Serendipitous discovery of peptide dialkyl peroxides

Article abstract of DOI:10.1002/1522-2675(200210)85:10<3099::AID-HLCA3099>3.0.CO;2-S

In an attempt to synthesize a homologous series of peptide peresters, we investigated the reaction of the oxazol-5(4H)-ones of Pht-(Aib)_n-OH (n=2-8) and tert-butyl hydroperoxide in the presence of 4-(dimethylamino)pyridine. Unexpectedly, the ma

Full text of DOI:10.1002/1522-2675(200210)85:10<3099::AID-HLCA3099>3.0.CO;2-S

Helvetica Chimica Acta Vol. 85 (2002)
3099
Serendipitous Discovery of Peptide Dialkyl Peroxides
by Alessandro Moretto, Marta De Zotti, Laura Scipionato, Fernando Formaggio, Marco Crisma,
and Claudio Toniolo*
Institute of Biomolecular Chemistry, CNR, Department of Organic Chemistry, University of Padova,
I-35131 Padova (tel.: (‡ 39) 049-827-5247; fax: (‡ 39) 049-827-5239; e-mail: claudio.toniolo@unipd.it)
and
Sabrina Antonello and Flavio Maran
Department of Physical Chemistry, University of Padova, I-35131 Padova
and
Quirinus B. Broxterman
DSM Fine Chemicals, Advanced Synthesis and Catalysis, P.O. Box 18, 6160 MD Geleen, The Netherlands
Dedicated to Professor Dieter Seebach on the occasion of his 65th birthday
In an attempt to synthesize a homologous series of peptide peresters, we investigated the reaction of the
oxazol-5(4H)-ones of Pht-(Aib)n-OH (n ˆ 2 8) and tert-butyl hydroperoxide in the presence of 4-
(dimethylamino)pyridine. Unexpectedly, the major product isolated in each case proved to be the peptide
dialkyl peroxide. This novel class of peptide derivatives was characterized by FT-IR, 1H-NMR, MS, cyclic
voltammetry, and X-ray diffraction. On the basis of the experimental data, a plausible mechanism is proposed
for this reaction.
Introduction. In the last few years, the study of intramolecular electron-transfer
(ET) reactions in donor-spacer-acceptor (D-spacer-A) molecules has provided
relevant information on how electrons are transferred through bonds and through
space (solvent). We have recently investigated intramolecular ET reactions in systems
in which A undergoes reductive cleavage of a s-bond (dissociative electron transfer,
DET). More specifically, as illustrated in Scheme 1, the phthalimido (Pht) moiety of
Aib (a-aminoisobutyric acid), which is easily reduced to its radical anion, was selected
as the donor D. In this system, the acceptor A is the Aib-O-OtBu perester function, a
well-characterized dissociative-type acceptor [1]. Interestingly, we found in our
electrochemical study that the DET rate of Pht-Aib-O-OtBu is several orders of
magnitude lower than the adiabatic limit.
Scheme 1
O
O
O
O
DET
N
O
N
O
+
O
O
e
O
O
e

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Helvetica Chimica Acta Vol. 85 (2002)
We also tackled the problem of the distance dependency of intramolecular DET
processes. To achieve this goal, we decided to exploit rigid 310-helical peptide spacers
[2] to control the electronic interaction between the D and A redox sites. Accordingly,
since Aib homo-oligomers are known to fold into this ordered secondary structure
[3][4], we planned the synthesis of the peptide perester series of type I. In the
published syntheses of I (n ˆ 0), activation of the COOH group and coupling of Pht-
Aib-OH [5] was achieved via the corresponding acid chloride and pyridine [6], or, in
higher yields, with 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (EDC) and 4-
(dimethylamino)pyridine (DMAP) [1], or with the acid chloride and DMAP [7]. It is
reasonable to assume that all these methods lead to the predominant formation of an
acylpyridinium intermediate [8] (in addition to a smaller amount of symmetrical
t
anhydride [6][9]), which, in the presence of BuOOH (tert-butyl hydroperoxide),
would eventually afford the Na-protected a-aminoacyl perester.
Any type of COOH activation of Na-protected Aib homo-peptides overwhelmingly
generates the corresponding oxazol-5(4H)-ones [10][11]. These reactive intermediates,
in the presence of DMAP/tBuOOH, were expected to afford the desired peptide
peresters. In this paper, we show that, surprisingly enough, this reaction leads instead to
a series of peptide dialkyl peroxides of type II as the major products.
O
O
N
O
O
I
N
H
O
O
O
n
O
H
N
N
O
II
N
O
H
O
O
n–1
Results and Discussion. Peptide Synthesis. The new Na-blocked Aib derivatives
and homo-peptide esters were prepared by known methodologies. In particular, Phac-
Aib-OtBu (Phac ˆ phenylacetyl) and Piv-Aib-OtBu (Piv ˆ pivaloyl) were synthesized
from H-Aib-OtBu [10] by the EDC/HOBt method [12] (HOBt ˆ 1-hydroxy-1,2,3-
benzotriazole) and the symmetrical anhydride method, respectively. The Pht-(Aib)n-
OtBu (n ˆ 2 8) homo-peptide series was obtained either from Pht-Aib-Cl [13] and the
pertinent H-(Aib)nÀ1-OtBu [10] amino component in CH2Cl2 in the presence of
(iPr)2NEt or by the segment-condensation approach in MeCN using a peptide oxazol-
5(4H)-one as the carboxy component [10][11][14][15]. The Na-deprotected H-
(Aib)nÀ1-OtBu homo-peptides were prepared by catalytic hydrogenation of the
corresponding benzyloxycarbonyl (Z) derivatives in MeOH [10]. The Na-protected,
C-terminally deprotected peptide acids [10][14] were obtained from the corresponding
tBu esters by treatment with dilute trifluoroacetic acid (TFA). The corresponding Na-
protected peptide oxazolones were synthesized by treatment of the Na-protected
peptide free acids with EDC in MeCN. The same synthetic steps were used to prepare

Helvetica Chimica Acta Vol. 85 (2002)
3101
the oxazolone from Pht-Aib-[l-(a-Me)Val]2-OH (Val ˆ valine). The syntheses and
characterizations of Z-Aib-OH [14][16], Piv-Aib-OH [17], Pht-Aib-OH [5], and Z-[l-
(a-Me)Val]2-OtBu [18] have already been reported.
Attempted Synthesis of Peptide Peresters. Three methods have been published for
the synthesis of Pht-Aib-O-OtBu [1][6][7]. To prepare the higher homologues of the
(Aib)n peptide perester series, we selected the method that exploits a carbodiimide
(EDC) and a pyridine derivative (DMAP) to activate the COOH function, followed by
addition of tBuOOH in CH2Cl2 (80% yield). The same approach was used to synthesize
the Z-, Phac-, and Piv-Na-blocked Aib derivatives. As mentioned above, in the
presence of a carbodiimide, Na-blocked Aib derivatives (except for Pht-Aib-OH) and
peptides are known to afford the corresponding oxazol-5(4H)-ones [10][11]. These
compounds were either isolated and characterized or prepared in situ. The X-ray
diffraction structure of the oxazol-5(4H)-one of Pht-(Aib)2-OH, solved in this work, is
shown in Fig. 1. For a comparison with the crystal structures of other oxazolones of
Aib-containing peptides and Pht-Na-blocked Aib derivatives, see [19] and [9],
respectively.
Fig. 1. X-Ray crystal structure (ORTEP view) and numbering of the oxazol-5(4H)-one of Pht-(Aib)2-OH.
Displacement ellipsoids are shown at the 30% probability level.
All our experiments led to a major final product that was purified by flash
chromatography. However, yields were modest (25 40%), except for the Z-Aib
derivative (68%). These compounds give rise to a reddish TLC spot when exposed to
NH4SCN/FeSO4/H2SO4 [20][21], which is typical for peroxy-containing compounds.
Characterization of Peptide Dialkyl Peroxides. The putative Na-blocked Aib
derivative and homo-peptide peresters were characterized by a number of analytical
1
and physico-chemical techniques, including FT-IR, H-NMR, MS, cyclic voltammetry,
and X-ray diffraction. The FT-IR absorption spectra of the Pht- and Z-protected amino
acid and peptide derivatives (KBr pellets) are complex in the carbonyl stretching
region and dominated by the contributions of the imide (two widely separated bands at
1780 1750 cmÀ1 and 1720 1700 cmÀ1) and urethane (ca. 1720 cmÀ1) absorptions,
respectively [22] (Table 1). However, in each of the simpler spectra of the Piv- and
Phac-derivatives, a single amide band is observed near 1660 cmÀ1. In other words, no

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Helvetica Chimica Acta Vol. 85 (2002)
evidence was obtained for the presence of a carbonyl stretching band indicative of the
perester function (1820 1750 cmÀ1) [22] in these two compounds.
The 1H-NMR resonances associated with the Me protons of the tBuO group of the
major reaction products (peroxides) in CD3CN are listed in Table 2 and compared with
those of the corresponding esters. While the latter are found at 1.43 1.35 ppm, the
former appear more upfield, between 1.19 and 1.11 ppm. Interestingly, the correspond-
ing signal for the perester Pht-Aib-O-OtBu is displayed in an intermediate region
(1.25 ppm). These findings further indicate that the major reaction products are distinct
compounds from esters and peresters.
Mass-spectral analysis afforded more informative data. All experimentally
determined molecular weights were consistently 28 mass units lower than calculated
for the putative peptide peresters (Table 3).
The chemical structures of the new compounds were finally solved by X-ray
diffraction analysis. We were able to grow suitable single crystals of the Pht-(Aib)3
derivative under two different conditions, namely by vapour diffusion from CHCl3/
petroleum ether and by slow evaporation from MeOH. In Figs. 2 and 3, the X-ray
structures of Pht-(Aib)2-NH-C(Me)2-O-OtBu and of an artifact thereof, [2-(methoxy-
carbonyl)]Bz-(Aib)2-NH-C(Me)2-O-OtBu (Bz ˆ benzoyl), are depicted, the latter
resulting from prolonged exposure to MeOH (alcoholysis of the phthalimide protecting
group). However, the most interesting observation is that the reaction product differs
from the putative tripeptide perester in the sense that the C-terminal CˆO moiety is
missing. Therefore, the resulting product is an Na-blocked Aib homopeptide dialkyl
peroxide. This finding is in full agreement with all our physico-chemical and analytical
investigations discussed above.
Fig. 2. X-Ray crystal structure (ORTEP view) and numbering of Pht-(Aib)2-NH-C(Me2)-O-OtBu. Displace-
ment ellipsoids are shown at the 30% probability level. An intramolecular CˆO ¥¥¥ HÀN H-bond is indicated by
a dashed line.

Helvetica Chimica Acta Vol. 85 (2002)
3103
Table 1. Physical and Analytical Properties of Synthesized Peptides (for abbreviations and conventions, see text
and Exper. Part)
Compound
M.p. [8]
Recryst.
solvent
TLC
IR [cmÀ1
]
RfI
RfII
RfIII
Phac-Aib-OtBu
Phac-Aib-OH
Piv-Aib-OtBu
83 84
177 178
82 83
AcOEt/PE
EtOH/Et2O
AcOEt/PE
0.80
0.35
0.85
0.95
0.85
0.95
0.60
0.20
0.75
3305, 1730, 1643, 1541
3374, 1722, 1613, 1540
3384, 1736, 1716, 1650,
1522
3387, 1775, 1713, 1681,
1517
3401, 1775, 1745, 1702,
1649, 1521
1818, 1777, 1708, 1679
Pht-(Aib)2-OtBu
Pht-(Aib)2-OH
oil
AcOEt/PE
EtOH/Et2O
Et2O/PE
0.90
0.60
0.95
0.75
0.50
0.70
0.40
0.70
0.35
0.65
0.30
0.65
0.25
0.65
0.20
0.95
0.85
0.95
0.95
0.95
0.85
0.80
0.85
0.80
0.85
0.75
0.85
0.75
0.85
0.75
0.65
0.35
0.65
0.50
0.20
0.40
0.15
0.35
0.15
0.35
0.10
0.35
0.10
0.35
0.10
208 209
85 86
Oxazol-5(4H)-one
of Pht-(Aib)2-OH
Pht-(Aib)3-OtBu
97 98
AcOEt/PE
EtOH/Et2O
AcOEt/PE
EtOH/Et2O
AcOEt/PE
EtOH/Et2O
AcOEt/PE
EtOH/Et2O
AcOEt/PE
EtOH/Et2O
AcOEt/PE
EtOH/Et2O
3374, 1776, 1705, 1683,
1653, 1535
3362, 1778, 1718, 1684,
1647, 1514
3351, 1776, 1712, 1682,
1665, 1526
3353, 3311, 1775, 1707,
1666, 1528
3359, 3339, 1776, 1710,
1670, 1523
3366, 1776, 1737, 1682,
1668, 1517
3334, 1776, 1731, 1712,
1666, 1527
3319, 1777, 1721, 1710,
1659, 1531
3326, 1777, 1731, 1712,
1662, 1529
3392, 3351, 1778, 1721,
1709, 1680, 1663, 1529
3323, 1777, 1709, 1664,
1529
3380, 3367, 1775, 1739,
1713, 1687, 1674, 1661,
1646, 1529
Pht-(Aib)3-OH
Pht-(Aib)4-OtBu
Pht-(Aib)4-OH
Pht-(Aib)5-OtBu
Pht-(Aib)5-OH
Pht-(Aib)6-OtBu
Pht-(Aib)6-OH
Pht-(Aib)7-OtBu
Pht-(Aib)7-OH
Pht-(Aib)8-OtBu
Pht-(Aib)8-OH
210 211
135 136
221 222
195 196
238 239
229 230
279 280
182 183
260 261
248 249
> 300
Pht-Aib-[l-(a-Me)Val]2-OtBu
Pht-Aib-[l-(a-Me)Val]2-OH
Pht-Aib-NH-C(Me2)-O-OtBu
Pht-(Aib)2-NH-C(Me2)-O-OtBu
Pht-(Aib)3-NH-C(Me2)-O-OtBu
Pht-(Aib)4-NH-C(Me2)-O-OtBu
Pht-(Aib)5-NH-C(Me2)-O-OtBu
oil
AcOEt/PE
0.80
0.50
0.90
0.85
0.80
0.70
0.70
0.95
0.90
0.95
0.95
0.95
0.90
0.90
0.90
0.45
0.60
0.55
0.50
0.45
0.45
3388, 1779, 1715, 1680,
1662, 1653, 1522
3389, 1777, 1710, 1678,
1656, 1509
3445, 3383, 1778, 1725,
1710, 1611, 1546
3401, 3301, 1780, 1715,
1692, 1668, 1616, 1522
3379, 1776, 1710, 1676,
1527
102 103EtOH/Et
2O
AcOEt/PE
AcOEt/PE
oil
143 144
oil
AcOEt/PE
AcOEt/PE
AcOEt/PE
98 99
272 273
3320, 1778, 1710, 1667,
1525
3333, 1777, 1721, 1711,
1667, 1528

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Helvetica Chimica Acta Vol. 85 (2002)
Table 1 (cont.)
Compound
M.p. [8]
Recryst.
solvent
TLC
RfI
IR [cmÀ1
]
RfII
RfIII
Pht-(Aib)6-NH-C(Me2)-O-OtBu
Pht-(Aib)7-NH-C(Me2)-O-OtBu
297 298
156 158
AcOEt/PE
AcOEt/PE
0.65
0.90
0.40
3321, 1777, 1712, 1663,
1529
3313, 1779, 1712, 1663,
1532
3299, 1660, 1560
3353, 1658, 1537
3389, 1727, 1531
0.65
0.90
0.40
Phac-NH-C(Me2)-O-OtBu
Piv-NH-C(Me2)-O-OtBu
Z-NH-C(Me2)-O-OtBu
85 86
57 58
oil
AcOEt/PE
AcOEt/PE
AcOEt/PE
0.95
0.95
0.95
0.95
0.95
0.95
0.85
0.90
0.90
Fig. 3. X-Ray crystal structure (ORTEP view) and numbering of [2-(methoxycarbonyl)]Bz-(Aib)2-NH-C(Me2)-
O-OtBu. Displacement ellipsoids are shown at the 30% probability level. Intramolecular CˆO ¥¥¥ HÀN H-
bonds are indicated by dashed lines.
t
Table 2. 1H-NMR Chemical Shifts of the Bu Groups of Synthesized Peptides in CD3CN
Esters
d(tBu) [ppm]
Dialkyl peroxides
d(tBu) [ppm]
Pht-(Aib)2-OtBu
Pht-(Aib)3-OtBu
Pht-(Aib)4-OtBu
Pht-(Aib)5-OtBu
Pht-(Aib)6-OtBu
Pht-(Aib)7-OtBu
Pht-(Aib)8-OtBu
Phac-Aib-OtBu
Piv-Aib-OtBu
1.43Pht-Aib-NH-C(Me
2)-O-OtBu
1.11
1.18
1.14
1.16
1.15
1.16
1.17
1.17
1.18
1.19
1.36
1.36
1.35
1.37
1.37
1.38
1.35
1.37
1.36
Pht-(Aib)2-NH-C(Me2)-O-OtBu
Pht-(Aib)3-NH-C(Me2)-O-OtBu
Pht-(Aib)4-NH-C(Me2)-O-OtBu
Pht-(Aib)5-NH-C(Me2)-O-OtBu
Pht-(Aib)6-NH-C(Me2)-O-OtBu
Pht-(Aib)7-NH-C(Me2)-O-OtBu
Phac-NH-C(Me2)-O-OtBu
Piv-NH-C(Me2)-O-OtBu
Z-NH-C(Me2)-O-OtBu
Z-Aib-OtBu

Helvetica Chimica Acta Vol. 85 (2002)
3105
Table 3. Mass Spectral (ESI-TOF) Data for Peptide Dialkyl Peroxides
‡
Compound
[M ‡ Na]
calc.
found
Pht-Aib-NH-C(Me2)-O-OtBu
Pht-(Aib)2-NH-C(Me2)-O-OtBu
Pht-(Aib)3-NH-C(Me2)-O-OtBu
Pht-(Aib)4-NH-C(Me2)-O-OtBu
Pht-(Aib)5-NH-C(Me2)-O-OtBu
Pht-(Aib)6-NH-C(Me2)-O-OtBu
Pht-(Aib)7-NH-C(Me2)-O-OtBu
Pht-Aib-[l-(a-Me)Val]-[d,l-NH-C(Me)(iPr)]-O-OtBua)
Phac-NH-C(Me2)-O-OtBu
385.1734
470.3390
555.4088
640.3317
725.3844
810.4372
895.4900
526.2888
254.1776
288.1570
304.1532
385.1939
470.2417
555.2869
640.3680
725.4503
810.5074
895.5178
526.3066
254.1807
288.1668
304.1625
Piv-NH-C(Me2)-O-OtBu
Z-NH-C(Me2)-O-OtBu
a) Mixture of diastereoisomers.
In both compounds, the characteristic peroxide O(T1)ÀO(T2) bond is only slightly
shorter than in classical dialkylperoxides (1.46 1.48 ä) (cf. Table 4) [23]. Both
compounds adopt a type III (III') b-turn conformation [24 26], i.e., the partial
structure of the 310-helix [2], as expected for Aib homopeptides [3][4]. The C-terminal
NH-C(Me)2-O-OtBu residue is also folded, but the signs of its backbone torsional angles
are opposite with respect to those of the preceding amino acids. The b-turn structure of
the Pht-protected peptide is stabilized by one weak intramolecular CˆO ¥¥¥ HÀN H-
bond (3.284(4) ä) [27]. The Bz-containing peptide exhibits two such H-bonds, with
distances of 2.972(5) and 3.216(4) ä, respectively. It is worth mentioning that these two
X-ray structures are quite promising for the further exploitation of (Aib)n homopep-
tides as rigid molecular rulers in the study of the Pht/dialkyl peroxide DET processes.
Further evidence as to the chemical nature of our new compounds was obtained
from cyclic voltammetry. The reduction of Piv-NH-C(Me)2-O-OtBu is characterized by
an irreversible and broad signal at 0.2 V sÀ1, with a peak potential (Ep) of À 2.26 V. The
main voltammetric features, e.g., the peak width and shift of Ep as a function of scan
rate, point to a very slow, heterogeneous ET process. As for many other peresters and
dialkyl peroxides [28], the analysis of the voltammetric data is in agreement with a
mechanism in which ET and fragmentation of the OÀO bond are concerted. Such a
fragmentation leads to the carboxylate or the alcoholate anion along with the alkoxy
.
t
radical, e.g., BuO , which is rapidly reduced (overall two-electron process). In Fig. 4,
the voltammetric curves of Piv-NH-C(Me)2-O-OtBu (curve b) and of two reference
compounds, Piv-O-OtBu (Ep ˆ À1.55 V (curve a) [1]) and di(tert-butyl) peroxide
(Ep ˆ À2.50 V (curve c) [29]) are shown. Our measurements are consistent with the
fact that dialkyl peroxides are reduced at potentials more negative than ca. À1.9 V,
whereas peresters are reduced at potentials more positive than À1.6 V [28].
Interestingly, the a-N-atom of the dialkyl peroxide lowers the reduction potential by
more than 0.2 V (cf. curve b vs. c). In summary, cyclic voltammetry confirmed that the
Piv-−Aib×-derivative (and by extension all other Aib derivatives and peptides
synthesized in this work) are terminated by a dialkyl peroxide rather than by a
perester function.

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Helvetica Chimica Acta Vol. 85 (2002)
Table 4. Selected Bond Lengths, Bond Angles, and Torsional Angles of Pht-(Aib)2-NH-C(Me2)-O-OtBu (cf.
Fig. 2) and [2-(Methoxycarbonyl)]Bz-(Aib)2-NH-C(Me2)-O-OtBu (cf. Fig. 3)
Parameter
Pht-(Aib)2-NH-
C(Me2)-O-OtBu
[2-(methoxycarbonyl)]Bz-
(Aib)2-NH-C(Me2)-O-OtBu
Bond lengths [ä]
C(3A)ÀO(T1)
O(T1)ÀO(T2)
O(T2)ÀC(T1)
1.428(5)
1.448(5)
1.447(6)
1.476(7)
1.436(7)
1.399(8)
Bond angles [8]
C(3A)ÀO(T1)ÀO(T2)
105.5(3)
108.1(3)
109.2(4)
108.6(5)
O(T1)ÀO(T2)ÀC(T1)
Torsional angles [8]
C(0)ÀN(1)ÀC(1A)ÀC(1) (−f1×)
N(1)ÀC(1A)ÀC(1)ÀN(2) (y1)
C(1A)ÀC(1)ÀN(2)ÀC(2A) (w1)
C(1)ÀN(2)ÀC(2A)ÀC(2) (f2)
N(2)ÀC(2A)ÀC(2)ÀN(3) (y2)
C(2A)ÀC(2)ÀN(3)ÀC(3A) (w2)
C(2)ÀN(3)ÀC(3A)ÀO(T1) (−f3×)
N(3)ÀC(3A)ÀO(T1)ÀO(T2) (−y3×)
C(3A)ÀO(T1)ÀO(T2)ÀC(T1) (−w3×)
À 48.1(5)
À 46.2(5)
À 161.5(3)
À 62.5(4)
À 32.3(5)
À 167.1(4)
61.6(5)
53.9(5)
37.1(5)
170.6(4)
63.9(5)
25.3(6)
166.7(5)
À 66.5(5)
À 69.0(5)
À 140.2(5)
68.0(5)
À 176.0(4)
Fig. 4. Cyclic voltammetry curves for the reduction of a) Piv-O-OtBu, b) Piv-NH-C(Me2)-O-OtBu, and c) ButO-
OtBu in DMF. Conditions: 0.1m Bu4NClO4, Tˆ 258, glassy C electrode (rel. to Standard Calomel Electrode),
scan rate: 0.2 V sÀ1, peptide concentration: 1 mm.
Mechanism of Peptide Dialkyl Peroxide Formation. We propose the following
mechanism for the formation of peptide dialkyl peroxides (Scheme 2). Activation of the
C-terminal COOH group of an Na-blocked Aib homopeptide affords the corresponding
oxazol-5(4H)-one [10][11]. Next, the pyridine N-atom of DMAP (the catalyst) attacks
the oxazolone, which leads to an acylpyridinium intermediate [8][30][31]. It is

Helvetica Chimica Acta Vol. 85 (2002)
3107
Scheme 2
O
O
O
H
N
EDC
Pht-N
Pht-N
N
N
H
OH
N
H
O
O
O
N
NMe2
O
O
O
O
H
N
H
tBuOOH
O
Pht-N
Pht-N
N
N
H
O
N
N
H
O
O
NMe2
– tBuO
O
– CO2
H
N
O
H
tBuOOH
Pht-N
Pht-N
N
O
N
H
N
H
O
O
O
t
reasonable to assume that the weakly nucleophilic BuOOH then attacks the terminal
reactive CˆO group of the acylpyridinium salt producing the peptide perester. If the
latter is unstable, it might fragment heterolytically under loss of CO2 and under
formation of an iminium salt, as described by R¸chardt and co-workers [6][32]. In the
t
final step, the iminium group is attacked by a second molecule of BuOOH, which
ultimately produces the peptide dialkyl peroxide.
To corroborate the proposed mechanism, we followed the progress of the reaction
between the oxazolone of Z-Aib-OH and tBuOOH by HPLC-MS. Fig. 5,a depicts the
chromatogram of the starting mixture in which the oxazolone 1 predominates. In
Fig. 5,b, the latter has practically disappeared, with the concomitant onset of the peptide
perester 2 and the imine 3. Then, the peptide dialkyl peroxide 4 starts to form (Fig. 5,c),
while, simultaneously, the peptide perester concentration decreases. In the final stage
(Fig. 5,d), only the peptide dialkyl peroxide 4 is present to a significant extent.
Provided the planar iminium compound shown in Scheme 2 is indeed an
intermediate, then the utilization of a peptide substrate with two C-terminal, chiral
a-amino acids should lead to epimerization and formation of a mixture of two
diastereoisomeric peptide dialkyl peroxides. We, therefore, synthesized the tripeptide
Pht-Aib-[l-(a-Me)Val]2-OH. Due to the presence of the two Ca-tetrasubstituted l-(a-
Me)Val residues, epimerization via direct a-H abstraction can be excluded, but not via
iminium salt formation. The 1H-NMR and HPLC data shown in Fig. 6 clearly indicate
that a mixture of two diastereoisomeric peptide dialkyl peroxides was indeed obtained.
1
The H-NMR signal of the C-terminal NH group (the one closer to the center of
epimerization) is split symmetrically (Fig. 6,a), and the same is true for the major
HPLC peak (Fig. 6,b). Unfortunately, chromatographic separation of the two
diastereoisomers was unsuccessful.

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Helvetica Chimica Acta Vol. 85 (2002)
Fig. 5. Progress of the reaction 1 ! 2 ! 3 ! 4 (analogous to the mechanism proposed in Scheme 2), as followed
by reversed-phase HPLC and MS
Conclusions.
The original aim of our work was the preparation of Pht-Na-
protected Aib peptides with perester groups for probing the fine details of the dynamics
of dissociative electron transfer (DET) processes [1]. However, our experiments
clearly show that peptide peresters, although formed in situ, are not stable enough to be
isolated or investigated. Since Pht-Aib-O-OtBu is fairly stable and can be easily
synthesized [1][6][7], we tend to attribute the instability of the Aib-containing
homologous peptide peresters to the presence of the C-terminal a-amide function. This
assumption is corroborated by the observation that, among the Na-blocked aminoacyl
derivatives studied so far, only Pht-Aib-OH and racemic, trans-oriented Na-phthaloyl-

Helvetica Chimica Acta Vol. 85 (2002)
3109
Fig. 6. Characterization of the diastereoisomeric mixture of Pht-Aib-[l-(a-Me)Val]-[d,l-NH-C(Me)(iPr)]-O-
1
OtBu. a) Partial H-NMR spectrum; b) analytical, reversed-phase HPLC chromatogram.
2-aminocyclohexanecarboxylic acid [33], which contain an a-imide or a b-amide
function, respectively, afforded the desired peresters. Following this working hypoth-
esis, we are currently trying to synthesize Pht-Na-protected Aib homo-oligomeric
peresters [33] that bear the above mentioned rigid b-amino acid residue [34][35] at the
C-terminus. At the same time, we are also actively investigating DET processes and
trying to solve the preferred three-dimensional structure of the peptide series based on
the Pht/dialkyl peroxide dyad.
Experimental Part
General. Abbreviations: NMM (N-methylmorpholine), PE (petroleum ether), DMF (N,N-dimethylform-
amide), TFA (trifluoroacetic acid). Anal. TLC and prep. FC: silica gel F 254 plates and silica gel 60 (0.040
0.063mm; Merck), resp.; TLC eluent systems: CHCl3/EtOH 9 :1 (RfI); BuOH/AcOH/H2O 3 :1:1 (RfII), and
toluene/EtOH 7 :1 (RfIII); UV fluorescence detection (254 nm) or chlorine-starch-potassium iodide develop-
ment. Anal. HPLC: C18 reversed-phase column Vydac 218TP54 with a Pharmacia LKB-LCC 2252 instrument
equipped with a Uvicord SD (l ˆ 226 nm). M.p. determination with a temp. raise of 38/min on a Leitz
Laborlux 12 apparatus; uncorrected. IR Spectra: KBr technique, Perkin-Elmer 580-B spectrophotometer
equipped with a Perkin-Elmer 3600 IR data station. 1H-NMR Spectra (250 MHz): in CD3CN (99.95% D;
Aldrich) or (D6)DMSO (99.96% D, Acros Organics); Bruker AC-250 spectrometer; d in ppm rel. to Me4Si as
internal standard. Mass spectra: Mariner ESI-TOF instrument (Perseptive Biosystems); positive and negative
ions accelerated to 10, 15, 20, or 30 keV and analyzed in the linear mode. Electrochemical apparatus and
procedures: DMF (99%, Acros Organics) was treated as previously described [29]. Electrochemical

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Helvetica Chimica Acta Vol. 85 (2002)
measurements were conducted in an all-glass cell, thermostated at 258. An EG&G-PARC 173/179 potentiostat-
digital coulometer, an EG&G-PARC 175 universal programmer, and a Nicolet 3091 12-bit resolution digital
oscilloscope were used. The feedback correction was applied to minimize the ohmic drop between the working
and the reference electrodes. Experiments were carried out inside a double-wall copper Faraday cage. Glassy
carbon, prepared and activated as previously described [29], was employed as the working electrode. The
reference electrode was Ag/AgCl, calibrated after each experiment against the ferrocene/ferricenium couple,
and finally converted to the KCl saturated calomel electrode. The counter electrode was a Pt plate (1 cm2). To
avoid father-son reaction between peroxide and tBuOÀ [36][37], one equivalent of acetanilide was added to the
soln. Detailed procedures representative of each type of reaction exploited are described below.
Phac-Aib-OtBu. To an ice-cold soln. of Phac-OH (0.50 g, 2.26 mmol) in CH2Cl2 (5 ml), HOBt (0.30 g,
2.26 mmol) and EDC hydrochloride (0.43g, 2.26 mmol) were added, followed by a soln. of H-Aib-O tBu [10]
(obtained by catalytic hydrogenation of 0.76 g, 2.26 mmol, of the corresponding Z-protected synthetic precursor
in MeOH) in CH2Cl2 (8 ml) and by NMM (0.28 ml, 2.26 mmol). The soln. was stirred at r.t. for 24 h and then
evaporated to dryness. The residue was dissolved in AcOEt, and the resulting soln. was washed with 10%
KHSO4 soln., H2O, 5% NaHCO3 soln., and H2O, dried (Na2SO4), filtered, and evaporated to dryness. The crude
product was purified by FC and crystallized from AcOEt/PE. Yield: 91%.
Piv-Aib-OtBu. To H-Aib-OtBu (3.4 mmol, prepared as described above) in CH2Cl2 (10 ml), (Piv)2O
(3.98 ml, 17.0 mmol) in CH2Cl2 (4 ml) was added. The soln. was stirred at r.t. for 4 h and evaporated several
times to dryness by adding toluene to remove the excess of (Piv)2O. The residue was crystallized from AcOEt/
PE. Yield: 89%.
Pht-(Aib)2-OtBu. Pht-Aib-Cl [13] (4.0 g, 16.5 mmol) was dissolved at 08 in CH2Cl2 (35 ml). Then, H-Aib-
OtBu (16.5 mmol, prepared as described above) in CH2Cl2 (55 ml) and (i-Pr)2NEt (2 ml, 16.5 mmol) were
added. The soln. was stirred at r.t. for 3h and evaporated to dryness. The residue was dissolved in AcOEt, and
the resulting soln. was washed with 10% KHSO4 soln., H2O, 5% NaHCO3 soln., and H2O, dried (Na2SO4),
filtered, and evaporated to dryness. The crude product was purified by FC, dissolved in AcOEt, and precipitated
by adding PE. Yield: 72%.
Pht-(Aib)2-OH. Pht-(Aib)2-OtBu (2.0 g, 5.20 mmol) was dissolved in a 1:1 mixture of TFA and CH2Cl2
(10 ml). The soln. was stirred at r.t. for 2 h. The solvent was evaporated several times to dryness by adding Et2O
to remove the excess of TFA. The crude product was dried over solid KOH in vacuo for 12 h and crystallized
from EtOH/Et2O. Yield: 90%.
Oxazol-5(4H)-one from Pht-(Aib)2-OH. To an iced soln. of Pht-(Aib)2-OH (0.50 g, 1.5 mmol) in MeCN
(15 ml), EDC hydrochloride (0.30 g, 1.7 mmol) was added. The soln. was stirred at r.t. for 2 h and evaporated to
dryness. The residue was dissolved in AcOEt, and the resulting soln. was washed with 10% KHSO4 soln. and
H2O, dried (Na2SO4), filtered, and evaporated to dryness. The crude product was crystallized from Et2O/PE.
Yield: 91%.
Pht-(Aib)4-OtBu. A soln. of the oxazol-5(4H)-one of Pht-(Aib)2-OH (2.0 g, 6.1 mmol) and H-(Aib)2-OtBu
(obtained by catalytic hydrogenation of 2.3g, 6.1 mmol of the corresponding Z-protected synthetic precursor in
MeOH [38]) in MeCN (20 ml) was refluxed for 48 h and evaporated to dryness. The residue was dissolved in
AcOEt, and the resulting soln. was washed with 10% KHSO4 soln., H2O, 5% NaHCO3 soln., and H2O, dried
(Na2SO4), filtered, and evaporated to dryness. The crude product was crystallized from AcOEt/PE. Yield: 78%.
Pht-Aib-NH-C(Me)2-O-OtBu. To an ice-cold soln. of the oxazol-5(4H)-one of Pht-(Aib)2-OH (0.5 g,
1.5 mmol) in CH2Cl2 (5 ml), tBuOOH (2.9 ml of a 5.5m soln. in decane, 16.0 mmol) and DMAP (0.78 g,
6.4 mmol) were added. The soln. was refluxed for 24 h and evaporated to dryness. The residue was dissolved in
AcOEt, and the resulting soln. was washed with 10% KHSO4 soln., H2O, 5% NaHCO3 soln., and H2O, dried
over Na2SO4, filtered, and evaporated to dryness. The crude product was purified by FC and precipitated from
an AcOEt soln. by adding PE. Yield: 32%.
X-Ray Diffraction. Colourless single crystals of the oxazol-5(4H)-one of Pht-(Aib)2-OH, Pht-(Aib)2-NH-
C(Me)2-O-OtBu, and [2-(methoxycarbonyl)]Bz-(Aib)2-NH-C(Me)2-O-OtBu were grown from Et2O by slow
evaporation, from CHCl3/PE by vapour diffusion, and from MeOH by slow evaporation, resp. Intensity data
were collected on a Philips PW-1100 four-circle X-ray diffractometer and corrected for Lorentz and polarization
effects. Data of Pht-(Aib)2-NH-C(Me)2-O-OtBu were additionally corrected for crystal decay, as suggested
by monitoring of three standard reflections (max decay 62%). Graphite-monochromated CuKa radiation
(l ˆ 1.54178 ä) and q 2q scan-mode were used. Cell parameters were obtained by least-square refinements
of the angular setting of 48 carefully centered high-angle reflections. The structures were solved by direct
methods with the SHELXS 97 program [39]. Refinements were carried out by the full-matrix block least-
squares procedure on F2 with SHELXL 97 [40], with all non-H-atoms anisotropic, and allowing their positional

Helvetica Chimica Acta Vol. 85 (2002)
3111
Table 5. Crystallographic Data and Details of Structure Determinations for the Oxazol-5(4H)-one of Pht-
(Aib)2-OH, for Pht-(Aib)2-NH-C(Me2)-O-OtBu, and for [2-(Methoxycarbonyl)]Bz-(Aib)2-NH-C(Me2)-O-
OtBu
Parameter
Oxazolone of Pht-
(Aib)2-OH
Pht-(Aib)2-NH-
C(CH3)2-O-OtBu
[2-(Methoxycarbonyl)]Bz-
(Aib)2-NH-C(CH3)2-O-OtBu
Empirical formula
Mr
C16H16N2O4
300.3
C23N33N3O6
447.5
C24H37N3O7
479.6
Crystal system
Space group
a [ä]
b [ä]
c [ä]
b [8]
V [ä3]
Z
orthorhombic
P212121
9.652(2)
10.628(3)
15.008(3)
90
1539.5(6)
4
1.296
monoclinic
P21/n
monoclinic
P21
13.210(3)
10.703(3)
18.288(4)
103.89(4)
2510.1(11)
4
9.333(3)
11.029(3)
13.547(4)
91.82(5)
1393.7(7)
2
D [g cmÀ3
]
1.184
0.706
1.143
0.693
m [mmÀ1
Radiation
]
0.782
CuKa (l ˆ 1.54178 ä) CuKa (l ˆ 1.54178 ä) CuKa (l ˆ 1.54178 ä)
0.60 Â 0.35 Â 0.30 0.50 Â 0.10 Â 0.10 0.45 Â 0.30 Â 0.20
2q 2q 2q
Crystal size [mm]
Mode of scan
F(000)
q
q
q
632
960
516
q Range [8]
Index ranges
5.5 60.0
3.7 60.1
3.3 60.0
À 1 ꢀ h ꢀ 10
À 14 ꢀ h ꢀ 14
À 10 ꢀ h ꢀ 10
0 ꢀ k ꢀ 11
0 ꢀ k ꢀ 12
0 ꢀ k ꢀ 12
0 ꢀ l ꢀ 16
0 ꢀ l ꢀ 20
0 ꢀ l ꢀ 15
Reflections collected
Independent reflections
Absorption correction
Refinement method
1487
3842
2288
1462 (R(int) ˆ 0.017)
3721 (R(int) ˆ 0.019)
2194 (R(int) ˆ 0.064)
none
none
none
full-matrix block
least-squares on F2
1462, 0, 200
full-matrix block
least-squares on F2
3721, 18, 305
full-matrix block
least-squares on F2
2194, 1, 307
Data, restraints, parame-
ters
Goodness-of-fit on F2
1.129
1.038
1.080
Final R indices [I > 2s(I)] R1 ˆ 0.0429
R1 ˆ 0.0824
wR2 ˆ 0.2276
R1 ˆ 0.1326
wR2 ˆ 0.2649
0.301, À0.264
R1 ˆ 0.0598
wR2 ˆ 0.1750
R1 ˆ 0.0651
wR2 ˆ 0.1829
0.392, À0.235
wR2 ˆ 0.1104
R Indices (all data)
R1 ˆ 0.0434
wR2 ˆ 0.1109
0.181, À0.247
Largest difference peak
and hole [e äÀ3
]
and anisotropic displacement parameters to refine at alternate cycles. The H-atoms were calculated at idealized
positions and refined by allowing them to ride on their carrying atom, with Uiso being 1.2 Â (1.5 Â for Me) as
large as Ueq of the parent atom. The C-terminal tBu group of Pht-(Aib)2-NH-C(Me)2-O-OtBu is distorted. Each
of its three Me groups was refined on two sites, with population parameters of 0.52 and 0.48, respectively.
Restraints were applied to the bond distances and angles of the disordered atoms. The relatively high final R
factors might be ascribed in part to the crystal decay and possible additional disorder at the C-terminal tBu group
that could not be modeled satisfactorily. Crystallographic data and details of the structure determinations are
presented in Table 5. Atomic coordinates, bond distances and angles, and anisotropic displacement parameters
have been deposited with the Cambridge Crystallographic Data Centre (CCDC) as deposition No. CCDC
184554, 184555, and 184556. Copies of the data can be obtained, free of charge, on application to the CCDC, 12
Union Road, Cambridge CB21EZ, UK (fax: ‡44(1223)336033; e-mail: deposit@ccdc.cam.ac.uk).
This work was in part financially supported by the University of Padova (research project A.OEEO.97)
¡
and the Ministero dell×Istruzione, dell×Universita e della Ricerca (MIUR) of Italy.

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Helvetica Chimica Acta Vol. 85 (2002)
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Received May 27, 2002