Is the backbone conformation of Cα-methyl proline restricted to a single region?

Article abstract of DOI:10.1002/chem.200900688

C^α-Methyl-L-proline, or L(αMe)Pro, is probably the most conformational constrained α-amino acid. In particular, its ω and o torsion angles are restricted to about 180 and -60°, respectively, and only three ranges of values are theoretically ava

Full text of DOI:10.1002/chem.200900688

FULL PAPER
DOI: 10.1002/chem.200900688
Is the Backbone Conformation of Ca-Methyl Proline Restricted to a Single
Region?
Matteo De Poli,[a] Alessandro Moretto,[a] Marco Crisma,[a] Cristina Peggion,[a]
Fernando Formaggio,[a] Bernard Kaptein,[b] Quirinus B. Broxterman,[b] and
Claudio Toniolo*[a]
Dedicated to the Centenary of the Italian Chemical Society
1
Abstract: Ca-Methyl-l-proline, or l-
(aMe)Pro, is probably the most confor-
mationally constrained a-amino acid.
In particular, its w and f torsion angles
are restricted to about 180 and ꢀ608,
respectively, and only three ranges of
values are theoretically available for y
in mono- or longer peptides, namely,
about ꢀ308 (cis’, 310/a-helical struc-
ture), 608 (inverse g turn), or 1408
Aib (a-aminoisoburyric acid), or l (or
d)-(aMe)Pro, long enough to fold into
intramolecularly hydrogen-bonded g or
b turns. The results are compared with
those obtained for the corresponding
dipeptides based on Pro, a well-known
turn-forming residue. For the crystal-
state 3D-structural analysis we used X-
ray diffraction, whereas our solution
conformational analysis was heavily
based on the FTIR absorption and H
and 13C NMR spectroscopy techniques.
We conclude that (aMe)Pro is able to
explore both trans’ and cis’ y areas of
the conformational space, but in
(aMe)Pro the latter is overwhelmingly
more populated, in marked contrast to
the Pro preference. This finding is a
clear indication that in (aMe)Pro the
major 3D-structural determinant is the
Ca-methyl group. The circular dichro-
ism (CD) signature of a peptide type
III’ b-turn conformation is also pro-
posed.
(trans’, polyACHTUNGTRENNUNG(l-Pro)n II structure). In
this work, we examined the tendency
of a number of Na-acyl dipeptide N’-al-
kylamides of the type RCO-(aMe)Pro-
Xxx-NHR’ or RCO-Xxx-(aMe)Pro-
NHR’, in which Xxx is l (or d)-Ala,
Keywords: circular dichroism · con-
formation analysis · IR spectrosco-
py · NMR spectroscopy · X-ray
diffraction
Introduction
bone substitution, producing Ca-methyl l-proline (l-
(aMe)Pro), would generate a f,y energy surface uniquely
restricted to a single region, namely, that of the right-
handed helical conformation.[1]
In 1974, in their theoretical study on the effect of Ca-methyl-
ation on the energetically preferred conformations of l-Pro
derivatives, Leach and co-workers predicted that this back-
[a] M. D. Poli, Dr. A. Moretto, Dr. M. Crisma, Dr. C. Peggion,
Prof. F. Formaggio, Prof. C. Toniolo
Institute of Biomolecular Chemistry, CNR
Padova Unit, Department of Chemistry
University of Padova
via Marzolo 1, 35131 Padova (Italy)
Fax : (+39)049-827-5239
E-mail: claudio.toniolo@unipd.it
[b] Dr. B. Kaptein, Dr. Q. B. Broxterman
DSM Pharmaceutical Products
A decade later, Flippen-Anderson et al.,[2] by use of X-ray
diffraction, showed that the simple, racemic “monopeptide”
Ac-dl-(aMe)Pro-NHMe (Ac=acetyl; NHMe=methylami-
no) is indeed helical in the crystalline state (without any sta-
Innovative Synthesis and Catalysis
P.O. Box 18, 6160 MD Geleen (The Netherlands)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200900688.
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ꢀ
bilization arising from a C=O···H N intramolecular hydro-
the course of 2,5-dioxopiperazine cyclization.[26] Using un-
natural amino acid mutagenesis, l-(aMe)Pro and other
modified Pro residues were incorporated in a member of
the Cys-loop receptor protein superfamily.[27] l-Pro ana-
logues, like l-(aMe)Pro, that strongly favor the w trans con-
former, were found to produce nonfunctional ion channels.
Finally, in a DFT calculation study on Ac-l-(aMe)Pro-
NHMe, in addition to not unexpected conclusions, such as
that the replacement of the Ca hydrogen with a methyl in
Pro destabilizes the w cis conformation, a surprising struc-
tural finding associated with l-Pro Ca methylation was re-
gen bond) and its l-enantiomer adopts right-handedness. On
the contrary, by use of conformational energy calculations,
Delaney and Madison[3] demonstrated that the total energy
for the l-enantiomer has a deep well at the right-handed C7’
(inverse g turn) conformation.[4,5] For this compound both
the semiextended (also termed polyACTHNUTRGNEN(UG l-Pro)n II) and right-
handed helical regions are less stable. However, the barrier
separating the C7’ and helical regions is rather low and the
population of the C7’ conformation tends to be overestimat-
ed in compounds too short to form b turns[6–8] or 310/a heli-
ces.[9,10] From a combined 13C NMR, IR absorption, and CD
analysis these authors[3] confirmed the preference of Ac-l-
(aMe)Pro-NHMe for the C7’ conformation, irrespective of
the solvent used.
ported, namely, the stabilization of the polyACTHNURTGNENG(U l-Pro)n II con-
formation, which was identified as an energy minimum for
the l-(aMe)Pro “monopeptide,” but not for those of the cor-
responding, unmethylated protein amino acid.[28]
Only a few other studies, scattered and nonsystematic,
have been subsequently reported on the conformational
preferences of (aMe)Pro. This observation is surprising in
that, among the Ca-methylated “monopeptides” of the
coded amino acids, (aMe)Pro is by far the most conforma-
tionally restricted (the f torsion angle is blocked; in linear
peptides the preceding tertiary amide torsion angle w can
adopt only the trans conformation; the side-chain cn torsion
angles are also rigidified).
Because of the published partially contradictory results
mentioned above on the preferred conformation(s) of
(aMe)Pro peptides, in this experimental work we decided to
synthesize and investigate a large set of Na-acylated, homo-
and heterochiral dipeptide monoalkylamide systems of the
type RCO-l
ACHTUNGTRENN(UNG or d)-(aMe)Pro-Xxx-NHR’ and RCO-Xxx-l
AHCTUNGTRENNUNG
ꢀ
or l (or d)-(aMe)Pro) long enough to fold into C=O···H N
intramolecularly hydrogen-bonded g or b turns. The results
are systematically compared with those obtained for the cor-
responding dipeptides based on the prototypical Pro, a well-
known turn-forming residue.[29–39] We have chosen ꢀNHiPr
(isopropylamino) as the C-terminal (and potential hydro-
gen-bonding donor) blocking group because it best mimics
the continuation of the peptide main chain. The (aMe)Pro
homo-dipeptides and the (aMe)Pro dipeptides containing
the helicogenic Aib[40–43] combine two amino acids with a
quaternary Ca atom. For the crystal-state 3D-structural anal-
ysis we used X-ray diffraction, whereas for our solution con-
formational study we relied heavily on FTIR absorption,
NMR, and CD spectroscopic techniques. A limited part of
this work has been reported in a preliminary form.[44,45]
Conformational potential-energy calculations suggested
that poly-(l-(aMe)Pro)n is locked in the type II polyACTHUNRGTNEUNG(l-Pro)n
conformation.[11] The CD spectrum of a solution of poly-(l-
(aMe)Pro)n of low molecular weight in alcohol, synthesized
by means of N-carboxyanhydride polymerization, is reminis-
cent of that of type II polyACHTNUGRTNEUNG
(l-Pro)n.[12] The preferred confor-
mations of the heterochiral dipeptides Z-l-Pro-d-(aMe)Pro-
NHMe (Z=benzyloxycarbonyl) and Z-d-(aMe)Pro-l-Pro-
NHMe were examined by IR absorption and 1H and
13C NMR spectroscopic techniques.[13] The former adopts a
type II b-turn conformation (in which d-(aMe)Pro is a left-
handed helix), whereas in the latter the coexistence of at
least four conformers was reported.
l-(aMe)Pro, inserted into a peptide antigen,[14,15] or at po-
sition 3 or 7 of the nonapeptide hormone bradykinin,[16–18] or
in the NPNA-repeating motif of the Plasmodium falcipari-
um protein,[19,20] was demonstrated by 2D NMR spectrosco-
py experiments to strongly stabilize b-turn conformations.
Analogous results (b-turn formation and trans-l-Xxx-L-
Results and Discussion
Synthesis and characterization: d-(aMe)Pro and l-
(aMe)Pro amide were obtained by amidase-catalyzed enzy-
matic resolution (Scheme 1).[46] Using the amidase from My-
cobacterium neoaurum ATCC 25795, a conversion of 45%
was obtained after 70 h (E ratio 240), whereas with the ami-
dase from Ochrobacterium anthropi NCIMB 40321 (overex-
pressed in E. coli)[47] 48% conversion was reached after 26 h
(E ratio 317). Note that whereas both amidases are in gener-
al l-selective for acyclic a-amino amides, for the (aMe)Pro
amide the stereoselectivity is reversed. The H-dl-(aMe)Pro-
NH2 racemic substrate was obtained in 61% overall yield by
base-catalyzed cyanoethylation of N-benzylidene-Ala amide,
followed by acidic workup and ring-closing hydrogenation
over palladium on charcoal.[48]
ACHTUNGTRENNUNG(aMe)Pro peptide bonds) were reported for l-(aMe)Pro-
containing analogues of an antigen mimotope peptide[21] and
the antimicrobial peptide buforin 2.[22] Interestingly, as op-
posed to the results from molecular-mechanics simulations,
it was experimentally shown that the sequence l-(aMe)Pro-
l-Pro is not tightly folded.[23] A combined molecular model-
ing and solution and crystal-state conformational study of
acyl-l-Val-l-Xxx-NHR peptides (in which Xxx is 4-methyl-
ꢀ
ene-l-(aMe)Pro) suggests the absence of any C=O···H N
intramolecular hydrogen bond in this sequence.[24,25] Accord-
ing to the X-ray diffraction data, the 4-substituted l-
(aMe)Pro residue is a right-handed helix.
Recently, the X-ray diffraction structure of c-(l-
(aMe)Pro)2 demonstrated the absence of epimerization in
tert-Butyloxycarbonyl (tBoc) and benzyloxycarbonyl (Z)
N-protected (aMe)Pro derivatives[49,50] were prepared with
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Ca-Methyl Proline
FULL PAPER
tBoc and Z-urethane groups
were removed by acidic treat-
ment (HCl in diethyl ether) and
by catalytic hydrogenation, re-
spectively. Na-Acetylation, iso-
butanoylation, and para-bromo-
benzoylation were performed
on
the
Na-deprotected
amide or
(aMe)Pro isopropylAHCTUNGTRENNUNG
(aMe)Pro-containing dipeptide
isopropylamides using the cor-
responding symmetrical anhy-
drides.
The physical properties and
analytical
data
for
the
Scheme 1. Chemoenzymatic synthesis of d-(aMe)Pro and l-(aMe)Pro amide with the conversion and enantio-
meric excess (ee) values.
(aMe)Pro derivatives and pep-
tides are listed in Table S1 in
the Supporting Information.
use of tert-butyldicarbonate and N-(benzyloxycarbonyl) suc-
cinimide, respectively (Table S1 in the Supporting Informa-
tion). The 3D structure of Z-d-(aMe)Pro-OH (crystals from
methanol) was solved by X-ray diffraction (Figure 1). Inter-
The syntheses and characterizations of tBoc-l-(aMe)Pro-
OH,[49] Z-l-(aMe)Pro-OH,[50] and Z-d-(aMe)Pro-OH[12,13]
have already been reported. All newly synthesized com-
1
pounds were also characterized by H NMR spectroscopy.
Crystal-state conformational analysis: We solved the X-ray
diffraction structures of three novel, Na-blocked, (aMe)Pro-
containing dipeptide alkylamides, namely, tBoc-l-Ala-l-
(aMe)Pro-NHiPr, Z-Aib-d-(aMe)Pro-NHiPr, and Z-d-
(aMe)Pro-d-(aMe)Pro-NHiPr. These crystal structures,
combined with the five recently published by us,[40,41] Ac-l-
Ala-l-(aMe)Pro-NHiPr (Ac=acetyl), Ac-d-(aMe)Pro-d-
Ala-NHiPr, Ac-d-(aMe)Pro-l-Ala-NHiPr, iBu-l-Ala-d-
(aMe)Pro-NHiPr (iBu=isobutanoyl), and Ac-d-(aMe)Pro-
Aib-NHiPr, offer an almost exhaustive overview of the con-
formations preferred by this sterically demanding amino
acid. (The only missing, noncrystalline, dipeptide sequence
in this list of X-ray diffraction structures is d-(aMe)Pro-l-
(aMe)Pro or its l,d enantiomer.) In this work we have also
solved the crystal structures of two Na-blocked dipeptide al-
kylamides based on the related, coded amino acid Pro: Ac-
Aib-l-Pro-NHiPr and Z-l-Pro-d-Pro-NHiPr. The molecular
structures of the five new structures are illustrated in Fig-
ures 2, 3, 4, 5, and 6. The relevant Na-blocking group and
backbone torsion angles,[56] and the intra- and intermolecu-
lar hydrogen-bond parameters have been deposited (Ta-
bles S2 and S3, respectively, in the Supporting Information).
Table 1 summarizes the f,y torsion angles for all published
(aMe)Pro-containing peptides and their related Pro ana-
logues.
Figure 1. X-ray diffraction structure of Z-d-(aMe)Pro-OH with atom
numbering.
estingly, the tertiary Z-urethane function[51] is in the
common trans conformation (Table S2 in the Supporting In-
formation) in the crystal state, but two rotamers are present
in a ratio of about (60–65)%:ACTHNUTRGNEUGN(40–35)% (ref. [13] and this
work) in CDCl3 and 55%:45% (this work) in CD3OH, using
the bCH3 signal as the NMR spectroscopy probe. In the
crystal the d-(aMe)Pro residue is a left-handed helix and
the molecules are held together by means of intermolecular
(carboxylic acid) OT···O0 (urethane) hydrogen bonds
(Table S3 in the Supporting Information). The pyrrolidine
ring is in a conformation intermediate between the 3T4
(twist) and the E4 (envelope).[52,53]
In the (aMe)Pro-based compounds, peptide and
Figure 7 shows the average bond lengths and bond angles
for the (aMe)Pro residue in comparison with those already
reported[29] for Pro. All bond lengths and most of the bond
angles closely match each other. Differences between 1.0
and 2.08 are found for the corresponding bond angles
around the Ca atom (trisubstituted for Pro, but tetrasubsti-
tuted for (aMe)Pro). The steric effects of Ca tetrasubstitu-
tion may also account for the widening of the Ca-C’-O bond
angle for (aMe)Pro as compared to Pro. The largest differ-
isopropylACHTUNGTRENNUNGamide bonds were formed by the 1-(3-dimethyl-
ACHTUNGTRENNUNGamino)propyl-3-ethylcarbodiimide (EDC)/1-hydroxy-1,2,3-
benzotriazole (HOBt)[54] or 7-aza-1-hydroxy-1,2,3-benzotria-
zole (HOAt)[55] C-activation method in CH2Cl2 in the pres-
ence of N-methylmorpholine. Despite the occurrence of the
severely sterically demanding (aMe)Pro residue, coupling
yields were from good to excellent (65–96%) with the single
exception of that of the Aib-d-(aMe)Pro bond (24%). The
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C. Toniolo et al.
Figure 4. X-ray diffraction structure of Ac-Aib-l-Pro-NHiPr with atom
numbering. The second occupancy site of the Pro Cg atom is omitted for
ꢀ
clarity. The C=O···H N intramolecular hydrogen bond is represented by
a dashed line.
Figure 2. X-ray diffraction structure of tBoc-l-Ala-l-(aMe)Pro-NHiPr
with atom numbering.
Figure 5. X-ray diffraction structure of Z-d-(aMe)Pro-d-(aMe)Pro-
ꢀ
NHiPr with atom numbering. The C=O···H N intramolecular hydrogen
bond is represented by a dashed line.
Figure 3. X-ray diffraction structures of the two independent molecules
(A and B) in the asymmetric unit of Z-Aib-d-(aMe)Pro-NHiPr with
ꢀ
atom numbering. In each structure the C=O···H N intramolecular hydro-
gen bond is represented by a dashed line.
Figure 6. X-ray diffraction structure of Z-l-Pro-d-Pro-NHiPr with atom
ꢀ
numbering. The C=O···H N intramolecular hydrogen bond is represent-
ed by a dashed line.
ence is observed for the endocyclic bond angle at Cg, which
is narrower by 3.38 for (aMe)Pro. However, this latter ob-
servation must be taken with caution, as it may be biased by
static structural disorder of the Pro Cg atom (as often high-
lighted by anisotropic displacement parameters higher than
those of the other ring atoms), which might result in an ap-
parent ring flattening. Indeed, the sum of the average endo-
cyclic bond angles is 525.28 for (aMe)Pro, but 529.48 for
Pro. For comparison, in a planar, regular pentagon, the sum
of the internal angles is 5408.
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Ca-Methyl Proline
FULL PAPER
Table 1. 3D-structural parameters in the crystal state for the known Ala/ACHTNUTRGENNUG(aMe)Pro, Ala/Pro, Aib/ACHTUNGTERN(NUGN aMe)Pro,
formation, which is stabilized
Aib/Pro, (aMe)Pro–(aMe)Pro, and Pro–Pro Na-blocked and C-amidated dipeptide sequences.
!
by an
amide)
i
i+3 ((urethane or
Dipeptide sequence
Backbone torsion angles
Type of turn
Reference
ꢀ
C0=O0···H NT
fi+1
yi+1
fi+2
yi+2
(amide)) hydrogen bond of
medium
strength[67–69]
(the
Ac-l-Ala-l-(aMe)Pro-NHiPr
tBoc-l-Ala-l-(aMe)Pro-NHiPr
iBu-l-Ala-l-Pro-NHiPr
ꢀ135
ꢀ76
ꢀ129
74
64
53
ꢀ66
ꢀ59
53
77
130
76
ꢀ150
ꢀ152
32
166
136
ꢀ129
ꢀ137
137
133
133
ꢀ140
37
139
32
39
ꢀ43
ꢀ40
34
138
134
139
141
ꢀ58
ꢀ59
ꢀ67
57
83
66
ꢀ37
ꢀ36
ꢀ22
ꢀ142
ꢀ156
25
154
14
ꢀ12
ꢀ14
3
15
0
9
28
25
31
22
ꢀ4
ꢀ25
28
ꢀ7
ꢀ7
ꢀ14
ꢀ3
–
–
–
–
[40]
this work
[57]
O···N distances are in the range
2.997(5)–3.162(4) ꢁ, and the
Piv-d-Ala-d-Pro-NHiPr[a]
[58]
ꢀ
O···H N angles are between
–
Ac-d-(aMe)Pro-d-Ala-NHiPr
Ac-l-Pro-l-Ala-NHtBu[b]
iBu-l-Pro-l-Ala-NHiPr
Ac-d-(aMe)Pro-l-Ala-NHiPr
Z-d-Pro-l-Ala-NHtBu
iBu-l-Pro-d-Ala-NHiPr
iBu-l-Pro-d-Ala-NHtBu
iBu-l-Ala-d-(aMe)Pro-NHiPr
Piv-d-Ala-l-Pro-NHiPr
b-III’
–
[40], [41]
[59]
[60], [61]
[40], [41]
[59]
[60], [61]
[62]
[40], [41]
[63]
148.6 and 162.38). The only
open structure is that of tBoc-l-
Ala-l-(aMe)Pro-NHiPr. The b
turns formed by the homochiral
-d-(aMe)Pro-d-(aMe)Pro- and
the heterochiral -l-Pro-d-Pro-
dipeptides are of type III’ and
type II, respectively, as expect-
ed from their sequence chirali-
ty.[66] In both -Aib-d-(aMe)Pro-
and -Aib-l-Pro- sequences, the
achiral, helical Aib adopts the
same screw sense as that of the
following chiral residue, thus
generating a type III’ b turn or
ꢀ71
66
b-II
b-II’
b-II’
b-II
b-II
b-II
b-II’
b-III’
b-II
b-III’
b-III’
b-I
b-III
b-III’
–
b-II
b-II
b-II
ꢀ77
ꢀ76
96
82
78
58
ꢀ62
ꢀ60
ꢀ55
60
ꢀ89
Ac-d-(aMe)Pro-Aib-NHiPr
53
61
[40], [41]
[64]
this work
Piv-l-Pro-Aib-NHMe[c]
ꢀ58
61
55
67
ꢀ80
ꢀ65
57
Z-Aib-d-(aMe)Pro-NHiPr
54
51
Ac-Aib-l-Pro-NHiPr
Z-Aib-l-Pro-NHMe
Z-d-(aMe)Pro-d-(aMe)Pro-NHiPr
Piv-l-Pro-l-Pro-NHMe
Piv-l-Pro-d-Pro-NHMe
ꢀ50
ꢀ51
50
ꢀ60
ꢀ58
ꢀ56
ꢀ58
this work
[65]
this work
[66]
ꢀ95
83
[66]
84
82
a
slightly distorted type I b
Z-l-Pro-d-Pro-NHiPr
this work
turn, respectively. The differ-
ence, albeit small, between
these two types of b turn points
to a higher propensity for a reg-
[a] Piv=pivaloyl. [b] NHtBu=tert-butylamino. [c] NHMe=methylamino.
ular helical conformation assignable to (aMe)Pro as com-
pared to Pro. The only major conformational difference ob-
served between molecules A and B of Z-Aib-d-(aMe)Pro-
NHiPr is found in the q2 torsion angle of the Z Na-protect-
ing group,[51] ꢀ69.5(6)8 for molecule A, and ꢀ175.5(4)8 for
molecule B. All (aMe)Pro residues in these structures, in-
cluding that of the open tBoc-l-Ala-l-(aMe)Pro-NHiPr, are
helical, with average f,y torsion angles, j57.68j, j30.28j, re-
markably close to those found experimentally for 310-helix
peptides (57, 308), but less near those of a-helix peptides
(63, 428).[9] It is worth pointing out that -d-(aMe)Pro-d-
(aMe)Pro- is the first linear (aMe)Pro homopeptide se-
quence ever solved by X-ray diffraction. The Ala residue in
tBoc-l-Ala-l-(aMe)Pro-NHiPr and the N-terminal Pro resi-
due in Z-l-Pro-d-Pro-NHiPr adopt the semiextended con-
formation, whereas a conformation in the “bridge” region of
the f,y space[70] is seen for the C-terminal Pro residues of
Ac-Aib-l-Pro-NHiPr and Z-l-Pro-d-Pro-NHiPr.
The pyrrolidine rings of the (aMe)Pro residues are
Figure 7. Average bond lengths in ꢁ (A and C) and bond angles in de-
[52,53]
4T3
3
3
in tBoc-l-Ala-l-(aMe)Pro-NHiPr, and T4 and T4/
E4 in molecules and B, respectively, of Z-Aib-d-
grees (B and D) for the (aMe)Pro and Pro[29,69] residues, respectively.
A
3
(aMe)Pro-NHiPr, whereas T4 for both residues 1 and 2 of
Z-d-(aMe)Pro-d-(aMe)Pro-NHiPr. On the other hand, the
pyrrolidine rings of the Pro residues are E5/1T5 and E1/2T1
for the two side-chain conformers of Ac-Aib-l-Pro-NHiPr,
In the five (aMe)Pro-/Pro-containing structures reported
in this paper, all (secondary and tertiary) urethane, amide,
and peptide bonds (w torsion angles) are in the usual trans
conformation with modest deviations from the (1808) pla-
narity (jDwjꢁ7.48), except for the amide w2 torsion angle
of molecule B in the asymmetric unit of Z-Aib-d-(aMe)Pro-
NHiPr, 163.1(4)8. Four structures are folded in a b-turn con-
3
4
and T4 and T3 for residues 1 and 2, respectively, of Z-l-
Pro-d-Pro-NHiPr.
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C. Toniolo et al.
Solution conformational analysis: We performed an exten-
sive conformational analysis of the Na-blocked dipeptide al-
kylamides based on (aMe)Pro and/or Pro residues in solu-
tion by use of FTIR absorption, NMR, and CD spectroscop-
ic techniques.
The effects of replacing Pro with (aMe)Pro, sequence
chirality, (aMe)Pro/Pro incorporation at position i+1 or i+
2 in the sequence, and presence of the weakly turn former
Ala or the much stronger turn former Aib[42–45] on the pre-
ferred conformation of model dipeptides are evident from
the FTIR absorption spectra in CDCl3 in the informative
ꢀ
N H stretching region, reported in Figures 8 and 9, as well
as Figures S1 and S2 in the Supporting Information. We first
Figure 9. FTIR absorption spectra (3500–3200 cmꢀ1 region) of: A) Ac-l-
(aMe)Pro-d-(aMe)Pro-NHiPr (solid line) and Ac-d-(aMe)Pro-d-
(aMe)Pro-NHiPr (dashed line); and B) Ac-l-Pro-d-Pro-NHiPr (solid
line) and Ac-d-Pro-d-Pro-NHiPr (dashed line) in CDCl3 ([peptide]=
1 mm).
-(aMe)Pro-(aMe)Pro- dipeptides, in which the opposite
trend is found. 2) Turn formation is enhanced when
(aMe)Pro replaces Pro in the sequence. 3) Positioning
(aMe)Pro or Pro as residue i+1 is more favorable for fold-
ing than as residue i+2, particularly for (aMe)Pro. 4) The
Aib/
ACHTUNGTRENNUNG
ducing a turn than the Ala/AHCTNUGTRENNNUG
5) In the homo-dipeptides, the (aMe)Pro d,d and the Pro
l,d stereoisomers appear to be folded to the highest extent
observed in the present study (100%). This latter finding re-
sembles that already reported for Z-l-Pro-d-(aMe)Pro-
NHMe.[13]
Figure 8. FTIR absorption spectra (3500–3200 cmꢀ1 region) of A) Ac-d-
(aMe)Pro-Aib-NHiPr (solid line) and Ac-Aib-d-(aMe)Pro-NHiPr
(dashed line); and B) Ac-l-Pro-Aib-NHiPr (solid line) and Ac-Aib-l-
Pro-NHiPr (dashed line) in CDCl3 ([peptide]=1 mm).
Our conformational study of samples in CDCl3 was ex-
tended to NMR spectroscopy. Not surprisingly, the NH
chemical-shift perturbation trends observed in the titrations
of the compounds studied in this work with the strong hy-
checked the concentration dependence (between 10 and
0.1 mm) in the spectra of all peptides investigated (not
shown). The results indicate that at 1 mm concentration
there is no evidence for significant self-association through
intermolecular hydrogen bonding, that is, that all NH
groups are either free (solvated by CDCl3) or intramolecu-
larly hydrogen bonded.
In general, the curves are characterized by two more or
less intense bands, one located above 3400 cmꢀ1 (free NHs)
and the other below 3400 cmꢀ1 (hydrogen-bonded NHs), re-
spectively.[71–73] The following considerations can be drawn
from our analysis: 1) As opposed to the homochiral sequen-
ces, the heterochiral sequences exhibit a higher tendency to
fold. However, this conclusion does not apply to the two
2
drogen-bond acceptor H6 DMSO (dimethyl sulfoxide)[74,75]
are often difficult to interpret, because in general the slopes
are significantly reduced if the peptide main chain is short
(as in our dipeptides)[73] and if the NH groups to be solvated
by the perturbing agent are significantly shielded, for exam-
ple, close to a sterically demanding a-amino acid, such as
Aib and (aMe)Pro.
The NMR spectra of both Ac-d-Pro-d-Pro-NHiPr and
Ac-l-(aMe)Pro-d-(aMe)Pro-NHiPr are complicated by
trans–cis isomerism that generates multiple resonances. The
differences in the chemical shifts observed for the isopropyl-
AHCTUNGTREGUNaNN mido NH proton of each conformer are Dd=0.9 ppm (d=
8020
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Chem. Eur. J. 2009, 15, 8015 – 8025

Ca-Methyl Proline
FULL PAPER
7.74 and 6.84 ppm) and Dd=0.7 ppm (d=6.55 and
5.85 ppm), respectively. In addition, the ratios of the con-
formers are 1:0.86 and 1:0.21, respectively. Conversely, their
diastereomers Ac-d-(aMe)Pro-d-(aMe)Pro-NHiPr and Ac-
l-Pro-d-Pro-NHiPr exhibit much simpler NMR spectra, thus
paralleling their FTIR absorption curves (Figure 9), indica-
tions of the ROESY spectra[76] of the homochiral (aMe)Pro
homo-dipeptide, the heterochiral Pro homo-dipeptide, and
the homo- and heterochiral -(aMe)Pro-Ala- dipeptides, re-
spectively.
A molecular model[13] and Figure 5 indicate that for the
homochiral Ac-d-(aMe)Pro-d-(aMe)Pro-NHiPr in a type
III’ b-turn conformation, a strong sequential connectivity is
expected between a dCH2 proton of (aMe)Pro1 and a dCH2
proton of (aMe)Pro2. This strong cross-peak is indeed ob-
served (Figure 10), accompanied by additional, weaker (se-
quential and medium-range) cross-peaks involving the sec-
ondary amide NH proton. The chemical shift difference be-
tween the Cb and Cg atoms (Ddbg) in Pro peptides has been
shown to be correlated with the y torsion angle.[2,3,77–80] In
this (aMe)Pro homo-dipeptide, we observed Ddbg values of
12.3 and 14.1 ppm for (aMe)Pro1 and (aMe)Pro2, respec-
tively. However, for (aMe)Pro-containing peptides this pa-
rameter is less conformationally indicative, as it is much
larger than that of Pro-containing peptides (8.0<Ddbg <
ꢀ7.0), in particular due to a significant downfield variation
in the chemical shift for the Cb atom associated with the
presence of the bCH3 substituent.[3]
ꢀ
tive of a 100% population of C=O···H N hydrogen-bonded
folded conformers. For these diastereomers the chemical
shifts of the isopropylamido NH proton single peak are seen
at d=7.11 and 7.06 ppm, respectively.
Complete assignments of the NMR signals were achieved
by COSY, TOCSY, and HMBC experiments at 400 MHz.
Figures 10 and 11, as well as Figure S3 in the Supporting In-
formation, show the most conformationally informative sec-
A molecular model[81] and Figure 6 show that for the het-
erochiral Ac-l-Pro-d-Pro-NHiPr in a type II b-turn confor-
mation, two strong sequential connectivities are expected
between the aCH
between the aCH
quential connectivity between a dCH
NH(iPr) proton should be also seen. In this conformation,
A
2ACHTUNGTREN(NGNU Pro2) protons and
A
ACHTUNGTRENNUNG
CTHUNGTRENNUNG
AHCTUNGTRENNUNG
the minimization of the pseudo A (1,3) strain between the
l-Pro1 aCH proton and the d-Pro2 d carbon restricts the ro-
tation of the Pro1 torsion angle y to approximately 1408
(semiextended conformation;[81] see also Table 1, and
Table S3 in the Supporting Information). All of the above-
mentioned cross-peaks are clearly apparent in Figure 11. In
this dipeptide Ddbg values of 3.1 and 5.5 ppm were seen for
Pro1 and Pro2, respectively.[2,3,77–80] A molecular model (not
shown) of Ac-l-(aMe)Pro-d-(aMe)Pro-NHiPr indicates
why this heterochiral dipeptide is not locked in the type II
b-turn conformation, typical of the -l-Pro-d-Pro- sequence.
The model shows a very short distance between the bCH3
substituent of residue 1 and the d carbon of residue 2.[13] Ro-
tation of the (aMe)Pro1 y torsion angle out of the region of
the semiextended conformation, required by the type II b
turn for the i+1 position, can relieve this interaction. This
finding is also consistent with the propensity of (aMe)Pro
for the helical region of the conformational space.
Figure 10. Sections of the ROESY spectrum of Ac-d-(aMe)Pro-d-
(aMe)Pro-NHiPr in CDCl3 ([peptide]=5 mm). The sequential connectivi-
ties bCH
G
2A
[(aMe)Pro1]!d1CH
A
and
d2CH
N
CHTUNGTRENNUNG
(inset) are indicated as well as the medium-range connectivity bCH3-
[(aMe)Pro1]!NHiPr.
ACHTUNGTRENNUNG
All connectivities involving the ring protons of the
(aMe)Pro2 residue seen in the ROESY spectrum of the
type III’ b turn forming Ac-d-(aMe)Pro-d-(aMe)Pro-NHiPr
(Figure 10) are clearly missing in the corresponding spec-
trum of Ac-d-(aMe)Pro-d-Ala-NHiPr (Figure S3, left, in the
Supporting Information). However, both spectra do exhibit
the only possible common connectivity, namely that between
a bCH3 proton of d-(aMe)Pro1 and the NHiPr proton.
Quite interestingly, if the structural restrictions imposed by
the presence of the dCH2 ring group of the (aMe)Pro2 resi-
Figure 11. Sections of the ROESY spectrum of Ac-l-Pro-d-Pro-NHiPr in
CDCl3 ([peptide]=5 mm). The sequential connectivities aCH
(Pro1)!
1,2CH
2A(Pro2), aCH 2A(Pro2)!
(Pro2)!NHiPr (inset, bottom), and d1CH
NHiPr (inset, top) are indicated as well as the medium-range connectivi-
ty aCH
(Pro1)!NHiPr (inset, bottom).
AHCTUNGTRENNUNG
d
N
G
CHTUNGTRENNUNG
ACHTUNGTRENNUNG
Chem. Eur. J. 2009, 15, 8015 – 8025
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8021

C. Toniolo et al.
due are removed, as in Ac-d-(aMe)Pro-l-Ala-NHiPr, then
d-(aMe)Pro1 can adopt the unusual semiextended confor-
mation and the related -d-(aMe)Pro-l-Ala- sequence can
fold in the type II’ b-turn conformation, typical of a -d,l-
heterochiral sequence. Figure S3 (right, in the Supporting
Information) shows that the cross-peak between a bCH3
proton of d-(aMe)Pro1 and the NHiPr proton is even more
intense than the corresponding one in the homochiral se-
quence (Figure S3, left, in the Supporting Information). This
observation is indeed expected on the basis of the related
distances seen in the X-ray diffraction structures of these
two -(aMe)Pro-Ala- dipeptides.[44,45] The Ddbg values for the
d-(aMe)Pro1 residue are 15.6 and 14.7 ppm for the
homochiral -d-(aMe)Pro-d-Ala- and the heterochiral
-d-(aMe)Pro-l-Ala- dipeptide sequences, respectively.
In summary, from our NMR spectroscopic data, it turns
out that the 3D structures adopted by the (aMe)Pro-con-
taining dipeptides in the crystalline state are highly (or ex-
clusively) populated in CDCl3 as well.
From our combined X-ray diffraction, FTIR absorption,
and NMR spectroscopy work on Ac-d-(aMe)Pro-d-
(aMe)Pro-NHiPr discussed above, it turns out clearly that
this simple dipeptide is unique in that its Z-protected ana-
logue is rigidly folded in a single, left-handed type III’ b turn
in the crystal state and it is fully folded in the same confor-
mation in CDCl3. Since the far-UV CD spectrum of a single
type II b turn is well established,[82–84] but the spectra of type
III and the closely related, nonhelical type I b turns have
been the matter of controversy for a long time, in part due
to the fact that not completely appropriate (long linear or
cyclic) model peptides were utilized in those studies, we de-
cided to take advantage of our short linear dipeptide to con-
clusively offer the correct CD spectrum for a type III/I b
turn. To avoid any ambiguity, we used acetonitrile (MeCN),
a solvent of low polarity (in this sense similar to CDCl3),
but (in contrast to CDCl3) compatible with a CD measure-
ment in the far-UV region. Figure 12 shows that the band of
free NH groups is absent in the FTIR absorption spectrum
in MeCN (only an intense band near 3330 cmꢀ1 is seen), cor-
roborating our data that this homochiral homo-dipeptide is
100% folded in an intramolecularly hydrogen-bonded b-
turn conformation in a solvent of low polarity. The corre-
sponding far-UV CD spectrum in MeCN (Figure 12) exhib-
its a very weak, positive shoulder at 225–230 nm followed by
a remarkably strong positive Cotton effect centered at
216 nm and a weak Cotton effect of opposite sign at 202 nm.
The crossover point between the two latter bands is seen at
205 nm. Obviously, in the CD spectrum of this d,d-config-
ured dipeptide, the signs of all Cotton effects are opposite
to those observed for the more common all-l peptides. Not
surprisingly, the overall shape of the CD spectrum in
Figure 12 closely resembles that of a 310 helix.[85–87] Indeed, a
type III/III’ b turn is the basic unit of the 310 helix.[9] Howev-
er, the positions of the Cotton effects and crossover point in
the spectrum of our model dipeptide are significantly shifted
(by about 8 nm) to longer wavelengths. This latter effect is
not associated with the nature of the solvent, as the CD
Figure 12. FTIR absorption spectrum (3500–3200 cmꢀ1 region) (top) and
far-UV CD spectrum (bottom) of Ac-d-(aMe)Pro-d-(aMe)Pro-NHiPr in
MeCN ([peptide]=1 mm).
spectrum of this same dipeptide in MeOH (not shown) ex-
hibits a very similar general shape.
Conclusion
An analysis of our experimental data allows us to draw the
following major conclusions:
1) Although the region of the (f,y) conformational map
overwhelmingly preferred by l-(aMe)Pro would indeed be
that typical of right-handed 310/a helices (with yꢂꢀ308 or
cis’), as suggested in 1974 by Leach and co-workers,[1] the
semiextended, type II poly
G
trans’) can also be explored (although rarely) by this ex-
tremely sterically hindered Ca-tetrasubstituted a-amino acid.
Conversely, the f,y region (yꢂ608), exactly halfway be-
tween the two regions discussed above and corresponding to
the C7’ (inverse g turn) conformation, does not seem to be
accessible to l-(aMe)Pro.
2) In addition to the dramatic restriction of the f torsion
angle (to ꢂꢀ608) by its five-membered pyrrolidine ring
structure, l-(aMe)Pro undergoes rigidification of the pre-
ceding tertiary peptide bond (trans, or 1808, w torsion angle)
as well, as shown by all Ca-methylated l-a-amino acids in-
vestigated to date.
3) The known high propensity of the l-Pro residue for b-
turn formation is even enhanced in peptides based on its Ca-
methylated derivative when it is located at the i+1 corner
position. Despite these characteristics, l-(aMe)Pro seems to
be unable to nucleate a b turn when it is located at the i+2
8022
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Chem. Eur. J. 2009, 15, 8015 – 8025

Ca-Methyl Proline
FULL PAPER
with the strongly basic resin Amberlyst A-26 was used to separate the
carboxylic acid and its amide. The amide was recovered by eluting with
water, and the carboxylic acid after eluting with 1n acetic acid. The two
fractions were evaporated to dryness, affording 7.3 g (49%) of l-amide
(79% ee) and 7.7 g (51%) of d-carboxylic acid (98% ee). The conversion
based on the enantiomeric excess (ee) was 45%. The carboxylic acid was
then stirred in cold iPrOH (50 mL) for 3 h, and filtered off as a solid
(4.6 g, 30%). 1H NMR (D2O, 300 MHz): d=3.31 (m, 2H;
CCH2CH2CH2), 2.27–1.88 (m, 4H; CCH2CH2CH2), 1.52 ppm (s, 3H;
CH3).
corner position of a homochiral dipeptide sequence (with a
coded amino acid at position i+1).
4) When incorporated at the i+1 corner position of the
dipeptide sequence, l-(aMe)Pro tends to bias the b turn to
its helical type (III), as opposed to the nonhelical type (II)
typically induced by l-Pro.
To obtain conclusive information on the (aMe)Pro con-
formational preferences, we are currently actively working
on the synthesis and 3D-structural characterization of the
(aMe)Pro homo-oligopeptides longer than dimers. Our goal
Enzymatic resolution of dl-(a-methyl)proline amide using Ochrobac-
trum anthropi: The amide (1.00 g, 7.8 mmol) was dissolved in an aqueous
solution of ZnSO4 (1 mm, 10 g) and the pH was adjusted to 6.5 by addi-
tion of acetic acid. The amidase of O. anthropi, overexpressed in E. coli
(20 mL cell-free extract), was added and the mixture was shaken at 378C.
After 26 h a conversion of 45% was reached, according to the ammonia
determination method. The reaction products were analyzed by HPLC:
d-carboxylic acid 98% ee and l-amide 91% ee The conversion based on
the ee was 48%. For the workup, see above.
is to assess whether the semiextended (type II polyACTHUNRGTNEUNG(l-Pro)n)
conformation would be populated at least under those “ex-
treme” conditions of sequence, as anticipated in earlier the-
oretical[11] and experimental[12] papers, and re-proposed in
recent conformational energy calculations.[28]
HPLC method for the enantiomeric excess determination: Column: Astec
CLC-L (150ꢂ4.6 mm i.d.); column temperature: 458C; eluant: 2 mm
CuSO4 in Mili-Q water; flow: 1.5 mLminꢀ1; detection UV: l=254 nm.
Experimental Section
X-ray diffraction: Colorless crystals of Z-d-(aMe)Pro-OH, tBoc-l-Ala-l-
(aMe)Pro-NHiPr, Z-Aib-d-(aMe)Pro-NHiPr, Ac-Aib-l-Pro-NHiPr, Z-d-
(aMe)Pro-d-(aMe)Pro-NHiPr, and Z-l-Pro-d-Pro-NHiPr were grown by
slow evaporation from MeOH, acetone, ethyl acetate, wet MeOH,
chloroform, and ethyl acetate, respectively. Diffraction data were collect-
ed at room temperature using a Philips PW1100 diffractometer in the q–
2q scan mode up to 2q=1208, using graphite-monochromated CuKa radi-
ation (l=1.54178 ꢁ). Intensities were corrected for Lorentz and polari-
zation effects, not for absorption. All structures were solved by direct
methods by use of the SIR 2002[88] program. Refinements were carried
out on F2 by full-matrix block least-squares, with use of all data, by appli-
cation of the SHELXL 97[89] program with all non-hydrogen atoms aniso-
tropic, and their positional parameters and the anisotropic displacement
parameters being allowed to refine at alternate cycles. Hydrogen atoms
of all peptide molecules were calculated at idealized positions and re-
fined using a riding model. The hydrogen atoms of the water molecule
cocrystallized with tBoc-l-Ala-l-(aMe)Pro-NHiPr were located on a DF
map and their positional parameters were not refined. The hydrogen
atoms of the water molecule cocrystallized with Ac-Aib-l-Pro-NHiPr
were located on a DF map and isotropically refined. The Pro Cg atom of
the same peptide was refined on two sites (atoms C2G and C2G’), each
with 0.5 occupancy.
Amino acid synthesis and resolution
N-Benzylidene-dl-(a-methyl)cyanoethylglycine amide: NaH (1.15 g, 0.04
mol, 60% in mineral oil) was added in small portions to a solution of N-
benzylidene-dl-alanine amide (50 g, 0.28 mol) and acrylonitrile (19.6 mL,
0.31 mol) in CH2Cl2 (400 mL). The temperature was kept at 208C with a
cooling bath. After 3 h no more starting material was present according
to TLC analyses. Then, the solution was washed with water (4ꢂ250 mL)
and the aqueous layer was extracted with CH2Cl2 (3ꢂ250 mL). The solu-
tion was stirred overnight and the precipitated side-product (3-(benzyli-
deamino)-3-methylglutaric imide) was filtered off. The solvent was re-
moved under reduced pressure, affording an oil that was dissolved in
methanol (120 mL). Addition of water (300 mL) then afforded a solid
(49.4 g, 76%). 1H NMR (CDCl3, 2H, 300 MHz), d=8.26 (s, 1H; NH=
CH), 7.81 (m, 2H; ortho-C6H5), 7.49 (m, 3H; meta- and para-C6H5), 7.35
(brs, 1H; CONH2), 6.05 (brs, 1H; CONH2), 2.44 (m, 3H; CH2CH2), 1.95
(m, 1H; CH2CH2), 1.54 ppm (s, 3H; CH3).
dl-(a-Methyl)cyanoethylglycine amide: N-Benzylidene-dl-(a-methyl)cya-
noethylglycine amide (48.55 g, 0.38 mol) was dissolved in CH2Cl2
(150 mL) and 4n HCl (70 mL, 1.2 equiv) was added under vigorous stir-
ring. After stirring for 1 h, the organic and aqueous layers were separat-
ed. The organic layer was extracted with a 0.1n HCl solution and the two
aqueous layers were combined. The acidic solution was made basic with
10n NaOH solution and concentrated under reduced pressure. The resi-
due was dissolved in CHCl3, and NaCl was filtered off. The organic layer
was dried over Na2SO4 and evaporated to dryness. Yield: 23.7 g, 80%;
1H NMR (CDCl3, 2H, 300 MHz), d=7.30 and 5.83 (2brs, 2H; CONH2);
2.44 (m, 2H; CH2CH2CN); 2.25 and 1.80 (m, 2H; CH2CH2CN); 1.49
(brs, 2H; CH3CNH2); 1.39 ppm (s, 3H; CH3).
Z-d-(aMe)Pro-OH: C14H17NO4; crystal size 0.50ꢂ0.45ꢂ0.35 mm; ortho-
rhombic; space group P212121; a=7.287(2), b=8.256(2), c=22.860(4) ꢁ;
V=1375.3(6) ꢁ3; Z=4; 1calcd =1.272 Mgmꢀ3; m=0.773 mmꢀ1; 1411 col-
lected reflections; 1377 independent reflections (Rint =0.048); data/pa-
rameters 1377/161; R1 =0.047 with Iꢃ2s(I); wR2 =0.139 (on F2, all data);
goodness-of-fit on F2 1.186; residual electron density 0.216/ꢀ177 eꢁꢀ3
.
tBoc-l-Ala-l-(aMe)Pro-NHiPr monohydrate: C17H33N3O5; crystal size
0.40ꢂ0.30ꢂ0.20 mm; monoclinic; space group P21; a=6.514(2), b=
dl-(a-Methyl)proline amide: A 25% solution of NH4OH (13 mL) and
5% Pd/C (4.7 g, Johnson Matthey type 39, 50% H2O) were added to a
solution of dl-(a-methyl)cyanoethylglycine amide (20 g, 0.14 mol) in
MeOH (120 mL). The mixture was hydrogenated at 508C and 30 bar of
H2 pressure in an autoclave. After 23 h the mixture was cooled down and
the catalyst was filtered off through decalite. The solvent was removed
under reduced pressure, affording the product as an oil. Yield: 18 g,
100%; 1H NMR (CDCl3, 2H, 300 MHz), d=7.70 and 5.25 (2brs, 2H;
CONH2); 3.10 and 2.85 (2m, 2H; CCH2CH2CH2); 2.25–1.60 (m, 5H;
CCH2CH2CH2NH); 1.41 ppm (s, 3H; CH3).
13.794(3), c=11.847(3) ꢁ; b=95.84(4)8; V=1059.0(5) ꢁ3; Z=2; 1calcd
=
1.127 Mgmꢀ3; m=0.678 mmꢀ1; 1864 collected reflections; 1788 independ-
ent reflections (Rint =0.087); data/parameters 1788/227; R1 =0.066 with
Iꢃ2s(I); wR2 =0.181 (on F2, all data); goodness-of-fit on F2 1.107; residu-
al electron density 0.267/ꢀ348 eꢁꢀ3
.
Z-Aib-d-(aMe)Pro-NHiPr: C21H31N3O4; crystal size 0.50ꢂ0.40ꢂ0.25 mm;
orthorhombic; space group P212121; a=9.172(2), b=10.921(2), c=
42.936(5) ꢁ; V=4300.8(13) ꢁ3; Z=8; 1calcd =1.203 Mgmꢀ3
;
m=
0.678 mmꢀ1
; 4172 collected reflections; 4070 independent reflections
(Rint =0.036); data/parameters 4070/482; R1 =0.054 with Iꢃ2s(I); wR2 =
Enzymatic resolution of dl-(a-methyl)proline amide using Mycobacteri-
um neoaurum: The above amide (15 g, 0.117 mol) was dissolved in H2O
and the pH was adjusted to 8.5 by addition of acetic acid. Freeze-dried
whole cells of M. neoaurum (1.5 g) were added and the mixture was
shaken at 378C. The reaction was stopped after 70 h when the conversion
was 48%, according to the ammonia determination method. The cell
mass was removed by centrifugation, and ion-exchange chromatography
0.159 (on F2, all data); goodness-of-fit on F2 1.079; residual electron den-
sity 0.189/ꢀ193 eꢁꢀ3
.
Ac-Aib-l-Pro-NHiPr monohydrate: C14H27N3O4; crystal size 0.50ꢂ0.25ꢂ
0.20 mm; monoclinic; space group P21; a=8.336(1), b=12.996(3), c=
8.816(2) ꢁ, b=116.91(4)8; V=851.7(3) ꢁ3; Z=2; 1calcd =1.175 Mgmꢀ3
;
m=0.707 mmꢀ1; 1523 collected reflections; 1439 independent reflections
Chem. Eur. J. 2009, 15, 8015 – 8025
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www.chemeurj.org
8023

C. Toniolo et al.
(Rint =0.134); data/parameters 1439/211; R1 =0.059 with Iꢃ2s(I); wR2 =
0.165 (on F2, all data); goodness-of-fit on F2 1.056; residual electron den-
[14] N. G. J. Richards, M. G. Hinds, D. M. Brennand, M. J. Glennie, J. H.
Welsh, J. A. Robinson, Biochem. Pharmacol. 1990, 40, 119–123.
[15] M. G. Hinds, J. H. Welsh, D. M. Brennand, J. Fisher, M. J. Glennie,
N. G. J. Richards, D. L. Turner, J. A. Robinson, J. Med. Chem. 1991,
34, 1777–1789.
[16] J. H. Welsh, O. Zerbe, W. von Philipsborn, J. A. Robinson, FEBS
Lett. 1992, 297, 216–220.
[17] O. Zerbe, J. H. Welsh, J. A. Robinson, W. von Philipsborn, Magn.
Reson. Chem. 1992, 30, 683–685.
[18] O. Zerbe, J. H. Welsh, J. A. Robinson, W. von Philipsborn, J. Magn.
Reson. 1992, 100, 329–335.
[19] C. Bisang, C. Weber, J. Inglis, C. A. Schiffer, W. F. van Gunsteren, I.
Jelesarov, H. R. Bosshard, J. A. Robinson, J. Am. Chem. Soc. 1995,
117, 7904–7915.
sity 0.264/ꢀ301 eꢁꢀ3
.
Z-d-(aMe)Pro-d-(aMe)Pro-NHiPr: C23H33N3O4; crystal size 0.45ꢂ0.35ꢂ
0.20 mm; monoclinic; space group P21; a=9.473(2), b=10.594(3), c=
12.082(3) ꢁ, b=108.40(8)8; V=1150.5(5) ꢁ3; Z=2; 1calcd =1.199 Mgmꢀ3
;
m=0.665 mmꢀ1; 2068 collected reflections; 1986 independent reflections
(Rint =0.063); data/parameters 1986/260; R1 =0.083 with Iꢃ2s(I); wR2 =
0.221 (on F2, all data); goodness-of-fit on F2 1.084; residual electron den-
sity 0.304/ꢀ290 eꢁꢀ3
.
Z-l-Pro-d-Pro-NHiPr: C21H29N3O4; crystal size 0.40ꢂ0.40ꢂ0.25 mm;
monoclinic; space group P21; a=10.321(3), b=9.746(3), c=11.465(3) ꢁ;
b=115.34(7)8; V=1042.3(5) ꢁ3; Z=2; 1calcd =1.235 Mgmꢀ3
;
m=
0.699 mmꢀ1
; 1925 collected reflections; 1839 independent reflections
[20] A. P. Nanzer, A. E. Torda, C. Bisang, C. Weber, J. A. Robinson,
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CCDC 711587, 711588, 711589, 711590, 711591, and 711592 contain the
supplementary crystallographic data for this paper. These data can be ob-
tained from The Cambridge Crystallographic Data Centre by visiting
www.ccdc.cam.ac.uk/data_request/cif.
FTIR absorption spectroscopy: FTIR absorption spectra were recorded
using a Perkin–Elmer 1720 X FTIR spectrophotometer, nitrogen-flushed,
with a sample shuttle device and at 2 cmꢀ1 nominal resolution, averaging
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were used. Spectrograde deuterochloroform (99.8%, 2H) was obtained
from Fluka.
NMR spectroscopy: 1H NMR spectra were recorded using a Bruker AM
400 spectrometer. Measurements were carried out in CDCl3 (99.96% 2H
from Acros Organics) and DMSO (99.96% 2H6 from Acros Organics).
Circular dichroism spectroscopy: The CD spectra were obtained using a
Jasco J-715 dichrograph. Cylindrical, fused quartz cells of 1.0, 0.2, and
0.1 mm path lengths (Hellma) were used. The values are expressed in
terms of [V]T, the total molar ellipticity (degcm2 dmolꢀ1). Spectrograde
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Acknowledgements
The authors are grateful to the Padova trainees Andrea Dalla Bona and
Erika Andreetto who developed the synthesis of (aMe)Pro during their
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