Mag: A Cα-methylated, side-chain unsaturated α-amino acid. Introduction into model peptides and conformational preference

Article abstract of DOI:10.1016/S0040-4020(00)00274-X

By a chemo-enzymatic approach we synthesized the chiral, C(α)- methylated α-amino acid Mag, characterized by a side-chain C(γ)=C(δ) bond. We also prepared a series of model peptides containing Mag in combination with Aib and Ala. All of the peptides were fully characterized and their conformational preference was determined in solution by FT-IR absorption and ¹H NMR investigations. X-Ray diffraction analyses of L-Mag, a derivative and three peptides are also presented. We find that this C(α)-methylated α- amino acid is an excellent β-turn and 3₁₀-helix former. A peptide with two Mag residues one on top of the other after one complete turn of the 3₁₀- helix has been synthesized and characterized. 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4020(00)00274-X

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 3589±3601
Mag: a Ca-Methylated, Side-chain Unsaturated a-Amino Acid.
Introduction into Model Peptides and Conformational Preference
Cristina Peggion,a Roberto Flammengo,a Eric Mossel,a Quirinus B. Broxterman,a
Bernard Kaptein,a Johan Kamphuis,b Fernando Formaggio,c Marco Crismac
and Claudio Tonioloc,
*
aDSM Research, Organic Chemistry and Biotechnology Section, 6160 MD Geleen, The Netherlands
bDSM Food Speciality, Nutritional Ingredients, P.O. Box 1, 2600 MA Delft, The Netherlands
cBiopolymer Research Centre, C.N.R., Department of Organic Chemistry, University of Padova, 35131 Padova, Italy
Received 22 October 1999; revised 14 March 2000; accepted 30 March 2000
AbstractÐBy a chemo-enzymatic approach we synthesized the chiral, Ca-methylated a-amino acid Mag, characterized by a side-chain
CgvCd bond. We also prepared a series of model peptides containing Mag in combination with Aib and Ala. All of the peptides were fully
1
characterized and their conformational preference was determined in solution by FT-IR absorption and H NMR investigations. X-Ray
diffraction analyses of l-Mag, a derivative and three peptides are also presented. We ®nd that this Ca-methylated a-amino acid is an excellent
b-turn and 310-helix former. A peptide with two Mag residues one on top of the other after one complete turn of the 310-helix has been
synthesized and characterized. q 2000 Elsevier Science Ltd. All rights reserved.
Introduction
linking reactions.7,8 Recently, syntheses of derivatives and
short peptides containing different types of allyl Ca-sub-
stituted a-amino acids have been published.9±15 Ca-Allyl
amino acid derivatives have proven to be useful inter-
mediates in the synthesis of conformationally restricted
peptidomimetics.9,10 More interestingly, derivatives and
peptides of Ca-allyl amino acids may step up to metathesis
reactions that lately have been improved by the develop-
ment of ef®cacious ruthenium catalysts.11±15 In particular,
ringclosing metathesis reactions form cycles of various
sizes. For example, Blackwell and Grubbs11 ef®ciently
stabilized a 310-helical peptide by ringclosing metathesis
between two homoserine O-allyl esters. Another useful
application of this family of amino acids dealt with their
incorporation into peptides followed by addition of an array
of thiol-based radicals to the alkene function (`built-into'
peptides).16
The relevance of stable secondary structure elements in
peptides and proteins has inspired considerable effort
toward the design of rigidi®ed arti®cial molecules. In par-
ticular, signi®cant progress has been made in engineering
and synthesizing turn and helix inducers and mimetics.
Indeed, turn and helical motifs are fundamental constituents
of many biologically active peptides and are often critical
for their biological activity. Among the many approaches to
the synthesis of rigidi®ed peptides a widely used strategy
has been represented by the exploitation of structurally
restricted Ca-substituted a-amino acids able to drastically
reduce the conformational freedom of the peptide in which
they are inserted.1±5 More speci®cally, the conformational
preferences of Ca-methylated a-amino acids have been
extensively studied and their tendency to fold into a
b-turn and the 310-helical structure unambiguously demon-
strated.4±6
Therefore, we decided to take advantage of Ca-methyl-Ca-
allylglycine (Mag), which combines the structural features
of a Ca-methylated a-amino acid with those of a Ca-allyl,
functionalizable side chain. In the last few years a variety of
methods aiming at synthesizing Mag and its derivatives has
been published.16±22 Mag has also been inserted into a
dipeptide.23 In the present work we describe a chemo-enzy-
matic synthesis of Mag and the preparation of a series of
Mag model peptides in combination with Aib (a-amino-
isobutyric acid) and Ala. Peptide preferred conformations
have been assessed in solution by FT-IR absorption and
Interest in allyl Ca-substituted a-amino acids arises from the
wide versatility of their CgvCd bond towards a number of
chemical reactions. Polypeptides have been prepared
containing a pendant unsaturation site as a handle for cross-
Keywords: amino acids and derivatives; NMR; peptides; X-ray crystal-
lography.
* Corresponding author. Tel.: 1390-49-827-5247; fax: 1390-49-827-
5239; e-mail: biop02@mail.chor.unipd.it
0040±4020/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00274-X

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C. Peggion et al. / Tetrahedron 56 (2000) 3589±3601
Scheme 1.
1H NMR techniques. In addition, the 3D-structures of the
performed a phase-transfer catalyzed allylation of Na-benzyl-
idene-d,l-alanine amide for the preparation of the racemic
a-amino amide (Scheme 1) which was puri®ed by distil-
lation. Then, we used a broadly speci®c amino amidase
from Mycobacterium neoaurum ATCC 25795 to achieve
optical resolution, affording the free l-amino acid and the
d-amino amide,16,19±21 which were separated by ion-
exchange chromatography. Alkaline hydrolysis of the
d-amino amide gave the corresponding free d-amino acid.
Since the enzymatic resolution is not fully enantioselective
(E-ratio: 40) a ®nal optical puri®cation was performed by
crystallization from iso-propanol/water 3:1. Thus, the l- and
d-Mag were obtained in enantiomeric excess .99%.
*
%
free amino acid H2-l-Mag-O , the derivative Piv-l-Mag-
NHtBu (Piv, pivaloyl; NHtBu, tert-butylamino), and the
peptides Boc-l-Mag-d-Ala-OMe (Boc, tert-butyloxycarbo-
nyl; OMe, methoxy), Boc-Aib-l-Mag-Aib-OMe, and Boc-
l-Mag-l-Ala-l-Ala-l-Mag-d-Ala-OMe have been solved
by X-ray diffraction analysis.
Results and Discussion
Amino acid and peptide synthesis
For the large-scale production of the enantiomerically pure
l-Mag we exploited an economically attractive and gener-
ally applicable chemo-enzymatic synthesis developed by
DSM Research19±21,24,25 a few years ago. At ®rst, we
The Boc Na-protected l-derivative was prepared by reacting
the free amino acid with (Boc)2O (di-tert-butyldicarbonate)
in an aqueous/dioxane mixture. For the synthesis of
Figure 1. FT-IR absorption spectra (3500±3200 cm21 region) in CDCl3 solution of (A) Boc-l-Mag-Aib-OMe (2), Boc-Aib-l-Mag-Aib-OMe (3), Boc-Aib-
Aib-l-Mag-Aib-OMe (4), and Boc-l-Mag-Aib-Aib-l-Mag-Aib-OMe (5); (B) Boc-l-Mag-l-Ala-OMe (2), Boc-l-Ala-l-Mag-l-Ala-OMe (3), Boc-l-Ala-l-
Ala-l-Mag-l-Ala-OMe (4), and Boc-l-Mag-l-Ala-l-Ala-l-Mag-l-Ala-OMe (5). Peptide concentration: 1.0 mM.

C. Peggion et al. / Tetrahedron 56 (2000) 3589±3601
3591
Figure 2. 1H NMR titrations of Boc-l-Mag-Aib-Aib-l-Mag-Aib-OMe. (A) Plot of NH chemical shifts as a function of increasing percentages of DMSO added
to the CDCl3 solution (v/v). (B) Plot of bandwidth of the NH signals as a function of increasing percentages of TEMPO (w/v) in CDCl3. Peptide concentration:
1.0 mM.
Piv-l-Mag-NHtBu l-Mag was ®rst treated with trimethyl-
silylchloride. Then, the O,N-bis-silyl Mag derivative was
reacted with Piv-Cl. Acidic hydrolysis gave Piv-l-Mag-
OH which was dehydrated to the corresponding 5(4H)-
oxazolone with N-ethyl, N0-[3-(dimethylamino)propyl]-
carbodiimide (EDC) and 1-hydroxybenzotriazole (HOBt).
Finally, the oxazolone was reacted with tert-butylamine in
N,N-dimethylformamide (DMF). Syntheses of the three
peptide series (to the pentamer level) were performed
step-by-step in solution, beginning from the C-terminal
amino acid methyl ester. Peptide bond formation was
achieved by the EDC/HOAt (1-hydroxy-7-azabenzotriazole
method)26 in CH2Cl2 in the presence of N-methylmorpho-
line (NMM). Using this approach, the sterically hindered
l-Mag-Aib and Aib-l-Mag peptide bonds were obtained
in satisfactory yields (70±90%). Removal of the Boc
Na-protecting group was performed by treatment with
diluted TFA (tri¯uoroacetic acid).
l-Mag peptides still exhibit a tendency to fold into a turn
or a helical conformation, but a higher ¯exibility is
observed. Indeed, the di-, tri-, and tetrapeptides show a
signi®cant amount of unordered conformation, while in the
pentapeptide (containing two l-Mag residues in positions 1
and 4) the helical character becomes predominant. No
signi®cant difference is observed in the spectra of the penta-
peptides with either l-Ala or d-Ala in position 5. All of the
peptides investigated tend to self-associate only marginally
in the concentration range examined (10±0.1 mM) (not
shown).
To better understand the type of helix that is formed, we
performed a H NMR investigation of the three pentapep-
1
tides. The analysis was carried out in CDCl3 at 1.0 mM
concentration where self-association is absent. All NH
proton resonances were assigned by means of 2D ROESY
experiments. The participation of speci®c NH groups in
H-bonding was established by examining the behaviour of
the NH resonances upon addition of perturbing agents. In
particular, we investigated the solvent dependence of NH
chemical shifts, by adding increasing amounts of the strong
H-bonding acceptor solvent dimethylsulphoxide (DMSO)28
to the CDCl3 solution, and the line broadening of NH
resonances induced by adding the free radical TEMPO
(2,2,6,6-tetramethylpiperidinyl-1-oxo).29 Fig. 2 illustrates
the behaviour of the NH resonances of the l-Mag/Aib
pentapeptide upon addition of DMSO and TEMPO. In this
peptide only the N(1)H and N(2)H proton chemical shifts, in
particular that of the N(1)H proton, are sensitive to the
addition of DMSO and their resonances broaden upon
addition of the paramagnetic TEMPO. All the other protons
display a behaviour characteristic of shielded protons, as
their chemical shifts appear relatively insensitive to solvent
composition and their linewidths are not in¯uenced by
addition of TEMPO.
Solution conformational analysis
A solution conformational analysis of the three series was
performed by using FT-IR absorption and H NMR in a
1
solvent of low polarity (CDCl3) at different peptide concen-
trations. Fig. 1A shows the FT-IR absorption spectra of the
Boc/OMe-protected l-Mag/Aib peptide series in the 3500±
3200 cm21 (N±H stretching) region. The curves are
characterized by two bands at about 3430 cm21, assigned
to free (solvated) NH groups, and at 3360±3340 cm21
,
assigned to H-bonded NH groups.27 The intensity of the
low-frequency band, relative to the high-frequency band,
increases linearly as the main-chain length increases.
Concomitantly, the absorption maximum markedly shifts
to lower wavenumbers. A similar trend is also observed
for the two Mag/Ala series (Fig. 1B shows the spectra of
the all-l-series). When in combination with l-Ala, the

3592
C. Peggion et al. / Tetrahedron 56 (2000) 3589±3601
Figure 3. 1H NMR titrations of Boc-l-Mag-l-Ala-l-Ala-l-Mag-l-Ala-OMe. (A) Plot of NH chemical shifts as a function of increasing percentages of DMSO
added to the CDCl3 solution (v/v). (B) Plot of bandwidth of the NH signals as a function of increasing percentages of TEMPO (w/v) in CDCl3. Peptide
concentration: 1.0 mM.
The two Mag/Ala pentapeptides behave in a similar manner,
more speci®cally as shown in Fig. 3 for the all-l diastereo-
mer. From our H NMR study it is reasonable to conclude
that the most populated conformation adopted in CDCl3
solution by the terminally protected Mag containing penta-
peptides is the 310-helix, where only the two N-terminal NH
protons do not participate in the intramolecular H-bonding
scheme.
1
Figure 4. Left: X-ray diffraction structure of %H2-l-Mag-O* monohydrate with numbering of the atoms. Only one of the two side-chain conformer is shown.
Right: X-ray diffraction structure of Piv-l-Mag-NHtBu with numbering of the atoms. The intramolecular H-bond is represented by a dashed line.
Figure 5. X-Ray diffraction structures of the two independent molecules A and B of Boc-l-Mag-d-Ala-OMe with numbering of the atoms.

C. Peggion et al. / Tetrahedron 56 (2000) 3589±3601
3593
Figure 6. X-Ray diffraction structures of the two independent molecules A and B of Boc-Aib-l-Mag-Aib-OMe with numbering of the atoms. The intramo-
lecular H-bonds are represented by a dashed line.
Crystal-state conformational analysis
chain, and side-chain torsion angles30 are given in Table
1. In Table 2, the intra- and intermolecular H-bond para-
meters are listed.
The molecular and crystal structures of the free amino acid
%H2-l-Mag-O* monohydrate, the derivative Piv-l-Mag-
NHtBu, the dipeptide Boc-l-Mag-d-Ala-OMe, the tripep-
tide Boc-Aib-l-Mag-Aib-OMe, and the pentapeptide Boc-
l-Mag-l-Ala-l-Ala-l-Mag-d-Ala-OMe were determined
by X-ray diffraction. The di- and tripeptides crystallize
with two independent molecules, A and B, in the asym-
metric unit. The molecular structures of the ®ve compounds
with the atomic numbering schemes are illustrated in Figs.
4±7, respectively. Relevant Na-protecting group, main-
Bond lengths and bond angles are in general agreement with
previously reported values for the geometry of the tert-butyl-
oxycarbonylamino moiety,31 the methylester group, the Aib
and Ala residues, and the peptide unit. The average bond
distances and bond angles for the Mag side-chain allyl
moiety (as obtained from the experimental values with
Ê
estimated standard deviations lower than 0.02 A and 28,
Ê
respectively) are: CDvCG 1.31 A, CG±CB 1.47 A, and
Ê
Figure 7. Left: X-ray diffraction structure of Boc-l-Mag-l-Ala-l-Ala-l-Mag-d-Ala-OMe with numbering of the atoms (a view parallel to the helix axis).
Right: A view of the same molecule along the helix axis. In both views of the molecule the intramolecular H-bonds are represented by a dashed line, the two
side-chain CvC bonds are marked, and only one of the side-chain conformers is shown.

3594
C. Peggion et al. / Tetrahedron 56 (2000) 3589±3601
Table 1. Selected Na-protecting group, backbone and side-chain torsion angles (the torsion angles for rotation about bonds of the peptide backbone (f, c, v)
and side chains (x) are described in Ref. 30. For the torsion angle for rotation about bonds of the Boc-protecting group (u) see Ref. 31 (deg) for Mag, its
derivative and peptides
Torsion angle
Amino acid monohydrate Amino acid derivative Dipeptide mol. A/mol. B Tripeptide mol. A/mol. B
Pentapeptide
u1
177.6(6)/2179.9(6)
172.9(5)/2172.8(5)
58.4(7)/256.0(7)
2177.6(5)/174.2(4)
2174.6(5)/177.2(4)
255.9(7)/58.5(7)
233.5(7)/27.9(7)
2174.9(5)/2177.7(5)
259.5(6)/50.2(6)
229.2(7)/37.9(7)
172.8(5)/2179.4(5)
50.4(8)/249.5(8)
49.6(8)a/249.4(8)a
175.9(5)b/2176.9(5)b
2173.7(6)
2170.1(6)
257.4(9)
v0
f1
c1
v1
f2
c2
v2
f3
c3
v3
f4
c4
v4
f5
c5
v5
x11
x12
x21
x22
x41
x42
2175.0(4)
180.0(4)
2179.5(4)
177.5(4)
42.8(7)/246.0(6)
171.5(5)/2175.6(5)
145.8(6)/95.1(6)
233.5(9)
2177.4(6)
263.4(10)
220.5(10)
2179.9(7)
263.9(9)
218.8(10)
175.0(6)
253.9(9)
224.4(9)a/2177.9(5)a
179.7(7)b/176.5(7)b
231.6(10)
179.0(8)
69.2(12)
2150.8(8)a
2174.8(10)b
2179.9(16)c/276.2(16)c
126(3)c/114(4)c
54(2)c/72(2)c
56.9(5)
2115.8(8)
254.5(9)/2171.7(8)
2122.7(9)/123.2(10)
2123(3)c/106(5)c
2158.2(8)/259.6(6)
110.5(13)/2129.6(8)
271.7(10)
2109.5(14)
a N-CA-C0-OT torsion angle.
b CA-C0-OT-CT torsion angle.
c The C1G atom is disordered over two sites.
CB±CGˆCD 123.38. The corresponding literature mean
values for the H2CvCH± and vCH±CH2± distances are
populate the helical region (A or Ap) of the conformational
space.34 However, the l-Mag residue of the amino acid
derivative is fully extended (region E or Ep). The average
value for the F, C backbone torsion angles of the Mag
residues forming helical turns are ^57.1, ^36.78, in the
range of those expected for a regular 310-helix (^57,
^308).35
32
Ê
1.30 and 1.50 A, respectively.
The conformationally informative t (N±CA±C0) bond
angle for the l-Mag residue of the amino acid derivative
is very narrow, 105.2(38), a preliminary indication of the
onset of the fully extended (C5) conformation.33
The (amide) N±H´´´OvC0 (amide) intramolecularly H-
bonded C5 conformation, exhibited by the Mag amino
All six l-Mag residues included in the peptides examined
Table 2. Intra- and intermolecular H-bond parameters for Mag, its derivative and peptides
Peptide
Donor
D±H
Acceptor
A
Symmetry
operation
Distance
Ê
(A) D¼A
Distance
Ê
(A) H¼A
Angle (deg)
D±H¼A
%H2-l-Mag-O* H2O
N1±HA
OW±HA
N1±HA
N1±HB
N1±HC
OW±HB
N1±H
O2
O1
O2
O1
OW
OW
O1
x, y, z
x, y, z
2.631(13)
2.702(13)
2.802(13)
2.785(11)
2.882(15)
2.82(2)
2.561(5)
3.093(5)
2.880(6)
2.910(5)
3.038(5)
2.843(6)
2.975(6)
2.961(6)
2.833(6)
2.811(6)
3.017(8)
2.47
1.94(6)
1.86
1.84
1.99
2.06(2)
2.10
2.30
2.02
2.27
2.19
2.10
2.22
2.18
1.98
1.96
2.19
89.3
155(15)
171.6
175.8
155.9
153(16)
113.1
154.2
173.7
131.8
168.3
143.7
145.9
151.4
174.0
171.0
162.1
x21, y11, 2z
2x11/2, y11/2, z21/4
x21, y, 2z
x, y, 2z
Piv-l-Mag-NHtBu
x, y, z
NT±H
O0
x11/2, 2y11/2, 2z11
x, y, z
x, y, z
x21, y, z
x21, y, z
x, y, z
x, y, z
x, y, z
x11, y, z
x, y, z
Boc-l-Mag-d-Ala-OMe
N1A±H
N2A±H
N1B±H
N2B±H
N3A±H
N3B±H
N1A±H
N1B±H
O0B
O1B
O0A
O1A
O0A
O0B
O2B
O2A
O0
Boc-Aib-l-Mag-Aib-OMe
Boc-l-Mag-l-Ala-l-Ala-l- N3±H
Mag-d-Ala-OMe
N4±H
N5±H
O1
O2
O4
x, y, z
x, y, z
x, y21, z
3.229(9)
3.103(10)
2.938(9)
2.39
2.28
2.11
165.9
160.6
161.0
N1±H

C. Peggion et al. / Tetrahedron 56 (2000) 3589±3601
3595
acid derivative, although uncommon,5 has been previously
reported for other chiral Ca-tetrasubstituted a-amino
acids.36 In addition to the narrow t bond angle mentioned
above, two other typical features of this conformation are:
(i) the very short intramolecular N1´´´O1 separation, and (ii)
the narrow N1±H´´´O1 angle. Interestingly, an intramolec-
ularly H-bonded C5 conformation, but of the ammonium ´´´
carboxylate type is also shown by the free amino acid
%H2-l-Mag-O*.
In the crystal the ®ve H-atoms of the ammonium and water
donors and the three oxygen atoms of the carboxylate and
water acceptors of %H2-l-Mag-O2 monohydrate are all
involved in
a complex network of intermolecular
H-bonds. The N´´´O and O´´´O H-bonds have normal
lengths and angles. The crystal structure of the fully
extended amino acid derivative is characterized by a single
intermolecular N±H´´´OvC0 H-bond between the C-termi-
nal amide NT-H group and the N-terminal amide O0vC00
group of a symmetry related molecule, generating rows
along the x-direction.
Two main conformational features distinguish molecule A
from molecule B of the dipeptide. First, the N-terminal
l-Mag residue is left-handed helical in molecule A, but
right-handed helical in molecule B. Secondly, the C-termi-
nal d-Ala residue, although in a partially extended confor-
mation in both molecules, has two sets of largely divergent
F, C torsion angles.
In the crystal packing mode of the dipeptide four inter-
molecular N±H´´´OvC0 H-bonds are observed, each
connecting a molecule A to a molecule B (and vice
versa). The urethane N1±H group is linked to the urethane
O0vC00 group, while the peptide N2±H group is linked to
the peptide O1vC10 group. In the unit cell of the turn-
forming tripeptide each of the two intermolecular
N±H´´´OvC0 H-bonds connects a molecule A to a mole-
cule B (and vice versa). The urethane N1 group forms a
H-bond with the peptide O2vC20 group. The molecules
of the helical pentapeptide are held together in a head-
to-tail fashion along the y-direction in rows stabilized
by (urethane) N1H´´´O4vC40 (peptide) intermolecular
H-bonds. Neither in the tripeptides, nor in the penta-
peptide does the N2±H group of the l-Mag2 residue
seem to be involved in the intermolecular H-bonding
scheme.
In both molecules A and B the 1±2 sequence of the tripep-
tide is folded in a 1Ã4 C0vO´´´H±N intramolecularly H-
bonded b-turn conformation of the helical (III/III0) type.37
The C00 ±O0´´´H±N3 intramolecular H-bond is of normal
strength. The b-turn conformation has opposite handedness
in the two molecules. In both molecules also the C-terminal
Aib residue is helical, but its handedness is opposite to that
of the preceding residues, a common observation for
Aib-based helical peptides.
The backbone of the pentapeptide adopts a regular right-
handed 310-helical structure. Peptide groups N3±H to
N5±H and O0vC00 to O2vC20 participate in three conse-
cutive 1Ã4 (type III b-turn) C0vO´´´H±N intramolecular
H-bonds, appropriate for a 310-helix. The range of observed
Conclusions
Ê
N´´´O distances is 3.017(8)±3.229(9) A, while that of
N±H´´´O angles is 160.6±165.98. The C-terminal d-Ala
residue is semi-extended.
By a chemo-enzymatic approach we have been able to
prepare both Mag enantiomers in a large amount and by
solution methods to incorporate l-Mag into a variety of
model peptides to the pentamer level. The results of the
solution conformational analysis described in this work,
combined with those extracted from an X-ray diffraction
study, also reported here, strongly favor the conclusion
that Na-acylated Mag-based tripeptide esters have a great
tendency to fold in a b-turn conformation, while the most
populated structures adopted by tetra and pentapeptides are
two consecutive b-turns (incipient 310-helix) and the
310-helix, respectively. These conclusions are in excellent
agreement with those already reported for other Ca-methyl-
ated a-amino acids.5 As for the relationship between Mag
chirality and peptide helix handedness, the available X-ray
diffraction data indicate that l-Mag can easily be accommo-
dated into a right-handed helix, but unfortunately do not
allow us to conclude that there is a univocal relationship
between these two con®gurational and conformational
properties. Hopefully, this issue will be solved by a con-
formational analysis of the l-Mag homo±oligomer series,
the synthesis of which is currently in progress in our labora-
tories.
In the amino acid derivative and peptides investigated no
signi®cant deviation of the v torsion angles (uDvu.108)
from the ideal values of the trans planar amide, urethane,
peptide, and ester units (1808) is observed. The trans
arrangement of the u1 torsion angle of the Boc-NH± moiety,
found for all of the molecules of di-, tri-, and pentapeptides,
is that commonly reported for Boc-protected peptides (type
b conformation).31
In the ten l-Mag residues of the ®ve compounds examined
(the di- and tripeptides are each characterized by two inde-
pendent molecules, and in the free amino acid and in l-Mag1
of the pentapeptide the side-chain CG atom is disordered
over two sites) the N±CA±CB±CG (x1) torsion angle is
either in the g1 conformation (three times) or in the g2
(four times) and t (three times) conformations, i.e. no
side-chain conformational bias is observed for this para-
meter. Conversely, the values for the x2 torsion angles are
in the range ^106±1308 (skew conformations). The two
Mag allyl side chains in positions 1 and 4 of the pentapeptide
seat one on top of the other after one complete turn of the 310-
helix (Fig. 7, right view). The intramolecular distances
C1A´´´C4A, C1B1´´´C4B1, C1G´´´C4G (or C1G0´´´C4G),
and C1D´´´C4D (or C1D0´´´C4D) are 6.13, 6.41, 6.49
In any case, we expect that Mag, with its remarkable confor-
mational bias towards b-turns and the 310-helix and the
concomitant chemical reactivity of its allyl side chain,
would become an important component in the arsenal of
peptide chemists.
Ê
Ê
(or 5.24 A), and 8.29 (or 5.97 A). It is worth remembering
35
Ê
that the reported pitch (CA´´´CA) for the 310-helix is 6.29 A.

3596
C. Peggion et al. / Tetrahedron 56 (2000) 3589±3601
Experimental
the ®rst eluant 120 g of H-d-Mag-NH2 (content 80 wt%,
yield 53%, e.e. 63%) were recovered as an oil. After elution
of the ion-exchange column with a 2 N acetic acid solu-
tion and evaporation of the eluant 89 g of H-l-Mag-OH
(content 77 wt%, yield 38%, e.e. 92%) were isolated as a
solid.
General
Amino acid and peptide synthesis. Melting points were
determined using a Leitz model Laborlux 12 apparatus
and are not corrected. Optical rotations were measured
using a Perkin±Elmer model 241 polarimeter equipped
with a Haake model D thermostat. Thin-layer chroma-
tography was performed on Merck Kieselgel 60/F254
precoated plates. The solvent systems used are: I, chloro-
form/ethanol (9:1); II, 1-butanol/acetic acid/water (3:1:1);
III, toluene/ethanol (7:1). The chromatograms were
developed by quenching of UV ¯uorescence, chlorine±
starch±potassium iodide or ninhydrin chromatic reaction,
as appropriate. Enantiomeric excesses (e.e.) were deter-
mined by HPLC after precolumn derivatization with
ophthaldehyde/(R)-3-mercaptoisobutyric acid according to
Duchateau et al.38 [eluant: 50 mM sodium acetate solution
(titrated to pH 6.0 with acetic acid)/methanol 7:3].
Ca-Methyl-l-allylglycine, H-l-Mag-OH. H-l-Mag-OH
(89 g, content 77 wt%, e.e. 92%), obtained from the enzy-
matic resolution, was ¯ushed with toluene to remove the
remaining water and acetic acid. The residue was recrystal-
lized from isopropanol/water 3:1 (950 mL). After 4 d the
chemically pure title compound was isolated by ®ltration
and dried on air (48 g, 77%; content .99 wt%, e.e.
99.3%); [a]2D0ˆ217.8 (c 1.3, 1 N HCl); dH (200 MHz,
DMSO d6) 7.40 (3H, br s, NH13 ), 5.78 (1H, m, gCH), 5.07
(2H, m, dCH2), 2.37 (2H, m, bCH2), 1.20 (3H, s, bCH3);
HRMS (EI): MH1, found 130.0873. C6H12NO2 requires
130.0868.
Ca-Methyl-d-allylglycine, H-d-Mag-NH2. A second enzy-
matic resolution was performed on the partially enriched
H-d-Mag-NH2 (105 g, content 80 wt%, e.e. 63%). The
amide was dissolved in 0.95 L of water (approximate pH
9.5), the pH was adjusted to 8.4 with acetic acid, and
Mycobacterium neoaurum (22 g) was added. The mixture
was shaken (170 rpm) at 408C for 3 d, after which the
conversion had reached 23% (HPLC). The enzyme was
removed by centrifugation, and the acid and amide were
separated by ion-exchange column chromatography and
isolated as described above. H-d-Mag-NH2 (62 g, 78%)
was obtained as an oil, e.e. .99.5%, [a]2D0ˆ4.7 (c 3.7,
1 N HCl); the NMR data were identical to those of H-d,l-
Mag-NH2. HRMS (EI): MH1, found 129.1021. C6H13N2O
requires 129.1028.
d,l-2-Amino-2-methyl-4-pentenoic acid amide or Ca-
methyl-d,l-allylglycine amide (H-d,l-Mag-NH2). To a
solution of 176 g of N-benzylidene-d,l-alanineamide19
(1.0 mol) and 35 g of Bu4N1´HSO42 (10 mol%) in 1 L of
CH2Cl2 in a 2 L Erlenmeyer ¯ask were added 700 mL of a
10 N NaOH solution and 96 mL of allyl bromide (1.1 mol).
The reaction mixture was stirred vigorously overnight at
room temperature. TLC (EtOAc/nhexane 7:3) revealed
that the reaction was at completion. The organic phase
was separated from the aqueous phase and washed with
water (3£500 mL). To the combined organic layers were
added 700 mL of a 2 N HCl solution and the mixture was
stirred at room temperature for 2 h. After separation of the
organic and the aqueous phases, the organic phase was
extracted with a 0.1 N HCl solution (3£350 mL). The
combined aqueous phases were washed with CH2Cl2
(3£600 mL) and added with a 10 N NaOH solution to pH
10 under stirring. After washing of the aqueous solution
with toluene and re-extraction of the toluene layer with
water, the combined aqueous phases were saturated with
NaCl and extracted with CHCl3/EtOH 9:1 (5£700 mL).
The combined CHCl3 phases were dried over anhydrous
Na2SO4 and concentrated in vacuo to give the title
compound (96 g, 75%) as a slowly crystallizing oil, mp
46±478C (after distillation, bp 115±1308C/0.2 Torr); RfI
0.55; dH (200 MHz, DMSO d6) 7.31 and 6.95 (2H, 2 br s,
amide NH2), 5.70 (1H, m, gCH), 5.00 (2H, m, dCH2), 2.35
and 2.12 (2H, 2m, bCH2), 1.75 (2H, s, NH2), 1.12 (3H, s,
bCH3).
Ca-Methyl-d-allylglycine, H-d-Mag-OH. H-d-l-Mag-OH
with e.e..99% was obtained in quantitative yield by alka-
line hydrolysis of H-d-Mag-NH2 with a 2 N KOH solution
at re¯ux for 2 h. The NMR data were identical to those of
H-l-Mag-OH.
N-Pivaloyl-Ca-methyl-l-allylglycyl tert-butylamide, Piv-
l-Mag-NHtBu. To a suspension of H-l-Mag-OH (0.516 g,
4.0 mmol) in anhydrous pyridine (10 mL) at room tempera-
ture were added under stirring Piv-Cl (1.446 g, 12.0 mmol)
and after 30 min NMM (0.44 mL, 4.0 mmol). The mixture
was stirred at room temperature overnight. Then, the solvent
was evaporated to dryness. The residue was dissolved in
EtOAc and the organic solution was quickly washed with
water, 5% NaHCO3, water, 0.5 M citric acid, and water,
dried over anhydrous Na2SO4, ®ltered, and evaporated to
dryness. The resulting 5(4H)oxazolone from Piv-l-Mag-
OH was treated without further puri®cation with tert-butyl-
amine (2.10 mL, 20.0 mmol) in DMF (10 mL) at 658C for
24 h. After evaporation of the solvent in vacuo, puri®cation
of the crude product by ¯ash chromatography gave the
title compound (0.68 g, 64%) as a solid, mp 120±1218C
(toluene/pentane); RfI 0.90, RfII 0.90, RfIII 0.60;
[a]2D0ˆ13.6 (c 0.5, MeOH); nmax (KBr) 3373, 3350, 1667,
1634, 1539 cm21; dH (250 MHz, CDCl3) 6.64 (1H, s, NH),
6.19 (1H, s, NH), 5.69 (1H, m, gCH), 5.14 (2H, m, dCH2),
2.84 (2H, m, bCH2), 1.56 (3H, s, bCH3), 1.34 (9H, s, NHtBu
Enzymatic resolution of H-d,l-Mag-NH2. To a solution of
180 g of H-d,l-Mag-NH2 (1.41 mol) in 1.45 L of water
(approximate pH 9.5), adjusted to pH 8.4 with acetic acid,
were added 20 g of freeze-dried Mycobacterium neoaurum
ATCC 25795.20 The mixture was shaken (150 rpm) at 408C
for 18 h. At a conversion of 40% (HPLC) the cell mass was
removed by centrifugation, followed by ®ltration over
Hy¯o. The ®ltrate was ®rst concentrated under reduced
pressure to remove NH3 and then diluted to 2.5 L with
water. The acid and the amide were separated by an ion-
exchange column chromatography (Amberlyst A26, a
strongly basic ion-exchange resin). After evaporation of

C. Peggion et al. / Tetrahedron 56 (2000) 3589±3601
3597
CH3), 1.18 (9H, s, Piv CH3). HRMS (EI): M1, found
268.2143. C15H28N2O2 requires 268.2151.
N-tert-Butyloxycarbonyl-a-aminoisobutyryl-Ca-methyl-
l-allylglycyl-a-aminoisobutyric acid methylester, Boc-
Aibl-Mag-Aib-OMe. TFA´H-l-Mag-Aib-OMe [obtained
by treatment with a 30% solution of TFA in CH2Cl2 of
the corresponding Boc-protected dipeptide methylester
(1.36 g, 4.15 mmol)] was dissolved in CH2Cl2 (20 mL)
and NMM (1.37 mL, 12.44 mmol) and the solution cooled
to 08C. To a solution of Boc-Aib-OH40 (1.35 g, 6.63 mmol)
in CH2Cl2 (20 mL) and NMM (1.37 mL, 12.44 mmol),
EDC´HCl (1.27 g, 6.64 mmol) and HOAt (0.90 g,
6.64 mmol) were added at 08C. After 15 min the two solu-
tions were combined and the resulting mixture was stirred at
room temperature for 4 d. Then, EtOAc was added and the
mixture was washed with 0.5 M citric acid, water, 5%
NaHCO3 and water, dried over anhydrous Na2SO4, and
concentrated. The title compound (1.32 g, 77%) was
obtained by precipitation with light petroleum ether as a
solid, mp 155±1568C; RfI 0.85, RfII 0.95, RfIII 0.40;
[a]2D0ˆ230.8 (c 0.5, MeOH); nmax (KBr) 3392, 3296,
1728, 1691, 1657, 1531 cm21; dH (250 MHz, CDCl3) 7.50
(1H, s, NH), 6.53 (1H, s, NH), 5.84 (1H, m, Mag gCH),
5.185.12 (2H, m, Mag dCH2), 4.84 (1H, s, NH), 3.69 (3H, s,
OMe CH3), 2.822.74 and 2.392.33 (2H, 2m, Mag bCH2),
1.591.40 (m, 15H, Mag bCH3 and 2 Aib 4 bCH3), 1.45 (9H,
s, Boc 3 CH3); HRMS (EI): M1, found 413.2527.
C20H35N3O6 requires 413.2526.
N-tert-Butyloxycarbonyl-Ca-methyl-l-allylglycine, Boc-l-
Mag-OH. A solution of H-l-Mag-OH (1.00 g, 7.74 mmol)
and NaOH (0.31 g, 7.74 mmol) in a 1:1 dioxane/water
solution (20 mL) was cooled to 08C. Boc2O (1.85 g,
8.51 mmol) was added in three portions and the reaction
was stirred at room temperature for 10 h and at 358C for
16 h. The pH was kept at 10 by addition of 2 N NaOH. Then,
dioxane was evaporated under reduced pressure. Excess of
Boc2O was extracted with diethyl ether (Et2O) and the
aqueous layer was acidi®ed to pH 3 with 10% KHSO4.
The product was extracted with EtOAc. The organic layer
was washed with water, dried over anhydrous Na2SO4, and
®ltered. Evaporation to dryness under reduced pressure gave
the title compound (1.59 g, 90%) as an oil; RfI 0.40, RfII
0.90, RfIII 0.20; [a]2D0ˆ219.0 (c 0.5, MeOH); nmax (®lm)
3434, 1712, 1635 cm21; dH (250 MHz, CDCl3) 10.62 (1H, s,
COOH), 5.72 (1H, m, gCH), 5.19 (1H, s, NH), 5.13 (2H, m,
dCH2), 2.66 (2H, m, bCH2), 1.54 (3H, s, bCH3), 1.44 (9H, s,
Boc 3 CH3); HRMS (EI): MH1, found 230.1383.
C11H20NO4 requires 230.1392.
N-tert-Butyloxycarbonyl-Ca-methyl-l-allylglycine methyl-
ester, Boc-l-Mag-OMe. To a solution of Boc-l-Mag-OH
(0.52 g, 2.26 mmol) and TEA (0.94 mL, 6.78 mmol) in
CH2Cl2, EDC´HCl (0.48 g, 2.48 mmol) and HOAt (0.34 g,
2.48 mmol) were added at 08C. After 1 h MeOH (about
20 equiv.) was added and the solution was stirred at room
temperature for 24 h. Then, the solvent was evaporated to
dryness. The residue was dissolved in EtOAc, the organic
solution washed with 10% KHSO4, water, 5% NaHCO3,
water, dried over anhydrous Na2SO4, and ®ltered. Evapo-
ration to dryness gave the title compound (0.58 g, 75%) as
an oil; RfI 0.90, RfII 0.90, RfIII 0.85; [a]2D0ˆ214.8 (c 0.5,
MeOH); nmax (®lm) 3428, 3376, 1742, 1718 cm21; dH
(250 MHz, CDCl3) 5.66 (1H, m, gCH), 5.15 (1H, s, NH),
5.13 (2H, m, dCH2), 3.74 (3H, s, OMe CH3), 2.69 and 2.56
(2H, 2m, bCH2), 1.53 (3H, s, bCH3), 1.43 (9H, s, Boc CH3);
HRMS (EI): M1, found 243.1465. C12H21NO4 requires
243.1471.
N-tert-Butyloxycarbonyl-a-aminoisobutyryl-a-aminoiso-
butyryl-Ca-methyl-l-allylglycyl-a-aminoisobutyric acid
methylester, Boc-Aib-Aib-l-Mag-Aib-OMe. TFA´H-
AiblMag-Aib-OMe [obtained by treatment with TFA of
the corresponding Boc-protected tripeptide methylester
(0.70 g, 1.69 mmol)] was dissolved in CH2Cl2 (15 mL)
and NMM (0.55 mL, 5.07 mmol) and the solution was
cooled to 08C. Boc-Aib-OH (0.55 g, 2.70 mmol) was
dissolved in CH2Cl2 (10 mL) and NMM (0.55 mL,
5.07 mmol), then EDC´HCl (518 mg, 2.70 mmol) and
HOAt (368 mg, 2.70 mmol) were added at 08C. The two
solutions were combined and the reaction mixture was
stirred at room temperature for 4 d. Then, EtOAc was
added and the mixture was washed with 0.5 M citric acid,
water, 5% NaHCO3 and water, dried over anhydrous
Na2SO4, and concentrated. The title compound (0.78 g,
93%) was obtained by precipitation with light petroleum
as a solid, mp 186±1878C; RfI 0.60, RfII 0.85, RfIII 0.20;
[a]2D0ˆ22.5 (c 0.5, MeOH); nmax (KBr) 3335, 3309, 1726,
1679, 1652, 1522 cm21; dH (250 MHz, CDCl3) 7.34 (1H, s,
NH), 7.27 (1H, s, NH), 6.53 (1H, s, NH), 5.68 (1H, m, Mag
gCH), 5.05 (2H, m, Mag dCH2), 4.89 (1H, s, Aib NH), 3.69
(3H, s, OMe CH3), 3.042.52 (2H, m, Mag bCH2), 1.571.38
(21H, m, Mag bCH3 and 3 Aib 6 bCH3), 1.46 (s, 9H, Boc 3
CH3); HRMS (EI): MH1, found 499.3120. C24H43N4O7
requires 499.3132.
N-tert-Butyloxycarbonyl-Ca-methyl-l-allylglycine-a-
aminoisobutyric acid methylester, Boc-l-Mag-Aib-
OMe. To a solution of BoclMag-OH (2.11 g, 9.18 mmol)
in CH2Cl2 (10 mL) and TEA (3.83 mL, 27.54 mmol) cooled
to 08C, HOAt (1.78 g, 12.85 mmol) and EDC´HCl (2.50 g,
12.85 mmol) were added. After 2 min HCl´H-Aib-OMe39
(2.30 g, 14.90 mmol) was added and the reaction was stirred
at room temperature for 2 d. Then, the solvent was removed
and the residue redissolved in EtOAc. The organic solution
was washed with 10% KHSO4, water, 5% NaHCO3 and
water, dried over anhydrous Na2SO4, and ®ltered. Evapo-
ration to dryness gave the title compound (2.71 g, 90%) as
an oil; RfI 0.80, RfII 0.90, RfIII 0.40; [a]2D0ˆ60.0 (c 0.5,
MeOH); nmax (KBr) 3359, 3330, 1743, 1731, 1684,
1524 cm21; dH (250 MHz, CDCl3) 7.04 (1H, s, Aib NH),
5.73 (1H, m, Mag gCH), 5.16 (2H, m, Mag dCH2), 4.97 (1H,
s, Mag NH), 3.73 (3H, s, OMe CH3), 2.722.48 (m, 2H, Mag
bCH2), 1.541.48 (9H, 3s, Mag bCH3 and Aib 2 bCH3), 1.43
(9H, s, Boc 3 CH3); HRMS (EI): M1, found 328.2004.
C16H28N2O5 requires 328.1998.
N-tert-Butyloxycarbonyl-Ca-methyl-l-allylglycyl-a-amino-
isobutyryl-a-aminoisobutyryl-Ca-methyl-l-allylglycyl-
a-aminoisobutyric acid methylester, Boc-l-Mag-Aib-
Aib-l-Mag-Aib-OMe. TFA´H-Aib-Aib-l-Mag-Aib-OMe
[obtained by treatment with TFA of the corresponding
Boc-protected tetrapeptide methylester (0.44 g, 0.89
mmol)] was dissolved in CH2Cl2 (15 mL) and NMM
(0.29 mL, 2.68 mmol) and the solution cooled to 08C.
Boc-l-Mag-OH (348 mg, 1.52 mmol) was dissolved in

3598
C. Peggion et al. / Tetrahedron 56 (2000) 3589±3601
CH2Cl2 (15 mL) and NMM (0.29 mL, 2.68 mmol),
EDC´HCl (291 mg, 1.52 mmol) and HOAt (207 mg, 1.52
mmol) were added at 08C. The two solutions were combined
and the reaction mixture was stirred at room temperature for
3 d. Then, EtOAc was added and the mixture was washed
with 0.5 M citric acid, water, 5% NaHCO3 and water, dried
over anhydrous Na2SO4, and concentrated. Addition of light
petroleum gave the title compound (379 mg, 70%) as a
solid, mp 240±2418C; RfI 0.65, RfII 0.85, RfIII 0.20;
[a]2D0ˆ26.3 (c 0.5, MeOH); nmax (KBr) 3310, 1727, 1661,
1530 cm21; dH (250 MHz, CDCl3) 7.72(1H, s, NH),
7.41(1H, s, NH), 7.10 (1H, s, NH), 6.47 (1H, s, NH), 5.74
(2H, m, 2 Mag 2 gCH), 5.06 (4H, m, 2 Mag 2 dCH2), 4.87
(1H, s, Mag NH), 3.69 (3H, s, OMe CH3), 3.052.51 (4H, m,
2Mag 2 bCH2), 1.551.39 (24H, m, 2 Mag 2 bCH3 and 3 Aib
6 bCH3), 1.46 (9H, s, Boc 3 CH3); HRMS (EI): M1, found
609.3739. C30H51N5O8 requires 609.3738.
1.41 (3H, d, Jˆ7.3 Hz, Ala bCH3), 1.36 (3H, d, Jˆ7.0 Hz,
Ala bCH3); HRMS (EI): M1, found 385.2227. C18H31N3O6
requires 385.2213.
N-tert-Butyloxycarbonyl-l-alanyl-l-alanyl-Ca-methyl-l-
allylglycyl-l-alanine methylester, Boc-l-Ala-l-Ala-l-
Mag-l-Ala-OMe. To a solution of Boc-l-Ala-OH (666
mg, 3.52 mmol) in CH2Cl2 (10 mL) and NMM (0.64 mL,
6.21 mmol) cooled to 08C, HOAt (479 mg, 3.52 mmol) and
EDC´HCl (675 mg, 3.52 mmol) were added. After 15 min
TFA´H-l-Ala-l-Mag-l-Ala-OMe [obtained by treatment of
the corresponding Boc-protected tripeptide methylester
(800 mg, 2.07 mmol) with a 30% solution of TFA in
CH2Cl2)] in CH2Cl2 (10 mL) and NMM (0.64 mL, 6.21
mmol) were added and the reaction was stirred at room
temperature for 2 d. Then, EtOAc was added and the
mixture was washed with 0.5 M citric acid, water, 5%
NaHCO3 and water, dried over anhydrous Na2SO4, and
evaporated to dryness. The title compound (680 mg, 72%)
was recrystallized from Et2O/light petroleum, mp 164±
1658C; RfI 0.90, RfII 0.90, RfIII 0.30; [a]2D0ˆ269.6 (c 0.5,
MeOH); nmax (KBr)3369, 3284, 1745, 1705, 1657,
1539 cm21; dH (250 MHz, CDCl3) 7.00 (1H, d, Jˆ7.0 Hz,
Ala NH), 6.61 (1H, d, Jˆ6.2 Hz, Ala NH), 6.60 (1H, s, Mag
NH), 5.64 (1H, m, Mag gCH), 5.14 (2H, m, Mag dCH2),
4.91 (1H, br d, Jˆ6.5 Hz, Ala NH), 4.52 (1H, dq, Jˆ7.0,
7.3 Hz, Ala aCH), 4.28 (1H, dq, Jˆ6.2, 7.3 Hz, Ala aCH),
4.13 (1H, dq, Jˆ6.5, 6.6 Hz, Ala aCH), 3.74 (3H, s, OMe
CH3), 2.772.66 (2H, m, Mag bCH2), 1.54 (3H, s, Mag
bCH3), 1.44 (9H, s, Boc 3 CH3), 1.40 (3H, d, Jˆ7.3 Hz,
Ala 3 bCH3), 1.39 (3H, d, Jˆ7.3 Hz, Ala 3 bCH3), 1.36 (3H,
d, Jˆ6.6 Hz, Ala 3 bCH3); HRMS (EI): MH1, found
457.2665. C21H37N4O7 requires 457.2662.
N-tert-Butyloxycarbonyl-Ca-methyl-l-allylglycyl-l-ala-
nine methylester, Boc-l-Mag-l-Ala-OMe. To a solution
of Boc-l-Mag-OH (750 mg, 3.27 mmol) in CH2Cl2 (10 mL)
and NMM (1.1 mL, 9.6 mmol) cooled to 08C, HOAt
(899 mg, 6.60 mmol) and EDC´HCl (1.26 g, 6.60 mmol)
were added. After 15 min HCl´H-l-Ala-OMe (1.0 g,
7.0 mmol) was added and the reaction was stirred at room
temperature for 20 h. Then, the solvent was removed and the
residue redissolved in EtOAc. The solution was extracted
with 0.5 M citric acid, water, 5% NaHCO3 and water, dried
over anhydrous Na2SO4, and concentrated. Addition of light
petroleum gave the title compound (0.98 g, 96%) as a solid,
mp 90±918C; RfI 0.95, RfII 0.95, RfIII 0.50; [a]2D0ˆ240.8 (c
1, MeOH); nmax (KBr) 3314, 1751, 1712, 1684, 1653,
1524 cm21; dH (250 MHz, CDCl3) 6.95 (1H, d, Jˆ7.0 Hz,
Ala NH), 5.72 (1H, m, Mag gCH), 5.15 (2H, m, Mag dCH2),
4.91 (1H, s, Mag NH), 4.57 (1H, dq, Jˆ7.0, 7.3 Hz, Ala
aCH), 3.74 (3H, s, OMe CH3), 2.762.53 (2H, m, Mag
bCH2), 1.48 (3H, s, Mag bCH3), 1.44 (9H, s, Boc 3 CH3),
1.40 (3H, d, Jˆ7.3 Hz, Ala bCH3); HRMS (EI): M1, found
314.1857. C15H26N2O5 requires 314.1842.
N-tert-Butyloxycarbonyl-Ca-methyl-l-allylglycyl-l-alanyl-
l-alanyl-Ca-methyl-l-allylglycyl-l-alanine methylester,
Boc-l-Mag-l-Ala-l-Ala-l-Mag-l-Ala-OMe. To a solu-
tion of Boc-l-Mag-OH (392 mg, 1.71 mmol) in CH2Cl2
(5 mL) and NMM (0.33 mL, 3.0 mmol) cooled to 08C,
HOAt (233 mg, 1.71 mmol) and EDC´HCl (328 mg, 1.71
mmol) were added. After 15 min TFA´H-l-Ala-l-Ala-l-
Mag-l-Ala-OMe [obtained by treatment of the correspond-
ing Boc-protected tetrapeptide methylester (520 mg,
1.14 mmol) with a 30% solution of TFA in CH2Cl2)] in
CH2Cl2 (5 mL) and NMM (0.33 mL, 3.0 mmol) were
added and the reaction was stirred at room temperature for
24 h. Then, EtOAc was added and the mixture was washed
with 0.5 M citric acid, water, 5% NaHCO3 and water, dried
over anhydrous Na2SO4, and concentrated. Addition of light
petroleum gave the title compound(582 mg, 90%) as a solid,
mp 132±1348C; RfI 0.90, RfII 0.90, RfIII 0.25; [a]2D0ˆ246.6
(c0.5, MeOH); nmax (KBr) 3314, 1742, 1665, 1526 cm21; dH
(200 MHz, CDCl3) 7.71 (1H, d, Jˆ7.0 Hz, Ala NH), 7.10
(1H, d, Jˆ7.3 Hz, Ala NH), 6.90 (1H, s, Mag NH), 6.42
(1H, d, Jˆ5.5 Hz, Ala NH), 5.78 (2H, m, 2Mag 2 gCH),
5.315.11 (4H, m, 2 Mag 2 dCH2), 5.03 (s, 1H, Mag NH),
4.51 (1H,dq, Jˆ7.3, 6.3 Hz, Ala aCH), 4.32 (1H, dq, Jˆ7.0,
7.0 Hz, Ala aCH), 4.24 (1H, dq, Jˆ5.5, 7.0 Hz, Ala aCH),
3.70 (s, 3H, OMe CH3), 2.812.42 (4H, m, 2 Mag 2 bCH2),
1.52 (6H, s, 2 Mag 2 bCH3), 1.45 (9H, s, Boc 3 CH3), 1.44
(6H, d, Jˆ7.0 Hz, 2 Ala 2 bCH3), 1.39 (3H, d, Jˆ6.3 Hz,
Ala bCH3); HRMS (EI): M1, found 567.3260. C27H45N5O8
requires 567.3268.
N-tert-Butyloxycarbonyl-l-alanyl-Ca-methyl-l-allylglycyl-
l-alanine methylester, Boc-l-Ala-l-Mag-l-Ala-OMe. To
a solution of Boc-l-Ala-OH (1.07 g, 5.4 mmol) in CH2Cl2
(10 mL) and NMM (0.89 mL, 8.1 mmol) cooled to 08C,
HOAt (734 mg, 5.4 mmol) and EDC´HCl (1.05 g, 5.4
mmol) were added. After 15 min TFA´H-l-Mag-l-Ala-
OMe [obtained by treatment of the corresponding Boc-
protected dipeptide methylester (850 mg, 2.70 mmol) with
a 30% solution of TFA in CH2Cl2)] in CH2Cl2 (10 mL) and
NMM (0.89 mL, 8.1 mmol) were added and the reaction
was stirred at room temperature for 24 h. Then, EtOAc
was added and the mixture was washed with 0.5 M citric
acid, water, 5% NaHCO3 and water, dried over anhydrous
Na2SO4, and concentrated. Addition of light petroleum gave
the title compound (1.00 g, 97%) as a solid, mp 154±1568C;
RfI 0.95, RfII 0.95, RfIII 0.35; [a]2D0ˆ264.8 (c 0.5, MeOH);
nmax (KBr) 3390, 3294, 1746, 1683, 1644, 1529 cm21; dH
(250 MHz, CDCl3) 7.11 (1H, d, Jˆ6.3 Hz, Ala NH), 6.60
(1H, s, Mag NH), 5.69 (1H, m, Mag gCH), 5.17 (2H, m,
Mag dCH2), 4.90 (1H, br d, Jˆ6.0 Hz, Ala NH), 4.52 (1H,
dq, Jˆ6.3, 7.3 Hz, Ala aCH), 4.05 (1H, dq, Jˆ6.0, 7.0 Hz,
Ala aCH), 3.73 (3H, s, OMe CH3), 2.822.62 (2H, m, Mag
bCH2), 1.57 (3H, s, Mag bCH3), 1.45 (9H, s, Boc 3 CH3),

C. Peggion et al. / Tetrahedron 56 (2000) 3589±3601
3599
N-tert-Butyloxycarbonyl-Ca-methyl-l-allylglycyl-d-ala-
nine methylester, Boc-l-Mag-d-Ala-OMe. To a solution
of Boc-l-Mag-OH (1.12 g, 4.88 mmol) in CH2Cl2 (10 mL)
and NMM (1.60 mL, 14.6 mmol) cooled to 08C, HOAt
(1.19 g, 8.78 mmol) and EDC´HCl (1.68 g, 8.78 mmol)
were added. After 15 min HCl´H-d-Ala-OMe (1.23 g,
8.78 mmol) was added and the reaction was stirred at
room temperature for 3 d. Then, the solvent was removed
and the residue redissolved in EtOAc. The organic solution
was washed with 10% KHSO4, water, 5% NaHCO3 and
water, dried over anhydrous Na2SO4, and concentrated.
Addition of light petroleum gave the title compound
(1.39 g, 91%) as a solid, mp 102±1038C; RfI 0.90, RfII
0.85, RfIII 0.50; [a]2D0ˆ1.2 (c 0.5, MeOH); nmax (KBr)
3318, 1752, 1713, 1685, 1652, 1522 cm21; dH (250 MHz,
CDCl3) 6.89 (1H, d, Jˆ7.1 Hz, Ala NH), 5.75 (1H, m, Mag
gCH), 5.17 (2H, m, Mag dCH2), 4.96 (1H, s, Mag NH), 4.57
(1H, dq, Jˆ7.1, 7.3 Hz, Ala aCH), 3.74 (3H, s, OMe CH3),
2.662.54 (2H, m, Mag bCH2), 1.51 (3H, s, Mag bCH3), 1.44
(9H, s, Boc 3 CH3), 1.40 (3H, d, Jˆ7.3 Hz, Ala bCH3);
HRMS (EI): M1, found 314.1856. C15H26N2O5 requires
314.1842.
mixture was washed with 0.5 M citric acid, water, 5%
NaHCO3 and water, dried over anhydrous Na2SO4, and
evaporated to dryness. The title compound (1.02 g, 78%)
was recrystallized from Et2O/lightpetroleum, mp 72±
748C; RfI 0.55, RfII 0.80, RfIII 0.25; [a]2D0ˆ23.3 (c 0.5,
MeOH); nmax (KBr) 3322, 1742, 1656, 1527 cm21; dH
(250 MHz, CDCl3) 6.96 (1H, d, Jˆ5.8 Hz, Ala NH), 6.73
(1H, br d, Jˆ6.0 Hz, Ala NH), 6.66 (1H, s, Mag NH), 5.81
(1H, m, Mag gCH), 5.13 (2H, m, Mag dCH2), 4.90 (1H, d,
Jˆ6.2 Hz, Ala NH), 4.52 (1H, dq, Jˆ5.8, 7.3 Hz, Ala aCH),
4.23 (1H, dq, Jˆ6.0, 7.1 Hz, Ala aCH), 4.14 (1H, dq,
Jˆ6.2, 7.0 Hz, Ala aCH), 3.73 (3H, s, OMe CH3), 2.71±
2.56 (2H, m, Mag bCH2), 1.57 (3H, s, Mag bCH3), 1.46
(9H, s, Boc 3 CH3), 1.42 (3H, d, Jˆ7.3 Hz, Ala bCH3), 1.39
(3H, d, Jˆ7.1 Hz, Ala bCH3), 1.37 (3H, d, Jˆ7.0 Hz, Ala
bCH3); HRMS (EI): M1, found 456.2595. C21H36N4O7
requires 456.2584.
N-tert-Butyloxycarbonyl-Ca-methyl-l-allylglycyl-l-alanyl-
l-alanyl-Ca-methyl-l-allylglycyl-d-alanine methylester,
Boc-l-Mag-l-Ala-l-Ala-l-Mag-d-Ala-OMe. To a solu-
tion of Boc-l-Mag-OH (624 mg, 2.72 mmol) in CH2Cl2
(5 mL) and NMM (0.56 mL, 5.10 mmol) cooled to 08C,
HOAt (370 mg, 2.72 mmol) and EDC´HCl (521 mg, 2.72
mmol) were added. After 15 min TFA´H-l-Ala-l-Ala-l-
Mag-d-Ala-OMe [obtained by treatment of the correspond-
ing Boc-protected tetrapeptide methylester (700 mg,
1.70 mmol) with a 30% solution of TFA in CH2Cl2)] in
CH2Cl2 (5 mL)and NMM (0.56 mL, 5.10 mmol) were
added and the reaction was stirred at room temperature for
4 d. Then, EtOAc was added and the mixture was washed
with 0.5 M citric acid, water, 5% NaHCO3 and water, dried
over anhydrous Na2SO4, and concentrated. Addition of light
petroleum gave the title compound (801 mg, 83%) as a
solid, mp 83±858C; RfI 0.90, RfII 0.90, RfIII 0.25;
[a]2D0ˆ7.2 (c 0.5, MeOH); nmax (KBr) 3313, 1742, 1658,
1530 cm21; dH (250 MHz, CDCl3) 7.74 (1H, d, Jˆ7.0 Hz,
Ala NH), 7.10 (1H, d, Jˆ6.7 Hz, Ala NH), 6.91 (1H, s, Mag
NH), 6.40 (1H, d, Jˆ5.1 Hz, Ala NH), 5.84 (2H, m, 2 Mag 2
gCH), 5.325.11 (4H, m, 2 Mag 2 dCH2), 4.97 (1H, s, Mag
NH), 4.51 (1H, dq, Jˆ6.7, 6.3 Hz, Ala aCH), 4.20 (2H, m,
2Ala 2 aCH), 3.70 (3H, s, OMe CH3), 2.722.42 (4H, m, 2
Mag 2 bCH2), 1.52 (6H, s, 2 Mag 2 bCH3), 1.46 (9H, s, Boc
3 CH3), 1.44 (3H, d, Jˆ7.0 Hz, Ala bCH3), 1.43 (3H, d,
Jˆ7.1 Hz, Ala bCH3), 1.42 (3H, d, Jˆ6.3 Hz, Ala bCH3);
HRMS (EI): M1, found 567.3292. C27H45N5O8 requires
567.3268.
N-tert-Butyloxycarbonyl-l-alanyl-Ca-methyl-l-allylglycyl-
d-alanine methylester, Boc-l-Ala-l-Mag-d-Ala-OMe. To
a solution of Boc-l-Ala-OH (1.24 g, 6.56 mmol) in CH2Cl2
(10 mL) and NMM (1.27 mL, 11.57 mmol) cooled to 08C,
HOAt (0.89 g, 6.56 mmol) and EDC´HCl (1.25 g, 6.56
mmol) were added. After 15 min TFA´H-l-Mag-d-Ala-
OMe [obtained by treatment of the corresponding Boc-
protected dipeptide methylester (1.21 g, 3.86 mmol) with a
30% solution of TFA in CH2Cl2] in CH2Cl2 (10 mL) and
NMM (1.27 mL, 11.57 mmol) were added and the reaction
was stirred at room temperature for 3 d. Then, EtOAc was
added and the mixture was washed with 0.5 M citric acid,
water, 5% NaHCO3 and water, dried over anhydrous
Na2SO4, and concentrated. Addition of light petroleum
gave the title compound (1.30 g, 88%) as a solid, mp
129±1318C; RfI 0.70, RfII 0.80, RfIII 0.30; [a]2D0ˆ22.5 (c
0.5, MeOH); nmax (KBr) 3355, 3310, 1735, 1711, 1667,
1648, 1541 cm21; dH (250 MHz, CDCl3) 7.20 (1H, br d,
Jˆ6.5 Hz, Ala NH), 6.65 (1H, s, Mag NH), 5.87 (1H, m,
Mag gCH), 5.18 (2H, m, Mag dCH2), 4.86 (1H, br d,
Jˆ6.2 Hz, Ala NH), 4.49 (1H, dq, Jˆ6.5, 7.3 Hz, Ala
aCH), 3.95 (1H, dq, Jˆ6.2, 7.2 Hz, Ala aCH), 3.71 (3H,
s, OMe CH3), 2.702.42 (2H, m, Mag bCH2), 1.62 (3H, s,
Mag bCH3), 1.46 (9H, s, Boc 3 CH3), 1.43 (3H, d,
Jˆ7.3 Hz, Ala bCH3), 1.36 (3H, d, Jˆ7.2 Hz, Ala bCH3);
HRMS (EI): M1, found 385.2214. C18H31N3O6 requires
385.2213.
FTIR absorption. The solid-state infrared absorption
spectra (KBr disk technique) were recorded with a
Perkin±Elmer model 580 B spectrophotometer equipped
with a Perkin±Elmer model 3600 IR data station. The
solution IR absorption spectra were recorded using a
Perkin±Elmer model 1720X FT-IR spectrophotometer,
nitrogen-¯ushed, equipped with a sample-shuttle device,
at 2 cm21 nominal resolution, averaging 100 scans. Solvent
(baseline) spectra were obtained under the same conditions.
Cell with path lengths of 0.1, 1.0 and 10 mm (with CaF2
windows) were used. Spectrograde deuterochloroform
(99.8% D) was purchased from Fluka.
N-tert-Butyloxycarbonyl-l-alanyl-l-alanyl-Ca-methyl-l-
allylglycyl-d-alanine methylester, Boc-l-Ala-l-Ala-l-
Mag-d-Ala-OMe. To
a solution of Boc-l-Ala-OH
(973 mg, 4.90 mmol) in CH2Cl2 (10 mL) and NMM (0.95
mL, 8.64 mmol) cooled to 08C, HOAt (666 mg, 4.90 mmol)
and EDC´HCl (938 mg, 4.90 mmol) were added. After
15 min TFA´H-l-Ala-l-Mag-d-Ala-OMe [obtained by
treatment of the corresponding Boc-protected tripeptide
methylester (1.11 g, 2.88 mmol) with a 30% solution of
TFA in CH2Cl2] in CH2Cl2 (10 mL) and NMM (0.95 mL,
8.64 mmol) were added and the reaction was stirred at room
temperature for 4 d. Then, EtOAc was added and the
1
Nuclear magnetic resonance. The H NMR spectra were
recorded with a Bruker model AM 400 spectrometer.

3600
C. Peggion et al. / Tetrahedron 56 (2000) 3589±3601
Measurements were carried out in deuterochloroform
(99.96% D; Aldrich) and deuterated DMSO (99.96% d6;
Acros Organics) with tetramethylsilane as the internal
standard. The free radical TEMPO was purchased from
Sigma.
free amino acid the positions of the H-atoms of the ±NH31
moiety and the co-crystallized water molecule were
recovered from a difference Fourier map. All other
H-atoms in this structure and the remaining structures
were calculated at idealized positions. During the re®ne-
ment these H-atoms were allowed to ride on their carrying
atom, with Uiso set equal to 1.2 (or 1.5 for methyl groups)
times the Ueq of the parent atom. The crystal of the penta-
Mass spectrometry. HRMS was carried out on a Micro-
mass AutoSpecE mass spectrometer tuned to resolution
6000 (at 5% peak height). Spectra were scanned at a
speed of 2 s/dec for the full mass range (m/z 20 900). Acqui-
sitions were performed by means of a water-cooled high
temperature solid probe with a constant bleed of per¯uoro-
kerosene (reference) via the septum inlet. The `secondary
reference correction' method was used to compensate for
the scan-to-scan errors which occur when acquiring data by
scanning the magnet (e.g. drift and hysteresis). This result
was achieved by looking for the peaks of the secondary
reference ®le, which have to be present during the acqui-
sition. When the data ®le was mass measured using
`accurate correction', we located all reference peaks within
the secondary reference ®le. The located secondary
reference peaks were then ¯agged as reference peaks and
their masses reassigned to their real mass. With a reference
peak on either side of an unknown mass peak, and
knowing the exponential scan law between the two
reference peaks, the mass of the unknown peak could be
calculated.
Ê
peptide did not signi®cantly diffract above uˆ508 (1.0 A
resolution). The relatively high values of Rint, as well as of
the R1 and wR2 factors might be ascribed to the far from
optimal crystal quality. However, we are con®dent that the
conformational features of the molecule, as discussed in this
work, are ®rmly established.
Acknowledgements
The authors thank Dr Gerard T. C. Kwakkenbos of DSM
Research for the HRMS determinations.
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