Versatile selective α-carboxylic acid esterification of N-protected amino acids and peptides by alcalase

Article abstract of DOI:10.1055/s-0028-1083362

Under continuous removal of water, the industrial protease Alcalase allows selective synthesis of α-carboxylic acid methyl, ethyl, benzyl, allyl, 2-(trimethylsilyl)ethyl, and tert-butyl esters of amino acids and peptides under mild conditions in very high

Full text of DOI:10.1055/s-0028-1083362

PAPER
809
Versatile Selective a-Carboxylic Acid Esterification of N-Protected Amino
Acids and Peptides by Alcalase
Versatile
Select
i
ive
a
-C
m
a
rboxylic Acid Este
o
rification Nuijens,a,b Claudia Cusan,a John A. W. Kruijtzer,b Dirk T. S. Rijkers,b Rob M. J. Liskamp,b
Peter J. L. M. Quaedflieg*a
a
DSM Pharmaceutical Products, Innovative Synthesis & Catalysis, P.O. Box 18, 6160 Geleen, MD, The Netherlands
Fax +31(46)4767604 ; E-mail: peter.quaedflieg@dsm.com
Medicinal Chemistry and Chemical Biology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University,
b
P.O. Box 80082, 3508 Utrecht, TB, The Netherlands
Received 3 October 2008; revised 3 November 2008
portantly, for activation in the case of a kinetically con-
trolled process. Most often, nonsterically hindered esters
as methyl, ethyl, or benzyl esters are used as activated
substrates.6
Abstract: Under continuous removal of water, the industrial pro-
tease Alcalase allows selective synthesis of a-carboxylic acid meth-
yl, ethyl, benzyl, allyl, 2-(trimethylsilyl)ethyl, and tert-butyl esters
of amino acids and peptides under mild conditions in very high
yields. The purified yields range from 72% to 92%.
The chemical synthesis of amino acid esters in which the
ester moiety is sterically demanding often requires harsh
conditions, which is troublesome for sensitive substrates.
For instance, tert-butyl esters are often synthesized using
a strong mineral acid with isobutene under high pressure,7
which is laborious in laboratory practice. Furthermore, se-
lective chemical a-carboxylic acid protection of aspartic
acid and glutamic acid is difficult due to the similar reac-
tivity of the side chain carboxylic acid functionalities. Al-
though several one step methods have been described for
(semi)selective side chain esterification,8 a-carboxylic
acid protection of aspartic acid and glutamic acid usually
needs multistep and/or low yielding protocols. For in-
stance, the internal anhydride method9 and the chlorofor-
mate method10 both give approximately 3:2 mixtures of a-
carboxy vs b/g-carboxy esterification.
Key words: enzymes, amino acids, esterification, esters, protecting
groups
There is a great demand for easily accessible amino acid
building blocks for their use in peptide synthesis. For dif-
ferent applications, a large variety of side chain, a-amino
and/or a-carboxylic acid protected amino acids is re-
quired. Orthogonality of the different protecting groups
used is pivotal for conventional peptide synthesis proto-
cols. Whereas the a-amino functions are most often pro-
tected as carbamates, the a-carboxylic acid functions are
most often protected as esters. Esters can be synthesized
by mineral acid catalysis using, for example, hydrochloric
or sulfuric acid in the presence of the (unprotected) amino
acid and the corresponding alcohol. However, this method
is restricted to n-alkyl esters such as methyl, ethyl, or ben-
zyl and for a subset of amino acids. More difficult to syn-
thesize, but very commonly used as protecting groups in
Fmoc- or Boc-based solid phase peptide synthesis, are the
orthogonal esters such as the tert-butyl, allyl (All) or
2-(trimethylsilyl)ethyl (TMSE) esters. 2-(Trimethylsi-
lyl)ethyl esters, cleavable with tetrabutylammonium fluo-
ride,1 and allyl esters, cleavable with palladium(0),2 are
orthogonal to other commonly used carboxylic acid pro-
tecting groups thus allowing selective deprotection and
modification of certain carboxylic acids during peptide
synthesis. Of special interest are a-carboxylic protected
aspartic acid (Asp) or glutamic acid (Glu) building blocks,
since these also contain a b- or g-carboxylic acid moiety,
respectively. These esters are used for (Fmoc- and Boc-
based) on-resin synthesis of head-to-tail cyclic peptides,3
side-chain lactam peptides,4 and branched peptides.5
To overcome the selectivity issue and to avoid racemiza-
tion, as is often encountered in chemical esterification, N-
protected amino acids have been esterified using en-
zymes.11 However, the water produced during the reaction
and the water required for enzyme activity prohibits a fa-
vorable position for the esterification equilibrium result-
ing in low to moderate yields.12 High yields were reported
using esterases or lipases in dry organic solvents, but these
enzymes are usually not selective for the a-carboxylic
moiety. Proteases such as papain,13 chymotrypsin,14 and
a-chymotrypsin15 are a-selective, but only demonstrated
esterification with sterically undemanding alcohols such
as methanol, ethanol, or benzyl alcohol. Esterification of
amino acids to aliphatic esters with Alcalase has, to our
knowledge, only been disclosed for Boc-Phe-OEt (65% in
48 h using subtilisin). Herein we demonstrate that the in-
expensive industrial protease Alcalase16 (from Novo-
zymes, 10 wt% Alcalase solution, 6.75 €/kg) can catalyze
the esterification of a wide range of N-protected proteino-
genic and nonproteinogenic amino acids in organic sol-
vents at low water content with high a-carboxylic
selectivity.
In chemoenzymatic peptide synthesis a-carboxylic acid
esters are not only used for protection, but also, more im-
SYNTHESIS 2009, No. 5, pp 0809–0814
x
x
.
x
x
.2
0
0
9
During an investigation of the Alcalase-catalyzed peptidic
bond formation between Cbz-Phe-OMe and H-Leu-Ot-Bu
in tert-butyl alcohol at low water content,17 we discovered
Advanced online publication: 11.02.2009
DOI: 10.1055/s-0028-1083362; Art ID: Z23408SS
© Georg Thieme Verlag Stuttgart · New York

810
T. Nuijens et al.
PAPER
O
O
CLEA-Alcalase
R2OH
H
H
N
N
R2
O
R1
OH
R1
O
H
N
Ot-Bu
Cbz
N
H
O
O
H
N
Alcalase
Cbz
OMe
H-Leu-Ot-Bu
1 R2 = Me
2 R2 = Et
3 R2 = Bn
4 R2 = All
a R1 = Cbz
b R1 = Boc
c R1 = Bz
d R1 = For
t-BuOH
+
O
H
5 R2 = TMSE e R1 = Fmoc
6 R2 = t-Bu
N
Cbz
Ot-Bu
Scheme 2
Table 1 Yields of Esterification Reactions of 1a–6e
Scheme 1
Product
HPLC yield Isolated yield
a small percentage of byproduct (Scheme 1). HPLC/MS
and NMR analysis proved this byproduct to be Cbz-Phe-
Ot-Bu.
(%)
92
95
98
95
97
96
98
94
99
93
(%)
83
85
90
86
87
85
85
90
92
88
1a
2a
3a
4a
5a
6a
6b
6c
6d
6e
Cbz-Phe-OMea
Cbz-Phe-OEta
To our knowledge, this transesterification to give a tert-
butyl ester had not been previously reported. This is much
to our surprise, because tert-butyl alcohol is generally the
solvent of choice for protease-catalyzed peptide synthesis,
and enzymes tend to have very good stability in this sol-
vent.18
Cbz-Phe-OBna
Cbz-Phe-OAlla
Cbz-Phe-OTMSEa
Cbz-Phe-Ot-Bub
Boc-Phe-Ot-Bub
Bz-Phe-Ot-Bub
For-Phe-Ot-Bub
Fmoc-Phe-Ot-Bub
Further investigation of the tert-butyl ester formation
from Cbz-Phe-OMe showed that, in the absence of the
amino acid nucleophile H-Leu-Ot-Bu, in neat tert-butyl
alcohol the tert-butyl ester could be obtained in 75%
yield. Upon addition of 4 Å molecular sieves Cbz-Phe-
OMe was converted into Cbz-Phe-Ot-Bu in 96% yield (50
°C, 48 h), indicating that the amounts of water and meth-
anol were critical for the reaction outcome. Most striking-
ly, equally high yields could be obtained starting from the
free carboxylic acid Cbz-Phe-OH (50 °C, 16 h, 96% yield)
using 3 Å molecular sieves whereas in the absence of mo-
lecular sieves only low yields (48 h, 37% yield) of Cbz-
Phe-Ot-Bu were obtained. Apparently, continuous remo-
val of water shifts the equilibrium towards product, with
the enzyme remaining active. We decided to investigate
the scope of the methodology using N-terminal protected
phenylalanine as the model substrate (Scheme 2). Cross-
linked enzyme aggregate (CLEA)19 Alcalase was used
since it could be easily dried and recovered after the reac-
tion by filtration. Due to the well-known inactivation of
Alcalase by methanol and polar aprotic solvents (such as
DMSO and DMF), apolar co-solvents were used (i.e., n-
heptane, toluene, or MTBE) in the esterification reactions.
All reported esterification reactions with primary alcohols
were performed using a mixture of the appropriate alcohol
with methyl tert-butyl ether (1:14, v/v, respectively).
a CLEA-Alcalase (300 mg), Cbz-Phe-OH (50 mg), R2OH (200 mL),
MTBE (2.8 mL), 3 Å MS (200 mg), shaken at 150 rpm, 50 °C.
b CLEA-Alcalase (300 mg), protected-Phe-OH (50 mg), t-BuOH (3.0
mL), 3 Å MS (200 mg), shaken at 150 rpm, 50 °C.
(Table 2). Only amino acids with a tertiary b-carbon, e.g.,
threonine, isoleucine, and valine were not efficiently ac-
cepted by Alcalase. The dipeptide Cbz-Phe-Leu-OH
proved to be a good substrate for esterification reactions
giving 8–10. Under the dry conditions used, neither hy-
drolysis nor alcoholysis of the peptidic bond was ob-
served.
Since the use of molecular sieves is inconvenient on large
scale, azeotropic water removal was investigated. Gratify-
ingly, the tert-butyl esters were formed in high yields
(>95%) when tert-butyl alcohol, which forms an azeo-
trope with water, was continuously evaporated and dry
tert-butyl alcohol was simultaneously added to keep the
reaction volume constant. Alcohols, which do not form an
azeotrope with water, such as allylic alcohol and 2-(tri-
methylsilyl)ethanol, could be combined with the azeotropic
solvent toluene (1:14, v/v, respectively), under reduced
pressure, to obtain ester yields of up to 98%. The CLEA-
Alcalase could be recycled with minimal activity loss; af-
ter filtration, new starting materials were added and only
1% activity loss occurred after five esterification cycles.
As is shown in Table 1, Cbz-protected phenylalanine was
smoothly converted into a variety of esters 1a–6e in very
high yields. The choice of N-terminal protection (6a–e)
did not influence the esterification reaction. As is known
from the literature, Alcalase accepts a wide variety of pro-
teinogenic (7a–e) and nonproteinogenic (7f) amino acids,
which was also demonstrated for tert-butyl ester synthesis
Synthesis 2009, No. 5, 809–814 © Thieme Stuttgart · New York

PAPER
Versatile Selective a-Carboxylic Acid Esterification
811
Table 2 Esterification Yields for Various Amino Acids and Pep-
tides 7–10
Table 3 Esterification Yields of Aspartic Acid and Glutamic Acid
Derivatives 11a–16b
O
O
Product
HPLC yield Isolated yield
R1HN
R1HN
CLEA-Alcalase
R3OH
(%)
98
95
97
96
97
94
96
92
94
93
96
93
(%)
84
86
88
88
89
88
87
85
88
85
89
86
OH
OR3
R2
R2
11a
11b
12a
12b
13a
13b
14a
14b
15a
15b
16a
16b
Cbz-Asp-OAll
7a–e,8–10
Cbz-Asp-OTMSE
Cbz-Glu-OAll
Product
HPLC yield Isolated yield
(%)
90
92
83
89
93
95
98
98
96
(%)
86
88
72
82
90
90
88
80
90
Cbz-Glu-OTMSE
Boc-Asp-OAll
7a
7b
7c
7d
7e
7f
Cbz-Ala-Ot-Bu
Cbz-Leu-Ot-Bu
Boc-Asp-OTMSE
Boc-Glu-OAll
Cbz-Ser-Ot-Bu
Cbz-Met-Ot-Bu
Boc-Glu-OTMSE
Fmoc-Asp-OAll
Fmoc-Asp-OTMSE
Fmoc-Glu-OAll
Fmoc-Glu-OTMSE
Cbz-Lys(Cbz)-Ot-Bu
Cbz-DOPA-Ot-Bu
Cbz-Phe-Leu-OMe
Cbz-Phe-Leu-OBn
Cbz-Phe-Leu-Ot-Bu
8
9
10
removed by azeotropic distillation and the CLEA-Alca-
lase could be recycled with minimal loss of activity.
To investigate the a-carboxylic acid selectivity of Alca-
lase we decided to apply the conditions as for phenylala-
nine to aspartic acid and glutamic acid derivatives with
various N-protecting groups (Scheme 3). Gratifyingly, all
aspartic acid and glutamic acid derivatives were convert-
ed into their corresponding monoesters 11a–16b in very
high yields and with complete a-selectivity (Table 3).
Unless stated otherwise, chemicals were obtained from commercial
sources and used without further purification. Before use CLEA-
Alcalase was dried as follows: CLEA-Alcalase (3 g, Codexis, 650
AGEU/g, 3.5 wt% H2O), was suspended in t-BuOH (100 mL) and
crushed with a spatula. After filtration, the enzyme was resuspended
in MTBE (50 mL) followed by filtration. 1H and 13C NMR spectra
were recorded on a Bruker Avance 300 MHz NMR spectrometer;
chemical shifts are given relative to TMS. TLC was performed on
pre-coated silica gel 60 F254 plates (Merck); spots were visualized
using UV light, ninhydrin, or permanganate soln. 3 Å molecular
sieves (8–12 mesh, Acros) were activated under reduced pressure at
200 °C and t-BuOH was stored on these activated molecular sieves.
t-BuOH was pre-heated to liquid (45 °C) before use. Column chro-
matography was carried out using silica gel, Merck grade 9385 60
Å. Analytical HPLC was performed on an HP1090 Liquid Chro-
matograph, using a reversed-phase column (Inertsil ODS-3, C18, 5
mm particle size, 150 × 4.6 mm internal diameter) at 40 °C. UV de-
tection was performed at 220 nm using a UV-Vis 204 Linear spec-
trophotometer (Varian). The gradient program was: 0–25 min linear
gradient from 5% to 98% eluent B and from 25.1–30 min 5% eluent
B (eluent A: 0.5 mL/L MsOH in H2O; eluent B: 0.5 mL/L MsOH in
MeCN). The flow was 1 mL/min from 0–25.1 min and 2 mL/min
from 25.2–29.8 min, then back to 1 mL/min until stop at 30 min. In-
jection volumes were 20 mL.
O
O
H
N
H
N
CLEA-Alcalase
R2OH
R2
R1
O
O
R1
O
OH
X
X
OH
OH
a R2 = Allyl
11 R1 = Cbz
X = 1
b R2 = TMSE
X = 2
X = 1
X = 2
X = 1
X = 2
12
13
14
15
16
R
R
R
R
R
1 = Cbz
1 = Boc
1 = Boc
1 = Fmoc
1 = Fmoc
Scheme 3
In conclusion, we have developed a general versatile
method for enzymatic a-carboxylic acid selective esterifi-
cation of N-protected amino acids and peptides towards a
variety of esters by Alcalase in very high to excellent
yields. Even esters that are difficult to synthesize chemi-
cally, e.g. tert-butyl esters, were obtained in high yields.
Furthermore, amino acids with an additional carboxylic
acid functionality in the side chain, e.g. aspartic acid and
glutamic acid, were esterified with complete selectivity
for the a-carboxylic moiety. The processes appeared ame-
nable to scale-up since the water could also efficiently be
The flow-injection analysis (FIA) experiments to determine the ex-
act mass were performed on an Agilent 1100 LC-MS system (Agi-
lent, Waldbronn, Germany), which consists of a binary pump,
degasser, autosampler, column oven, diode-array detector, and a
TOF-MS. The ESI-MS was run in positive mode, with the follow-
ing conditions: m/z 50–3200, 175 V fragmentor, 0.94 cycl/sec, 350
°C drying gas temperature, 10 L N2/min drying gas, 45 psig nebu-
lizer pressure and 4 kV capillary voltage. The exact mass was deter-
mined using an internal reference to recalibrate the m/z axis for each
measurement. The samples were directly introduced into the ESI by
Synthesis 2009, No. 5, 809–814 © Thieme Stuttgart · New York

812
T. Nuijens et al.
PAPER
injection of 5 mL into the eluent flow of 0.5 mL/min (MeOH–H2O,
1:1).
Enzymatic Esterification with tert-Butyl Alcohol; General Pro-
cedure
CLEA-Alcalase (300 mg) was added to N-protected amino acid
(0.167 mmol), t-BuOH (3.0 mL), and 3 Å molecular sieves (200
mg). The mixture was shaken at 50 °C with 150 rpm for 16 h. After
filtration, the enzyme was washed three times by resuspension in
EtOAc (20 mL) followed by filtration. The combined organic layers
were concentrated in vacuo and the resulting oil redissolved in
EtOAc (50 mL). This soln was washed with sat. aq NaHCO3 (2 × 40
mL), aq HCl (pH 1, 2 × 40 mL), and brine (40 mL) and subsequently
dried (Na2SO4), concentrated in vacuo, and co-evaporated with tol-
uene (2 × 20 mL) and CHCl3 (2 × 20 mL). For Cbz-Ser-Ot-Bu (7c)
additional column chromatography (n-heptane–EtOAc, 1:1) was re-
quired.
Preparative HPLC was performed on a Varian PrepStar system us-
ing a stationary-phase column (Pursuit XRs, C18, 10 mm particle
size, 500 × 41.4 mm internal diameter) at room temperature. UV
detection was performed at 220 nm and 254 nm using a UV-Vis
Varian ProStar spectrometer. The isocratic program was 85% eluent
B and 15% eluent A (eluent A: 0.1 mL/L HCO2H in H2O; eluent B:
0.1 mL/L HCO2H in MeCN) with a flow rate of 80 mL/min, injec-
tion volume of 10 mL and stop time after 30 min. Pure fractions
were pooled and concentrated in vacuo and co-evaporated with tol-
uene (2 × 25 mL) and CHCl3 (2 × 25 mL).
Spectral data of Cbz-Phe-OEt (2a),20 Cbz-Phe-OAll (4a),21 Boc-
Phe-Ot-Bu (6b),22 Bz-Phe-Ot-Bu (6c),23 For-Phe-Ot-Bu (6d),24
Fmoc-Phe-Ot-Bu (6e),25 Cbz-Ala-Ot-Bu (7a),26 Cbz-Ser-Ot-Bu
(7c),27 Cbz-Phe-Leu-OMe (8),28 Cbz-Asp-OAll (11a),29 Boc-Asp-
OTMSE (13b),30 and Boc-Glu-OTMSE (14b)31 were consistent
with those reported in the literature. The identity of Cbz-Phe-OMe
(1a), Cbz-Phe-OBn (3a), Cbz-Phe-Ot-Bu (6a), Fmoc-Asp-OAll
(15a), and Fmoc-Glu-OAll (16a) was ascertained by NMR spec-
troscopy and by comparison with commercially available samples
(Bachem or Senn, Switzerland).
N-(Benzyloxycarbonyl)leucine tert-Butyl Ester (7b)
1H NMR (300 MHz, CDCl3): d = 0.86 (d, J = 6.3 Hz, 6 H, CdH3),
1.38 (s, 9 H, CCH3), 1.39–1.70 (m, 3 H, CgH, CbH2), 4.19–4.22 (m,
1 H, CaH), 5.03 (s, 2 H, CH2OCO), 5.12 (d, J = 7.2 Hz, 1 H, NH),
7.24–7.28 (m, 5 H, CArH).
13C NMR (75 MHz, CDCl3): d = 22.6, 25.4, 28.6, 42.5, 53.7, 67.4,
82.4, 128.2, 128.7, 137.0, 156.5, 172.9.
MS [FIA-ESI(+)-TOF]: m/z [M + Na]+ calcd for C18H27NNaO4:
Enzymatic Esterification with Primary Alkyl Alcohols; General
Procedure
344.1838; found: 344.1828.
CLEA-Alcalase (300 mg) and 3 Å molecular sieves (200 mg) were
added to N-protected amino acid (0.167 mmol) dissolved in MTBE
(2.8 mL) and the appropriate alcohol (200 mL). The mixture was
shaken at 50 °C with 150 rpm for 16 h. After filtration, the enzyme
was washed three times by resuspension in EtOAc (20 mL) fol-
lowed by filtration. The combined organic layers were washed with
sat. aq NaHCO3 (2 × 40 mL), aq HCl (pH 1, 2 × 40 mL), and sat. aq
NaCl (40 mL) and subsequently dried (Na2SO4), concentrated in
vacuo, and co-evaporated with toluene (2 × 20 mL) and CHCl3 (2 ×
20 mL). Additional preparative HPLC was required for Cbz-Phe-
OBn (3a) and Cbz-Phe-Leu-OBn (9).
N-(Benzyloxycarbonyl)methionine tert-Butyl Ester (7d)
1H NMR (300 MHz, CDCl3): d = 1.39 (s, 9 H, CCH3), 1.80–1.89
(m, 2 H, CbH2), 2.01 (s, 3 H, SCH3), 2.40–2.48 (m, 2 H, CgH2),
4.28–4.30 (m, 1 H, CaH), 5.03 (s, 2 H, CH2OCO), 5.33 (d, J = 7.5
Hz, 1 H, NH), 7.26–7.29 (m, 5 H, CArH).
13C NMR (75 MHz, CDCl3): d = 22.2, 23.0, 28.2, 42.1, 53.3, 67.0,
82.0, 128.2, 129.1, 136.6, 156.1, 171.4.
MS [FIA-ESI(+)-TOF]: m/z [M + Na]+ calcd for C17H25NNaO4S:
362.1396; found: 362.1334.
N2,N6-Bis(benzyloxycarbonyl)lysine tert-Butyl Ester (7e)
1H NMR (300 MHz, CDCl3): d = 1.25–1.45 (m, 13 H, CCH3, CgH2,
CdH2), 1.46–1.71 (m, 2 H, CbH2), 3.01–3.12 (m, 2 H, CeH2), 4.13–
4.19 (m, 1 H, CaH), 4.81–4.85 (m, 1 H, NH), 5.00 (s, 2 H,
CH2OCO), 5.01 (s, 2 H, CH2OCO), 5.42 (d, J = 7.2 Hz, 1 H, NH),
7.18–7.26 (m, 10 H, CArH).
N-(Benzyloxycarbonyl)phenylalanine 2-(Trimethylsilyl)ethyl
Ester (5a)
1H NMR (300 MHz, CDCl3): d = 0.00 (s, 9 H, SiCH3), 0.94–0.89
(m, 2 H, CH2Si), 3.09–3.05 (dd, J = 6.0 Hz, 2 H, CbH2), 4.07–4.20
(m, 2 H, COOCH2), 4.57–4.60 (dd, J = 3.3, 9.0 Hz, 1 H, CaH), 5.06
(s, 2 H, CH2OCO), 5.23 (d, J = 9.0 Hz, 1 H, NH), 7.06 (dd, J = 3.3,
5.7 Hz, 2 H, CArH), 7.19–7.34 (m, 8 H, CArH).
13C NMR (75 MHz, CDCl3): d = 22.4, 28.2, 29.9, 32.6, 40.9, 54.3,
66.8, 67.1, 82.3, 128.2, 128.7, 136.6, 136.8, 156.2, 156.7, 171.7.
13C NMR (75 MHz, CDCl3): d = –1.2, 17.2, 29.1, 55.1, 64.0, 67.1,
MS [FIA-ESI(+)-TOF]: m/z [M + Na]+ calcd for C26H34N2NaO6:
493.2309; found: 493.2216.
127.2, 128.2, 128.6, 129.6, 136.0, 136.5, 155.8, 171.8.
MS [FIA-ESI(+)-TOF]: m/z [M + Na]+ calcd for C22H29NNaO4Si:
422.1758; found: 422.1757.
N-(Benzyloxycarbonyl)-3,4-dihydroxyphenylalanine tert-Butyl
Ester (7f)
1H NMR (300 MHz, CDCl3): d = 1.34 (s, 9 H, CCH3), 2.83–2.88
(dd, J = 9.3, 4.2 Hz, 2 H, CbH2), 4.37–4.40 (dd, J = 13.0, 4.5 Hz, 1
H, CaH), 5.00 (s, 2 H, CH2OCO), 5.25 (d, J = 8.1 Hz, 1 H, NH)
5.90–6.05 (m, 2 H, OH), 6.45 (d, J = 7.2 Hz, 1 H, CArH), 6.58–6.65
(m, 2 H, CArH), 7.19–7.30 (m, 5 H, CArH).
N-(Benzyloxycarbonyl)phenylalanylleucine Benzyl Ester (9)
1H NMR (300 MHz, CDCl3): d = 0.78 (d, J = 6.0 Hz, 6 H, CdLeuH3),
1.37–1.57 (m, 3 H, CgLeuH, CbLeuH2), 2.96–3.01 (dd, J = 6.3 Hz, 2
H, CbPheH2), 4.41–4.49 (dd, J = 12.0, 3.3 Hz, 1 H, CaPheH), 4.49–
4.55 (m, 1 H, CaLeuH), 5.00 (s, 2 H, CH2OCO), 5.05 (s, 2 H,
COOCH2), 5.25 (d, J = 7.8 Hz, 1 H, NH), 6.12 (d, J = 8.1 Hz, 1 H,
NH), 7.07–7.29 (m, 15 H, CArH).
13C NMR (75 MHz, CDCl3): d = 21.9, 22.6, 24.7, 38.2, 41.7, 51.2,
56.3, 67.2, 127.2, 128.1, 128.2, 128.5, 128.7, 128.9, 129.4, 135.5,
136.4, 156.1, 170.7, 172.3.
13C NMR (75 MHz, CDCl3): d = 28.2, 38.0, 55.7, 67.3, 82.8, 115.4,
116.5, 121.9, 127.2, 128.2, 128.4, 128.7, 136.3, 143.2, 144.0, 156.2,
171.2.
MS [FIA-ESI(+)-TOF]: m/z [M + Na]+ calcd for C21H25NNaO6:
410.1574; found: 410.1530.
MS [FIA-ESI(+)-TOF]: m/z [M + Na]+ calcd for C30H34N2NaO5:
525.2359; found: 525.2324.
N-(Benzyloxycarbonyl)phenylalanylleucine tert-Butyl Ester
(10)
1H NMR (300 MHz, CDCl3): d = 0.80 (d, J = 6.0 Hz, 6 H, CdLeuH3),
1.36–1.50 (m, 12 H, CgLeuH, CbLeuH2, CCH3), 3.00 (d, J = 6.3 Hz, 2
H, CbPheH2), 4.32–4.39 (m, 2 H, CaLeuH, CaPheH), 5.00 (s, 2 H,
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Versatile Selective a-Carboxylic Acid Esterification
813
CH2OCO), 5.29 (d, J = 5.4 Hz, 1 H, NH), 6.23 (d, J = 6.9 Hz, 1 H,
(d, J = 8.4 Hz, 1 H, NH), 5.82–5.92 (m, 1 H, CH=), 8.89 (s, 1 H,
NH), 7.09–7.25 (m, 10 H, CArH).
COOH).
13C NMR (75 MHz, CDCl3): d = 22.1, 22.6, 24.8, 28.0, 38.4, 41.8,
51.4, 56.0, 67.0, 81.9, 127.0, 128.0, 128.1, 128.7, 128.8, 129.5,
136.3, 155.8, 170.3, 171.5.
13C NMR (75 MHz, CDCl3): d = 28.4, 36.8, 50.1, 66.0, 80.7, 119.0,
131.7, 155.9, 171.1, 175.9.
MS [FIA-ESI(+)-TOF]: m/z [M + Na]+ calcd for C12H19NNaO6:
296.1134; found: 296.1117.
MS [FIA-ESI(+)-TOF]: m/z [M + Na]+ calcd for C27H36N2NaO5:
491.2516; found: 491.2525.
N-(tert-Butyloxycarbonyl)glutamic Acid a-Allyl Ester (14a)
1H NMR (300 MHz, CDCl3): d = 1.42 (s, 9 H, CCH3), 1.94–2.03
(m, 1 H, CgH2), 2.15–2.21 (m, 1 H, CgH2), 2.41–2.47 (m, 2 H, CbH2),
4.34–4.36 (m, 1 H, CaH), 4.61 (d, J = 5.7 Hz, 2 H, COOCH2), 5.26–
5.35 (m, 3 H, NH, =CH2), 5.83–5.94 (m, 1 H, CH=), 9.44 (s, 1 H,
COOH).
13C NMR (75 MHz, CDCl3): d = 27.1, 28.2, 30.1, 52.9, 66.0, 80.1,
118.9, 131.5, 155.5, 172.0, 177.5.
MS [FIA-ESI(+)-TOF]: m/z [M + Na]+ calcd for C13H21NNaO6:
310.1261; found: 310.1266.
Enzymatic a-Selective Esterification of N-Protected Aspartic
Acid and Glutamic Acid; General Procedure
CLEA-Alcalase (3 g) was added to N-protected amino acid (0.5 g),
MTBE (28.0 mL), the appropriate alcohol (2.0 mL), and 3 Å molec-
ular sieves (2.0 g). The mixture was shaken at 50 °C with 150 rpm
for 16 h. After filtration, the enzyme was washed three times by re-
suspension in aq HCl (pH 1, 50 mL) and three times by resuspen-
sion in EtOAc (50 mL) followed by filtration. The combined EtOAc
and HCl phases were separated and the organic phase was washed
with aq HCl (pH 1, 100 mL), dried (Na2SO4), and concentrated in
vacuo. The resulting oil was redissolved in CH2Cl2–MeOH–AcOH
(89.9:10:0.1, 20 mL) followed by silica gel filtration. The mixture
was concentrated in vacuo and co-evaporated with toluene (2 × 50
mL) and CHCl3 (2 × 50 mL).
N-(9H-Fluoren-9-ylmethoxycarbonyl)aspartic Acid a-[2-(Tri-
methylsilyl)ethyl] Ester (15b)
1H NMR (300 MHz, CDCl3): d = 0.00 (s, 9 H, SiCH3), 0.80–0.96
(m, 2 H, CH2Si), 2.91 (dd, J = 3.9, 16.8 Hz, 1 H, CbH2), 3.06 (dd,
J = 3.6, 16.8 Hz, 1 H, CbH2), 4.19–4.57 (m, 6 H, CH2OCO,
COOCH2, CaH, fluorenyl CH), 5.79 (d, J = 7.8 Hz, 1 H, NH), 7.22–
7.73 (m, 8 H, CArH).
N-(Benzyloxycarbonyl)aspartic Acid a-[2-(Trimethylsilyl)eth-
yl] Ester (11b)
1H NMR (300 MHz, CDCl3): d = 0.00 (s, 9 H, SiCH3), 0.92–1.01
(m, 2 H, CH2Si), 2.85 (dd, J = 4.5, 17.4 Hz, 1 H, CbH2), 2.90 (dd,
J = 4.2, 16.8 Hz, 1 H, CbH2), 4.18–4.25 (m, 2 H, COOCH2), 4.58–
4.64 (m, 1 H, CaH), 5.10 (s, 2 H, CH2OCO), 5.84 (d, J = 8.1 Hz, 1
H, NH), 7.30–7.39 (m, 5 H, CArH).
13C NMR (75 MHz, CDCl3): d = –1.3, 17.5, 30.0, 36.6, 47.4, 64.9,
67.7, 120.3, 125.4, 127.3, 128.1, 141.6, 144.0, 156.1, 170.3, 175.5.
MS [FIA-ESI(+)-TOF]: m/z [M + Na]+ calcd for C24H29NNaO6Si:
478.1656; found: 478.1651.
13C NMR (75 MHz, CDCl3): d = –1.5, 17.3, 36.7, 50.4, 53.5, 64.5,
128.0, 128.7, 136.2, 159.9, 170.9, 175.4.
MS [FIA-ESI(+)-TOF]: m/z [M + Na]+calcd for C17H25NNaO6:
390.1343; found: 390.1336.
N-(9H-Fluoren-9-ylmethoxycarbonyl)glutamic Acid a-2-(Tri-
methylsilyl)ethyl Ester (16b)
1H NMR (300 MHz, CDCl3): d = 0.00 (s, 9 H, SiCH3), 0.86–1.02
(m, 2 H, CH2Si), 1.91–2.06 (m, 1 H, CgH2), 2.17–2.28 (m, 1 H,
CgH2), 2.39–2.45 (m, 2 H, CbH2), 4.18–4.21 (m, 3 H, CH2OCO,
fluorenyl CH), 4.35–4.42 (m, 3 H, COOCH2, CaH), 5.42 (d, J = 6.6
Hz, 1 H, NH) 7.23–7.74 (m, 8 H, CArH).
N-(Benzyloxycarbonyl)glutamic Acid a-Allyl Ester (12a)
1H NMR (300 MHz, CDCl3): d = 1.91–2.20 (m, 2 H, CgH2), 2.38–
2.45 (m, 2 H, CbH2), 4.37–4.45 (m, 1 H, CaH), 4.60 (d, J = 5.7 Hz,
2 H, COOCH2), 5.07 (s, 2 H, CH2OCO), 5.20–5.26 (m, 2 H, =CH2),
5.54 (d, J = 7.5 Hz, 1 H, NH), 5.80–5.91 (m, 1 H, CH=), 7.23–7.30
(m, 5 H, CArH), 8.95 (s, 1 H, COOH).
13C NMR (75 MHz, CDCl3): d = –1.3, 17.4, 22.3, 27.9, 47.5, 53.8,
63.4, 64.4, 67.5, 120.0, 125.2, 127.2, 128.3, 141.4, 144.2, 156.4,
171.5, 177.4.
13C NMR (75 MHz, CDCl3): d = 27.8, 30.2, 53.2, 66.6, 67.6, 119.6,
MS [FIA-ESI(+)-TOF]: m/z [M + Na]+ calcd for C25H31NNaO6Si:
492.1812; found: 492.1824.
128.5, 128.6, 128.9, 131.7, 136.5, 171.9, 178.1.
MS [FIA-ESI(+)-TOF]: m/z [M + Na]+calcd for C16H19NNaO6:
344.1104; found: 344.1134.
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N-(tert-Butoxycarbonyl)aspartic Acid a-Allyl Ester (13a)
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Synthesis 2009, No. 5, 809–814 © Thieme Stuttgart · New York