Exploring new dipeptides based on phenylglycine and Cα- methyl phenylglycine as hosts in inclusion resolutions

Article abstract of DOI:10.1016/j.tetasy.2004.04.044

Twelve homo-dipeptides derived from phenylglycine, Phg, and C ^α-methyl phenylglycine, (αMe)Phg, were synthesized and tested as resolving agents in resolutions through selective crystallization of inclusion compounds. The 3D-structure of a hydra

Full text of DOI:10.1016/j.tetasy.2004.04.044

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 15 (2004) 1919–1927
Exploring new dipeptides based on phenylglycine and Ca-methyl
phenylglycine as hosts in inclusion resolutions
a
a
b,
b
*
€
Simona Muller, Gerry J. A. Ariaans, Bernard Kaptein, Quirinus B. Broxterman,
Fernando Formaggio,c,* Elisa Battan,c Marco Crisma,c Claudio Tonioloc and
Alle Brugginka,b
aDepartment of Organic Chemistry, University of Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands
bDSM Pharma Chemicals, Advanced Synthesis and Catalysis, PO Box 18, 6160 MD Geleen, The Netherlands
cInstitute of Biomolecular Chemistry, CNR, Department of Chemistry, University of Padova, via Marzolo 1, 35131 Padova, Italy
Received 5 April 2004; accepted 20 April 2004
Abstract—Twelve homo-dipeptides derived from phenylglycine, Phg, and Ca-methyl phenylglycine, (aMe)Phg, were synthesized and
tested as resolving agents in resolutions through selective crystallization of inclusion compounds. The 3D-structure of a hydrated
(aMe)Phg dipeptide host was also solved by single crystal X-ray diffraction. These dipeptides were examined in the co-crystallization
with 15 different racemic guests, mainly alcohols and sulfoxides. Next to confirming the literature results for the resolution of
methylphenylsulfoxide, a rather limited scope was found for new resolutions. Only racemic solketal could be resolved with H-(S)-
(aMe)Phg-(S)-(aMe)Phg-OH in modest efficiency using various experimental techniques. This resolution was complicated by the
formation of polymorphic host–guest crystals. Whereas a wide array of similar dipeptides could be explored as resolving agents, it is
expected to be difficult to rationally design potentially successful molecular structures. Compared to resolution by diastereomeric
salt formation, inclusion complexes are less readily formed and therefore of a more limited scope and preparative applicability.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
R,R
Ar
R
R
methyl
phenyl
o-tolyl
p-tolyl
Access to pure enantiomers through selective crystalli-
zation of diastereomeric inclusion compounds would
extend the scope of traditional racemate resolution
beyond salt forming molecules. The pioneering work by
Toda and co-workers1;2 showed a promising potential of
this approach. Using a limited number of diols (Fig. 1)
derived from tartaric acid, almost 200 successful inclu-
sions were reported and about 100 racemates were
resolved. In more than half of the examples ee’s of 80%
or higher were obtained, combined, in about 20 cases, to
yields of 40% or higher (based on the racemate).
O
O
Ar
Ar
Ar
Ar
(CH2)4
(CH2)5
phenyl
phenyl
OH HO
Figure 1. Successful taddols (2,2-dialkyl-a,a,a0,a0-tetraaryldioxolane-
4,5-dimethanols) for inclusion resolutions.
alkyl phenyl sulfoxides,3–5 1-arylethylamines,6 a-hydr-
oxy esters,7 and ethers.8 With the aim at investigating
the scope and limitations of inclusion resolutions we
extended the selective crystallization of diastereomeric
inclusion compounds to new racemic guests. In addi-
tion, we tested new host dipeptides, based on the con-
formationally restricted Ca-methyl phenylglycine
[(aMe)Phg] a-amino acid residue.9–11 The easy access to
a wide range of simple peptides would greatly expand
the potential applications of the inclusion resolution
methods.
Ogura and co-workers3–8 reported that some phenyl-
glycine (Phg) dipeptides, such as 1 (Fig. 2), are highly
efficient hosts for the inclusion resolution of racemic
* Corresponding authors. Tel.: +31-46-4767416; fax: +31-46-4767604
(B.K.); tel.: +39-049-8275277; fax: +39-049-8275239 (F.F.); e-mail
addresses:
unipd.it
bernard.kaptein@dsm.com;
fernando.formaggio@
0957-4166/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2004.04.044

1920
S. M€uller et al. / Tetrahedron: Asymmetry 15 (2004) 1919–1927
R1
R2
R3
Entry
( ,#)
*
(R,R)
(S,S)
(R,S)
(S,R)
H
H
H
H
H
H
H
H
OH
OH
OH
OH
1
2
3
4
R2
O
H
N
R3
(R,R)
(S,S)
(R,S)
(S,R)
Z
Z
Z
Z
H
H
H
H
OBn
OBn
OBn
OBn
5
6
7
8
R1
N
#
*
H
R2
O
(S,S)
(S,R)
(R,R)
(S,R)
H
H
H
H
CH3
CH3
CH3
CH3
OH
OH
NH2
NH2
9
10
11
12
Figure 2. Dipeptide hosts used in this study.
2. Results and discussion
2.1. Selection of hosts and guests
this latter obtained, in turn, by treating (S)- or (R)-Z-
(aMe)Phg-OH with EDC in anhydrous CH2Cl2.
We were able to solve the crystal structure of H-[(S)-
(aMe)Phg]2-OH dihydrate by X-ray diffraction. The
molecular structure is illustrated in Figure 3. As
expected, the molecule of the free homo-dipeptide is
zwitterionic. Indeed, the C2–O2 and C2–OT bond
We decided to prepare 12 dipeptides (Fig. 2) to be
investigated for their inclusion capabilities. Known
synthetic procedures were followed for the preparation
of the Phg-containing peptides 1–8: the carboxyl group
was protected as benzyl ester (–COOBn), while the
amino group was masked by the benzyloxycarbonyl (Z)
moiety. The N-protected residue was coupled to the C-
protected residue by means of the DCC/HOBt (DCC,
1,3-dicyclohexylcarbodiimide; HOBt, 1-hydroxy-1,2,3-
benzotriazole) method.12 Simultaneous N- and C-de-
protection was achieved through catalytic hydrogena-
tion.5 Purification of intermediates and desired products
was found to be laborious but necessary to obtain pure
compounds for inclusion experiments. The tendency of
the Phg-peptides to include solvents13–15 appeared a
promising feature in view of the inclusion experiments.
ꢀ
lengths, 1.239(5) and 1.251(5) A, respectively, differing
by less than 3r, strongly point to the carboxylate form
of the C-terminal group. In addition, three H-atoms
bound to the N1 atom could be located on a difference
Fourier map. The peptide backbone is fully extended,
with w1 ¼ )168.5(3)ꢁ and /2 ¼ )174.8(4)ꢁ.20;21 As a
consequence, two consecutive, intra-residue H-bonded
C5-ring structures are observed, one between the pro-
tonated N1 amino group and the peptide carbonyl
oxygen O1 atom, and the other between the peptide
N2–H group and the C-terminal (carboxylate) O2 atom,
ꢀ
with Nꢀ ꢀ ꢀO separations of 2.645(4) and 2.560(4) A,
respectively. In each molecule the phenyl rings of the
two (aMe)Phg residues are nearly parallel to each other
(the angle between the normals to their average planes is
20ꢁ), and roughly perpendicular to the peptide back-
bone.
The four Ca-methyl substituted dipeptides 9–12 were
prepared starting from H-(S)-(aMe)Phg-OH and H-(R)-
(aMe)Phg-NH2. Both starting materials were synthe-
sized by a chemo-enzymatic process and available at
DSM Pharma Chemicals on a multi-ton scale.16;17 For
the synthesis of dipeptides 9 and 10 two orthogonal N-
and C-protecting groups, Z and tert-butoxy (–Ot-Bu),
respectively, were selected. This choice, implying a
double step for the protecting group removal, allowed
us to obtain peptides of higher purity. As (aMe)Phg is a
slow-reacting residue in peptide bond formation,18 to
prepare dipeptides 9–12 we looked for a more effective
activation procedure, as compared to the DCC/HOBt
method employed for peptides 1–8. Initially, the dipep-
tides were prepared by the EDC/HOAt [EDC, N-
ethyl,N0-(3-dimethylamino)propylcarbodiimide; HOAt,
7-aza-1-hydroxy-1,2,3-benzotriazole] coupling meth-
od.19 However, on large scale the symmetrical anhydride
method proved to be more convenient and cost effective.
The symmetrical anhydride of Z-protected (S)- or (R)-
(aMe)Phg was prepared in good yield (about 80%) by
mixing in anhydrous CH3CN an equimolar amount of
(S)- or (R)-Z-(aMe)Phg-OH and its 5-(4H)oxazolone,
In the packing mode of this peptide dihydrate (Fig. 4)
there is only one direct peptideꢀ ꢀ ꢀpeptide intermolecular
H-bond, between the protonated N1 amino group and
the (carboxylate) OT oxygen atom of a (1/2)x, )y,
1/2+z) symmetry related molecule, with an Nꢀ ꢀ ꢀO sep-
ꢀ
aration of 2.772(5) A, which links molecules head-to-tail
in a zig-zag motif parallel to the c direction. All other
intermolecular H-bonds involve the co-crystallized
water molecules, as bridges between peptide molecules
related through one of the crystallographic twofold
screw axes. More specifically, the O1W water molecule
acts as H-bond donor to the (carboxylate) O2 atom
within the same asymmetric unit with an O1Wꢀ ꢀ ꢀO2
ꢀ
separation of 2.731(5) A, and to the (peptide) O1 atom
of a (1/2)x, )y, )1/2+z) symmetry related molecule with
ꢀ
an O1Wꢀ ꢀ ꢀO1 separation of 2.886(4) A, thus bridging
peptide molecules along the c direction. In addition,
O1W is the acceptor of a H-bond from the N1 amino

S. M€uller et al. / Tetrahedron: Asymmetry 15 (2004) 1919–1927
1921
Figure 3. ORTEP view of the X-ray diffraction structure of H-[(S)-(aMe)Phg]2-OH dihydrate with atom numbering. Anisotropic displacement
ellipsoids are drawn at the 30% probability level. The two intramolecular H-bonds are indicated by dashed lines.
between symmetry related peptide molecules along the b
direction.
The remaining H atom of the O2W molecule is involved
in a O–Hꢀ ꢀ ꢀp interaction with the phenyl ring of the N-
terminal residue of a ()1/2+x, 1/2)y, 1)z) symmetry re-
lated peptide molecule. The distances between the O2W
oxygen atom and the ring carbon atoms range from
ꢀ
ꢀ
3.284(5) A (C1G1) to 3.518(5) A (C1D2), while the re-
ꢀ
lated Hꢀ ꢀ ꢀC distances vary from 2.57(5) to 3.21(5) A.
This latter interaction occurs along the a direction.
Conversely, all of the water-mediated and the single di-
rect peptide–peptide H-bonds connect molecules in the
bc plane. This layered packing motif is similar to the
‘water-buried sheet’ mode reported by Ogura and
co-workers14
for
(1-naphthyl)glycyl-phenylglycine
hydrated inclusion complexes. Interestingly, the latter
complexes and the structure described in this work,
although differing in significant details, share the same
symmetry as they belong to the same space group
(P212121).
A selection of 15 racemates, depicted in Table 1, as
potential guests was used to test the resolving capability
of dipeptides 1–12 through inclusion compound for-
mation. Priority was given to chiral alcohols, as many
amines were already successfully resolved by Ogura and
co-workers6 using dipeptide 1. Chiral separation of
alcohols by inclusion resolution is of special interest
because this class of compounds cannot be resolved
directly via diastereomeric salt formation.
Figure 4. Packing mode of the H-[(S)-(aMe)Phg]2-OH dihydrate
molecules in the crystal state as viewed down the a axis. Intermolecular
H-bonds are represented by dashed lines.
In order to investigate the possible influence of the
oxidation state of the sulfur atom in the inclusion phe-
nomenon, we extended our investigation to the reduced
(methylphenylsulfide, also called thioanisole) and oxi-
dized (methylphenylsulfone) achiral forms of methyl-
phenylsulfoxide tested by Ogura and co-workers.4
group of a (1)x, )1/2+y, 1/2)z) symmetry related pep-
ꢀ
tide molecule [N1ꢀ ꢀ ꢀO1W distance 2.785(5) A]. This
latter interaction provides water-mediated connection of
peptide molecules along the b direction. The second
water molecule, O2W, is the acceptor of a H-bond from
the protonated N1 amino group within the same
asymmetric unit, the N1ꢀ ꢀ ꢀO2W distance being
ꢀ
2.675(5) A, and the H-bond donor to the (carboxylate)
2.2. Inclusion experiments
OT atom of a ()x, 1/2+y, 1/2)z) symmetry related
peptide molecule with an O2Wꢀ ꢀ ꢀOT separation of
Given our experience with the sometimes poor repro-
ducibility of crystallizations, in particular in resolution
ꢀ
2.700(5) A. As a consequence, O2W acts as a bridge

1922
S. M€uller et al. / Tetrahedron: Asymmetry 15 (2004) 1919–1927
Table 1. Selected examples of racemic guests tested in the inclusion resolution with hosts 1–12
OH
OH
O
trans-2-Methoxycyclohexanol
trans-2-Methylcyclohexanol
Tetrahydrofurfurylalcohol
O
OCH3
OH
OH
OH
1-Phenoxy-2-propanol
H3C
CH3
OH
O
2,2-Dimethyl-1,3-dioxolane-4-
methanol (solketal)
1-tert-Butoxy-2-propanol
O
O
H3C
H3C CH3
NO2
OH
OH
CH3
3-Nitro-2-butanol
1-Phenyl-1,2-ethanediol
H3C
OH
NO2
O
H3C
H3C
CH3
3-Nitro-2-pentanol
2-Butanol
2-Methylcyclohexanone
Methylphenylsulfoxide
Methyl-p-tolylsulfoxide
CH3
OH
O
S
CH3
CH3
OH
OH
O
S
CH3
1-Phenylethylalcohol
CH3
H3C
OH
1-p-Tolylethylalcohol
CH3
H3C
studies, we initially re-investigated a literature experi-
ment. Ogura and co-workers3–5 found a high (R)-
enantioselectivity (92–93% ee) for methylphenylsulfox-
ide when forming an inclusion complex with dipeptide
host 1. Accordingly, in a reproduction of this experi-
ment we obtained an ee of 90%. Apparently, the sulf-
oxide group is of crucial importance for complex
formation, as inclusions with the corresponding sulfone
and sulfide as guests did not occur. Ogura and co-
workers3–5 showed that in the crystal state the sulfoxide
molecules are accommodated in a cavity between adja-
cent layers of dipeptide 1, linked by theþintermolecular
salt bridges formed between the –NH3 and –COOꢁ
groups. In this way host and guest can experience three
different types of interactions: hydrogen bonding be-
were used as guests. Surprisingly, also all other racemic
guests depicted in Table 1 failed to form inclusion
crystals, not only when employing 1 as host, but with all
other dipeptide hosts as well. All these inclusion exper-
iments were performed according to the ‘sorption’
method,4 that is by stirring at room temperature in
water for one or more days a suspension of host (usually
sparingly soluble in water) and 1 or 2 equiv of guest. As
shown above, host 9 tends to include water and there-
fore in all experiments only hydrated crystals of 9 were
found. Various other practical procedures were tested,
that is crystallization from solution, employing in-
creased pressure, or grinding the substrates in a mortar,
again without success. Another known alternative ap-
proach is to prepare the inclusion compound through a
slurry of the crystalline dipeptide and racemic guest
compounds in heptane at room temperature. After one
or two days the solid is filtered off. Treatment with water
and methylene chloride separates the unreacted host
peptide from the racemic guest or inclusion product.
Testing this ‘heptane sorption’ method with all other
hosts and guests allowed us to identify two new inclu-
sion complexes: dipeptide 9 with (i) methylphenylsulf-
þ
tween a –NH3 and the sulfoxide group, phenyl–phenyl
edge-to-face interactions and C–Hꢀ ꢀ ꢀp interactions be-
tween phenyl and alkyl groups.3 Small structural chan-
ges can easily upset these subtle interactions, thus
hampering a successful resolution. This observation may
probably account for the lack of inclusion crystal for-
mation when the methylphenylsulfide and methylphen-
ylsulfone (and also the racemic methyl-p-tolylsulfoxide)

S. M€uller et al. / Tetrahedron: Asymmetry 15 (2004) 1919–1927
1923
oxide [36% ee with a (R)-enantiomer preference] and (ii)
solketal [48% ee, also with a preference for the (R)-
enantiomer]. As the latter inclusion complex appeared
to be novel and the most promising one, it has been
analyzed in more detail.
2.3. Inclusion resolution of solketal
The resolution of solketal with dipeptide host 9 through
the heptane sorption method could be repeated using
various slurrying agents such as hexane, methanol, and
also water in host–guest ratios from 1:2 to 1:10. The ee
values for the (R)-enantiomer range from 0% to 2% in
water (employing 10 equiv of solketal; with 2 equiv no
crystals were obtained) to 30–33% in methanol and 45–
48% in hexane. Starting from a solution with excess of
solketal no resolution was obtained: from methanol (not
dried; 2–10 equiv of solketal) only crystals of host hy-
drates were formed, whereas with neat solketal inclusion
crystals with racemic solketal were obtained. These also
formed when dry methanol was used. Interestingly,
when neat (R)- and (S)-solketal were employed sepa-
rately, 1:1 host–guest crystals were obtained in both
cases.
4
9
14
19
24
(deg)
29
34
39
2
θ
Figure 6. X-ray powder diffractions of pure dipeptide host 9 (green),
its inclusion compound with racemic solketal (red), and its inclusion/
sorption complex enriched in (R)-solketal (blue).
dipeptide 9 and solketal were not suitable for a single
crystal X-ray diffraction analysis and, therefore, further
details of the 3D-structures could not be evaluated.
The powder X-ray diffraction (XPRD) patterns (Fig. 5)
of the two latter types of crystals are quite different, as
expected for diastereomers. XPRD also showed that the
crystals enriched in (R)-solketal, obtained through the
sorption method, are quite different from the diaste-
reomeric inclusion complex containing (S)-solketal (Fig.
6). Remarkably, none of the two diastereomers showed
any resemblance with the inclusion complex with (RS)-
solketal (no eutectic crystallization). In addition the
XPRD pattern of the pure host 9 (not the hydrate) is
almost featureless, most likely as a result of the forma-
tion of an amorphous solid. We believe that polymor-
phism, quite common in this type of resolutions, might
explain these varying results. Only the polymorph ob-
tained via sorption showed some enantioselectivity.
Unfortunately, the inclusion crystals obtained from
Final proof for the presence of polymorphs was ob-
tained by using induced crystallization. A solution of
dipeptide 9 was prepared in racemic solketal. After
cooling, the clear solution was seeded with pure dipep-
tide 9, that did not dissolve, but formed a slurry, which
acted as a template for further crystallization. The
inclusion complex formed revealed an ee of the (R)-
solketal of 33%, a value similar to that observed in the
sorption experiments using methanol as the slurry
medium.
It has to be noted that in the packing mode of the
dipeptide host H-[(S)-(aMe)Phg]2-OH (9) dihydrate the
co-crystallized O2W water molecule is located at dis-
ꢀ
tances of 3.69 and 3.59 A, respectively, from two dif-
ferent symmetry equivalents of O1W (Fig. 7b). A model
of the preferred guest (R)-solketal (Fig. 7a), built with
standard values of bond distances and bond angles and
assuming a trans disposition of the hydroxyl group rel-
ative to the closer ring oxygen atom, showed that the
distances between the hydroxyl oxygen atom and the
ꢀ
two ring oxygen atoms are both in the 3.60–3.64 A
range. This observation may suggest the possibility that
in the inclusion crystals solketal could replace the co-
crystallized water molecules, with its hydroxyl group
replacing O2W and the ring oxygen atoms replacing the
two symmetry equivalents of O1W. However, the dis-
tance between the two symmetry equivalents of O1W
ꢀ
(5.88 A) is much larger than that occurring between
ꢀ
the two ring oxygen atoms in solketal (2.32 A). In
addition: (i) the (ether) ring oxygen atoms of solketal, at
variance with the water molecules, can only act as
H-bond acceptors, and (ii) the rather close packing
of the dipeptide molecules observed for 9 dihydrate
would need to be significantly relaxed in order to
4
9
14
19
24
2θ (deg)
29
34
39
44
Figure 5. X-ray powder diffractions of dipeptide host 9 with (R)- and
(S)-solketal [(blue) and (red), respectively].

1924
S. M€uller et al. / Tetrahedron: Asymmetry 15 (2004) 1919–1927
4. Experimental section
4.1. Peptide synthesis and characterization
Well-known procedures were used for the preparation of
peptides 1–8.6;12 Repeated recrystallizations are required
after each reaction step to allow for efficient subsequent
manipulations. Experimental details for dipeptides 9–12
are given below.
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 ther-
mostat. Thin-layer chromatography was performed on
Merck Kieselgel 60/F254 precoated plates. The solvent
systems used are: (1) chloroform/ethanol (9:1); (2)
1butanol/acetic acid/water (3:1:1); (3) toluene/ethanol
(7:1); (4) ethyl acetate/petroleum ether (1:1). The chro-
matograms were developed by quenching of UV fluo-
rescence, chlorine–starch–potassium iodide or ninhydrin
chromatic reaction, as appropriate. The IR absorption
spectra were recorded on a Perkin–Elmer 1720X FTIR
spectrophotometer using the KBr disk technique. The
1H NMR spectra were obtained with a Bruker AC 250
spectrometer. Measurements were carried out in deu-
terochloroform (99.96% d, Merck) or in deuterated
dimethylsulfoxide (DMSO, 99.96% d6 Acros Organics)
with tetramethylsilane as the internal standard. High-
resolution mass spectra were obtained by electrospray
ionization (ESI) on a Perseptive Biosystem Mariner
API-TOF spectrometer. A 1 nM solution of neuroten-
sin, angiotensin I, and bradykinin in an acetonitrile/
water 1:1 mixture, containing 1% formic acid, was used
for calibration.
Figure 7. (a) Model of (R)-solketal. (b) Portion of the packing mode of
ꢀ
H-[(S)-(aMe)Phg]2-OH dihydrate. Only atoms within 4.6 A from O2W
are shown. The (1/2)x, )y, 1/2+z) and (1)x, 1/2+y, 1/2)z) symmetry
equivalents of O1W are indicated as (i) and (ii), respectively.
easily accommodate the solketal guest, particularly its
hydrophobic gem-dimethyl group. The geometrical
considerations reported above are based on the spatial
relationships among the three oxygen atoms of (R)-
solketal, which define a plane. The same considerations
also hold for its mirror image, (S)-solketal. Therefore, at
the present stage our analysis is unable to provide any
significant clue on the observed enantioselectivity in
the formation of the inclusion compounds of 9 with
solketal.
3. Conclusions
The limited scope of resolution through selective crys-
tallization of inclusion compounds, found in all our
studies thus far,22 has been confirmed in this work.
Compared to selective crystallization of diastereomeric
salts, in which ionic interactions are determining the
energy minima of the crystals, the substrates in inclusion
resolutions have far more possibilities to reach stable
crystal forms, that is single host crystals and dimers,
trimers, etc., or solvation forms thereof. Inclusions
of a racemic or chiral guest are other options, but
not necessarily adding to crystal stability. In fact, a
rational approach to the design of inclusion resolutions
is even more complicated than in diastereomeric
salts.23;24
4.1.1. Z-(S)-(aMe)Phg-OH. H-(S)-(aMe)Phg-OH (5.00 g,
30.0 mmol) was suspended in dioxane (30 mL) and cooled to
0ꢁC. Then, a solution of 2.0 M NaOH (16.0 mL, 32 mmol)
and NaHCO3 (2.68 g, 32 mmol) was added. Then, Z–OSu
(5.00 g, 20 mmol) was added to the solution, and a suspen-
sion formed. The reaction was stirred at rt for 4 d. Then,
again Z–OSu (5.00 g, 20 mmol) was added and the reaction
was stirred for additional 12 d. The solvent mixture was
evaporated under reduced pressure. The oily product was
dissolved in 5% NaHCO3 (100 mL) and the unreacted Z–
OSu was extracted with Et2O. The aqueous layer was acid-
ified to pH 3 with KHSO4 and the product was extracted
with EtOAc. The organic layer was washed with H2O, dried
over Na2SO4, and evaporated to dryness under reduced
pressure. Oil; yield 95%; Rf1 0.50, Rf2 0.90, Rf3 0.30;
20
We have found a new resolving homo-dipeptide, (9),
characterized by an aromatic Ca-tetrasubstituted a-
amino acid, although with limited efficiency and some
practical limitations. Whereas a wide array of similar
dipeptides could be explored as resolving agents, it
will be rather difficult to design a rational approach
toward potentially successful molecular structures. We
confirmed Ogura’s three-points interaction hypothesis
required for an effective inclusion3–8;13–15 by extending
the analysis of a sulfoxide to its reduced and oxidized
forms.
½aꢂ ¼ +24.9 (c 0.5, MeOH); IR (film) mmax 3300, 1717,
D
1654 cmꢁ1; 1H NMR (250 MHz, CDCl3) d 7.45 (m, 5H, Z
phenylCH),7.32[m,5H,(aMe)Phg phenyl CH], 6.35 (s, 1H,
NH), 5.06 (s, 2H, Z CH2), 2.03 (s, 3H, b-CH3). MS (ESI-
TOF) m=z calcd for C17H18NO4: 300.1235; found: 300.1301
[M+H]þ.
4.1.2. Z-(R)-(aMe)Phg-OH. This compound was pre-
pared from H-(R)-(aMe)Phg-OH and Z–OSu as de-
scribed above for its (S)-enantiomer. Oil; yield 93%; Rf1

S. M€uller et al. / Tetrahedron: Asymmetry 15 (2004) 1919–1927
1925
20
0.50, Rf2 0.90, Rf3 0.30; ½aꢂ ¼ )24.8 (c 0.5, MeOH); IR
under reduced pressure and the aqueous phase was ex-
tracted with EtOAc. The organic layer was washed with
5% NaHCO3 and H2O, dried over Na2SO4, filtered, and
D
1
(film) mmax 3303, 1718, 1652 cmꢁ1; H NMR (250 MHz,
CDCl3) d 7.45 (m, 5H, Z phenyl CH), 7.32 [m, 5H,
(aMe)Phg phenyl CH], 6.38 (s, 1H, NH), 5.06 (s, 2H, Z
CH2), 2.03 (s, 3H, b-CH3). MS (ESI-TOF) m=z calcd for
C17H18NO4: 300.1235; found: 300.1286 [M+H]þ.
evaporated to dryness. Yield 81%; mp 61–63 ꢁC (from
20
EtOAc); Rf1 0.90, Rf2 0.85, Rf3 0.90; ½aꢂ ¼ +9.5 (c 0.5,
D
MeOH); IR (KBr) mmax 3345, 1714 cmꢁ1
;
1H NMR
(250 MHz, CDCl3) d 7.41 (m, 5H, Z phenyl CH), 7.35
[m, 5H, (aMe)Phg phenyl CH], 6.27 (s, 1H, NH), 5.03
(dd, 2H, Z CH2), 2.00 (s, 3H, b-CH3), 1.33 (s, 9H, Ot-Bu
CH3). MS (ESI-TOF) m=z calcd for C21H26NO4:
356.1860; found: 356.1811 [M+H]þ.
4.1.3. 5-(4H)Oxazolone from Z-(S)-(aMe)Phg-OH. Z-
(S)-(aMe)Phg-OH (40.7 g, 136 mmol) was dissolved in
anhydrous CH2Cl2 and EDCꢀHCl (26.3 g, 137 mmol)
was added. The reaction was stirred at rt for 2 h. Then,
the solvent was removed in vacuo and the residue dis-
solved in EtOAc. The solution was washed with 10%
KHSO4, H2O, 5% NaHCO3 and H2O, dried over
4.1.8. Z-(R)-(aMe)Phg-Ot-Bu. This compound was
prepared from Z-(R)-(aMe)Phg-OH and isobutylene as
described above for its (S)-enantiomer. Yield 83%; mp
Na2SO4, and evaporated to dryness. Oil; yield 96%; Rf4
20
0.95; ½aꢂ ¼ +49.0 (c 0.5, MeOH); IR (film) mmax 1833,
59–61 ꢁC (from EtOAc); Rf1 0.90, Rf2 0.85, Rf3 0.90;
D
20
1688 cmꢁ1; 1H NMR (250 MHz, CDCl3) d 7.55–7.25 [m,
10H, Z and (aMe)Phg phenyl CH], 5.49 (s, 2H, Z CH2),
1.80 (s, 3H, b-CH3).
½aꢂ ¼ )9.3 (c 0.5, MeOH); IR (KBr) mmax 3347,
D
1
1715 cmꢁ1; H NMR (250 MHz, CDCl3) d 7.41 (m, 5H,
Z phenyl CH), 7.35 [m, 5H, (Me)Phg phenyl CH], 6.31
(s, 1H, NH), 5.03 (dd, 2H, Z CH2), 2.00 (s, 3H, b-CH3),
1.33 (s, 9H, Ot-Bu CH3). MS (ESI-TOF) m=z calcd for
C21H26NO4: 356.1860; found: 356.1912 [M+H]þ.
4.1.4. 5-(4H)Oxazolone from Z-(R)-(aMe)Phg-OH. This
compound was prepared as described above for its (S)-
enantiomer starting from Z-(R)-(aMe)Phg-OH. Oil;
20
D
yield 94%; Rf4 0.95; ½aꢂ ¼ )45.5 (c 0.5, MeOH); IR
4.1.9. Z-(S)-(aMe)Phg-(S)-(aMe)Phg-Ot-Bu. To a solu-
tion of [Z-(S)-(aMe)Phg]2O (71.4 g, 123 mmol) in anhy-
drous CH2Cl2, H-(S)-(aMe)Phg-Ot-Bu [obtained by
catalytic hydrogenation of the corresponding Z-deriva-
tive (44 g, 123 mmol) in anhydrous CH2Cl2] and
0.5 equiv of NMM were added. The reaction mixture
was stirred at rt for 5 d. Then, the solvent was removed
in vacuo and the residue dissolved in EtOAc. The
solution was washed with 10% KHSO4, H2O, 5%
NaHCO3 and H2O, dried over Na2SO4, and evaporated
to dryness. The product was crystallized from EtOAc/
(film) mmax 1835, 1688 cmꢁ1
;
1H NMR (250 MHz,
CDCl3) d 7.55–7.25 [m, 10H, Z and (aMe)Phg phenyl
CH], 5.49 (s, 2H, Z CH2), 1.80 (s, 3H, b-CH3).
4.1.5. [Z-(S)-(aMe)Phg]2O. Z-(S)-(aMe)Phg-OH (40.7 g,
136 mmol) was added to a solution of the 5-(4H)oxa-
zolone from Z-(S)-(aMe)Phg-OH (38.2 g, 136 mmol) in
anhydrous CH3CN. After stirring the reaction mixture
overnight at rt the solvent was removed in vacuo and the
residue dissolved in EtOAc. The solution was washed
with 5% NaHCO3 and H2O, dried over Na2SO4, and
PE. Yield 54%; mp 128–130 ꢁC (EtOAc/PE); Rf1 0.95,
20
Rf2 0.95, Rf3 0.90; ½aꢂ ¼ +7.5 (c 0.5, MeOH); IR (KBr)
D
1
evaporated to dryness. Oil; yield 82%; Rf4 0.90;
mmax 3386, 1735, 1722, 1681 cmꢁ1; H NMR (250 MHz,
20
D
½aꢂ ¼ +28.5 (c 0.5, MeOH); IR (film) mmax 3404, 3330,
CDCl3) d 7.55–7.15 [m, 15H, Z and (aMe)Phg phenyl
CH], 6.88 (s, 1H, NH), 6.65 (s, 1H, NH), 5.00 (m, 2H, Z
CH2), 2.00 and 1.89 (2s, 6H, b-CH3), 1.25 (s, 9H, Ot-Bu
CH3). MS (ESI-TOF) m=z calcd for C30H35N2O5:
503.2540; found: 503.2514 [M+H]þ.
1
1822, 1721 cmꢁ1; H NMR (250 MHz, CDCl3) d 7.55–
7.08 [m, 20H, Z and (aMe)Phg phenyl CH], 5.85 (2s, 2H,
NH), 5.15–4.90 (m, 4H, Z CH2), 1.85 (s, 6H, b-CH3).
4.1.6. [Z-(R)-(aMe)Phg]2O. This compound was pre-
pared as described above for its (S)-enantiomer starting
4.1.10. Z-(S)-(aMe)Phg-(R)-(aMe)Phg-Ot-Bu. This
compound was prepared as described above for Z-(S)-
(aMe)Phg-(S)-(aMe)Phg-Ot-Bu starting from [Z-(S)-
(aMe)Phg]2O and H-(R)-(aMe)Phg-Ot-Bu. The product
was isolated by flash chromatography (eluant EtOAc/
from Z-(R)-(aMe)Phg-OH and the corresponding 5-
20
D
(4H)oxazolone. Oil; yield 81%; Rf4 0.90; ½aꢂ ¼ )33.5 (c
0.5, MeOH); IR (film) mmax 3407, 3328, 1822, 1721 cmꢁ1
;
1H NMR (250 MHz, CDCl3) d 7.55–7.10 [m, 20H, Z and
(aMe)Phg phenyl CH], 5.85 (2s, 2H, NH), 5.15–4.90 (m,
4H, Z CH2), 1.85 (s, 6H, b-CH3).
PE 1:1). Oil; yield 53%; Rf1 0.95, Rf2 0.95, Rf3 0.90;
20
½aꢂ ¼ )5.6 (c 0.25, MeOH); IR (KBr) mmax 3385, 1732,
D
1684 cmꢁ1; 1H NMR (250 MHz, CDCl3) d 7.55–6.90 [m,
16H, Z and (aMe)Phg phenyl CH, and 1NH], 6.68 (s,
1H, NH), 5.00 (m, 2H, Z CH2), 2.05 and 1.95 (2s, 6H, b-
CH3), 1.27 (s, 9H, Ot-Bu CH3). MS (ESI-TOF) m=z
calcd for C30H35N2O5: 503.2540; found: 503.2614
[M+H]þ.
4.1.7. Z-(S)-(aMe)Phg-Ot-Bu. Isobutylene (20 mL) was
slowly bubbled into a solution of Z-(S)-(aMe)Phg-OH
(4.00 g, 13.4 mmol) in anhydrous CH2Cl2 (50 mL) and
cooled to ꢁ60 ꢁC. Concentrated H2SO4 (0.1 mL) was
added and the pressure resistant reaction flask was
hermetically closed. After keeping the reaction vessel at
rt for 7 d, the content was poured into a 5% aqueous
solution of NaHCO3 (50 mL). CH2Cl2 was removed
4.1.11.
Z-(S)-(aMe)Phg-(S)-(aMe)Phg-OH.
Z-(S)-
(aMe)Phg-(S)-(aMe)Phg-Ot-Bu (14.7 g, 29.2 mmol) was

1926
S. M€uller et al. / Tetrahedron: Asymmetry 15 (2004) 1919–1927
1
dissolved in a 1:1 CH2Cl2/TFA mixture. After stirring at
rt for 1 h, the solvent was removed in vacuo, Et2O was
added, and the compound collected by filtration. Yield
mmax 3386, 1735, 1722, 1681 cmꢁ1; H NMR (250 MHz,
DMSO, d6) d 8.14 (s, 1H, NH), 7.87 (s, 1H, NH), 7.45–
7.15 [m, 17H, Z and (aMe)Phg phenyl CH, and 2 NH],
5.07 (m, 2H, Z CH2), 1.81 and 1.67 (2s, 6H, b-CH3). MS
(ESI-TOF) m=z calcd for C26H28N3O4: 446.2074; found:
446.2058 [M+H]þ.
90%; mp 191–193 ꢁC (from MeOH/Et2O); Rf1 0.65, Rf2
20
0.95, Rf3 0.20; ½aꢂ ¼ +22.5 (c 0.5, MeOH); IR (KBr)
D
1
mmax 3373, 3351, 1738, 1675 cmꢁ1; H NMR (250 MHz,
CDCl3) d 7.55–7.15 [m, 16H, Z and (aMe)Phg phenyl
CH, and 1NH], 6.32 (s, 1H, NH), 5.05 (m, 2H, Z CH2),
1.92 and 1.82 (2s, 6H, b-CH3). MS (ESI-TOF) m=z calcd
for C26 H27N2O5: 447.1914; found: 447.2001 [M+H]þ.
4.1.16. Z-(S)-(aMe)Phg-(R)-(aMe)Phg-NH2. This com-
pound was prepared as described above for Z-(S)-
(aMe)Phg-(S)-(aMe)Phg-NH2 starting from [Z-(S)-
(aMe)Phg]2O and H-(R)-(aMe)Phg-NH2. The product
was isolated by flash chromatography (eluant EtOAc/
PE 1:1) and crystallized from EtOAc/PE. Yield 62%; mp
4.1.12. Z-(S)-(aMe)Phg-(R)-(aMe)Phg-OH. This com-
pound was prepared as described above for Z-(S)-
(aMe)Phg-(S)-(aMe)Phg-OH starting from the corre-
sponding dipeptide tert-butyl ester. Yield 89%; mp 66–
75–76 ꢁC (EtOAc/PE); Rf1 0.85, Rf2 0.95, Rf3 0.30;
20
½aꢂ ¼ )9.0 (c 0.2, MeOH); IR (KBr) mmax 3360, 1726,
D
1
68 ꢁC (from MeOH/Et2O); Rf1 0.50, Rf2 0.95, Rf3 0.15;
1673 cmꢁ1; H NMR (250 MHz, DMSO, d6) d 8.19 (s,
20
½aꢂ ¼ )9.0 (c 0.5, MeOH); IR (KBr) mmax 3381, 1730,
1H, NH), 7.74 (s, 1H, NH), 7.40–7.10 [m, 17H, Z and
(aMe)Phg phenyl CH, and 2NH], 5.04 (m, 2H, Z CH2),
1.83 and 1.72 (2s, 6H, b-CH3). MS (ESI-TOF) m=z calcd
for C26 H28N3O4: 446.2074; found: 446.2138 [M+H]þ.
D
1682 cmꢁ1; 1H NMR (250 MHz, CDCl3) d 7.55–6.95 [m,
16H, Z and (aMe)Phg phenyl CH, and 1NH], 6.40 (s,
1H, NH), 5.00 (m, 2H, Z CH2), 1.98 and 1.90 (2s, 6H, b-
CH3). MS (ESI-TOF) m=z calcd for C26 H27N2O5:
447.1914; found: 447.1982 [M+H]þ.
4.1.17. H-(R)-(aMe)Phg-(R)-(aMe)Phg-NH2 (11). Z-
(R)-(aMe)Phg-(R)-(aMe)Phg-NH2 (20.8 g, 46.7 mmol)
was dissolved in MeOH containing Pd/C. Under stir-
ring, the mixture was flushed with N2 and then H2 was
bubbled for 30 min. The catalyst was filtered off and the
4.1.13. H-(S)-(aMe)Phg-(S)-(aMe)Phg-OH (9). Z-(S)-
(aMe)Phg-(S)-(aMe)Phg-OH (11.7 g, 26.3 mmol) was
dissolved in MeOH containing Pd/C. Under stirring the
mixture was flushed with N2 and then H2 was bubbled
for 30 min. The catalyst was filtered off and the solvent
was removed in vacuo. Yield 92%; mp 310 ꢁC (phase
solvent was removed in vacuo. Yield 98%; mp 69–70 ꢁC;
20
Rf1 0.70, Rf2 0.70, Rf3 0.20; ½aꢂ ¼ )27.6 (c 0.5, MeOH);
D
1
IR (KBr) mmax 3327, 1662 cmꢁ1; H NMR (250 MHz,
transition around 145 ꢁC, formation of crystals at about
DMSO, d6) d 8.69 (s, 1H, NH), 7.48–7.25 [m, 10H,
(aMe)Phg phenyl CH], 5.92 (s, 1H, NH), 5.43 (s, 1H,
NH), 2.24 (br s, 2H, 2NH), 1.90 and 1.76 (2s, 6H, b-
CH3). MS (ESI-TOF) m=z calcd for C18H22N3O2:
312.1707; found: 312.1801 [M+H]þ.
20
160 ꢁC); Rf1 0.05, Rf2 0.80, Rf3 0.05; ½aꢂ ¼ +82.9 (c 0.5,
D
MeOH); IR (KBr) mmax 3415, 3357, 1683 cm1 ; 1H NMR
(250 MHz, DMSO, d6) d 7.55–7.05 [m, 13H, (aMe)Phg
phenyl CH and NH], 1.78 (s, 6H, b-CH3). MS (ESI-
TOF) m=z calcd for C18H21N2O3: 313.1547; found:
313.1609 [M+H]þ.
4.1.18. H-(S)-(aMe)Phg-(R)-(aMe)Phg-NH2 (12). Z-(S)-
(aMe)Phg-(R)-(aMe)Phg-NH2 (46 g, 103 mmol) was
treated as described above for 11. Yield 88%; mp 86–
4.1.14. H-(S)-(aMe)Phg-(R)-(aMe)Phg-OH (10). This
compound was prepared as described above for 9
starting from Z-(S)-(aMe)Phg-(R)-(aMe)Phg-OH. Yield
20
88 ꢁC; Rf1 0.80, Rf2 0.75, Rf3 0.25; ½aꢂ ¼ )72.1 (c 0.5,
D
MeOH); IR (KBr) mmax 3449, 3314, 1660 cmꢁ1; 1H NMR
(250 MHz, DMSO, d6) d 9.25 (s, 1H, NH), 7.65–7.05 [m,
12H, (aMe)Phg phenyl CH and 2NH], 3.12 (br s, 2H,
2NH), 1.83 and 1.55 (2s, 6H, b-CH3). MS (ESI-TOF)
m=z calcd for C18H22N3O2: 312.1707; found: 312.1764
[M+H]þ.
96%; mp 158–160 ꢁC; Rf1 0.05, Rf2 0.75, Rf3 0.05;
20
½aꢂ ¼ +58.9 (c 0.5, MeOH); IR (KBr) mmax 3416,
D
1680 cmꢁ1; 1H NMR (250 MHz, DMSO, d6) d 7.65–7.05
[m, 13H, (aMe)Phg phenyl CH and NH], 1.82 and 1.78
(2s, 6H, CH3). MS (ESI-TOF) m=z calcd for
C18H21N2O3: 313.1547; found: 313.1603 [M+H]þ.
4.2. X-ray diffraction
4.1.15. Z-(R)-(aMe)Phg-(R)-(aMe)Phg-NH2. To
a
solution of [Z-(R)-(aMe)Phg]2O (64.9 g, 112 mmol) in
CH2Cl2 H-(R)-(aMe)Phg-NH2 (18.3 g, 112 mmol), ob-
tained by the partial Strecker synthesis of the a-amino
acid,16;17 and 0.5 equiv of NMM were added. The reac-
tion was stirred at rt for 6 d. Then, the solvent was re-
moved in vacuo and the residue dissolved in EtOAc. The
solution was washed with 10% KHSO4, H2O, 5%
NaHCO3 and H2O, dried over Na2SO4, and evaporated
to dryness. The product was crystallized from EtOAc/
Single crystals of H-[(S)-(aMe)Phg]2-OH dihydrate were
grown from an ethyl acetate–methanol solution by dif-
fusion of petroleum ether vapour. C18H20N2O3 · 2H2O.
Orthorhombic, space group (P212121). Unit cell
ꢀ
parameters a ¼ 9.559(2), b ¼ 11.680(2), c ¼ 16.417(3) A.
3
V ¼ 1832.9(6) A ; Z ¼ 4; Dcalcd ¼ 1.262 Mg mꢁ3. Data
ꢀ
collection was performed on a Philips PW1100 diffrac-
tometer, using graphite monochromated Cu Ka radia-
ꢀ
tion (k ¼ 1.54178 A) in the h-2h scan mode up to h ¼
PE. Yield 52%; mp 189–190 ꢁC (EtOAc/PE); Rf1 0.85, Rf2
60ꢁ. Limiting indices: 0 6 h 6 10; 0 6 k 6 13; 0 6 l 6 18.
20
0.95, Rf3 0.30; ½aꢂ ¼ )19.0 (c 0.2, MeOH); IR (KBr)
A total of 1593 reflections were collected, 1575 of which
D

S. M€uller et al. / Tetrahedron: Asymmetry 15 (2004) 1919–1927
1927
independent. Three standard reflections, periodically
References and notes
monitored, showed a linear decay that reached 20% at
the end of data collection. Data were rescaled accord-
ingly. The structure was solved by direct methods of the
SHELXS 97 program,25 and refined by full-matrix block
least-squares on F2, using all data, by application of the
SHELXL 97 program,26 with all non-H atoms aniso-
tropic, and allowing the positional parameters and the
anisotropic displacement parameters of the non-H
atoms to refine at alternate cyclesþ. The positions of the
H-atoms of the N-terminal NH3 group and the two
co-crystallized water molecules were recovered from a
difference Fourier map. All other H-atoms were calcu-
lated at idealized positions. H-atoms of the peptide
molecule were refined as riding, while the positional
parameters of H-atoms bound to the water molecules
were refined with a common bond distance restraint of
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ꢀ
0.82 A. Data/restraints/parameters: 1575/4/214. Refine-
ment converged to R1 ¼ 0.0672 [on F P 4ðrÞF ] and
wR2 ¼ 0.1771 (on F 2 all data). Goodness-of-fit on F 2
ꢁ3
ꢀ
1.047. Dq max. and min. +0.330 and )0.352 e A
.
CCDC-232008 contains the supplementary crystallo-
graphic data for this paper. These data can be obtained
free of charge at www.ccdc.cam.ac.uk/conts/retriev-
ing.html [or from the Cambridge Crystallographic Data
Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax:
+44-1223-336-033; e-mail: deposit@ccd.cam.ac.uk].
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pp 511–521.
Several experimental techniques were used to reach
successful crystallization in resolutions, including all
common methods to induce nucleation and crystal for-
mation. Most techniques employed for inclusion reso-
lutions have already been described.6 A representative
description is given below.
4.3.1. General procedure for crystallization experiments
using slurry systems. The dipeptide was stirred together
with the respective guest compound in water or in an
organic solvent. Most experiments were performed in
1:1, 1:2 and 1:10 host/guest ratios at room temperature.
In those cases in which crystals were obtained, they were
filtered off, washed with heptane, and analyzed for the
presence of the respective guest compound by 1H NMR.
If an inclusion complex was obtained, the ee of the in-
cluded guest was determined using HPLC (Chiralcel
OB) or GC (b-CD). In other cases a H2/CH2Cl2 work-
up was used to separate non-included guest from host
compounds.
€
22. Muller, S.; Afraz, M. C.; de Gelder, R.; Ariaans, G. J. A.;
Kaptein, B.; Broxterman, Q. B.; Bruggink, A., submitted.
23. Gervais, C.; Grimbergen, R. F. P.; Markovits, I.; Ariaans,
G. J. A.; Kaptein, B.; Bruggink, A.; Broxterman, Q. B.
J. Am. Chem. Soc. 2004, 126, 655–662.
24. Dunitz, J. A. Chem. Commun. 2003, 545–548.
25. Sheldrick, G. M. SHELXS 97. Program for Crystal Struc-
€
ture Determination. University of Gottingen, Gottingen,
Germany, 1997.
26. Sheldrick, G. M. SHELXL 97. Program for Crystal
€
Acknowledgements
The authors are grateful to Dr. Barbara Biondi for mass
spectral analyses.
€
Structure Refinement. University of Gottingen, Gottingen,
Germany, 1997.
€