Synthesis and enzymatic resolution of Cα-dialkylated α-azido carboxamides: New enantiopure α-azido acids as building blocks in peptide synthesis

Article abstract of DOI:10.1055/s-2004-834947

α-Azido carboxylic acids have recently emerged as versatile N-protected equivalents for α-amino acids, especially valuable when the sterically hindered C^α-dialkylated α-amino acids have to be incorporated. Unsymmetrically substituted C^α

Full text of DOI:10.1055/s-2004-834947

272
PAPER
Synthesis and Enzymatic Resolution of Ca-Dialkylated a-Azido Carbox-
amides: New Enantiopure a-Azido Acids as Building Blocks in Peptide
Synthesis
S
ynthesis and Enzyma
i
tic Rescolution of a
C
h
-Dialkylate
a
d
a
-AzidoCarbox
J
a
mides ost,a,1 Theo Sonke,b Bernard Kaptein,b Quirinus B. Broxterman,b Norbert Sewald*a
a
b
Received 11 August 2004; revised 28 September 2004
um falciparum.9 Recently, we reported on the synthesis of
2H-labelled a-azidoisobutyryl chloride (D6-Azib-Cl) as
D6-Aib equivalent building block in peptide synthesis.10
Thus it was shown that a-azido carboxylic acids are ver-
Abstract: a-Azido carboxylic acids have recently emerged as ver-
satile N-protected equivalents for a-amino acids, especially valu-
able when the sterically hindered Ca-dialkylated a-amino acids
have to be incorporated. Unsymmetrically substituted Ca-dialkylat-
ed a-azido carboxylic acids can be obtained in enantiomerically satile building blocks especially useful for the synthesis of
pure form by enzymatic resolution of a-azido carboxamides. A L-
amidase from Ochrobactrum anthropi NCIMB 40321 accepts 2-
azido-2,4-dimethylpentanamide as the substrate and provides both
the corresponding S-configured a-azido carboxylic acid and the R-
configured a-azido carboxamide in excellent enantiomeric purity.
The former is a valuable synthetic precursor of a-methylleucine [(a-
Me)Leu] in peptide synthesis, as demonstrated by the successful
synthesis of a (a-Me)Leu containing efrapeptin C analogue.
sterically hindered peptide sequences, i.e. for the incorpo-
ration of Ca-dialkylated amino acids.
For the synthesis of peptides containing chiral Ca-dialkyl-
ated amino acids, however, the necessary building blocks
have to be available in enantiopure form. Ca-Dialkylated
amino acids can be obtained by enzymatic resolution of
the racemic amino acid carboxamides.11 They can be con-
verted into the a-azido acids by diazo transfer with triflyl
azide.2
Key words: azides, bioorganic chemistry, enantiomeric resolution,
enzymes, peptides
We wondered whether the enantiopure a-azido acids
could be obtained alternatively by direct enzymatic reso-
lution of a-azido carboxamides. The enzymatic resolution
of a-H-a-azido carboxylic acids with an amidase from
Pseudomonas putida has already been reported for two
substrates,12 but the resolution of Ca-dialkylated a-azido
carboxamides has not yet been described.
a-Azido carboxylic acids may be used as synthetic precur-
sors for a-amino acids and are thus equivalent to N-pro-
tected a-amino acids, which are the standard building
blocks in peptide synthesis for the stepwise elongation of
the peptide chain from the C-terminus to the N-terminus.
There are several examples for the application of a-azido
carboxylic acids in peptide synthesis. The solid phase syn-
thesis of a large number of small peptides has been report-
ed by Pelletier et al.2 using a-azido-substituted derivatives
as substitutes for amino acids. The azido acids are coupled
following the DCC–HOBt protocol (N,N¢-dicyclohexyl-
carbodiimide/N-hydroxybenzotriazole) and the azido
group is reduced on the solid support by treatment with
trimethylphosphine in aqueous dioxane. DalPozzo et al.3
reported the solution phase synthesis of a homooctamer of
a-methylvaline using the corresponding azido acid bro-
mide. The use of a-azidoisobutyryl chloride (Azib-Cl) as
building block for the incorporation of Aib-residues in
solid phase peptide synthesis has been reported by Meldal
et al.4 We used a similar protocol for the total synthesis of
the naturally occurring peptide antibiotic efrapeptin C.5
The efrapeptins are Aib-rich peptides produced by the
fungus Tolypocladium niveum. They are inhibitors of F1-
ATPase,6 show insecticidal7 and antiproliferative8 proper-
ties and are active against the malaria pathogen Plasmodi-
In this paper we report on the synthesis and enzymatic res-
olution of two Ca-dialkylated a-azido carboxamides, 2-
azido-2,4-dimethylpentanamide (6a) and 2-azido-2-meth-
yl-3-phenylpropanamide (6b). The enantiopure (S)-2-azi-
do-2,4-dimethylpentanoic acid [(S)-5a] thus obtained was
used as a building block for the introduction of an a-
methylleucine residue into an analogue of efrapeptin.
The synthesis of the racemic substrates 6a and 6b
(Scheme 1) for the enzymatic resolutions started from
simple ethyl esters 1 bearing already the side chains at the
a-carbon atom. The a-methyl groups were introduced by
alkylation of the ester enolates of 1 with iodomethane.
The esters 2 obtained were again converted to the enolates
and treated with CBr4,13 thus yielding a-bromo esters 3.
Substitution of the bromide in 3 by treatment with sodium
azide in dimethyl sulfoxide at room temperature overnight
proceeded smoothly and gave rise to the a-azido esters 4.
The ethyl esters 4 were saponified by reaction with KOH
in EtOH. The carboxylic acids 5 were activated by con-
version to the mixed anhydride with ethyl chloroformate
and reacted with NH3 to yield the desired Ca-dialkylated
a-azido carboxamides 6, which were obtained in high pu-
rity after recrystallization.
SYNTHESIS 2005, No. 2, pp 0272–0278
x
x
.
x
x
.2
0
0
5
Advanced online publication: 03.12.2004
DOI: 10.1055/s-2004-834947; Art ID: T09704SS
© Georg Thieme Verlag Stuttgart · New York

PAPER
Synthesis and Enzymatic Resolution of Ca-Dialkylated a-Azido Carboxamides
273
R
amidase from Ochrobactrum anthropi NCIMB 40321,
which was obtained by expression of the lamA gene in E.
coli. The carboxylic acid (S)-5a and the carboxamide (R)-
6a were isolated in yields of 48% and 50%, respectively.
The enantiomeric excess values of (S)-5a and (R)-6a were
determined by HPLC analysis on a chiral stationary phase
as being 96% and 95%, respectively, indicating enzymatic
conversion slightly higher than 50% (E-ratio 150). The
proof of the absolute configuration of (S)-5a as well as a
confirmation of the enantiomeric excess of >98% was ob-
tained by conversion to the corresponding a-methylleu-
cine by catalytic hydrogenation and comparison with
authentic samples of (S)-a-methylleucine15 by chiral
HPLC.
1. LDA
2. CBr4
1. LDA
2. CH3I
R
O
O
H3C
THF
THF
O
O
2a: R= i-Bu, 80%
2b: R= Bn, 69%
1a,b
R
R
1. KOH, EtOH
2. HCl aq
NaN3
N3
Br
O
O
H3C
DMSO
H3C
O
O
3a: R= i-Bu, 87%
3b: R= Bn, quant.
4a: R= i-Bu, 97%
4b: R= Bn, 99%
H3C
N3
R
H3C
N3
R
1. ClCOOEt, NEt3
2. NH3 aq
OH
NH2
L-amidase from
H3C
O
O
Ochrobactrum anthropi
NH2
N3
5a: R= i-Bu, 81%
5b: R= Bn, 59%
6a: R= i-Bu, 82%
6b: R= Bn, 62%
pH 8, 55 °C
O
1 mM ZnSO4
7 d
Scheme 1 Synthesis of a-azido carboxamides 6
rac-6a
1.39 g
As it was planned to use the enantiomerically pure car-
boxylic acids 5 as building blocks in peptide syntheses,
we investigated the coupling of the racemic mixtures to L-
alanine methyl ester. Best results were obtained by activa-
tion of the carboxylic acids with BTC (bis(trichlorometh-
yl) carbonate, triphosgene) in the presence of sym-
collidine (Scheme 2).14 After overnight reaction and chro-
matographic workup, the diastereomeric mixtures 7a and
7b were isolated in good yields.
H3C
N3
H3C
N3
NH2
OH
+
O
O
(S)-5a
(R)-6a
>98% ee
[α]D –19.1 (c 1.6, CHCl3)
0.67 g, 48%
95% ee
[α]D –38.5 (c 1.1, CHCl3)
0.69 g, 50%
Scheme 3 Enzymatic resolution of a-azido carboxamide 6a
H3C
N3
R
1. BTC, sym-collidine
2. H-Ala-OMe
OH
Coupling of the enantiomerically pure 2-azido-2,4-di-
methylpentanoic acid [(S)-5a] with L-alanine methyl ester
under the same conditions as described above for the ra-
cemic mixtures yielded the diastereomerically pure dipep-
tide (S,S)-7a (Scheme 4). There are no traces of (R,S)-7a
or (S,R)-7a detectable in the NMR spectra of crude (S,S)-
7a.
CH2Cl2, overnight
O
5a,b
O
O
R
R
H3C
N3
H3C
N3
H
N
H
N
+
OCH3
OCH3
O
CH3
O
CH3
(S,S)-7a, (S,S)-7b
(R,S)-7a, (R,S)-7b
7a: R = iBu, 57%
7b: R = Bn, 71%
O
H3C
N3
H3C
N3
H
N
1. BTC, sym-collidine
2. H-Ala-OMe
OH
OCH3
Scheme 2 Coupling of the racemic carboxylic acids 5 to L-alanine
69%
O
CH3
O
methyl ester
(S,S)-7a
(S)-5a
Scheme 4 Synthesis of diastereomerically pure dipeptide (S,S)-7a
Preliminary studies on the enzymatic resolution of 2-azi-
do-2,4-dimethylpentanamide (6a) and 2-azido-2-methyl-
3-phenylpropanamide (6b) on an analytical scale revealed
that the enzymatic activities are very low. This was ex-
pected as compounds 6 profoundly differ from the natural
substrates of amidases.11 In the case of 6b only a moderate
stereoselectivity was observed, while the resolution of 6a
proceeded with very high stereoselectivity. Therefore, the
resolution of this compound was also performed on a pre-
parative scale (Scheme 3). The carboxamide rac-6a was
incubated at 55 °C and pH 8 for several days with the L-
Recently, we reported on the first total synthesis of the
peptide antibiotic efrapeptin C 14a (Scheme 6).5 The full
Aib-rich sequence was assembled by fragment condensa-
tions of an N-terminal fragment (Pip1-Gly8), a central
fragment (Aib9-Gly13) and a C-terminal fragment contain-
ing the residues Leu14-Aib15 as well as the unusual cation-
ic head group derived from DBN and leucinol. In the
course of our structure-activity relationship studies, we in-
tended to incorporate (S)-5a in an analogue of efrapeptin
Synthesis 2005, No. 2, 272–278 © Thieme Stuttgart · New York

274
M. Jost et al.
PAPER
C containing a-methylleucine instead of leucine in posi- tion of 11 with 10 followed by hydrogenolytic cleavage of
tion 14. The synthesis of the modified C-terminal frag- the Z group gave rise to compound 12. The final segment
ment 10 is shown in Scheme 5. The Boc protected amino condensation of 12 and 13 completed the synthesis of the
amidinium salt 8 was deprotected with TFA and one Aib efrapeptin analogue 14b.
residue was added by N-HATU mediated condensation
with Boc-Aib-OH.
In conclusion we developed an efficient synthetic proce-
dure for the synthesis of Ca-dialkylated a-azido carboxyl-
ic acids and the corresponding carboxamides starting
from simple ethyl esters with overall yields up to 55% (4
1. TFA
steps) for the a-azido carboxylic acids and up to 45% (5
steps) for the corresponding carboxamides. We demon-
strated that compounds 6a and 6b are substrates of an L-
amidase from Ochrobactrum anthropi NCIMB 40321 and
we could obtain the a-azido arboxylic acid (S)-5a in enan-
tiopure form by enzymatic resolution of the carboxamide
6a. Furthermore, we demonstrated the usefulness of Ca-
dialkylated a-azido carboxylic acids as building blocks in
peptide synthesis by the synthesis of small peptides and
the incorporation of (S)-5a into an analogue of the peptide
antibiotic efrapeptin.
O
H
N
2. Boc-Aib-OH,
N-HATU,
DIPEA
CF3COO
Boc
I
N
H
Boc
N
H
N
N
9
8
N
N
72%
1. HClaq
O
2. (S)-5a, BTC,
sym-collidine
3. H2, Pd/C
H
N
CF3COO
N
.
H2N
N
TFA
H
O
15%
N
10
All moisture and air-sensitive reactions were performed in flame-
dried glassware under Ar or N2. THF was distilled from sodium
benzophenone ketyl immediately prior to use. CH2Cl2 was distilled
over CaH2. All other reagents were used as received. CAUTION:
Low molecular weight azido compounds should be handled with
great care. Differential scanning calorimetry analyses (DSC) of
compounds 5 and 6 revealed that they start to decompose at temper-
atures around 100 °C with evolution of large amounts of heat. Flash
column chromatography was carried out with silica gel 60 (40–63
mm) from Merck. IR spectra were recorded on a FT/IR-410 (Jasco)
spectrometer from the neat oil film (NaCl plates) or from a solid in
a KBr pellet. 1H NMR spectra were recorded on AC-250-P or DRX-
500 instruments (Bruker) in CDCl3 solution, unless otherwise not-
ed, at 250 MHz or 500 MHz; 13C NMR spectra were recorded at 63
MHz or 126 MHz. TMS was used as an internal standard, shift val-
ues are reported in ppm. CI mass spectra were recorded on an Au-
tospec X instrument (Vacuum Generators) using NH3 as the
reactant gas. ESI mass spectra were recorded on an Esquire 3000
ion trap instrument (Bruker). MALDI-ToF mass spectra were re-
corded on a Voyager-DE BioSpectrometry instrument (PerSeptive
Biosystems) using 2,5-dihydroxybenzoic acid (DHB) as a matrix.
Microanalyses were performed on a CHNS-932 (Leco) apparatus.
Preparative RP-HPLC was performed with a Vydac 218TP102 effi-
ciency protein and peptide C18-column (10 mm particle size, 250 ×
Scheme 5 Synthesis of the modified C-terminal fragment 10 of ef-
rapeptin C
It was found that the use of TFA for the removal of the
Boc group in 9 leads to the formation of trifluoroacetylat-
ed products in the following coupling reaction. Therefore,
it was necessary to remove the Boc group in 9 by treat-
ment with hydrochloric acid. In the next step the a-meth-
ylleucine residue was introduced by coupling of (S)-5a
with deprotected 9 followed by catalytic hydrogenation
giving rise to 10.
With the modified fragment 10 in hand, the synthesis of
the complete efrapeptin analogue 14b was completed in
the same way as described for the natural product 14a
(Scheme 6).5 The N-terminally Z-protected central frag-
ment 11 and the N-terminal fragment 13 were synthesized
by solid phase methods employing a-azidoisobutyryl
chloride as carboxy activated Aib equivalent. Condensa-
1. N-HATU, DIPEA
.
TFA
H-Aib-Aib-Pip-Aib-Gly-(α-Me)Leu-Aib
2. H2, Pd/C, MeOH/AcOH
CF3COO
N
Z-Aib-Aib-Pip-Aib-Gly-OH
N
H
+
10
11
12
N
50%
Ac-Pip-Aib-Pip-Aib-Aib-Leu-β-Ala-Gly-OH
13
Ac-Pip-Aib-Pip-Aib-Aib-Leu-β-Ala-Gly-Aib-Aib-Pip-Aib-Gly-Xaa-Aib
CF3COO
N
N-HATU, DIPEA
N
H
14a Xaa = Leu
14b Xaa = (α−Me)Leu, 67%
N
Scheme 6 Synthesis of the analogue 14b of efrapeptin C (14a) containing a-methylleucine instead of leucine in position 14
Synthesis 2005, No. 2, 272–278 © Thieme Stuttgart · New York

PAPER
Synthesis and Enzymatic Resolution of Ca-Dialkylated a-Azido Carboxamides
275
22 mm) using mixtures of water and MeCN containing 0.1% TFA
as the mobile phase.
IR (neat): 2961, 2935, 2906, 2872, 1735, 1467, 1381, 1368, 1305,
1228, 1154, 1125, 1067, 1040, 1021, 866, 669 cm–1.
Abbreviations: BTC: bis(trichloromethyl)carbonate; DBN: 1,5-di-
1H NMR (250 MHz): d = 0.88 (d, J = 6.7 Hz, 3 H), 0.97 (d, J = 6.7
Hz, 3 H), 1.31 (t, J = 7.1 Hz, 3 H), 1.76 (m, 1 H), 1.93 (s, 3 H), 2.09
(dd, J = 14.1, 5.3 Hz, 1 H), 2.25 (dd, 2J = 14.1, 7.1 Hz, 1 H), 4.23
(q, J = 7.1 Hz, 2 H).
13C NMR (63 MHz): d = 13.9, 22.7, 24.0, 26.7, 28.1, 50.9, 61.1,
62.0, 171.7.
azabicyclo[4.3.0]non-5-ene; DCC: N,N¢-dicyclohexylcarbodiim-
ide;
DIPEA:
diisopropylethylamine;
N-HATU:
1-[bis-
(dimethylamino)methyliumyl]-1H-1,2,3-triazolo[4,5-b]pyridine-3-
oxide hexafluorophosphate; HOBt: N-hydroxybenzotriazole; TFA:
trifluoroacetic acid.
MS (CI): m/z (%) = 254, 256 (100), 237, 239 (4).
Ethyl 2,4-Dimethylpentanoate (2a)
A solution of n-BuLi in hexane (1.6 M, 82.5 mL, 132 mmol, 1.1
equiv) was added dropwise at 0 °C to a stirred solution of diisopro-
pyl amine (20.2 mL, 144 mmol, 1.2 equiv) in THF (200 mL). The
mixture was stirred for 30 min at this temperature, cooled to –78 °C
and ethyl 4-methylpentanoate (1a; Fluka, 19.9 mL, 120 mmol, 1.0
equiv) was added dropwise. The mixture was stirred for 30 min and
then iodomethane (12.0 mL, 192 mmol, 1.6 equiv) was added slow-
ly. The reaction mixture was allowed to warm up slowly to r.t. and
was stirred overnight. The mixture was quenched with a sat. solu-
tion of NH4Cl (200 mL) and petroleum ether (150 mL) was added.
The phases were separated, the organic phase was dried (Na2SO4)
and filtered. After evaporation of the solvent the residue was puri-
fied by vacuum distillation to afford a colorless liquid (15.1 g, 96
mmol, 80%); bp 99 °C/150 mbar [Lit.16 bp 105 °C/175 mbar].
Ethyl 2-Bromo-2-methyl-3-phenylpropanoate (3b)
Following the procedure described for 3a, starting from 2b (10.0 g,
52.0 mmol) 3b was obtained as a colorless liquid (14.3 g, 52.8
mmol, quantitative yield); bp 115 °C/1.6 × 10–1 mbar [Lit:18 85 °C/
2 × 10–2 Torr].
IR (neat): 3987, 3063, 3031, 2980, 2935, 2872, 1735, 1496, 1453,
1380, 1299, 1279, 1254, 1223, 1195, 1166, 1101, 1055, 1022, 744,
702 cm–1.
1H NMR (250 MHz): d = 1.31 (t, J = 7.1 Hz, 3 H), 1.82 (s, 3 H), 3.36
(d, J = 13.8 Hz, 1 H), 3.62 (d, J = 13.8 Hz, 1 H), 4.24 (q, J = 7.1 Hz,
2 H), 7.18–7.32 (m, 5 H).
13C NMR (63 MHz): d = 13.9, 27.4, 48.0, 60.2, 62.1, 127.3, 128.3,
130.5, 135.9, 171.0.
IR (neat): 2959, 2873, 1736, 1467, 1376, 1336, 1279, 1256, 1222,
1181, 1152, 1090, 1047, 1027 cm–1.
MS (CI): m/z (%) = 288, 290 (100).
1H NMR (250 MHz): d = 0.87 (d, J = 6.3 Hz, 3 H), 0.91 (d, J = 6.4
Hz, 3 H), 1.13 (d, J = 6.9 Hz, 3 H), 1.20 (m, 1 H), 1.25 (t, J = 7.1
Hz, 3 H), 1.50–1.68 (m, 2 H), 2.49 (m, 1 H), 4.12 (q, J = 7.1 Hz, 2
H).
13C NMR (63 MHz): d = 14.3, 17.5, 22.5, 26.0, 37.7, 43.1, 60.1,
177.2.
Ethyl 2-Azido-2,4-dimethylpentanoate (4a)
A mixture of NaN3 (6.3 g, 98 mmol, 1.5 equiv), 3a (15.4 g, 65
mmol, 1.0 equiv) and DMSO (100 mL) was stirred at r.t. overnight.
The mixture was poured carefully into water (400 mL) and Et2O
(200 mL) was added. The phases were separated and the organic
phase was washed with water (100 mL) and brine (100 mL), dried
(Na2SO4) and filtered. After evaporation of the solvent a light yel-
lowish liquid (12.5 g, 63 mmol, 97%) was obtained, which was used
in the following step without further purification.
MS (CI): m/z (%) = 176 (100), 159 (45).
Ethyl 2-Methyl-3-phenylpropanoate (2b)
Following the procedure described for 2a, starting from 3-phenyl
propionic acid ethyl ester 1b (Merck, 17.6 mL, 100 mmol, 1.0
equiv) 2b was obtained as a colorless liquid (13.3 g, 69 mmol, 69%)
after vacuum distillation; bp 57–60 °C/1 × 10–1 mbar [Lit.17 bp 68
°C/8 ×10–1 mbar].
IR (neat): 2960, 2872, 2116 (vs, N3), 1737, 1465, 1388, 1378, 1367,
1258, 1227, 1152, 1091, 1075, 1020 cm–1.
1H NMR (250 MHz): d = 0.90 (d, J = 6.3 Hz, 3 H), 0.94 (d, J = 6.3
Hz, 3 H), 1.32 (t, J = 7.1 Hz, 3 H), 1.48 (s, 3 H), 1.57–1.85 (m, 3 H),
4.23 (q, J = 7.1 Hz, 2 H).
13C NMR (63 MHz): d = 14.1, 23.0, 23.5, 23.8, 24.6, 46.3, 61.8,
66.2, 172.9.
IR (neat): 3086, 3063, 3029, 2978, 2936, 2876, 1733, 1605, 1496,
1454, 1376, 1350, 1284, 1252, 1174, 1117, 1028, 745, 700 cm–1.
1H NMR (250 MHz): d = 1.15 (d, J = 6.6 Hz, 3 H), 1.18 (t, J = 7.1
Hz, 3 H), 2.60–2.80 (m, 2 H), 3.02 (m, 1 H), 4.08 (q, J = 7.1 Hz, 2
H), 7.10–7.32 (m, 5 H).
13C NMR (63 MHz): d = 14.1, 16.8, 39.8, 41.5, 60.3, 126.3, 128.3,
129.0, 139.5, 176.1.
MS (CI): m/z (%) = 217 (100), 200 (5), 172 (68).
Ethyl 2-Azido-2-methyl-3-phenylpropanoate (4b)
Following the procedure described for 4a, azide 4b was obtained
starting from 3b (14.3 g, 52.8 mmol) as a light yellowish liquid
(12.2 g, 52.4 mmol, 99%), which was used in the subsequent step
without further purification.
MS (CI): m/z (%) = 210 (100), 193 (50).
Ethyl 2-Bromo-2,4-dimethylpentanoate (3a)
IR (neat): 3088, 3064, 3032, 2983, 2937, 2874, 2114 (vs, N3), 1739,
1497, 1454, 1378, 1254, 1195, 1109, 1019, 860, 767, 743, 702 cm–1.
1H NMR (250 MHz): d = 1.27 (t, J = 7.1 Hz, 3 H), 1.45 (s, 3 H), 2.98
(d, J = 13.7 Hz, 1 H), 3.09 (d, J = 13.7 Hz, 1 H), 4.21 (q, J = 7.1 Hz,
2 H), 7.17–7.32 (m, 5 H).
13C NMR (63 MHz): d = 14.1, 22.3, 43.8, 62.0, 66.9, 127.2, 128.2,
130.4, 135.1, 172.1.
A solution of n-BuLi in hexane (1.6 M, 68.8 mL, 110 mmol, 1.1
equiv) was added dropwise at 0 °C to a stirred solution of diisopro-
pyl amine (16.9 mL, 120 mmol, 1.2 equiv) in THF (200 mL). The
mixture was stirred for 30 min at this temperature, cooled to –78 °C
and ethyl ester 2a (15.8 g, 100 mmol, 1.0 equiv) was added drop-
wise. The mixture was stirred for 30 min and then a solution of tet-
rabromomethane (33.2 g, 100 mmol, 1.0 equiv) in THF (20 mL)
was added slowly. The mixture was warmed to r.t., quenched with
a sat. solution of NH4Cl (200 mL) and Et2O (200 mL) was added.
The phases were separated, the organic phase was washed with wa-
ter (100 mL) and brine (100 mL), dried (Na2SO4) and filtered. After
evaporation of the solvent a black liquid was obtained, which was
purified by vacuum distillation to afford a colorless liquid (20.6 g,
87 mmol, 87%); bp 57–60 °C (1.5 × 10–1 mbar).
MS (CI): m/z (%) = 91 (100).
2-Azido-2,4-dimethylpentanoic Acid (5a)
A mixture of KOH (9.3 g, 125 mmol, 2.0 equiv) and 4a (12.5 g, 63
mmol, 1.0 equiv) in EtOH (150 mL) and water (15 mL) was re-
fluxed for 40 min. The mixture was cooled in an ice bath and acid-
ified to pH 2 with concd HCl. The precipitated inorganic salts were
Synthesis 2005, No. 2, 272–278 © Thieme Stuttgart · New York

276
M. Jost et al.
PAPER
dissolved by addition of water (300 mL) and the mixture was ex-
tracted with Et2O (3 × 60 mL). The combined organic layers were
dried (Na2SO4) and filtered. After evaporation of the solvent a vis-
cous, colorless oil (8.7 g, 51 mmol, 81%) was obtained, which was
used in the following step without further purification.
Anal. Calcd for C10H12N4O (204.23): C, 58.81; H, 5.92; N, 27.43.
Found: C, 58.83; H, 5.89; N, 27.35.
Enzymatic Resolution of Ca-Dialkylated-a-azidocarboxamides;
General Description of the Screening Reactions
The amidase activity and stereoselectivity for 2-azido-2,4-dimeth-
ylpentanamide (6a) and 2-azido-2-methyl-3-phenylpropanamide
(6b) were tested according to the following general protocol:
IR (neat): 2960, 2873, 2119 (vs, N3), 1713, 1463, 1256, 1156, 1050,
920, 781 cm–1.
1H NMR (250 MHz): d = 0.94 (d, J = 6.7 Hz, 3 H), 0.97 (d, J = 6.6
Hz, 3 H), 1.54 (s, 3 H), 1.61–1.90 (m, 3 H), 10.1 (s, 1 H).
A concentrated enzyme solution (500 mL) containing the L-amidase
(1 mg) from Ochrobactrum anthropi NCIMB 40321 (produced via
expression of the lamA gene in E. coli XL-1 Blue MRF¢/p-
TrcLAM19) was added at 55 °C to a suspension (15 mL) of the sub-
strate (concentration 30 mM in 100 mM HEPES-NaOH buffer, pH
8.0, and 1 mM ZnSO4). The reaction mixture was stirred at 55 °C in
an orbital shaker. The time course of the reaction was monitored by
taking 1 mL samples where the enzyme activity was stopped by ad-
dition of 500 mL of 1 M H3PO4 solution. The samples were analyzed
by HPLC [column Chirobiotic R (250 × 4.6 mm); eluent: 15 mM
NH4OAc (pH 4.1)–MeOH (80:20); flow rate: 1.0 mL/min; T 20 °C;
UV detection at 215 nm] with respect to conversion and ee of the
azido acid 5a/5b. With substrate 6a conversion was 11% with an ee
of 97% after 30 h, and 30% with an ee of 96% after 198 h, corrected
for 0.5% racemic a-azido acid 5a in the substrate 6a. Identically,
with substrate 6b a conversion of 45% with an ee of 33% was ob-
tained after 30 h. No work-up of these screening reactions was per-
formed.
13C NMR (63 MHz): d = 23.0, 23.4, 23.8, 24.7, 46.2, 66.0, 179.2.
MS (CI): m/z (%) = 189 (47), 100 (100).
2-Azido-2-methyl-3-phenylpropionic Acid (5b)
Following the procedure described for 5a, azide 5b was obtained
starting from 4b (10.2 g, 43.8 mmol) as a colorless and highly vis-
cous oil (5.3 g, 25.7 mmol, 59%), which was used in the following
step without further purification.
IR (neat): 3032, 2110 (vs, N3), 1711, 1497, 1454, 1413, 1257, 1120,
700 cm–1.
1H NMR (250 MHz): d = 1.51 (s, 3 H), 3.01 (d, J = 13.7 Hz, 1 H),
3.15 (d, J = 13.7 Hz, 1 H), 7.20–7.35 (m, 5 H), 9.55 (s, 1 H).
13C NMR (63 MHz): d = 22.1, 43.5, 66.7, 127.4, 128.4, 130.4,
134.6, 178.0.
MS (CI): m/z (%) = 223 (1), 134 (100).
In a blank reaction (identical conditions; no enzyme added) no hy-
drolysis occurred for either substrate.
2-Azido-2,4-dimethylpentanamide (6a)
Et3N (5.9 mL, 42.6 mmol, 1.0 equiv) and ethyl chloroformate (5.1
mL, 42.6 mmol, 1.0 equiv) were added at –10 °C to a solution of 5a
(7.3 g, 42.6 mmol, 1.0 equiv) in THF (100 mL). The resulting sus-
pension was stirred for 10 min and was subsequently poured into an
ice cooled aqueous solution of NH3 (25%, 70 mL). The reaction
mixture was warmed up to r.t. within 1 h and was extracted with
Et2O (200 mL). The organic phase was washed with brine (80 mL),
dried (Na2SO4) and filtered. After evaporation of the solvent a solid
was obtained, which was purified by crystallization from hexane to
give a colorless, crystalline solid (5.9 g, 34.8 mmol, 82%); mp 69
°C.
Preparative Enzymatic Resolution of 2-Azido-2,4-dimethyl-
pentanamide (6a)
The same concentrated L-amidase solution (27 mL) as used in the
screening reaction was added to a solution of racemic 6a (1.4 g, 8.2
mmol) in water [518 mL, containing 100 mM HEPES-NaOH buffer
(pH 8.0) and 1 mM ZnSO4]. After stirring for 2 d at 55 °C in an or-
bital shaker a conversion of 25% was reached. Additional L-ami-
dase solution (27 mL) was added and stirring was continued for 5 d
(total reaction time 166 h). At that time a constant conversion of
45% was reached which did not change after addition of fresh L-
amidase solution. Precipitated proteins were removed by centrifu-
gation (30 min, 28,000 × g) and the supernatant (pH 8.0) was ex-
tracted with CHCl3 (3 × 100 mL). The combined organic layers
were washed with water, dried over Na2SO4 and concentrated under
reduced pressure. Thus, NMR-pure (R)-6a (690 mg, 4.1 mmol,
50%) was obtained as cream-colored crystals with an ee of 95% (R).
The ee was determined by chiral HPLC: column Chiralpak AD (250
× 4.6 mm); eluent: n-hexane–i-PrOH–TFA, 98.8:1.2:0.05; flow rate
2.0 mL/min; T 20 °C; UV detection at 210 nm.
IR (KBr): 3518, 3474, 3402, 2964, 2936, 2873, 2119 (vs, N3), 1688,
1570, 1523, 1388, 1159, 1046, 929 cm–1.
1H NMR (250 MHz): d = 0.92 (d, J = 6.6 Hz, 3 H), 1.00 (d, J = 6.6
Hz, 3 H), 1.54 (s, 3 H), 1.59 (dd, J = 14.1, 6.2 Hz, 1 H), 1.78 (m, 1
H), 1.97 (dd, J = 14.1, 6.3 Hz, 1 H), 6.18 (s, 1 H), 6.50 (s, 1 H).
13C NMR (63 MHz): d = 23.3, 23.6, 24.5, 24.9, 46.1, 67.2, 174.9.
MS (CI): m/z (%) = 188 (49), 171 (100).
Anal. Calcd for C7H14N4O (170.21): C, 49.39; H, 8.29; N, 32.92.
Found: C, 49.60; H, 8.01; N, 32.64.
The aqueous layer after extraction was acidified to pH 1 with concd
HCl solution and again extracted with CHCl3 (3 × 100 mL). The
combined organic layers were washed with water, dried over
Na2SO4. After removal of the solvent under reduced pressure (S)-5a
(670 mg, 3.9 mmol, 48%) was obtained as a NMR-pure oil. The ee
of 96% (S) was determined by chiral HPLC (column Chirobiotic R
(250 × 4.6 mm) + Chirobiotic T (50 × 4.6 mm) in line; eluent: 15
mM NH4OAc (pH 4.1)–MeOH, 80:20; flow rate: 1.0 mL/min; T 20
°C; UV detection at 215 nm).
2-Azido-2-methyl-3-phenylpropanamide (6b)
Following the procedure described for 6a, 6b was obtained starting
from 5b (4.5 g, 22.0 mmol) as a colorless solid (2.8 g, 13.6 mmol,
62%) after crystallization from a mixture of CHCl3 and hexane; mp
100 °C.
IR (KBr): 3473, 3416, 3294, 3220, 2120 (vs, N3), 1687, 1627, 1495,
1455, 1217, 1099, 909, 757, 737, 704 cm–1.
1H NMR (250 MHz): d = 1.62 (s, 3 H), 2.95 (d, J = 13.7 Hz, 1 H),
3.24 (d, J = 13.7 Hz, 1 H), 5.77 (s, 1 H), 6.24 (s, 1 H), 7.20–7.35 (m,
5 H).
13C NMR (63 MHz): d = 22.4, 44.2, 68.0, 127.2, 128.3, 130.2,
135.2, 174.1.
The absolute stereochemistry of the resolved azido acid 5a was de-
termined by transferring the a-azido acid into the corresponding a-
amino acid: Ammonium formate (310 mg, 5 mmol) and Pd/C (250
mg, 5 wt%) were added to a solution of the azido acid (30 mg, 0.18
mmol) in MeOH (5 mL). The mixture was stirred for 30 min at am-
bient temperature followed by heating to reflux for 5 min. The Pd/
C was filtered and washed with MeOH (10 mL). The combined
MeOH layers were concentrated under reduced pressure yielding
(S)-a-methylleucine (52 mg) as a white solid. The ee and absolute
MS (CI): m/z (%) = 222 (96), 205 (89).
Synthesis 2005, No. 2, 272–278 © Thieme Stuttgart · New York

PAPER
Synthesis and Enzymatic Resolution of Ca-Dialkylated a-Azido Carboxamides
277
configuration of > 98% of the (S)-enantiomer were determined with
chiral HPLC by comparison with authentic samples of (S)- and
(RS)-a-methylleucine.15 HPLC conditions: column Sumichira OA
5000 (150 × 4.6 mm); eluent: 2 mM aq CuSO4–i-PrOH, 90:10; flow
rate: 1.0 mL/min; T 35 °C; post-column reaction o-phthalic alde-
hyde (OPA)/mercaptoethanol (MCE); fluorescence detection with
excitation at 338 nm and emission at > 420 nm).
(both diastereomers), 130.19, 130.25, 135.18, 135.33, 170.36,
170.89, 172.55, 172.89.
MS (CI): m/z (%) = 308 (22), 291 (100).
Anal. Calcd for C14H18N4O3 (290.32): C, 57.92; H, 6.25; N, 19.30.
Found: C, 57.90; H, 5.95; N, 19.06.
2-[N-tert-Butyloxycarbonyl)amino]isobutyryl-N¢-{(2S)-1-(1,5-
diazabicyclo[4.3.0]non-5-ene-5-yliumyl)-4-methylpent-2-
yl}amide Trifluoroacetate (9)
N-[2-Azido-2,4-dimethylpentanoyl]-L-alanine Methyl Ester
(7a)
TFA (5 mL) was added at 0 °C to a solution of amidinium salt 85
(600 mg, 1.33 mmol, 1.0 equiv) in CH2Cl2 (3 mL). The mixture was
warmed to r.t. and after 30 min the volatiles were removed in vacuo
and the residue was lyophilized from water. DIPEA (1.36 mL, 7.98
mmol, 6.0 equiv) was added at 0 °C to a stirred solution of Boc-Aib-
OH (Novabiochem, 352 mg, 1.73 mmol, 1.3 equiv) and N-HATU
(657 mg, 1.73 mmol, 1.3 equiv) in DMF. After 10 min a solution of
the amino component obtained above in CH2Cl2 (6 mL) was added.
After 6 h the volatiles were removed in vacuo and the product was
isolated by RP-HPLC as a colorless solid (504 mg, 0.96 mmol,
72%).
1H NMR (500 MHz): d = 0.86 (d, J = 6.3 Hz, 3 H), 0.89 (d, J = 6.3
Hz, 3 H), 1.13 (m, 1 H), 1.39 (s, 9 H), 1.40 (s, 3 H), 1.52 (s, 3 H),
1.61–1.72 (m, 2 H), 2.03–2.29 (m, 4 H), 3.02 (m, 1 H), 3.26 (m, 1
H), 3.31–3.46 (m, 4 H), 3.66–3.84 (m, 4 H), 4.31 (m, 1 H), 5.41 (s,
1 H), 7.67 (d, J = 8.5 Hz, 1 H).
sym-Collidine (616 mL, 4.7 mmol, 4.0 equiv) was added at 0 °C to
a solution of rac-5a (200 mg, 1.17 mmol, 1.0 equiv) and BTC (139
mg, 0.47 mmol, 0.4 equiv) in CH2Cl2 (4 mL). The resulting suspen-
sion was stirred for 10 min and then L-alanine methyl ester hy-
drchloride (181 mg, 1.8 mmol, 1.5 equiv), dissolved in DMF, (1
mL) was added. After stirring overnight Et2O (40 mL) was added
and the mixture was washed with water (2 × 10 mL). The organic
layer was dried (Na2SO4) and filtered. After evaporation of the sol-
vent, the residue was purified by flash column chromatography (pe-
troleum ether–EtOAc, 10:1) to afford the product as a colorless oil.
IR (neat): 3348, 2957, 2873, 2116 (vs, N3), 1746, 1670, 1517, 1454,
1275, 1215, 1151, 1058 cm–1.
1H NMR (250 MHz): d = 0.87 (d, J = 6.6 Hz, 3 H, S,S), 0.88 (d, J =
5.7 Hz, 3 H, R,S), 0.99 (d, J = 6.6 Hz, 3 H, S,S), 1.00 (d, J = 6.6 Hz,
3 H, R,S), 1.42 (d, J = 7.2 Hz, 3 H, S,S), 1.43 (d, J = 7.2 Hz, 3 H,
R,S), 1.53 (s, 6 H, both diastereomers), 1.58 (dd, J = 14.3, 7.1 Hz, 1
H, R,S), 1.59 (dd, J = 14.4, 6.3 Hz, 1 H, S,S), 1.73 (m, 1 H, S,S), 1.75
(m, 1 H, R,S), 1.98 (dd, J = 14.3, 6.0 Hz, 1 H, R,S), 2.00 (dd, J =
14.4, 6.3 Hz, S,S), 3.75 (s, 3 H, R,S), 3.76 (s, 3 H, S,S), 4.53 (dq,
J = 7.3, 7.3 Hz, 1 H, R,S), 4.54 (dq, J = 7.2, 7.2 Hz, 1 H, S,S), 7.06
(d, J = 6.9 Hz, 1 H, R,S), 7.10 (d, J = 6.9 Hz, 1 H, S,S).
13C NMR (126 MHz): d = 18.2, 18.8, 21.3, 23.2, 24.6, 24.9, 26.3,
28.3, 30.9, 40.2, 42.4, 44.8, 45.2, 54.5, 56.5, 57.3, 79.1, 154.8,
165.3, 176.1.
MS (MALDI–ToF): m/z = 409.29 [M]+, monoisotopic mass calcu-
lated for the cation [C22H41N4O3]+: 409.32.
13C NMR (63 MHz): d = 18.16 (R,S), 18.25 (S,S), 23.26 (S,S), 23.40
(R,S), 23.49 (R,S), 23.57 (S,S), 24.51 (S,S), 24.60 (R,S), 24.78 (R,S),
24.88 (S,S), 46.14 (both diastereomers), 48.10 (R,S), 48.19 (S,S),
52.42 (R,S), 52.49 (S,S), 67.29 (S,S), 67.37 (R,S), 171.24 (S,S),
171.28 (R,S), 172.92 (R,S), 173.07 (S,S).
(S)-2-Methylleucyl-2-aminoisobutyryl-N-[(2S)-1-(1,5-diazabi-
cyclo[4.3.0]non-5-ene-5-yliumyl)-4-methylpent-2-yl]amide Tri-
fluoroacetate (10)
Concd HCl (3 mL) was added at 0 °C to a suspension of 9 (120 mg,
230 mmol) in water (0.5 mL). Water (20 mL) was added after 1 h
and the mixture was lyophilized. sym-Collidine (121 mL, 915 mmol)
was added slowly at 0 °C to a stirred solution of (S)-5a (30 mg, 172
mmol) and BTC (21 mg, 72 mmol) in CH2Cl2 (1 mL). After 5 min a
solution of the amino component obtained above, dissolved in DMF
(1 mL) and CH2Cl2 (1 mL) was added. The mixture was warmed to
r.t. and after 2 h another portion of BTC (11 mg, 36 mmol) was add-
ed. The mixture was stirred 72 h at r.t., the volatiles were removed
in vacuo and the coupling product was isolated from the residue by
RP-HPLC (39 mg, 68 mmol, 30%). Palladium on activated charcoal
(10%, 10 mg) was added to a solution of a part of this coupling
product (10 mg, 17 mmol) in MeOH (4 mL). The mixture was stirred
vigorously under an atmosphere of H2 for 2 h, the catalyst was fil-
tered off and washed with MeOH. The volatiles were removed in
vacuo and the product was isolated by RP-HPLC as a colorless solid
(6.0 mg, 8.3 mmol, 49%).
1H NMR (500 MHz, CD2Cl2): d = 0.84 (d, J = 6.3 Hz, 3 H), 0.88 (d,
J = 6.3 Hz, 3 H), 0.91 (d, J = 6.3 Hz, 3 H), 0.94 (d, J = 6.6 Hz, 3 H),
1.13 (m, 1 H), 1.44 (s, 3 H), 1.46 (s, 3 H), 1.53 (s, 3 H), 1.53–1.64
(m, 2 H), 1.71–1.81 (m, 2 H) 1.85 (dd, J = 14.0, 5.8 Hz, 1 H), 2.02
(m, 1 H), 2.08–2.20 (m, 2 H), 2.26 (m, 1 H), 2.83 (ddd, J = 17.0,
10.4, 6.0 Hz, 1 H), 3.19–3.30 (m, 3 H), 3.31–3.45 (m, 2 H), 3.61–
3.70 (m, 2 H), 3.79–3.89 (m, 2 H), 4.32 (m, 1 H), 7.07 (d, J = 9.1
Hz, 1 H), 7.74 (s, 1 H), 8.96 (br s, 3 H).
MS (CI): m/z (%) = 274 (5), 257 (100).
Anal. Calcd for C11H20N4O3 (256.30): C, 51.55; H, 7.87; N, 21.86.
Found: C, 51.60; H, 7.60; N, 21.47.
N-[(S)-2-Azido-2,4-dimethylpentanoyl]-L-alanine Methyl Ester
[(S,S)-7a]
Following the procedure described for the mixture of diastereomers
7a, the pure (S,S)-diastereomer was obtained starting from (S)-5a
(50 mg, 0.29 mmol) as a colorless oil (50 mg, 0.20 mmol, 69%);
[a]D25 +23.2 (c = 0.3, CHCl3)
N-[2-Azido-2-methyl-3-phenylpropanoyl]-L-alanine Methyl Es-
ter (7b)
Following the procedure described for 7a the diastereomeric mix-
ture 7b was obtained starting from rac-5b (205 mg, 1.00 mmol) as
a colorless oil (207 mg, 0.71 mmol, 71%) after flash column chro-
matography (petroleum ether–EtOAc, 4:1).
IR (neat): 3406, 3354, 3062, 3030, 2989, 2953, 2879, 2848, 2116
(vs, N3), 1745, 1674, 1518, 1454, 1377, 1271, 1213, 1167, 1090,
1061, 743, 702 cm–1.
1H NMR (250 MHz): d = 1.15 (d, J = 7.2 Hz, 3 H), 1.37 (d, J = 7.2
Hz, 3 H), 1.58 (s, 3 H), 1.63 (s, 3 H), 2.94 (d, J = 13.8 Hz, 1 H), 2.97
(d, J = 13.8 Hz, 1 H), 3.20 (d, J = 13.8 Hz, 1 H), 3.23 (d, J = 13.8
Hz, 1 H), 3.67 (s, 3 H), 3.70 (s, 3 H), 4.44 (dq, J = 7.2, 7.2 Hz, 1 H),
4.45 (dq, J = 7.3, 7.3 Hz, 1 H), 6.77 (d, J = 7.8 Hz, 1 H), 6.86 (d,
J = 7.1 Hz, 1 H), 7.16–7.32 (m, 10 H).
13C NMR (126 MHz): d = 18.6, 19.3, 21.5, 21.7, 23.2, 24.0, 24.1,
24.3, 25.1, 26.8, 31.2, 40.4, 42.8, 44.8, 45.4, 46.9, 55.0, 57.0, 58.2,
60.9, 165.7, 170.5, 175.1.
13C NMR (63 MHz): d = 17.99, 18.27, 22.12, 22.58, 44.21, 44.26,
48.00, 48.19, 52.39, 52.43, 67.85, 68.12, 127.14, 127.16, 128.24
MS (ESI): m/z = 436.3, monoisotopic mass calculated for the cation
[C24H45N5O2]+: 435.36.
Synthesis 2005, No. 2, 272–278 © Thieme Stuttgart · New York

278
M. Jost et al.
PAPER
2-Aminoisobutyryl-2-aminoisobutyryl-(S)-pipecolinyl-2-ami-
noisobutyryl-glycyl-(S)-2-methylleucyl-2-aminoisobutyryl-N-
[(2S)-1-(1,5-diazabicyclo[4.3.0]non-5-ene-5-yliumyl)-4-methyl-
pent-2-yl]amide Bis(trifluoroacetate) (12)
(4) (a) Meldal, M.; Juliano, M. A.; Jansson, A. M. Tetrahedron
Lett. 1997, 38, 2531. (b) Tornøe, C. W.; Davis, P.; Porreca,
F.; Meldal, M. J. Pept. Sci. 2000, 6, 549.
(5) Jost, M.; Greie, J.-C.; Stemmer, N.; Wilking, S. D.;
Altendorf, K.; Sewald, N. Angew. Chem. Int. Ed. 2002, 41,
4267; Angew. Chem. 2002, 114, 4438.
[(S)-a-Methylleucine14]efrapeptin C-(9-15)-peptide bis(trifluoroac-
etate) (12)
(6) Abrahams, J. P.; Buchanan, S. K.; van Raau, M. J.; Fearnley,
I. M.; Leslie, A. G. W.; Walker, J. E. Proc. Natl. Acad. Sci.
U.S.A. 1996, 93, 9420.
DIPEA (16.1 mL, 94.5 mmol, 4.2 equiv) was added at 0 °C to a
stirred solution of Z-Aib-Aib-Pip-Aib-Gly-OH 115 (14.2 mg, 24.7
mmol, 1.1 equiv) and N-HATU (10.3 mg, 27.0 mmol, 1.2 equiv) in
DMF (1.5 mL). After 10 min a solution of 10 (14.9 mg, 22.5 mmol,
1.0 equiv) in CH2Cl2 (1.5 mL) was added. The mixture was left
overnight at 0 °C. The volatiles were removed in vacuo and the res-
idue was redissolved in MeOH (3 mL), then HOAc (0.3 mL) and
palladium on activated charcoal (10%, 10 mg) were added and the
mixture was stirred vigorously under an atmosphere of H2 for 1 h.
The catalyst was removed by filtration and washed carefully with
MeOH (15 mL). After removal of the solvent in vacuo, the product
was isolated by RP-HPLC as a colorless solid (12.3 mg, 11.3 mmol,
50%).
(7) (a) Gupta, S.; Krasnoff, S. B.; Roberts, D. W.; Renwick, J.
A. A.; Brinen, L. S.; Clardy, J. J. Am. Chem. Soc. 1991, 113,
707. (b) Gupta, S.; Krasnoff, S. B.; Roberts, D. W.;
Renwick, J. A. A.; Brinen, L. S.; Clardy, J. J. Org. Chem.
1992, 57, 2306. (c) Matha, V.; Weiser, J.; Olejnicek, J. Folia
Parasitol. 1988, 35, 379. (d) Bandani, A. R.; Khambay, B.
P. S.; Faull, J. L.; Newton, R. M.; Deadman, M.; Butt, T. M.
Mycol. Res. 2000, 104, 537.
(8) Papathanassiu, A. E. (Ergon Pharmaceuticals LLC) U. S.
Patent, 6528489, 2003.
(9) Nagaraj, G.; Uma, M. V.; Shivayogi, M. S.; Balaram, H.
Antimicrob. Agents Chemother. 2001, 45, 145.
MS (ESI): m/z = 859.7, monoisotopic mass calculated for the cation
[C44H79N10O7]+: 859.61.
(10) Weigelt, S.; Sewald, N. Synlett 2004, 726.
(11) Sonke, T.; Kaptein, B.; Boesten, W. H. J.; Broxterman, Q.
B.; Schoemaker, H. E.; Hans, E.; Kamphuis, J.; Formaggio,
F.; Toniolo, C.; Rutjes, F. In Stereoselective Biocatalysis;
Patel, R. N., Ed.; Marcel Dekker: New York, 2000, 23.
(12) Tornøe, C. W.; Sonke, T.; Maes, I.; Schoemaker, H. E.;
Meldal, M. Tetrahedron: Asymmetry 2000, 11, 1239.
(13) Arnold, R. T.; Kulenovic, S. T. J. Org. Chem. 1978, 43,
3687.
(14) (a) Falb, E.; Yechezkel, T.; Salitra, Y.; Gilon, C. J. Pept.
Res. 1999, 53, 507. (b) Thern, B.; Rudolph, J.; Jung, G.
Tetrahedron Lett. 2002, 43, 5013. (c) Thern, B.; Rudolph,
J.; Jung, G. Angew. Chem. Int. Ed. 2002, 41, 2307; Angew.
Chem. 2002, 114, 2401. (d) Sewald, N. Angew. Chem. Int.
Ed. 2002, 41, 4661; Angew. Chem. 2002, 114, 4855.
(15) Kaptein, B.; Boesten, W. H. J.; Broxterman, Q. B.; Peters, P.
H. J.; Schoemaker, H. E.; Kamphuis, J. Tetrahedron:
Asymmetry 1993, 4, 1113.
[(S)-a-Methylleucine14]efrapeptin C (14b)
To a stirred solution of 135 (3.1 mg, 4.0 mmol, 1.3 equiv) and N-
HATU (1.5 mg, 4.0 mmol, 1.3 equiv) in DMF (0.5 mL) was added
at 0 °C DIPEA (1.7 L, 10.2 mmol, 3.3 equiv). After 10 min a solution
of 12 (3.4 mg, 3.1 mmol, 1.0 equiv) in CH2Cl2 (0.5 mL) was added.
The mixture was left at 0 °C overnight, the volatiles were removed
in vacuo and the product was isolated by RP-HPLC as a colorless
solid (3.6 mg, 2.1 mmol, 67%).
MS (ESI): m/z = 1620.4, monoisotopic mass calculated for the cat-
ion [C81H139N18O16]+: 1620.06.
Acknowledgment
This project was supported by the Deutsche Forschungsgemein-
schaft and the Fonds der Chemischen Industrie. Mrs. Ilse Maes and
Mr. Math Boesten are thanked for the determination of the ee values
by HPLC.
(16) Göttlich, R.; Schopfer, U.; Stahl, M.; Hoffmann, R. W.
Liebigs Ann./Recl. 1997, 1757.
(17) Meyers, A. I.; Tempel, D. L.; Nolen, R. L.; Mihelich, E. D.
J. Org. Chem. 1974, 39, 2778.
(18) Roberts, A.; Jaguelin, S.; Guinamant, J. L. Tetrahedron
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Synthesis 2005, No. 2, 272–278 © Thieme Stuttgart · New York