Full text of DOI:10.1016/S0040-4020(01)00548-8

TETRAHEDRON
Pergamon
Tetrahedron 57 22001) 6567±6577
Enantiopure Ca-tetrasubstituted a-amino acids. Chemo-
enzymatic synthesis and application to turn-forming peptides
Bernard Kaptein,a Quirinus B. Broxterman,a Hans E. Schoemaker,a Floris P. J. T. Rutjes,b
Johan J. N. Veerman,c Johan Kamphuis,d Cristina Peggion,e Fernando Formaggioe
and Claudio Tonioloe,p
aDSM Research, Organic Chemistry and Biotechnology Section, P.O. Box 18, 6160 MD Geleen, The Netherlands
bDepartment of Organic Chemistry, University of Nijmegen, 6525 ED Nijmegen, The Netherlands
cInstitute of Molecular Chemistry, University of Amsterdam, 1018 WS Amsterdam, The Netherlands
dDSM Food Specialties, Nutritional Ingredients, P.O. Box 1, 2600 MA Delft, The Netherlands
eDepartment of Organic Chemistry, Biopolymer Research Centre, C.N.R., University of Padova, Via Marzolo 1, 35131 Padova, Italy
Received 2 January 2001; revised 15 February 2001; accepted 23 February 2001
AbstractÐBy a chemo-enzymatic approach we carried out a large-scale synthesis of four enantiopure, sterically constrained, Ca-tetra-
substituted a-amino acids, all characterized by a sidechain CgCd double bond. By using one of them 2l-Mag), we prepared an Na-protected
tetrapeptide benzylamide which was shown to adopt a b-turn conformation and to ef®ciently undergo ring-closing ole®n metathesis. q 2001
Elsevier Science Ltd. All rights reserved.
1. Introduction
pounds25±35 and cyclic peptides via intramolecular
carbon±carbon formation36±43 has been already reported
for allyl-based, chiral Ca-trisubstituted a-amino acids and
peptides. For the ®rst time, Verdine and coworkers44 have
recently extended the exploding ®eld of ring-closing
metathesis to peptides characterized by Ca-methyl-l-allyl-
glycine 2l-Mag) 2l-2a) and related residues.
Chiral Ca-tetrasubstituted a-amino acids are increasingly
attracting attention as useful building blocks for the produc-
tion of naturally occurring and tailor-made derivatives of
pharmaceutical and agricultural interest.1±4 In addition, it
has been unambiguously demonstrated that these synthons,
in analogy with their achiral prototype a-aminoisobutyric
acid 2Aib) 21),5±10 are able to dramatically reduce the
conformational freedom of the peptides in which they are
incorporated. Indeed, detailed experimental investi-
gations11±13 have shown that peptides rich in chiral Ca-tetra-
substituted a-amino acids either fold in screw-sense biased
b-turns14±16 and 310-helical structures17 or adopt planar,
fully-extended 2C5) conformations.15,18,19 The stabilization
of speci®c ordered secondary structures has recently
become a major issue in peptide science, with special
emphasis to the design of rigidi®ed catalysts, spacers, and
templates 2for leading review articles see Refs. 20±22).
In this context, allyl-type, chiral Ca-tetrasubstituted
a-amino acids are of emerging relevance for the additional
potential ability of their peptides to undergo a wide range of
transformations at the CgvCd bond, including Grubbs'
intramolecular, side-chain to side-chain, ring-closing ole®n
metathesis.23,24 The preparation of `built-into' com-
Keywords: amino acids and derivatives; metathesis; NMR; peptides and
polypeptides.
p
Corresponding author. Tel.: 139-49-8275247; fax: 139-49-8275239;
e-mail: biop02@chor.unipd.it
0040±4020/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020201)00548-8

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B. Kaptein et al. / Tetrahedron 57 62001) 6567±6577
Scheme 1.
As part of a programme dealing with chemistry and struc-
tural characterization of allyl-type, Ca-tetrasubstituted
a-amino acids, and their derivatives and peptides,25,44±63
in this article we describe the large-scale synthesis of the
four enantiopure a-amino acids l-2a±d. We also prepared
and investigated the preferred conformation of: 2i) a linear,
Na-protected tetrapeptide benzylamide containing one
residue of l-Mag 2l-2a) and one of its unmethylated
counterpart l-allylglycine 2l-Agl) 2l-3), and 2ii) its side-
chain to side-chain, ring-closing ole®n metathesis/
hydrogenation derivative.
been cloned and overexpressed in an E. coli K-12 host
microorganism. For the resolution of Ca-tetrasubstituted
a-amino amides two amidases 2from Mycobacterium
neoaurum ATCC 25795 and Ochrobactrum anthropi
NCIMB 40321, respectively) are available, both exhibiting
a high l-stereoselectivity. The amidase from the latter
species is also able to stereoselectively hydrolyze
Ca-trisubstituted a-amino amides.
Racemic Ca-tetrasubstituted a-amino amides 27b±d) were
prepared by PTC 2phase-transfer catalyzed) alkylation of
N-benzylidene-d,l-alaninamide 4 2Scheme 1), as previously
described for 7a.46 With allyl-type bromides 5a, c and d
Ca-alkylation is fast and proceeds to high conversion.
After acidic work-up of the racemic Schiff bases 6a, c and
d, the racemic Ca-tetrasubstituted a-amino amides 7a, c and
d were all obtained in 72±73% yield. In the alkylation
reactions with prenyl 23,3-dimethallyl) bromide 25c) or
cinnamyl bromide 25d) a complete SN2 reaction occurred
and no SN20 product was observed. Ca-Alkylation of 4 with
3-bromo-2-methyl-1-propene 5b 2or 3-chloro-2-methyl-1-
propene) proceeded only to low conversion. In addition, a
substantial amount of byproducts occurred, which made the
puri®cation of the reaction mixture dif®cult. All attempts to
improve the yield of this reaction failed.
2. Results and discussion
2.1. Amino acid synthesis and enzymatic resolution
A variety of methods for the synthesis of enantiomerically
pure Ca-tetrasubstituted a-amino acids 2recently reviewed
in Ref. 64) have been described. At DSM Research we
concentrated on the preparation of this type of amino
acids via enzymatic resolution of racemic a-amino amides.
This route is especially attractive for large-scale preparation
since the racemic a-amino amide substrates are readily
accessible and the enzymatic resolution can be easily
scaled-up and proceeds with high stereoselectivity.
Currently, we have three amidases available for a-amino
amide resolution.65 The l-aminopeptidase from Pseudomonas
putida ATCC 12633 is used for the enzymatic resolution of
Ca-trisubstituted a-amino amides. This enzyme has recently
The four racemic substrates 7a±d were subjected to enzym-
atic hydrolysis using the amidases from M. neoaurum48,66
and O. anthropi67 2Scheme 2). Thus, the stereoselectivity
of the two enzymes could be compared. In general, the
Scheme 2.

B. Kaptein et al. / Tetrahedron 57 62001) 6567±6577
6569
Table 1. Results of the enzymatic resolutions
Substrate
R
Amidase
Conversion 2%)
e.e.amide 2%)
e.e.acid 2%)
E-ratio
7a
±CH2±CHvCH2
M. neoaurum
O. anthropi
40
64
63
50
92
27
46
2.7
7b
7c
7d
a
±CH2±C2CH3)vCH2
±CH2±CHvC2CH3)2
±CH2±CHvCH-Ph
M. neoaurum
O. anthropi
46
46
85
72
98
85
.250
27
M. neoaurum
O. anthropi
45
62
80
98
98
61
.250
17
M. neoaurum
O. anthropi
51
31
$98
45
95
96
$180
79a
At 40±50% conversion the E-ratio is 65±69.
enzymatic resolutions are performed on a 10% aqueous
solution of the substrate. With the M. neoaurum amidase
the initial pH was set at 8.0±8.5. With the amidase from
O. anthropi a starting pH between 6 and 8 was applied as
this enzyme has a much broader pH optimum. Since the
enzyme reactions are not buffered, the pH during the
reaction increased to 9.6 due to the formation of ammonia.
The conversion of the resolution reactions was measured
using an ammonia sensitive electrode. After the appropriate
reaction time, in general between 6 and 24 h, the crude cell
mass was removed by centrifugation and the amide and acid
were separated by ion-exchange chromatography 2strongly
basic resin). The results of these enzymatic resolutions are
listed in Table 1. If necessary, the e.e. values of the l-acids
can be improved by recrystallization from isopropanol/
water.
to contain the Ca-methyl substituent of the amides
7a±d.48,62,66 Larger substituents, like an ethyl or an allyl
group, are accepted with great dif®culty, which results in
a slow hydrolysis of the d-enantiomer. In general, the
enantiomeric ratio E68 improves with increasing size of
the large substituent. As already known from earlier
results,48,66 branching at the a-amino amide b-position
results in a complete stereoselectivity 2E.250) for the
amidase from M. neoaurum, as well as for that from O.
anthropi. From the current results it can be concluded that
increasing the branching at the g-27b) and to a somewhat
lesser extent also at the d-position 27c and 7d) has a similar
effect.
Hydrogenation of the double bond of Ca-methyl-d,l-allyl-
glycinamide 27a) substantially improves the stereoselec-
tivity of both enzymes. Indeed, Ca-methyl-d,l-norva-
linamide is hydrolysed by M. neoaurum amidase with a
nearly complete stereoselectivity 2E.232), while the
enantiomeric ratio with the amidase from O. anthropi is
enhanced from 27 to 97.69 Similarly, Ca-methyl-d,l-leu-
cinamide, the hydrogenated analogue of 7b, is hydrolysed
with full stereoselectivity by the amidase from both
species.48,65,66,70
In all resolution experiments the l-enantiomer of the amide
was preferably hydrolysed to the l-amino acid, l-2a±d,
leaving the d-enantiomer of the amide, d-7a±d, behind.
The observed stereoselectivity is clearly a result of steric
interactions during the enzyme reaction. For the M.
neoaurum amidase an active site model was suggested in
which a small hydrophobic pocket is present and able
Figure 1. Representation of Grubbs' alkene-stabilized -l-Pro-Aib- b-turn peptide 8, our linear b-turn peptide Boc-l-Agl-l-Pro-Aib-l-Mag-NHBzl 9,
Balaram's disul®de-stabilized -l-Pro-Aib- b-turn peptide 10, and our alkane-stabilized -l-Pro-Aib- b-turn peptide 17.

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B. Kaptein et al. / Tetrahedron 57 62001) 6567±6577
Scheme 3.
On the other hand, replacing of the Ca-methyl group of 7a
by a phenyl group 2i.e. Ca-phenyl-d,l-allylglycinamide)
results in the complete loss of activity of the M. neoaurum
amidase,48 whereas the S-enantiomer is still hydrolysed by
the O. anthropi amidase 2E.200).71
three sterically hindered peptide bonds were obtained in
satisfactory yields. Removal of the Boc Na-protecting
group was performed by treatment with diluted tri¯uoro-
acetic acid 2TFA).
While stirring of the linear Agl/Mag tetrapeptide diene 9 in
the presence of 5 mol% of the conventional, Ru-based
Grubbs' catalyst77 resulted in complete recovery of the start-
ing material 2even at high temperatures), subjection of 9
to the newly developed, very active, thermally stable,
dihydroimidazolylidene catalyst analogue 215)78 led, after
16 h at 808C, to the ring-closed, ole®n product 16 as a
mixture of two CvC bond diastereomers in 82% isolated
yield 2Scheme 3). Unfortunately, the isomers were not
separable by regular column chromatography nor by
HPLC. Subsequent hydrogenation of the isomeric mixture
16 with H2/PtO2 in ethyl acetate resulted in a clean reduction
of the CvC bond to the saturated carbon±carbon cyclic
tetrapeptide 17 in 85% isolated yield.
In summary, the active site of the O. anthropi amidase can
bind larger substrates than those of the amidase from M.
neoaurum. Therefore, the more open active site of O.
anthropi results in a lower stereoselectivity for the
substrates 7a±d as compared to M. neoaurum.
2.2. Peptide synthesis, metathesis and conformational
characterization
In this work we exploited the simplest of the four allyl-type,
Ca-tetrasubstituted a-amino acids discussed above, i.e.
l-Mag 2l-2a), in the CvC covalent stabilization of a tetra-
peptide b-turn conformation involving a ring-closing
metathesis/hydrogenation approach. To this aim we decided
to partly modify the terminally blocked -l-Agl-l-Pro-Aib-
l-Agl- sequence, shown to undergo ole®n ring-closing
metathesis to 8 2Fig. 1) in 60% yield by Grubbs and co-
workers,36 by replacing the l-Agl 2l-3) residue in position 4
with its Ca-methylated counterpart l-Mag 2l-2a). The
reasons for our choice of the -l-Agl-l-Pro-Aib-l-Mag-
sequence 9 were based on the following observations:
2i) The -l-Pro-Aib- dipeptide is expected to easily fold
into a b-turn conformation.36,72,73 2ii) l-Mag 2l-2a) is
known to overwhelmingly prefer f,c backbone torsion
angles in the right-handed helical region of the confor-
mational space.62,63 2iii) An X-ray diffraction analysis of
10, the disul®de analogue of 8, carried out by Balaram
and coworkers,72 clearly showed that, while Cys1 is semi-
extended, Cys4 is helical.
To ascertain the type of conformation preferentially formed
in solution by the cyclic compound 17 and its linear pre-
1
cursor 9 we performed a H NMR investigation of the two
tetrapeptides. The analysis was carried out in CDCl3 at
1.0 mM concentration. All NH proton resonances were
assigned by means of 2D ROESY experiments. The par-
ticipation of speci®c NH groups in H-bonding was estab-
lished by examining the behaviour of the NH resonances
upon addition of perturbing agents. In particular, we investi-
gated the solvent dependence of NH chemical shifts, by
adding an increasing amount of the strong H-bonding
acceptor solvent dimethylsulphoxide 2DMSO)79,80 to the
CDCl3 solution, and the line broadening of NH resonances
induced by adding the free radical TEMPO 22,2,6,6-
tetramethylpiperidinyl-1-oxo).81 Fig. 2 shows the behaviour
of the NH resonances of the linear tetrapeptide 9 upon addi-
tion of DMSO and TEMPO. In this peptide only the N23)H
proton chemical shift is sensitive to the addition of DMSO
and its resonance broadens upon addition of the para-
magnetic TEMPO. All the other protons display a behaviour
characteristic of shielded protons, as their chemical shifts
appear relatively insensitive to solvent composition and
The Boc 2tert-butoxycarbonyl) Na-protected derivatives,
including those of l-Agl 211)74,75 and l-Mag,62 were
prepared in excellent yield by reacting the free amino acid
with 2Boc)2O 2di-tert-butyldicarbonate) in an aqueous/
dioxane mixture. The Boc-l-Mag-NHBzl 2NHBzl, benzyl-
amino) 212) C-terminal derivative was synthesized by using
N-ethyl,N0-[3-2dimethylamino)propyl]-carbodiimide 2EDC)
as the dehydrating agent. Synthesis of the linear tetrapeptide
9 was performed step-by-step in solution via the inter-
mediates Boc-Aib-l-Mag-NHBzl 213) and Boc-l-Pro-Aib-
l-Mag-NHBzl 214), beginning from the C-terminal H-l-
Mag-NHBzl. Peptide bond formation was achieved by
the EDC/HOAt 21-hydroxy-7-aza-1,2,3-benzotriazole)
method76 in CH2Cl2 in the presence of the tertiary amine
N-methylmorpholine 2NMM). Using this approach, the

B. Kaptein et al. / Tetrahedron 57 62001) 6567±6577
6571
1
Figure 2. H NMR titrations of the linear tetrapeptide Boc-l-Agl-l-Pro-Aib-l-Mag-NHBzl 9. 2A) Plot of NH chemical shifts as a function of increasing
percentages of DMSO added to the CDCl3 solution 2v/v). 2B) Plot of bandwidth of the NH signals as a function of increasing percentages of TEMPO 2w/v) in
CDCl3 solution. Peptide concentration: 1.0 mM.
their linewidths are not in¯uenced by addition of TEMPO.
The cyclic tetrapeptide 17 behaves in a similar manner 2Fig. 3)
intense cross-peak between the Ca22)H and N23)H protons
2Fig. 4), the typical signature of a type-II b-turn confor-
mation.14±16 The overall preferred structure of both peptides
must be quite compact, as the N21)H and benzylamide NH
protons as well appear to be highly solvent shielded.
1
From our H NMR study and the known conformational
tendencies of the Pro82,83 and Aib5±10 corner residues it is
reasonable to conclude that the most populated confor-
mation adopted in CDCl3 solution by both terminally
protected tetrapeptides 9 and 17 involves a b-turn centred
at the -l-Pro2-Aib3- sequence with an intramolecular trans-
annular H-bond connecting the N24)H and C01vO1 groups.
The ROESY spectra of both peptides show a remarkably
3. Conclusions
By a chemo-enzymatic approach we have been able to
synthesize in a large amount four enantiopure Ca-tetra-
Figure 3. 1H NMR titrations of the cyclized/reduced tetrapeptide Boc-l-Aglp-l-Pro-Aib-l-Magp-NHBzl 17 2starred residues refer to those with the side chain
modi®ed by cyclization). 2A) Plot of NH chemical shifts as a function of increasing percentages of DMSO added to the CDCl3 solution 2v/v). 2B) Plot of
bandwidth of the NH signals as a function of increasing percentages of TEMPO 2w/v) in CDCl3 solution [the NH2Bzl) proton is under the benzyl aromatic
protons]. Peptide concentration: 1.0 mM.

6572
B. Kaptein et al. / Tetrahedron 57 62001) 6567±6577
Figure 4. Partial ROESY spectra of 2A) the linear tetrapeptide Boc-l-Agl-l-Pro-Aib-l-Mag-NHBzl 9 and 2B) its cyclized/reduced derivative 17 in CDCl3
solution.
substituted a-amino acids carrying a side chain of the allyl
type. One of them, l-Mag 2l-2a), was incorporated in a
terminally blocked tetrapeptide 29), a model compound for
ring-closing ole®n metathesis. This reaction proceeds
smoothly with a newly developed Grubbs' catalyst.78
Reduction of the cyclized ole®n diastereomeric mixture
16 afforded 17, a peptide with a fully hydrogenated all-
hydrocarbon cross-linking system.
60/F254 precoated plates. The solvent systems used are: 2I)
chloroform/ethanol 29:1); 2II) 1-butanol/acetic acid/water
23:1:1); 2III) toluene/ethanol 27:1); 2IV) ethyl acetate
2EtOAc). The chromatograms were developed by quenching
of UV ¯uorescence, chlorine±starch±potassium iodide or
ninhydrin chromatic reaction, as appropriate. Enantiomeric
excesses 2e.e.) were determined by HPLC after precolumn
derivatization with o-phthaldehyde/2R)-3-mercaptoiso-
butyric acid according to Duchateau et al.84 [eluent:
50 mM sodium acetate solution 2titrated to pH 6.0 with
acetic acid)/methanol 7:3]. Grubbs' catalyst 15 was
prepared according to the literature procedure.78 The syn-
thesis and characterization of H-d,l-Mag-NH2 27a), H-l-
Mag-OH 2l-2a), and H-d-Mag-NH2 2d-7a) have already
been described.46
The linear tetrapeptide diene precursor 9 and the ®nal cyclic
product 17 share a common conformational preference, in
particular an intramolecularly H-bonded type-II b-turn
motif. We conclude that preorganization in the linear
precursor 9, characterized by two allyl-based residues sepa-
rated by two turn-forming residues in the amino acid
sequence, favours ring closure and generates peptide 17,
in which the b-turn conformation is frozen by a side-chain
to side-chain, 4-carbon cross-link. Compared to Grubbs'
ole®n cyclopeptide 8 and Balaram's disul®de analogue 10,
our linear and cyclic peptides 29 and 17, respectively)
appear to be more markedly intramolecularly H-bonded,
and hence, presumably, more extensively folded. We attri-
bute this structural propensity to the presence of the severely
conformationally constrained, turn/helix forming, Ca-tetra-
substituted a-amino acid Mag in 9 and its metathesized
side-chain derivative in 17.
4.1.2. Ca-Methyl-d,l-.g-methyl)allylglycinamide .7b).
This compound was prepared as described for H-d,l-Mag-
NH2 27a),46 starting from N-benzylidene-d,l-alaninamide
24) 2470 g, 2.7 mol) and 3-chloro-2-methyl-1-propene
2285 mL, 2.9 mol). The title compound, obtained as a
colourless oil after vacuum distillation 239 g, 10%),
crystallized on standing, mp 70±728C, bp 115±1308C
20.2 mmHg); dH 2200 MHz, CDCl3) 7.48 and 6.00 22H, 2
br s, amide NH2), 4.82 and 4.66 22H, 2m, allyl dCH2), 2.80
and 1.99 22H, 2 d, allyl bCH2), 1.64 23H, s, allyl dCH3), 1.45
22H, s, amine NH2), 1.25 23H, s, bCH3); dC 250 MHz,
CDCl3) 178.78 2s, CO), 141.71 2gC), 114.63 2t, dCH2),
55.84 2s, aC), 48.28 2t, bCH2), 28.41 2q, dCH3), 22.79 2q,
bCH3); HRMS 2FAB): MH1, found 143.1169. C7H15N2O
requires 143.1184.
4. Experimental
4.1. General
4.1.3. Ca-Methyl-d,l-prenylglycinamide .7c). This com-
pound was prepared as described for H-d,l-Mag-NH2
27a),46 starting from N-benzylidene-d,l-alaninamide 24)
217.2 g, 0.09 mol) and prenyl bromide 21-bromo-3-methyl-
2-butene)85 216.0 g, 0.11 mol). The title compound 27c)
27.0 g, 50%) was obtained as a colourless oil after vacuum
distillation, bp 131±1348C 21.8 mmHg); dH 250 MHz,
4.1.1. Amino acid and peptide synthesis. l-Allylglycine
22-amino-l-4-pentenoic acid) 2l-3) is an ACROS product.
Melting points were determined using a Leitz model Labor-
lux 12 apparatus and are not corrected. Optical rotations
were measured using a Perkin±Elmer model 241 polari-
meter equipped with a Haake model D thermostat. Thin-
layer chromatography was performed on Merck Kieselgel

B. Kaptein et al. / Tetrahedron 57 62001) 6567±6577
6573
CDCl3) 7.40 and 5.82 22H, 2 br s, amide NH2), 5.13 21H, t,
allyl gCH), 2.56 and 2.21 22H, 2 dd, allyl bCH2), 1.83 and
1.74 26H, 2 s, eCH3), 1.44 22H, br s, amine NH2), 1.34 23H,
s, bCH3); dC 250 MHz, CDCl3) 179.49 2s, CO), 135.15 2s,
dC), 117.71 2d, gCH), 56.74 2s, aC), 38.42 2t, bCH2), 26.16
and 25.01 22 q, 2 eCH3), 17.00 2q, bCH3); HRMS 2FAB):
MH1, found 157.1328. C8H17N2O requires 157.1341.
allyl dCH3), 0.62 23H, s, allyl bCH3); dC 250 MHz,
D2O1DCl) 173.36 2s, CO), 137.96 2s, gC), 118.16 2t,
dCH2), 58.89 2s, aC), 44.18 2t, bCH2), 23.33 and 22.21 22
q, dCH3 and bCH3); HRMS 2FAB): MH1, found 144.1003.
C8H16NO2 requires 144.1025.
Ca-Methyl-d-6g-methyl)allylglycinamide 6d-7b) 220 g,
content 86 wt%, e.e. 85%) was recovered as a pale yellow
oil.
4.1.4. Ca-Methyl-d,l-cinnamylglycinamide .7d). This
compound was prepared as described for H-d,l-Mag-NH2
27a),46 except for the toluene extraction that was omitted,
and the ®nal extraction performed with neat CHCl3
22£250 mL). Starting from N-benzylidene-d,l-alaninamide
24) 227.6 g, 0.16 mol) and cinnamyl bromide 224 mL,
0.16 mol), the title compound 222.7 g, 71%) was obtained
as a white crystalline solid after crystallization from EtOAc/
pentane, mp 112±1148C; dH 2200 MHz, CDCl3) 7.49 and
5.78 22H, 2 s, amide NH2), 7.35 2m, 5H, phenyl CH), 6.52
21H, dd, allyl dCH), 6.17 21H, m, allyl gCH), 2.94 and 2.30
22H, 2 dd, allyl bCH2), 1.51 22H, s, amine NH2), 1.43 23H, s,
bCH3); dC 250 MHz, CDCl3) 177.87 2s, CO), 135.07 2s,
phenyl C), 132.54 2d, gCH), 126.64, 125.54 and 124.30
23d, phenyl CH), 122.98 2d, dCH), 55.67 2s, aC), 43.07 2t,
bCH2), 25.68 2q, bCH3); HRMS 2FAB): MH1, found
205.1330. C12H17N2O requires 205.1341.
4.2.2. Resolution of Ca-methyl-d,l-prenylglycinamide
.7c). The enzymatic resolution of Ca-methyl-d,l-prenylgly-
cinamide 261 g, 0.39 mol) was stopped after 45% conver-
sion.
Ca-Methyl-l-prenylglycine 6l-2c) 221 g, 34%, e.e. 98%)
was recovered as a pale yellow solid; dH 2200 MHz,
D2O1DCl) 4.77 2br s, allyl gCH and solvent signal), 2.31
22H, 2dd, allyl bCH2), 1.41 23H, s, allyl e-CH3), 1.34 23H, s,
allyl e-CH3), 1.27 23H, s, bCH3); dC 250 MHz, D2O1DCl)
174.00 2s, CO), 140.91 2s, dC), 114.41 2d, gCH), 60.61 2s,
aC), 35.53 2t, bCH2), 25.47 2q, bCH3), 21.45 and 17.58 22 q,
2 eCH3); HRMS 2FAB): MH1, found 158.1186. C8H16NO2
requires 158.1181.
Ca-Methyl-d-prenylglycinamide 6d-7c) [47 g, approxi-
mately 50% 2still containing some water), e.e. 80%] was
obtained as a pale yellow oil.
4.2. General procedure for the enzymatic resolution of
7a±d with Mycobacterium neoaurum
4.2.3. Resolution of Ca-methyl-d,l-cinnamylglycinamide
.7d). Due to the poor solubility of Ca-methyl-d,l-cinnamyl-
glycinamide 215 g, 0.074 mol), the enzymatic resolution
was performed on a 5% 2w/w) suspension. The reaction
was stopped after 51% conversion.
To a solution of racemic a-amino amides 27a±d) 2100 g) in
water 21 L), adjusted to pH 8.0±8.5 with acetic acid, the
freeze-dried M. neoaurum ATCC 2579548,66 28±10 g) was
added. The mixture was shaken 2150 rpm) at 408C until a
40±50% conversion was reached 215±24 h, depending on
the substrate) as determined by ammonia development or
HPLC analysis. After removing the cell mass by centrifu-
gation, the reaction mixture was ®ltered over Hy¯o, concen-
trated under reduced pressure 2to remove NH3) and, ®nally,
diluted to 500 mL with water. The l-amino acids 2l-2a±c)
were separated from the d-amino amides 2d-7a±c) by ion-
exchange column chromatography 2Amberlyst A26,
strongly basic ion-exchange resin). The d-amino amides
were eluted ®rst and recovered by evaporating the aqueous
solution. The l-amino acids were obtained by eluting the
ion-exchange column with a 2N acetic acid solution and
evaporating the eluent to dryness. The l-amino acid l-2d
was separated from its d-amino amide 2d-7d) by extracting
with CHCl3 25£100 mL) the aqueous solution which was
basi®ed to pH 9 with 5N NaOH. The d-amino amide pre-
cipitated from the organic phase on concentrating and cool-
ing, while the l-amino acid crystallized from the aqueous
layer after neutralization to pH 7 with 5N HCl and concen-
tration under reduced pressure.
Ca-Methyl-l-cinnamylglycine 6l-2d) 27.04 g, 47%, e.e.
95%) was obtained as a white solid; dH 2200 MHz, D2O)
7.39 25H, m, phenyl CH), 6.55 21H, d, allyl dCH), 6.21 21H,
m, allyl g-CH), 2.65 and 2.40 22H, 2 dd, allyl bCH2), 1.36
23H, s, bCH3); dC 250 MHz, D2O1DCl) 173.65 2s, CO),
136.57 2d, gCH), 136.50 2s, phenyl C), 129.08, 128.41 and
126.66 23d, phenyl CH), 120.85 2d, dCH), 60.18 2s, aC),
40.20 2t, bCH2), 21.43 2q, bCH3); HRMS 2FAB): MH1,
found 206.1176. C12H16NO2 requires 206.1181.
Ca-Methyl-d-cinnamylglycinamide 6d-7d). 26.98 g, 46%,
e.e.$98%) was obtained as a pale yellow solid.
4.3. General procedure for the enzymatic resolution of
7a±d with Ochrobactrum anthropi
To a solution of racemic a-amino amides 27a±d) 210 g) and
MnSO4´H2O 215 mg) in H2O 2100 mL), adjusted to pH 6.0±
8.0 with acetic acid or H2SO4, a crude cell suspension
210 wt%) of O. anthropi NCIMB 4032167 21 g) was
added. The mixture was shaken 2150 rpm) at 508C until a
conversion of 40±50% was reached 2ammonia determi-
nation). The work-up of the enzymatic resolutions was
performed as described above for the enzymatic resolutions
with M. neoaurum.
4.2.1. Resolution of Ca-methyl-d,l-.g-methyl)allylgly-
cinamide .7b). The enzymatic resolution of Ca-methyl-
d,l-2g-methyl)allylglycinamide 232 g, 0.22 mol) was
stopped after 46% conversion.
Ca -Methyl-l-6g-methyl)allylglycine 6l-2b) 215.0 g, 46%,
e.e. 98%) was obtained as a pale yellow solid; dH
2200 MHz, D2O1DCl) 4.06 and 3.90 22H, 2 br s, allyl
dCH2), 1.77 and 1.58 22H, 2 d, allyl bCH2), 0.71 23H, s,
4.3.1. Resolution of Ca-methyl-d,l-.g-methyl)allylgly-
cinamide .7b). After a conversion of 46% was reached,

6574
B. Kaptein et al. / Tetrahedron 57 62001) 6567±6577
chiral HPLC analysis revealed 85% and 72% e.e. values for
Ca-methyl-l-2g-methyl)allylglycine 2l-2b) and Ca-methyl-
d-2g-methyl)allylglycinamide 2d-7b), respectively. Further
work-up of the reaction was omitted.
methyl-l-allylglycine benzylamide, Boc-Aib-l-Mag-
NHBzl .13). To a solution of Boc-Aib-OH86 2565 mg,
2.78 mmol) in CH2Cl2 210 mL) cooled to 08C, EDC´HCl
2533 mg, 2.78 mmol) and HOAt 2379 mg, 2.78 mmol)
*
were added. After 30 min CF3CO2 %H2-l-Mag-NHBzl
4.3.2. Resolution of Ca-methyl-d,l-prenylglycinamide
.7c). After a conversion of 62% was reached, chiral
HPLC analysis revealed 61% and 98% e.e. values for
Ca-methyl-l-prenylglycine 2l-2c) and Ca-methyl-d-prenyl-
glycinamide 2d-7c), respectively. Further work-up of the
reaction was omitted.
[obtained by treatment of the corresponding Boc-protected
amino acid benzylamide 212) 2590 mg, 1.85 mmol) with a
30% solution of TFA in CH2Cl2 for 30 min], neutralized
with NMM 21.23 mL, 11.13 mmol), was added. The reac-
tion mixture was stirred for 2 days. Then, EtOAc was added
and the solution was extracted with 10% KHSO4, H2O, 5%
NaHCO3, H2O, dried over anhydrous Na2SO4, and evapo-
rated to dryness. The title compound 2373 mg, 50%) was
4.3.3. Resolution of Ca-methyl-d,l-cinnamylglycinamide
.7d). After a conversion of 31% was reached, the work-up
of the reaction yielded Ca-methyl-l-cinnamylglycine 2l-2d)
2e.e. 96%) and Ca-methyl-d-cinnamylglycinamide 2d-7b)
2e.e.$98%) as white solids. Further work-up of the reaction
was omitted. The e.e. of l-2d improves by re-crystallization
from an iPrOH/H2O mixture.
crystallized as a white solid from EtOAc/petroleum ether,
mp 178±1798C; RfI 0.85, RfII 0.95, RfIII 0.35; [a]D
20
ˆ
219.9 2c 0.5, MeOH); nmax 2KBr) 3411, 3319, 3294,
1693, 1677, 1646, 1547 cm21; dH 2250 MHz, CDCl3) 7.76
21H, br s, NHBzl), 7.30 25H, m, Bzl CH), 6.47 21H, s, Mag
NH), 5.66 21H, m, Mag gCH), 5.15 22H, m, Mag dCH2),
4.61 21H, s, AibN H), 4.52 21H, dd, Jˆ7.5, 18.2 Hz, Bzl
CH2), 4.38 21H, dd, Jˆ7.0, 18.2 Hz, Bzl CH2), 2.86 21H, dd,
Jˆ8.3, 16.2 Hz, Mag bCH2), 2.42 21H, dd, Jˆ10.6, 16.2 Hz,
Mag bCH2), 1.59 23H, s, Aib bCH3), 1.57 23H, s, Aib
bCH3), 1.41 23H, s, Mag bCH3), 1.27 29H, s, Boc CH3);
HRMS 2FAB): MH1, found 404.2555. C22H33N3O4 requires
404.2549.
4.3.4. N-tert-Butyloxycarbonyl-l-allylglycine, Boc-l-Agl-
OH .11).74,75 H-l-Agl-OH 20.50 g, 4.34 mmol) and NaOH
20.21 g, 5.21 mmol) in 30 mL of a 1:1 mixture of dioxane/
H2O was cooled to 08C. Di-tert-butyl-dicarbonate 2Boc2O)
21.14 g, 5.21 mmol) was added in three portions and the
reaction was stirred at room temperature for 24 h. Then,
dioxane was evaporated under reduced pressure. Excess of
Boc2O was extracted with diethyl ether and the aqueous
layer was acidi®ed to pH 3 with 10% KHSO4. The aqueous
solution was extracted with EtOAc. The organic solution
was washed with 10% KHSO4 and H2O, dried over
anhydrous Na2SO4, and evaporated to dryness to give the
title compound 20.90 g, 96%) as a colourless oil; RfI 0.35,
4.3.7. N-tert-Butyloxycarbonyl-l-prolyl-a-aminoisobu-
tyryl-Ca-methyl-l-allylglycine benzylamide, Boc-l-Pro-
Aib-l-Mag-NHBzl .14). To a solution of Boc-Pro-OH
2267 mg, 1.24 mmol) in CH2Cl2 25 mL) cooled to 08C,
EDC´HCl 2237 mg, 1.24 mmol) and HOAt 2168 mg,
1.24 mmol) were added. After 30 min CF3CO2* %H2-Aib-
l-Mag-NHBzl [obtained by treatment of the corresponding
Boc-protected dipeptide benzylamide 213) 2250 mg,
0.619 mmol) with a 30% solution of TFA in CH2Cl2 for
1 h], neutralized with NMM 2409 mL, 3.71 mmol), was
added. The reaction mixture was stirred for 2 days. Then,
EtOAc was added and the solution was extracted with
10% KHSO4, H2O, 5% NaHCO3, H2O, dried over anhy-
drous Na2SO4, and evaporated to dryness. The title
compound 2294 mg, 95%) was crystallized as a white
solid from EtOAc/petroleum ether; mp 58±628C; RfI 0.80,
20
RfII 0.95, RfIII 0.15; [a]D ˆ19.5 2c 0.5, MeOH); nmax
2KBr) 3363, 1715, 1698 cm21; dH 2250 MHz, CDCl3) 5.71
21H, m, gCH), 5.21 22H, m, dCH2), 5.14 21H, d, Jˆ8.3 Hz,
NH), 4.39 21H, m, aCH), 2.58 22H, m, bCH2), 1.45 29H, s,
Boc CH3); HRMS 2FAB): MH1, found 215.2496.
C10H17NO4 requires 215.2493.
4.3.5. N-tert-Butyloxycarbonyl-Ca-methyl-l-allylglycine
benzylamide, Boc-l-Mag-NHBzl .12). To a solution of
Boc-l-Mag-OH62 2900 mg, 3.9 mmol) in CH2Cl2 210 mL)
cooled to 08C, EDC´HCl 2752 mg, 3.9 mmol) was added.
After 20 min, benzylamine 24.5 mL, 39 mmol) was added
and the reaction was stirred at room temperature for 2 days.
Then, the solvent was removed and the residue dissolved in
EtOAc. The solution was washed with 10% KHSO4, H2O,
5% NaHCO3 and H2O, dried over anhydrous Na2SO4, and
evaporated to dryness. The title compound 2559 mg, 48%)
was crystallized as a white solid from EtOAc/petroleum
ether, mp 79±808C; RfI 0.95, RfII 0.95, RfIII 0.55;
20
RfII 0.90, RfIII 0.30; [a]D ˆ257.9 2c 0.5, MeOH); nmax
2KBr) 3307, 1672, 1541 cm21; dH 2250 MHz, CDCl3) 7.73
21H, br s, NHBzl), 7.31 25H, m, Bzl CH), 7.09 21H, s, NH),
6.50 21H, s, NH), 5.67 21H, m, Mag gCH), 5.13 22H, m,
Mag dCH2), 4.46 22H, m, Bzl CH2), 4.09 21H, m, Pro aCH),
3.36 22H, m, Pro dCH2), 2.94 21H, dd, Jˆ8.1, 17.6 Hz, Mag
bCH2), 2.46 21H, dd, Jˆ11.0, 17.6 Hz, Mag bCH2), 2.06±
1.84 24H, m, Pro bCH2 and gCH2), 1.61 23H, s, bCH3), 1.54
23H, s, bCH3), 1.44 29H, s, Boc CH3), 1.43 23H, s, bCH3);
HRMS 2FAB): MH1, found 501.3060. C27H40N4O5 requires
501.3077.
20
[a]D ˆ215.3 2c 0.5, MeOH); nmax 2KBr) 3332, 1732,
1682, 1650, 1540 cm21; dH 2250 MHz, CDCl3) 7.30 25H,
m, Bzl CH), 6.73 21H, br s, NHBzl), 5.74 21H, m, Mag
gCH), 5.16 22H, m, Mag dCH2), 4.88 21H, s, Mag NH),
4.45 22H, d, Jˆ5.5 Hz, Bzl CH2), 2.73 21H, dd, Jˆ7.3,
14.1 Hz, Mag bCH2), 2.55 21H, dd, Jˆ7.6, 14.1 Hz, Mag
bCH2), 1.51 23H, s, Mag bCH3), 1.40 29H, s, Boc CH3);
HRMS 2FAB): MH1, found 319.2023. C18H35N3O6 requires
319.2022.
4.3.8. N-tert-Butyloxycarbonyl-l-allylglycyl-l-prolyl-a-
aminoisobutyryl-Ca-methyl-l-allylglycine benzylamide,
Boc-l-Agl-l-Pro-Aib-l-MagNHBzl .9). To a solution of
Boc-l-Agl-OH 211) 2223 mg, 1.04 mmol) in CH2Cl2
25 mL) cooled to 08C, EDC´HCl 2199 mg, 1.04 mmol) and
HOAt 2141 mg, 1.04 mmol) were added. After 30 min
*
CF3CO2
%H2-l-Pro-Aib-l-Mag-NHBzl [obtained by
treatment of the corresponding Boc-protected tripeptide
4.3.6. N-tert-Butyloxycarbonyl-a-aminoisobutyryl-Ca-

B. Kaptein et al. / Tetrahedron 57 62001) 6567±6577
6575
benzylamide 214) 2260 mg, 0.519 mmol) with a 30% solu-
4.5. Nuclear magnetic resonance
tion of TFA in CH2Cl2 for 1 h], neutralized with NMM
2343 mL, 3.11 mmol), was added. The reaction mixture
was stirred for 2 days. Then, EtOAc was added and the
solution was extracted with 10% KHSO4, H2O, 5%
NaHCO3, H2O, dried over anhydrous Na2SO4, and evapo-
rated to dryness. The title compound 2239 mg, 77%) was
crystallized as a white solid from EtOAc/petroleum ether,
1
The H NMR spectra were recorded by means of Bruker
model AC 200, AC250 or AM 400 spectrometers. Deuter-
ated chloroform 299.96% D) and water 299.98% D) were
purchased from Aldrich, while deuterated DMSO 299.96%
D6) was obtained from Acros Organics. The free-radical
TEMPO was a Sigma product.
20
mp 87±888C; RfI 0.75, RfII 0.90, RfIII 0.30; [a]D ˆ263.2
2c 0.5, MeOH); nmax 2KBr) 3310, 1710, 1674, 1640,
1539 cm21; dH 2400 MHz, CDCl3) 7.63 21H, br t, Jˆ
5.6 Hz, NHBzl), 7.31 25H, m, Bzl CH), 6.90 21H, s, Aib
NH), 6.54 21H, s, Mag NH), 5.71 21H, m, Agl gCH), 5.66
21H, m, Mag gCH), 5.12 24H, m, Agl and Mag dCH2), 5.05
21H, d, Jˆ8.8 Hz, Agl NH), 4.55 21H, dd, Jˆ6.5, 14.9 Hz,
Bzl CH2), 4.42 21H, m, Agl aCH), 4.31 21H, dd, Jˆ5.3,
14.9 Hz, Bzl CH2), 4.28 21H, m, Pro aCH), 3.67 and 3.47
22H, 2m, Pro dCH2), 3.01 21H, dd, Jˆ6.8, 14.0 Hz, Mag
bCH2), 2.48 21H, dd, Jˆ8.2, 14.0 Hz, Mag bCH2), 2.30
22H, m, Agl bCH2), 1.91 24H, m, Pro bCH2 and gCH2),
1.53 23H, s, Mag bCH3), 1.44 23H, s, Aib bCH3), 1.43
29H, s, Boc CH3), 1.40 23H, s, Aib bCH3); HRMS
2FAB): MH1, found 598.3598. C32H47N5O6 requires
598.3605.
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