 84.6 mmol), and AcOH (1.13 g, 18.8 mmol,
1.2 equiv) in MeOH (150 mL) for 2 h. Toluene (200 mL) was added,
and the mixture was extracted with sat. aq NaHCO3 (150 mL). The
separated aqueous layer was extracted with toluene (100 mL) and
the organics were washed with H2O (500 mL), and concentrated to
afford the product as a pale yellow oil (23.7 g, 96%) that eventually
crystallized after refrigeration. This material was used directly in
the next step.
HRMS: m/z calcd for C8H15NO3: 174.1131; found: 174.1132
[M + H].
Anal. Calcd for C8H15NO3: C, 55.47; H, 8.73; N, 8.09. Found: C,
55.57; H, 8.85; N, 7.96.
IR (CCl4): 3440, 3360, 3060, 3040, 2980, 2940, 1700, 1500, 1460,
1400, I370, 1250, 1170, 1090, 1050, 1030 cm–1.
1H NMR (CDCl3): d = 7.38 (s, 5 H), 5.63 (m, 0.6 H), 5.58 (m, 0.4
H), 5.32 (m, l H), 4.95 (m, 1 H), 4.70 (m, 0.6 H), 4.63 (m, 0.4 H),
1.45 (s, 2.7 H), 1.43 (s, 6.3 H).
13C NMR (CDCl3): d = 156.0, 136.2, 135.8, 129.1, 128.9, 128.8,
127.9, 127.3, 127.0, 118.4, 117.9, 81.5, 81.0, 67.1, 65.1, 59.1, 57.9,
28.2.
(R)-N-(tert-Butoxycarbonyl)-2-phenylglycinal (6c)
To a chilled slurry of N-Boc-(R)-phenylglycinol (23.7 g, 100 mmol)
in H2O-saturated CH2Cl2 (300 mL) was added portionwise, Dess–
Martin periodinane (89.1 g, 210 mmol) over 0.5 h. The stirred mix-
ture was allowed to warm to r.t. over 2 h. More H2O-saturated
CH2Cl2 (50 mL) was added and stirring was continued for 0.5 h. The
reaction was quenched by the addition of a solution of
Na2S2O3·5H2O (573 g, 2.31 mol) in sat. aq NaHCO3 (1.5 L). When
the mixture tested negative to starch-iodide paper, it was diluted
with MTBE (300 mL), and the phases separated. The aqueous layer
was extracted with MTBE (50 mL), and the combined organic phas-
es were washed with H2O (1 L). Concentration under reduced pres-
sure and chasing the residual H2O with EtOAc (100 mL) afforded a
semi-solid yellow product (24.0 g, 102%) suitable for use directly
in the next step; [a]D20 –24.0 (c = 5.16, MeOH).
HRMS: m/z calcd for C14H18N2O3: 263.1396; found: 263.1392
[M + H].
Anal. Calcd for C14H18N2O3: C, 64.10; H, 6.92; N, 10.68. Found: C,
64.39; H, 7.11; N, 10.57
N-Fmoc-3-(R)-amino-2-(R,S)-hydroxy Acids 3; General Proce-
dure
Crude 7 (243 mmol, 1.00 equiv) and anisole (34 mL) were dissolved
in 1,4-dioxane (350 mL). Concd aq HCl (12.1 N, 350 mL) was add-
ed slowly and the mixture was heated at a gentle reflux for 4.5 h.
Volatiles were removed under reduced pressure and the resulting
residue was dissolved in H2O (250 mL). After extraction with 50%
toluene–EtOAc (100 mL), the pH was adjusted to 10.5–11 with sol-
id NaOH. Water and NH3 were removed under reduced pressure
and the residual (3R,2RS)-3-amino-2-hydroxy acid sodium salt 8
was dissolved in 50% aq acetone (700 mL). This mixture was buff-
ered with NaHCO3 (243 mmol, 1.00 equiv) and Fmoc-O-succinim-
idyl ester (243 mmol, 1.00 equiv) and stirred at r.t. for 18 h. The
reaction mixture was carefully poured into a mixture of aq NaHSO4
(0.5 M, 1 L) and EtOAc (1 L), the layers were separated, and the
aqueous phase was extracted with EtOAc (500 mL). The combined
organic layers were washed with H2O (500 mL) and concentrated to
a residue, which was dissolved in 15% H2O in acetone and applied
to a column of Amberlyst A-21 ion exchange resin (210 g). Elution
of impurities with the same mixture, followed by passage of a
3:4:16 H2O–AcOH–acetone mixture through the column afforded
essentially pure product (139–192 mmol, 57–79% yield, 54–74%
from aldehyde) after concentration and drying under reduced pres-
sure at 55 °C.
IR (neat): 3360, 3045, 3020, 2980, 2940, 2720, 1700, 1500, 1450,
1390, 1365, 1240, 1170, 1040, 860, 750, 700 cm–1.
1H NMR (CDCl3): d = 9.54 (s, 1 H), 7.34 (m, 5 H), 5.78 (br s, 1 H),
5.33 (d, J = 6.3 Hz, 1 H), 1.45 (s, 9 H).
13C NMR (CDCl3): d = 191.8, 155.1, 133.0, 129.2, 128.7, 127.7,
81.1, 64.8, 28.3.
HRMS: m/z calcd for C13H17NO3: 236.1287; found: 236.1275 [M +
H].
Cyanohydrins 7; General Procedure
Crude N-Boc-(R)-amino aldehyde (250 mmol, 1.00 equiv) was dis-
solved in EtOAc (300 mL) and treated with a solution of KCN (275
mmol, 1.10 equiv) and AcOH (300 mmol, 1.2 equiv) in MeOH (425
mL) for 2 h. Concentration afforded a semi-solid, from which traces
of AcOH and MeOH were removed by chasing with 1,4-dioxane
(100 mL) under reduced pressure. The residue obtained was slurried
in 1,4-dioxane (200 mL) and the solids were removed via filtration
prior to use directly in the next step. Concentration gave the oily cy-
anohydrin (230–243 mmol, 92–97%) that typically crystallized af-
ter rigorous removal of solvent.
(R)-tert-Butyl 1-Cyano-1-hydroxypropan-2-ylcarbamate (7a)
Mp 74–82 °C.
IR (Nujol): 3460, 3330, 2900, 1680, 1250, 1170, 960 cm–1.
(R)-3-{[(9H-Fluoren-9-yl)methoxy]carbonylamino}-2-hy-
droxybutanoic Acid (3a)
Mp 120–132 °C.
1H NMR (CDCl3): d = 5.53 (d, J = 7.7 Hz, 0.3 H), 4.87 (m, 1 H),
4.6 (s, 0.7 H), 4.58 (s, 0.6 H), 4.48 (dd, J = 2.5, 5.0 Hz, 0.4 H), 4.05
(m, 0.3 H), 3.88 (m, 0.7 H), 1.46 (s, 3.15 H), 1.45 (s, 5.85 H), 1.35
(d, J = 6.9 Hz, 2 H), 1.30 (d, J = 6.9 Hz, 1 H).
13C NMR (CDCl3): d = 157.3, 156.0, 118.4, 117.9, 81.4, 80.9, 67.4,
65.0, 50.4, 49.5, 28.2, 28.1, 16.1, 14.9.
IR (Nujol): 3500, 3310, 2900, 1680, 1530, 1250 cm–1.
1H NMR (acetone-d6): d = 7.85–7.29 (m, 8 H), 6.51 (d, J = 8.4 Hz,
0.4 H), 6.30 (d, J = 8.4 Hz, 0.6 H), 4.44–4.05 (m, 5 H), 1.28 (d,
J = 6.8 Hz, 1.8 H), 1.16 (d, J = 6.8 Hz, 1.2 H).
13C NMR (acetone-d6): d = 174.3, 174.0, 156.6, 145.3, 145.0, 144.9,
142.0, 128.4, 127.9, 127.8, 126.1, 126.0, 73.7, 73.4, 67.1, 50.3,
47.9, 17.8, 14.7.
HRMS: m/z calcd for C9H16N2O3: 201.1240; found: 201.1233
[M + H].
HRMS: m/z calcd for C19H19NO5: 342.1342; found: 342.1352
Anal. Calcd for C9H16N2O3: C, 53.98; H, 8.05; N, 13.99. Found: C,
54.26; H, 7.79; N, 13.88
[M + H].
Anal. Calcd for C19H19NO5: C, 66.85; H, 5.61; N, 4.10. Found: C,
66.70; H, 5.52; N, 3.85
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E. P. Johnson et al.
PAPER
Supporting Information for this article is available online at
http://www.thieme-connect.com/ejournals/toc/synthesis.
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Synthesis 2011, No. 24, 4023–4026 © Thieme Stuttgart · New York