The structural characterization of folded peptides containing the conformationally constrained β-amino acid residue β 2,2Ac6c

Article abstract of DOI:10.1002/hlca.201200537

Backbone alkylation has been shown to result in a dramatic reduction in the conformational space that is sterically accessible to α-amino acid residues in peptides. By extension, the presence of geminal dialkyl substituents at backbone atoms also restricts available conformational space for β and γ residues. Five peptides containing the achiral β^2,2- disubstituted β-amino acid residue, 1-(aminomethyl)cyclohexanecarboxylic acid (β^2,2Ac₆c), have been structurally characterized in crystals by X-ray diffraction. The tripeptide Boc-Aib-β ^2,2Ac₆c-Aib-OMe (1) adopts a novel fold stabilized by two intramolecular H-bonds (C₁₁ and C₉) of opposite directionality. The tetrapeptide Boc-[Aib-β^2,2Ac ₆c]₂-OMe (2) and pentapeptide Boc-[Aib-β ^2,2Ac₆c]₂-Aib-OMe (3) form short stretches of a hybrid αβ C₁₁ helix stabilized by two and three intramolecular H-bonds, respectively. The structure of the dipeptide Boc-Aib-β^2,2Ac₆c-OMe (5) does not reveal any intramolecular H-bond. The aggregation pattern in the crystal provides an example of an extended conformation of the β^2,2Ac₆c residue, forming a 'polar sheet' like H-bond. The protected derivative Ac-β^2,2Ac₆c-NHMe (4) adopts a locally folded gauche conformation about the C_βi?￡?C _α bonds (θ=-55.7°). Of the seven examples of β^2,2Ac₆c residues reported here, six adopt gauche conformations, a feature which promotes local folding when incorporated into peptides. A comparison between the conformational properties of β^2,2Ac₆c and β^3,3Ac₆c residues, in peptides, is presented. Backbone torsional parameters of H-bonded αβ/βα turns are derived from the structures presented in this study and earlier reports. Copyright

Full text of DOI:10.1002/hlca.201200537

Helvetica Chimica Acta – Vol. 95 (2012)
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The Structural Characterization of Folded Peptides Containing the
Conformationally Constrained b-Amino Acid Residue b2,2Ac6c
by Krishnayan Basuroya), Vasantham Karuppiahb), Narayanaswamy Shamala*a), and
Padmanabhan Balaram*b)
a) Department of Physics, Indian Institute of Science, Bangalore-560012, India
(fax: þ 91-80-2360-2602/-0683, e-mail: shamala@physics.iisc.ernet.in)
b) Molecular Biophysics Unit, Indian Institute of Science, Bangalore-560012, India
(fax: þ 91-80-2360-0535/-0683, e-mail : pb@mbu.iisc.ernet.in)
Dedicated to Prof. Dieter Seebach on the occasion of his 75th birthday
Backbone alkylation has been shown to result in a dramatic reduction in the conformational space
that is sterically accessible to a-amino acid residues in peptides. By extension, the presence of geminal
dialkyl substituents at backbone atoms also restricts available conformational space for b and g residues.
Five peptides containing the achiral b2,2-disubstituted b-amino acid residue, 1-(aminomethyl)cyclohex-
anecarboxylic acid (b2,2Ac6c), have been structurally characterized in crystals by X-ray diffraction. The
tripeptide Boc-Aib-b2,2Ac6c-Aib-OMe (1) adopts a novel fold stabilized by two intramolecular H-bonds
(C11 and C9) of opposite directionality. The tetrapeptide Boc-[Aib-b2,2Ac6c]2-OMe (2) and pentapeptide
Boc-[Aib-b2,2Ac6c]2-Aib-OMe (3) form short stretches of a hybrid ab C11 helix stabilized by two and
three intramolecular H-bonds, respectively. The structure of the dipeptide Boc-Aib-b2,2Ac6c-OMe (5)
does not reveal any intramolecular H-bond. The aggregation pattern in the crystal provides an example
of an extended conformation of the b2,2Ac6c residue, forming a ꢀpolar sheetꢁ like H-bond. The protected
derivative Ac-b2,2Ac6c-NHMe (4) adopts a locally folded gauche conformation about the CbꢀCa bonds
(q ¼ ꢀ55.78). Of the seven examples of b2,2Ac6c residues reported here, six adopt gauche conformations,
a feature which promotes local folding when incorporated into peptides. A comparison between the
conformational properties of b2,2Ac6c and b3,3Ac6c residues, in peptides, is presented. Backbone torsional
parameters of H-bonded ab/ba turns are derived from the structures presented in this study and earlier
reports.
Introduction. – Short peptides composed of the 20 a-amino acids that occur
naturally in proteins are characterized by an ensemble of conformational states in
solution. In the solid state, short a-peptide sequences invariably adopt extended
conformations, with intermolecular H-bonds, favoring sheet-like arrangements of
peptides. The incorporation of proline residues into short sequences facilitates local
folding, permitting characterization of b-turn conformations, stabilized by 4 ! 1 H-
bond in Pro-Xxx sequences [1]. This feature is a consequence of the locking of the
rotation about the NꢀCa (f) bond by side-chain backbone cyclization, necessitated by
the pyrrolidine ring of the Pro residue. Secondary-structure formation resulting in
helical folds have been shown to be chain-length-, solvent-, and sequence-dependent
[2][3]. In large polypeptides, exemplified by the remarkable range of folded structures
in proteins, local secondary structures are often stabilized by long-range tertiary
interactions [4]. The design of shorter a-peptide sequences with well-defined
ꢂ 2012 Verlag Helvetica Chimica Acta AG, Zꢃrich

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conformational preferences is readily achieved by the incorporation of guest residues
which impose local conformational restraints, thereby facilitating the nucleation of
secondary structures [5][6]. In works dating back to the 1970s [7][8], the a,a-
dialkylated residues, of which a-aminoisobutyric acid (Aib) residue is a prototype, were
shown to be extraordinarily efficient promoters of helix formation in short sequences
composed of a-amino acids [9 – 15]. Limiting conformational heterogeneity in solution
had an unanticipated effect of promoting peptide crystallizability, permitting definitive
structural characterization by X-ray diffraction [10][12]. Over the last 35 years, Aib-
containing linear peptide sequences constitute the largest group of peptide crystal
structures available in the Cambridge Crystallographic Data Centre, Cambridge,
England.
The principle of stereochemical constraints to engineer local folding nuclei may be
D
extended to the construction of peptide hairpins, where Pro-Xxx sequences strongly
promote hairpin formation with H-bond-registered antiparallel strands [16 – 19].
Considering the local conformational flexibility of the 19 non-proline a-amino acids
found in proteins, conventional wisdom in the field of peptide structures might have
suggested an even greater backbone conformational variability for b-amino acids, in
which an additional degree of torsional freedom has been introduced. While
conformational space for a-residues may be defined, by using the Ramachandran
angles f (NꢀCa) and y (CaꢀC¼O) [20], three torsional variables, f (NꢀCb), q
(CbꢀCa), and y (CaꢀC¼O), define the structure space for b residues [21]. In the early
1990s, few researchers in the field would have ventured to suggest that well-ordered
folded structures could indeed be formed in sequences containing backbone-
homologated b-amino acid residues. Dieter Seebachꢁs 1996 report on the folded
conformation of a hexapeptide composed of six b-amino acids in solution raised the
intriguing possibility that the backbone-homologated residues, especially b- and g-
residues, may indeed have an intrinsic propensity to support folded structures [22].
Locally folded conformation require gauche or near gauche conformations about the
CbꢀCa (q) bonds in b-residues. Sam Gellmanꢁs use of constrained b-residues, in which
the CbꢀCa (q) bond was locked into five- and six-membered rings, resulted in the first
characterization of helical structures in b oligopeptides in the crystalline state [23][24].
Most importantly, the early reports on b-peptides raised the intriguing possibility that
H-bonded helical structures, hitherto unknown in a-peptides, may be constructed using
b- and g-residues [25 – 30].
Can backbone-homologated w-amino acid residues be incorporated into designed
secondary structures formed by a-amino acid sequences? Accommodattion of
additional backbone CH2 groups into canonical helical folds was realized in solution,
in the peptide Boc-Leu-Aib-Val-d-Ava-Leu-Aib-Val-OMe [31]. The construction of
helical structures with hybrid backbones was demonstrated in the crystal structures of
eleven and fourteen residue peptides containing internal b-Ala-g-Abu segments [32].
(The term b-alanine (b-Ala) was used in the literature for the amino acid b-
aminopropionic acid. In subsequent years, following the explosion of interest in b-
homologs of protein amino acids, it is preferable to use the description b-Gly or b-hGly
suggested by Seebach et al. [28].) These structures contained unsubstituted b- and g-
residues which may be formally viewed as higher homologs of the Gly residue. By
analogy with a-amino acids, it may then be anticipated that the introduction of

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backbone substituents would significantly reduce sterically accessible conformational
space, a feature best understood by comparing Ramachandran-allowed regions for Gly
and Ala residues [33]. Furthermore, geminal dialkyl substituents on backbone atoms
may be expected to dramatically limit allowed regions of conformational space, a
feature apparent by comparing the Ramachandran maps for Ala and Aib residues
[9][34]. In the extensive work that emanated from Seebachꢁs laboratory, multiply
substituted b-amino acids quickly became the objects of study. A 1998 paper first
reported the synthesis and structural characterization of peptides containing geminally
disubstituted b2,2- and b3,3-amino acids [35]. The residues chosen were the homologs of
the well-characterized, conformationally constrained amino acid 1-aminocyclohexane-
1-carboxylic acid (Ac6c) [36][37]. Fig. 1 shows the structures of the three related
residues, Ac6c, b2,2Ac6c, and b3,3Ac6c. In all three amino acid residues, both possible
chair conformations of the cyclohexane ring are observed. Seebach et al. reported
crystal structures of the protected amino acid Boc-b2,2Ac6c-OH and tripeptide Boc-
b2,2Ac6c-b2,2Ac6c-b2,2Ac6c-OMe. A novel ten-atom H-bond NHi ··· COiþ1(1 ! 2) with a
directionality opposite to that normally observed in a-peptide structures was
established in the tripeptide ester.
Fig. 1. Chair conformations of the 1,1-disubstituted cyclohexane moieties in the amino acid residues Ac6c,
b3,3Ac6c, and b2,2Ac6c. Conformations represented are from previously published crystal structures. a)
Boc-Aib-Ac6c-OMe [37], b) N-[1-(3,5-Dimethylbenzoyl)cyclohexyl]-3-methoxy-2-methylbenzami-
deꢀmethanol solvate [41], c) Boc-Phe-b3,3Ac6c-NHMe [40], d) Boc-b3,3Ac6c-b3,3Ac6c-NHMe [38], e)
Boc-b2,2Ac6c-b2,2Ac6c-b2,2Ac6c-OMe [35], f) Boc-b2,2Ac6c-OH [35].

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A later study from in our laboratory provided several crystal structures of short
peptides containing the b3,3Ac6c residue [38][39]. Only one example of an internally H-
bonded ab-turn, which is a backbone-expanded analog of b-turn in the peptide was
observed [39]. We, therefore, turned to a more extensive structural characterization of
peptides containing the b2,2Ac6c residue [40]. Fig. 2 shows the structures of the peptides
studied. In this paper, intramolecularly H-bonded folded structures of hybrid ab-
peptides, Boc-Aib-b2,2Ac6c-Aib-OMe (1), Boc-[Aib-b2,2Ac6c]2-OMe (2), and Boc-
[Aib-b2,2Ac6c]2-Aib-OMe (3), are described. In addition, structures of Ac-b2,2Ac6c-
NHMe (4) and Boc-Aib-b2,2Ac6c-OMe (5) are also reported.
Fig. 2. Structures of the peptides 1 – 5
Results. – Fig. 3 shows the molecular conformations determined in crystals for the
peptides Boc-Aib-b2,2Ac6c-Aib-OMe (1), Boc-[Aib-b2,2Ac6c]2-OMe (2), and Boc-[Aib-
b2,2Ac6c]2-Aib-OMe (3). In all three cases, the molecules adopt folded conformations
in the solid state, stabilized by intramolecular C¼O ··· HN H-bonds. Fig. 4, shows the
structures of the protected amino acid derivative Ac-b2,2Ac6c-NHMe (4) and protected
dipeptide ester Boc-Aib-b2,2Ac6c-OMe (5), both of which adopt conformations lacking
intramolecular H-bonds. The backbone torsion-angle parameters are compiled in
Table 1, and the H-bond parameters are collected in Table 2.
Boc-Aib-b2,2Ac6c-Aib-OMe (1). In this protected tripeptide, two internal H-bonds
of opposite directionality are observed. The Aib(1)-b2,2Ac6c(2) (ab) segment forms a
C11 H-bonded turn, which is a backbone-expanded analog of 4 ! 1 H-bonded C10 b-
turns observed in (aa)n sequences. The C-terminus dipeptide segment b2,2Ac6c(2)-
Aib(3) (ba) forms a C9 H-bonded structure (b2,2Ac6c(2)NH ··· O¼C Aib(3)). This H-
bonded conformational feature has been previously characterized in solution [42][43]
and in the solid state [44] for short peptides containing b-amino acids. A notable feature
of this structure is that both Aib(1) and Aib(3) residues adopt polyproline (PII)
conformations (f ꢁꢀ 608,y ꢁ 1208). PII Conformations at Aib are extremely rare in
folded oligopeptides [12], where helical conformations (aL/aR) (f ꢁꢂ 608,y ꢁꢂ 308)
are invariably observed [5][6]. Clearly, the energy penalty for forcing Aib residues into

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Fig. 3. Molecular conformations in crystals. a) Boc-Aib-b2,2Ac6c-Aib-OMe (1), b) Boc-[Aib-b2,2Ac6c]2-
OMe (2), c) Boc-[Aib-b2,2Ac6c]2-Aib-OMe (3) (a lone solvent molecule (CHCl3) is also observed in the
crystal).
Fig. 4. Molecular conformations in crystals. a) Ac-b2,2Ac6c-NHMe (4), b) Boc-Aib-b2,2Ac6c-OMe (5).
an unfavorable PII conformation has been paid by the simultaneous formation of the C11
and C9 intramolecular H-bonds.

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Table 1. Backbone Torsion Angles of Peptides 1 – 5
Peptides
Residues
Backbone torsion angles [8]
f
q
y
Boc-Aib-b2,2Ac6c-Aib-OMe (1)
Aib(1)
ꢀ 56.9
97.5
ꢀ 51.5
122.1
b2,2Ac6c(2)
Aib(3)
61.9
80.7
ꢀ 94.3
141.8
Boc-[Aib-b2,2Ac6c]2-OMe (2)
Aib(1)
ꢀ 58.4
ꢀ 36.7
ꢀ 72.4
ꢀ 44.6
94.6
b2,2Ac6c(2)
Aib(3)
ꢀ 108.6
ꢀ 53.2
93.4
b2,2Ac6c(4)
ꢀ 68.8
Boc-[Aib-b2,2Ac6c]2-Aib-OMe (3)
Aib(1)
ꢀ 67.0
ꢀ 108.5
ꢀ 53.1
ꢀ 103.5
49.2
ꢀ 27.2
ꢀ 68.8
ꢀ 34.4
ꢀ 98.1
42.7
b2,2Ac6c(2)
Aib(3)
82.9
70.6
b2,2Ac6c(4)
Aib(5)
Ac-b2,2Ac6c-NHMe (4)
Boc-Aib-b2,2Ac6c-OMe (5)
b2,2Ac6c
ꢀ 100.8
ꢀ 55.7
ꢀ 63.0
Aib(1)
b2,2Ac6c(2)
ꢀ 65.5
ꢀ 26.8
ꢀ 102.5
170.0
81.1
Boc-(Aib-b2,2Ac6c)2-OMe (2). The structure of the tetrapeptide reveals two
consecutive C11 H-bonded ab/ba-turns, corresponding to a single turn of a C11 helix.
This is a backbone-expanded analog of the incipient 310-helical structures characterized
in short a-peptides [7 – 9][45]. In this case, both Aib residues adopt the anticipated
helical conformations, in sharp contrast to the observations for the peptide 1.
Boc-(Aib-b2,2Ac6c)2-Aib-OMe (3). Extension of peptide chain length results in a
further propagation of the C11 helix, with three consecutive C11 H-bonds being observed
in the abab-segment. This structure is analogous to previously determined short C10 H-
bonded 310 helices in all a-pentapeptides [8]. Here again, all three Aib residues adopt
helical conformation with a helix sense reversal at the C-terminus, a feature commonly
observed in helical Aib peptides [12].
Ac-b2,2Ac6c-NHMe (4) and Boc-Aib-b2,2Ac6c-OMe (5). The protected derivative 4
and dipeptide ester 5 do not possess any intramolecular H-bonds in the observed
conformations (Fig. 4), with all donor and acceptor groups participating in intermo-
lecular interactions in the crystals. Fig. 5,a, shows a view of the molecular packing in the
crystals of 5. A pair of intermolecular H-bonds link adjacent molecules in rows in a
direction parallel to the crystallographic ꢀcꢁ axis (Fig. 5,b). The b2,2Ac6c residue adopts
a trans conformation about the CbꢀCa bond (q ¼ 1708), which orients the C¼O groups
of the N-terminus peptide unit and C-terminus ester group in the same direction,
facilitating formation of a motif refered to as a ꢀpolar sheetꢁ arrangement [46][47]. The
helical conformation of the Aib(1) residue orients the NH groups of the N-terminus
urethane and C-terminus amide in the same direction, resulting in intermolecular H-
bonds between adjacent molecules, forming an infinite chain along the direction of the
crystallographic ꢀcꢁ axis.
Conformation of b2,2Ac6c Residues. With the exception of the dipeptide ester Boc-
Aib-b2,2Ac6c-OMe (5), the b2,2Ac6c residues in all other cases adopt gauche

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Fig. 5. Packing of the molecules in the crystal of Boc-Aib-b2,2Ac6c-OMe (5). a) A projection down the
crystallographic ꢀbꢁ axis, b) Intermolecular H-bonding network, in a direction parallel to the
crystallographic ꢀcꢁ axis, c) A different projection, showing the intermolecular H-bonding network.
conformations, with q values ranging from 55.78 to 82.98. Thus far, a total of 13 b2,2Ac6c
residues have been crystallographically characterized from nine independent mole-
cules (eight different peptides and derivatives). Three of these peptide structures
containing b2,2Ac6c residues were reported earlier, two by Seebach et al. in 1998 [35]
and one from Bangalore [40]. Interestingly, of these 13 residues twelve reveal gauche
conformations about the CbꢀCa bond (q). The sole example of a trans conformation
was observed in the dipeptide ester 5, discussed above. This may be compared with the
case of the isomeric b3,3Ac6c residue, where a survey of 23 examples revealed 18 cases
of gauche and five cases of trans conformations. Folding into the intramolecularly H-
bonded structures, is facilitated by adoption of gauche conformation about the CbꢀCa
bond (q). The gem-dialkyl substituents at Ca in b2,2Ac6c may also be expected to restrict
the range of the torsion angle y about the CaꢀCO bond. A scatter plot in f, y space for
crystallographically characterized b2,2Ac6c residues is shown in Fig. 6. Twelve out of the
thirteen residues represented, adopt y values which are clustered about ꢂ 608 to ꢂ 908.
The sole example of an extended value of y ꢁ 1808 is observed in the tripeptide
Boc-b2,2Ac6c-b2,2Ac6c-b2,2Ac6c-OMe, reported by Seebach et al [35]. In this case a C10 H-
bond, b2,2Ac6c(2) NH ··· OC b2,2Ac6c(3), is observed. This reverse directionality 1 ! 2
C10 H-bond in the bb-segment is analogous to the 1 ! 2 C9 H-bond discussed earlier, for
the ba segment in the tripeptide 1. Presumably, the formation of the H-bond
compensates energetically for an unfavorable value of the y torsion angle.
H-Bonded Turns Involving b2,2Ac6c Residues. Fig. 7,a and b, illustrate two distinct
families of 4 ! 1 H-bonded C11 turns observed in the peptides 1 to 3, described in the
present study. Of these, the C11-helical ab/ba-turn is formed for backbone torsion-angle

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Fig. 6. Scatter plot in f,y space of crystallographically characterized b2,2Ac6c residues. For three distinct
!
*
*
classes of torsion-angle values about the CbꢀCa bond (q), with : q ꢁþ 608; : q ꢁꢀ 608, and : q ꢁ 1808.
Fig. 7. Two different types of 4 ! 1 C11 (ab) H-bonded turns observed in the present study. a) Helical turn
(torsion-angle values averaged over all helical peptides of the present study are shown). b) Non-helical
turn (torsion-angle values are taken from peptide 1, the sole example of this type discussed in the present
study). c) Non-helical turn in Boc-Phe-b3,3Ac6c-NHMe [40] (torsion-angle values are shown).
values of f ꢁꢀ 1008, q ꢁ 808, and y ꢁꢀ 808 at the b2,2Ac6c residue. The C11 ab non-
helical turn observed in the tripeptide 1 (Fig. 7,b) may be compared with the C11 turn
observed in the peptide Boc-Phe-b3,3Ac6c-NHMe (Fig. 7,c), in which the geminal-

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dialkyl substitution is effected at Cb (Fig. 7,c). In both cases, the a-residue adopts a
polyproline (PII) conformation. The b2,2Ac6c residues at the I þ 2 position in the two
peptides may be considered to belong to the same conformational family: f ꢁ 708 ꢂ 308,
q ꢁ 608 ꢂ 108, and y ꢁꢀ 808 ꢂ 208. The two classes of C11 turns shown in Fig. 7 may be
formally viewed as backbone expanded analogs of type-I/III and type-II b-turn
structures commonly observed for aa-sequences [48][49]. While the C11 turn in
Fig. 7,a, upon repetition generates a continuous C11 helix, the C11 conformations
depicted in Fig. 7,b and c, can serve as isolated chain reversals in larger sequences.
Conclusions. – The structural studies presented in this report point to the utility of
the b2,2Ac6c residue in generating folded conformations in ab-hybrid sequences. The
comparison with previously reported structural work on the related b3,3Ac6c residue
suggests that the positioning of the geminal dialkyl substituents on the b-residue
backbone can be used to modulate local conformational preferences. Synthetically
accessible achiral geminally disubstituted b residues may prove valuable in the design
of hybrid peptide foldamers.
This work was supported by a Program Grant from the Department of Biotechnology. K. V. thanks
the University Grant Commission for a D. S. Kothari postdoctoral fellowship.
Experimental Part
General. Abbreviations: Boc2O, di(tert-butyl)dicarbonate; DCC, N,N’-dicyclohexylcarbodiimide;
BtOH, 1-hydroxy-1H-benzotriazole; HATU, 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyuro-
niumhexafluorophosphate; NMM, N-methylmorpholine; Aib, a-aminoisobutyric acid; b2,2Ac6c, 1-
(aminomethyl)cyclohexanecarboxylic acid. THF was distilled over Na/benzophenone before use. CHCl3
employed for the coupling reactions was filtered over Al2O3. All other reagents were used as received
from Fluka and Sigma-Aldrich. The peptides were synthesized following standard solution-phase
procedures. TLC: silica gel 60 F254 plates (SiO2; Merck) using hexanes/AcOEt as eluent; visualization on
exposure to I2 vapor or UV light. HPLC: Reversed-phase (RP) C18 column (5 – 10 mm, 7.8 mm ꢃ
250 mm) using MeOH/H2O gradients. M.p.: Stuart Biocote SMP10 melting-point apparatus; uncorrected.
IR Spectra: JASCO spectrometer; ˜n in cmꢀ1. NMR Spectra: Bruker AV400 FT-NMR spectrometer
(400 MHz); d in ppm rel. to Me4Si as internal standard, J in Hz. ESI-MS: Bruker Daltonics Esquire-3000
instrument; in m/z.
Synthesis of N- and C-Protected b2,2Ac6c Residue (Scheme). Methyl 1-(aminomethyl)cyclohexane-
carboxylate (H-b2,2Ac6c-OMe; 9) was prepared using a previously published procedure via bisalkylation
of methyl 2-cyanoacetate (6) with 1,5-dibromopentane (7) [50 – 53]. The cyano ester 8 thus obtained was
hydrogenated using CoCl2 · 6 H2O to yield 9 [52][53]. Boc-Protection of 9 under solvent-free condition
in the presence of cat. amount of I2 [54], followed by saponification with 1n NaOH in MeOH, furnished
the Boc-protected amino acid 10a.
Peptide Synthesis. General Procedure (GP). i) Peptides 1 and 5 were synthesized via coupling
mediated by DCC/BtOH. DCC (1 equiv.) was added to an ice cold soln. of the Boc-protected amino acid
and peptide acid (1 equiv.) in dry THF/CHCl3, followed by the addition of BtOH (1.1 equiv.), and the
soln. was stirred for 30 min. After complete activation of the acid, the amino component (free amino acid
ester or dipeptide ester) was added dropwise, and the mixture was allowed to attain r.t. with stirring
continued for 24 h under N2. After completion of the reaction (TLC), dicyclohexylurea obtained as by-
product was filtered, and the filtrate was concentrated, diluted with CHCl3, and washed with 1n Na2CO3,
followed by sat. aq. NaCl soln. The org. layer was dried (MgSO4) and evaporated under vacuo.
ii) Peptides 2, 3, and 4 were synthezised via the HATU/BtOH-mediated coupling method. HATU
(1 equiv.) was added to an ice cold soln. of Boc-protected amino acid (1 equiv.) in dry DMF, followed by

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Scheme
the addition of BtOH (1 equiv.) and EtNiPr2 or NMM (3 equiv.). After complete activation of the acid,
the free amino ester (1.2 equiv.) was added dropwise, and the mixture was allowed to stir at r.t. for 24 h
under N2. The reaction was monitored using TLC. After completion of the reaction, DMF was
evaporated under reduced pressure. The crude product was dissolved in CHCl3, and washed with sat. aq.
NaHCO3, KHSO4 and NaCl solns. The org. layer was dried (MgSO4) and evaporated in vacuo. Following
the procedure described above, 9 was coupled with Boc-Aib-OH to yield the dipeptide ester 5.
Saponification of 5, followed by coupling with H-Aib-OMe, provided the tripeptide ester 1. The
dipeptide ester 5 was deprotected at the N-terminus and coupled with N-protected dipeptide acid to
afford the tetrapeptide ester 2. The Boc-protected dipeptide acid derived from 5 was coupled with the
free tripeptide ester derived from 1 to provide the pentapeptide 3. Deprotections of N- and C-termini
were achieved with 98% HCOOH and 2n NaOH/MeOH, resp.
Boc-Aib-b2,2Ac6c-OMe (5). The ester 9 (0.84 g, 4.9 mmol) was coupled with Boc-Aib-OH (0.5 g,
2.5 mmol) according to GP. Flash chromatography (FC) yielded 5 (0.684 g, 78%). Crystalline white solid.
M.p. 108 – 1108. Rf (Hexane/AcOEt 8 :2) 0.34. IR (CHCl3): 3344w, 2979m, 2934s, 2862w, 1718s, 1667s,
1520s, 1453m, 1367m, 1162s, 1078m, 1019w, 755w. 1H-NMR (400 MHz, CDCl3): 1.41 (s, 3 Me); 1.47 (s, 2
Me); 3.40 (d, J ¼ 6.4, CH2); 3.67 (s, MeO); 4.97 (s, NH); 6.78 (s, NH). ESI-MS: 379 ([M þ Na]þ), 395
([M þ K]þ).
Boc-Aib-b2,2Ac6c-Aib-OMe (1). H-Aib-OMe (0.21 g, 1.8 mmol) was coupled with Boc-Aib-b2,2Ac6c-
OH (0.5 g, 1.5 mmol) derived by saponification of 5 according to GP. FC yielded 1 (0.432 g, 67%). White
powder. M.p. 168 – 1698. Rf (Hexane/AcOEt 6 :4) 0.38. IR (CHCl3): 3365w, 3306w, 2931s, 2863w, 1745m,
1693s, 1526s, 1455m, 1364m, 1286s, 1078m, 1018w, 756w. 1H-NMR (400 MHz, CDCl3): 1.42 (s, 3 Me); 1.51
(s, 2 Me); 1.54 (s, 2 Me); 3.30 (d, J ¼ 6.4, CH2); 3.42 (d, J ¼ 6.4, CH2); 3.70 (s, MeO); 5.19 (s, NH); 6.73 (s,
NH); 7.4 (s, NH). ESI-MS: 464 ([M þ Na]þ), 480 ([M þ K]þ).
Boc-Aib-b2,2Ac6c-Aib-b2,2Ac6c-OMe (2). H-Aib-b2,2Ac6c-OMe (0.45 g, 1.8 mmol) derived by depro-
tection of Boc group of 5 was coupled with Boc-Aib-b2,2Ac6c-OH (0.5 g, 1.5 mmol) derived by
saponification of 5 according to GP. FC yielded 2 (0.458 g, 54%). White powder. M.p. 209 – 2108. Rf
(Hexane/AcOEt 1:1) 0.31. IR (CHCl3): 3274w, 2936m, 1726m, 1692m, 1647s, 1527, 1447m, 1384w, 1157m,
1080m, 1021w, 715w. 1H-NMR (400 MHz, CDCl3): 1.42 (s, 3 Me); 1.51 (s, 2 Me); 1.54 (s, 2 Me); 3.39 (d,
J ¼ 5.6, CH2); 3.43 (d, J ¼ 5.6, CH2); 3.70 (s, MeO); 5.19 (s, NH); 6.73 (s, NH); 7.02 (s, NH); 7.4 (s, NH).
ESI-MS: 603 ([M þ Na]þ), 619 ([M þ K]þ).
Boc-Aib-b2,2Ac6c-Aib-b2,2Ac6c-Aib-OMe (3). H-Aib-b2,2Ac6c-Aib-OMe (0.55 g, 1.6 mmol) derived
by deprotection of Boc group of 1 was coupled with Boc-Aib-b2,2Ac6c-OH (0.5 g, 1.5 mmol) derived by
saponification of 5 according to the general procedure. FC yielded 3 (0.21 g, 21%). White powder. M.p.
220 – 2218. Rf (Hexane/AcOEt 1:1) 0.31. IR (CHCl3): 3393w, 2934s, 2862w, 1716s, 1668s, 1521s, 1455m,
1367m, 1170s, 1076m, 1048w, 782w. 1H-NMR (400 MHz, CDCl3): 1.41 (s, 3 Me); 1.44 (s, 2 Me); 1.55 (s, 2

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Me); 3.22 (d, J ¼ 6.4, CH2); 3.28 (d, J ¼ 6.4, CH2); 3.75 (s, MeO); 5.23 (s, NH); 6.94 (s, NH); 7.09 (s, NH);
7.33 (s, NH); 7.50 (s, NH). ESI-MS: 688 ([M þ Na]þ), 704 ([M þ K]þ).
Ac-b2,2Ac6c-NHMe (4). Acetylation of 9 (1 equiv.) with Ac2O (1 equiv.) in the presence of cat.
amount of I2 (10 mol %) under solvent-free conditions furnished Ac-b2,2Ac6c-OMe. Saponification
followed by coupling of the resulting acid 10b (0.5 g, 2.5 mmol) with MeNH2 · HCl (0.12 g, 3.8 mmol)
mediated by HATU in the presence of EtNiPr2 afforded 4 (0.15 g, 36%). White solid. M.p. 199 – 2008. Rf
(Hexane/AcOEt 1:1) 0.45. IR (CHCl3): 3321w, 2934s, 2862w, 1723s, 1535s, 1249s, 1161s, 1355m, 1367m,
1
1170s, 976m, 620w. H-NMR (400 MHz, CDCl3): 1.97 (s, Me); 2.82 (d, J ¼ 4.8, CH2); 3.67 (d, J ¼ 4.8,
CH2); 5.96 (s, NH); 6.22 (s, NH). ESI-MS: 235 ([M þ Na]þ), 251 ([M þ K]þ).
X-Ray Diffraction. Suitable single crystals of all the peptides and amino acid derivatives 1 – 5 were
obtained by the slow-evaporation method. Single crystals for peptides 1 and 5 were grown by dissolving
ca. 8 mg of the peptide in 200 ml of AcOEt and 50 ml of hexane. Colorless single crystals of tetrapeptide 2
were obtained by dissolving 6 – 8 mg of the peptide in 300 ml of MeOH and 50 ml of H2O. Pentapeptide 3
and 4 were crystallized by dissolving 6 – 8 mg of the peptides in 400 ml and 300 ml of CHCl3, resp., and
then 2 – 3 drops of hexane were added to both solns. Peptides 1 and 3 crystallized in the monoclinic space
group P21/n. Ac-b2,2Ac6c-NHMe (4) and the dipeptide 5, both crystallized in the monoclinic space group
Cc, with one molecule in the asymmetric unit. The tetrapeptide 2 crystallized with one peptide molecule
in the asymmetric unit, in the orthorhombic space group Pbca. A cocrystallized solvent molecule
(CHCl3) was observed only in the case of 3.
For peptides 1, 2, and 4, X-ray data were collected on Bruker AXS ULTRA APEXII CCD (rotating
anode X-ray generator) with CuKa (l 1.54178 ꢄ) radiation. For peptides 3 and 5, the X-ray data were
collected on Bruker AXS KAPPA APEXII CCD with MoKa (l 0.71073 ꢄ) radiation. All the X-ray
diffraction data sets were collected at r.t. (296 K). In all these cases, the X-ray data were acquired in f
and w scan mode. The structures were solved by using iterative dual-space direct methods in SHELXD
[55]. The structures were refined against F2 isotropically, followed by full-matrix anisotropic least-
squares refinement using SHELXL-97 [56][57]. The solvent molecule in peptide 3 were located from
difference Fourier map. All H-atoms, with the exception of the terminal Me groups (Boc, MeO, Ac,
MeNH), were located from difference Fourier maps. For the terminal Me groups, the H-atoms were fixed
geometrically in idealized position and allowed to ride with the respective C-atoms, to which each H-
atom was bonded, in the final cycles of refinement. Details of crystal data and structure refinement
parameters are compiled in Table 3.
CCDC Deposition Nos. for peptides and derivative are 899142 (1), 899143 (2), 899144 (3), 899139
(4), and 899141 (5), which contain the crystallographic data of the peptides mentioned in this manuscript
and can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.
cam.ac.uk/data_request/cif.
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Received September 22, 2012