Total synthesis of sequential retro-peptide oligomers

Article abstract of DOI:10.1002/ejoc.200400242

The total synthesis of sequential oligomers of retro-peptides of the general formula Boc(-gGly-mAib)_n-OBn, starting from dimethylmalonic acid, has been carried out in good overall yield. The key step is the formation of the gGly unit >N-CH

Full text of DOI:10.1002/ejoc.200400242

FULL PAPER
Total Synthesis of Sequential Retro-Peptide Oligomers
Simone Ceretti,[a] Gianluigi Luppi,[a] Silvia De Pol,[b] Fernando Formaggio,[b]
Marco Crisma,[b] Claudio Toniolo,[b] and Claudia Tomasini*[a]
Keywords: Conformation analysis / Curtius rearrangement / Peptidomimetics / Retro-peptides
The total synthesis of sequential oligomers of retro-peptides
of the general formula Boc(-gGly-mAib)n-OBn, starting from
dimethylmalonic acid, has been carried out in good overall
yield. The key step is the formation of the gGly unit
ϾN−CH2−NϽ, which is easily obtained in a one-pot/three-
step reaction from BnO-mAib-Gly-OH and diphenylphos-
phoryl azide. The synthesis of the sequential oligomers of
RCO(-gAla-mAib)n-ORЈ was also attempted, but the oligo-
merization step failed because the gAla moiety readily de-
composes or racemizes.
( Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2004)
Introduction
involves the prefixes g (geminal) and m (malonic) for the
α,α-diamino residue and for the α,α-dicarboxy residue,
respectively. For instance, the dipeptide unit shown in Fig-
ure 1 is termed gGly-mGly.
Backbone modifications of peptides were driven by their
use as pharmaceuticals, since the physicochemical proper-
ties of most peptides preclude their exploitation as drugs.
As the mammalian body presents many barriers to the en-
try of macromolecules, peptides are affected by poor ab-
sorption because they do not readily pass across biological
membranes, while swift metabolism by proteolytic enzymes
and rapid excretion through the liver and kidneys also take
place.[1,2,3] These barriers result in peptides suffering from
low bioavailability and short biological half-lives.
To the best of our knowledge, a variety of compounds
incorporating single retro-peptide units have been pre-
pared,[7,8,9,10,11,12] but no strictly sequential oligomeric
chains of retro-peptides have ever been synthesized. Aleman
and co-workers[13,14,15,16,17] calculated the conformational
preferences of poly(retro-peptides), including poly(retro-
Gly) and its fully Cα-methylated analogue poly(retro-Aib)n
(Aib, α-aminoisobutyric acid). These authors concluded
that the use of retro-modified peptide units might be useful
for the stabilization of specific helical foldamers, the natures
of which, however, do not always match those preferred by
the parent polypeptides.
In this paper we describe an approach to the synthesis of
the poly(retro-peptide) series Boc-(gGly-mAib)n-OBn (Boc,
tert-butyloxycarbonyl; OBn, benzyloxy) with n ϭ 1Ϫ4 (Fig-
ure 2), which may provide information on the preferred
conformations of these compounds.
Goodman and Chorev[4,5,6] introduced the family of re-
tro-peptides, regioisomers of a parent peptide in which the
direction of the amino acid sequence is reversed (peptidom-
imetics), as a variation of the linear main chain (Figure 1).
The nomenclature currently utilized for retro-peptides
Figure 1. Chemical structures of the parent Gly-Gly (a) and retro-
modified peptide gGly-mGly (b) dipeptide units
[a]
Department of Chemistry ‘‘G. Ciamician’’, Alma Mater
Figure 2. Chemical structure of the sequential Boc-(gGly-mAib)n-
OBn (n ϭ 1Ϫ4; 4, 7, 9, 11) peptide oligomers
Studiorum, University of Bologna,
40126 Bologna, Italy
Fax: (internat.) ϩ39-051-2099456
E-mail: claudia.tomasini@unibo.it
[b]
Institute of Biomolecular Chemistry, CNR, Department of
Results and Discussion
Chemistry, University of Padova,
35131 Padova, Italy
Fax: (internat.) ϩ39-049-8275239
The synthesis of the retro-dipeptide unit Boc-gGly-mAib-
OBn (4) was achieved by starting from 2,2-dimethylmalonic
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
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DOI: 10.1002/ejoc.200400242
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Total Synthesis of Sequential Retro-Peptide Oligomers
FULL PAPER
acid, which was transformed into the corresponding benzyl duces a carbamate unit. As tert-butyl alcohol behaves both
ester 1 by partial hydrolysis of the dibenzyl ester in a well as a reagent and as a solvent, a large excess is crucial for
known procedure (Scheme 1).[18] All attempts to obtain the the production of 4 in a satisfactory yield. An alternative
benzyl ester 1 in good yield directly from dimethylmalonic approach allowing 4 to be obtained from the rearrangement
acid failed. The easiest way to introduce a gGly unit was of the amide Boc-mAib-GlyϪNH2 by treatment with [bis-
envisaged as by Curtius rearrangement of the correspond- (trifluoroacetoxy)iodo]benzene (TIB)[24Ϫ27] was also exam-
ing Gly unit, itself in turn obtained by oxidation of an al- ined, but it afforded the desired compound in an unsatisfac-
lylamine moiety. To this end the benzyl ester 1 was trans- tory yield.
formed into the corresponding allylamide 2 in very high
Formation of the sequential oligomers Boc-(gGly-
yield by treatment with allylamine in the presence of O-(6- mAib)n-OBn (7, 9 and 11) was achieved by consecutive
chloro-1H-benzotriazol-1-yl)-N,N,NЈ,NЈ-tetramethyluroni- couplings of Boc-(gGly-mAib-)n-OH (n ϭ 1Ϫ3) (6, 8 and
um hexafluorophosphate (HCTU)[19] and triethylamine 10) with TFA (trifluoroacetate)·ϩH2-gGly-mAib-OBn (5) in
(TEA). Compound 2 was transformed into BnO-mAib-Gly- acetonitrile in the presence of HCTU and TEA (Scheme 2).
OH 3 by oxidation of the CϭC double bond with potas- Yields were generally good, although Boc-(gGly-mAib)4-
sium permanganate[20] in acetone. The product was ob-
tained as a solid in good yield.
Scheme 1. Synthetic routes for Boc-gGly-mAib-OBn (4); reagents
and conditions: (i) BnBr (2.5 equiv.), DIEA (3.5 equiv.), DMAP
(0.13 equiv.), CH2Cl2, 24 h, r.t.; (ii) KOH (1.1 equiv.), BnOH, 72
h, r.t.; (iii) allylamine (1 equiv.), HCTU (1 equiv.), Et3N (2 equiv.),
CH3CN, 20 min, r.t.; (iv) KMnO4 (3.5 equiv.), acetone/water/AcOH
82:12:6; (v) DPPA (1 equiv.), Et3N (1 equiv.), tBuOH,
4 h, reflux
Formation of the gGly unit was achieved in a one-pot/
three-step reaction between BnO-mAib-Gly-OH 3 and di-
phenylphosphoryl azide (DPPA) (Figure 3), in an approach
involving the formation of an acylazide, which spon-
taneously decomposes to afford N2 and a nitrene, which in
turn rearranges to the corresponding isocyanate.[21Ϫ23]
Upon addition of tert-butyl alcohol, the isocyanate pro-
Scheme 2. Synthetic routes for Boc-(gGly-mAib)n-OBn sequential
oligomers; reagents and conditions: (i) TFA (18 equiv.), CH2Cl2, 4
h, r.t.; (ii) H2 (3 atm), C/Pd (10%), MeOH, 12 h, r.t.; (iii) TEA (3.5
equiv.), HCTU (1 equiv.), acetonitrile, 20 min, r.t.
Figure 3. Reaction mechanism for the synthesis of Boc-gGly-mAib-
OBn (4) by a one-pot/three-step mechanism
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OBn (11) was obtained only in moderate yield (40%), prob-
ably due to the low solubility of Boc-(gGly-mAib)3-OH (10)
in acetonitrile. All oligomers show decreasing solubility in
organic solvents as the main-chain length increases.
Initial investigation of the preferred conformations ad-
opted by the terminally protected oligomers 4, 7, 9 and 11
was carried out by IR absorption spectroscopy in the NϪH
stretching region in chloroform solution (Figure 4). As Cϭ
O···HϪN-mediated intermolecular aggregation is of minor
significance for 4, 7, and 9 at 1 m concentrations (results
not shown), only intramolecular hydrogen bonds should
need to be considered for these oligomers. Non-hydrogen-
bonded NϪH bands are typically seen above 3400 cmϪ1
,
while hydrogen-bonded NϪH bands fall in the 3390Ϫ3330
cmϪ1 region.[28,29,30] The figure also shows a remarkable
concentration effect taking place above 1 m concentration
for the longest oligomer 11. Two additional bands at 3300
Ϯ 20 cmϪ1, indicative of the occurrence of strongly H-
bonded NH groups, stand out clearly. In any case, the IR
absorption spectra suggest an increasing amount of intra-
molecularly hydrogen-bonded NH groups with increasing
main-chain length.
1
Analogously, no clearcut results were obtained from H
NMR titrations of the NH protons by addition of the per-
turbing agent DMSO (dimethyl sulfoxide) to the CDCl3
solution.[31,32] Figure 5 highlights modest variations for
most of the NH proton chemical shifts, which suggest that
most of the NH groups are intramolecularly hydrogen
bonded, but only weakly.
The above results suggest that the introduction of a sub-
stituent on the flexible gGly unit could restrict the confor-
mational space available to the retro-peptides and favour
the formation of a helix, as proposed by Aleman and co-
workers.[13Ϫ17] Unfortunately, insertion of an alkyl moiety
into the gGly unit is not a simple task, because an alkyl
Figure 5. Plot of NΗ chemical shifts in the 1H NMR spectra of
oligomers Boc-(gGly-mAib)n-OBn 4, 7, 9 and 11 as a function of
increasing percentages of DMSO added to the CDCl3 solution
(v/v); peptide concentration: 1 m for 4, 7, and 9; 0.1  for 11
moiety in an aminal function makes it much less stable. based on the gGly-mAib unit (Scheme 3). The benzyl ester
Nonetheless, the preparation of oligomers containing the 1 was coupled with a commercial sample of HCl·H--Ala-
gAla-mAib dipeptide unit was attempted, by an approach OtBu (OtBu, tert-butyl) in acetonitrile in the presence of
similar to that utilized for the preparation of the oligomers HCTU and TEA. The product BnO-mAib--Ala-OtBu (13)
was achieved in high yield, and this was quantitatively hy-
drolysed to the corresponding carboxylic acid 14. The syn-
thesis of the gAla unit was achieved by treatment of 14 with
DPPA in the one-pot/three-step procedure[21Ϫ23] success-
fully used for the synthesis of Boc-gGly-mAib-OBn (4).
More specifically, Boc-gAla-mAib-OBn (15) was prepared
in 43% yield by treatment of BnO-mAib--Ala-OH (14)
with DPPA in tert-butyl alcohol at reflux. Again, a large
excess of tert-butyl alcohol is required for the formation of
15 in good yield. The synthesis of the sequential oligomers
Boc-(gAla-mAib)n-OBn by selective deprotection was at-
tempted. The benzyl group at the mAib end was success-
fully removed by hydrogenolysis with H2 in the presence of
Pd (10%) on charcoal in a quantitative yield. Unfortunately,
removal of the Boc group at the gAla end did not afford
the desired product, but instead gave the benzyl dimeth-
Figure 4. FT-IR absorption spectra in the 3550Ϫ3200 cmϪ1 region
ylmalonate amide (H-mAib-OBn). This unwanted result
of the oligomers Boc-(gGly-mAib)n-OBn (4, 7, 9 and 11) in CDCl3
was probably due to the spontaneous decomposition of
solution (peptide concentration: 1 m); the spectrum of 11 at
0.1 m concentration is also shown (11Ј)
ϩH2-gAla-mAib-OBn, possibly favoured by traces of water,
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Total Synthesis of Sequential Retro-Peptide Oligomers
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with formation of acetaldehyde and ammonia as by-prod-
ucts.
Scheme 3. Synthetic routes for Boc-gAla-mAib-OBn (15); (i)
HCl•H-L-Ala-OtBu (1 equiv.), Et3N (3 equiv.), HCTU (1 equiv.),
acetonitrile, 20 min, r.t.; (ii) TFA (9 equiv.), CH2Cl2, 4 h, r.t.; (iii)
DPPA (1 equiv.), Et3N (1.1 equiv.), tBuOH, 5 h, reflux
We were thus forced to try an alternative approach to
circumvent the acidic deprotection step of the sensitive gAla
unit. Dimethylmalonic acid was transformed into the corre-
sponding di-tert-butyl ester by treatment with a large excess
of isobutene under acidic conditions, and this was partially
hydrolysed with TFA to afford the mono tert-butyl ester 16
(Scheme 4). The -Ala residue was introduced by treatment
of 16 with H--Ala-NH2 in the presence of 1-hydroxy-7-
aza-1,2,3-benzotriazole (HOAt), 1-(3-dimethylamino)pro-
pyl-3-ethylcarbodiimide hydrochloride (EDC·HCl) and N-
methylmorpholine (NMM) to afford 17 in very good yield.
Formation of the gAla unit was achieved in satisfactory
yield by treatment of 17 with TIB.[24Ϫ27] As this reagent
promotes the Curtius rearrangement of a primary amide
under very mild conditions, we judged that it might be ap-
propriate for this sensitive compound. Formation of 18 by
this reaction was confirmed by X-ray diffraction analysis
(Figure 6), which clearly showed the presence of an aminal
function.
Scheme 4. Synthetic routes for Ac-(gAla-mAib)2-OtBu (22); re-
agents and conditions: (i) isobutene (35 equiv.), H2SO4 (0.24
equiv.), CH2Cl2, 240 h, r.t.; (ii) TFA (8 equiv.), CH2Cl2, 20 min,
r.t;. (iii) EDC·HCl (1.1 equiv.), HOAt (1 equiv.), NMM (2 equiv.),
CH2Cl2, 48 h, r.t.; (iv) TIB (1.4 equiv.), CH3CN/H2O (6:4), 48 h,
r.t.; (v) Z-OSu (1.3 equiv.), NMM (2 equiv.), CH2Cl2, 48 h, r.t.; (vi)
TFA (18 equiv.), CH2Cl2, 35 min, 0 °C; (vii) H2 (3 atm), C/Pd
(10%), MeOH, 12 h, r.t.; (viii) Ac2O (10 equiv.), CH2Cl2, 24 h, r.t.
Compound 18 contains a free amino group, ready for the
formation of the oligomer Z-(gAla-mAib)2-OtBu (21). To
prepare the carboxylic unit for the coupling reaction, 18
was transformed into the corresponding carbobenzoxy (Z)
derivative Z-gAla-mAib-OtBu (19) in high yield, by treat-
ment with ZϪOSu (OSu, oxysuccinimide). Subsequently,
the tert-butyl ester of 19 was cleaved under acidic con-
ditions to afford Z-gAla-mAib-OH (20).
The synthesis of the oligomer Z-(gAla-mAib)2-OtBu (21)
was achieved in high yield by coupling of 18 with 20 in the
presence of HOAt, EDC·HCl and NMM under standard
1
conditions. However, HPLC and H NMR analyses of 21,
Figure 6. X-ray diffraction structure of TFA·ϩH2-gGly-mAib-OtBu
(18) with numbering of the atoms; the NϪH···OϭC H-bonds are
represented by dashed lines
containing two gAla units, suggested that a mixture of dia-
stereomers might be present. It is reasonable to assume that
the formation of a set of diastereomeric products could
have originated from (partial) racemization either during
the rearrangement of the -Ala residue to the gAla unit
or during the subsequent deprotonation step required for obtained even from a solution of a racemate. In addition,
coupling. The result of a X-ray diffraction study of com- we have been unable to assign the absolute configuration
pound 18 does not clash with this hypothesis, because a of the single gAla stereogenic centre of 18 from our X-ray
single crystalline enantiomer, as found in our case, can be diffraction analysis, as this compound lacks atoms heavier
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C. Tomasini et al.
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than oxygen, thus preventing determination by the anomal- (gAla-mAib)n was therefore attempted. Unfortunately, al-
ous dispersion method. though two different approaches were used, neither was
Analytical HPLC of the more polar oligomer Ac-(gAla- successful, owing to the presence of the very sensitive gAla
mAib)2-OtBu (22) obtained by hydrogenolysis of the car- unit, which has a high tendency to decompose and racem-
bobenzoxy N-protecting group of 21, followed by acety- ize. Further studies directed towards the synthesis of this
lation of the resulting free amino function, indicated more series of sequential retro-modified peptides are currently in
clearly that two peaks (most probably each corresponding progress in the Bologna laboratory.
to a diastereomer formed by a pair of unseparated enanti-
omers) were present in the product mixture. However, even
with the more promising 22, HPLC separation of the dia-
Experimental Section
stereomeric compounds at the preparative level proved im-
possible. Nevertheless, the 13C NMR spectrum of the prod-
Synthesis and Chemical Characterization: Materials and reagents
uct mixture confirmed the presence of two diastereomers,
as each carbon signal was doubled (Figure 7).
were of the highest commercially available grade and were used
without further purification. Reactions were monitored by thin-
layer chromatography on Merck 60 F254 silica gel covered plastic
plates. Compounds were viewed under UV light and by use of ceric
ammonium molybdate. Flash chromatography was performed on a
Merck 60 silica gel stationary phase. 1H and 13C NMR spectra
were recorded on Varian Gemini 300 or Varian Mercury 400 spec-
trometers. Chemical shifts are reported in δ values relative to the
solvent peak of CHCl3, set at δ ϭ 7.27 ppm. Infrared absorption
spectra were recorded with a Nicolet 210 FT-IR absorption spec-
trometer. Melting points were determined in open capillaries and
are uncorrected.
Phenylmethyl Dimethylmalonate (1): DIEA (266 mmol, 44 mL) and
DMAP (10 mmol, 1.22 g) were added to a stirred solution of 2,2-
dimethylmalonic acid (76 mmol, 10 g) in dichloromethane (60 mL)
under inert atmosphere. Benzyl bromide (190 mmol, 22 mL) was
then added and the mixture was stirred at room temp. for 24 h.
Water (100 mL) and dichloromethane (40 mL) were added, and the
organic layer was separated, washed with brine, aqueous HCl (1 ,
3 ϫ 30 mL) and 5% aqueous NaHCO3 (1 ϫ 30 mL), dried over
sodium sulfate and concentrated in vacuo. Dibenzyl dimethylma-
lonate was obtained pure in 84% yield (19.9 g) as a liquid, after
silica gel chromatography (cyclohexane/ethyl acetate, 9:1). 1H
NMR (200 MHz, CDCl3): δ ϭ 1.48 (s, 6 H, 2 ϫ CH3), 5.12 (s, 4
H, 2 ϫ OCH2Ph), 7.20Ϫ7.39 (m, 10 H). 13C NMR (50 MHz,
CDCl3): δ ϭ 22.7, 49.8, 66.7, 127.6, 127.9, 128.2, 135.4, 172.2. IR
(film): ν˜ ϭ 1729 (CϭO) cmϪ1. A solution of KOH (70 mmol, 4 g)
in benzyl alcohol (96 mL) was added to a stirred of dibenzyl di-
methylmalonate (64 mmol, 20 g) and the mixture was stirred for
72 h. The mixture was diluted with diethyl ether (1.5 L) and washed
with water (2 ϫ 100 mL). HCl (6 ) was added to the aqueous
layer to pH 2, and the product 1 was extracted with ethyl acetate
(2 ϫ 150 mL). The ethyl acetate solution was dried over sodium
sulfate and concentrated in vacuo. Benzyl dimethylmalonate was
obtained pure in 72% yield (10.2 g) as a solid after recrystallization
from cyclohexane/ethyl acetate; m.p. 48Ϫ50 °C. 1H NMR
(200 MHz, CDCl3): δ ϭ 1.51 (s, 6 H, 2 ϫ CH3), 5.20 (s, 2 H,
OCH2Ph), 7.27Ϫ7.38 (m, 5 H, Ph), 13C NMR (50 MHz, CDCl3):
δ ϭ 22.6, 49.9, 67.1, 127.7, 128.1, 128.4, 135.3, 172.1, 178.8. IR
(nujol): ν˜ ϭ 1747, 1703 cmϪ1 (CϭO).
Figure 7. Partial 13C NMR spectrum of Ac-(gAla-mAib)2-OtBu
(22) in CDCl3 solution; only the spectral regions of the α-carbons
(NϪCϪN and COϪCϪCO) (a) and carbonyl carbons (b) are
shown
Conclusions
The sequential oligomers Boc-(gGly-mAib)n-OBn (n ϭ
1Ϫ4) have been synthesized in good yields. The key step
was the formation of the ϾNϪCH2ϪNϽ gGly unit, which
was easily obtained in a one-pot/three-step reaction starting
from BnO-mAib-Gly-OH and DPPA. The reaction mecha-
nism involves formation of an acylazide, which spon-
taneously decomposes to generate N2 and a nitrene, which
in turn rearranges to provide the corresponding isocyanate.
This latter intermediate reacts with tert-butyl alcohol to af-
ford a carbamate moiety. The oligomers were easily pre-
pared, but a conformational analysis, carried out by IR ab-
sorption and 1H NMR techniques in a structure-supporting
solvent, did not afford clear-cut results. The synthesis of
Phenylmethyl 2-Methyl-2-(2-propenyl)amidopropanoate (2): HCTU
(4.5 mmol, 1.86 g), allylamine (4.5 mmol, 0.34 mL) and triethyl-
amine (9 mmol, 1.25 mL) were added at room temp. to a stirred
solution of benzyl dimethylmalonate (1, 4.5 mmol, 1 g) in aceto-
nitrile (20 mL). After 20 min the mixture was washed with brine,
aqueous HCl (1 , 3 ϫ 30 mL) and 5% aqueous NaHCO3 (1 ϫ
30 mL), dried over sodium sulfate and concentrated in vacuo.
Compound 2 was obtained pure as a liquid in 92% yield (1.08 g)
the more conformationally restricted, sequential oligomers after silica gel chromatography (cyclohexane/ethyl acetate, 8:2). 1H
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Total Synthesis of Sequential Retro-Peptide Oligomers
FULL PAPER
NMR (300 MHz, CDCl3): δ ϭ 1.49 (s, 6 H, 2 ϫ CH3), 3.83 [tt, two solutions were mixed dropwise and the mixture was stirred
3JH,H ϭ 1.5, 5.5 Hz; 2 H, CH2ϪCHϭCH2], 5.04Ϫ5.16 (m, 2 H,
for an additional 20 min. The acetonitrile was then removed under
CH2ϪCHϭCH2), 5.18 (s, 2 H, OCH2Ph), 5.68Ϫ5.82 (m, 1 H, reduced pressure and replaced with ethyl acetate (50 mL). The re-
CH2ϪCHϭCH2), 6.37 (br. s, 1 H, NH), 7.25Ϫ7.40 (m; 5 H). 13C sulting solution was washed with aqueous HCl (1 , 3 ϫ 30 mL)
NMR (75 MHz, CDCl3): δ ϭ 23.4, 41.9, 49.9, 66.9, 115.9, 127.8, and aqueous NaHCO3 (5%, 1 ϫ 30 mL), dried over sodium sulfate
128.2, 128.4, 133.7, 135. 3, 171.4, 174.3. IR (film): ν˜ ϭ 3430, 3337
and concentrated in vacuo.
(NϪH), 1733, 1653 (CϭO) cmϪ1
.
1
Boc-gGly-mAib-OH (6): Quantitative yield; m.p. 138Ϫ139 °C. H
NMR (300 MHz, CDCl3): δ ϭ 1.42 (s, 9 H, tBu), 1.47 (s, 6 H, 2 ϫ
CH3), 4.53 [t, 3JH,H ϭ 6.3 Hz; 2 H, NCH2CO], 5.99 [br. t, 3JH,H ϭ
6.3 Hz; 1 H, NH], 8.00 (br. s, 1 H). 13C NMR (100 MHz, CD3OD):
δ ϭ 22.6, 27.5, 45.9, 49.8, 79.5, 157.0, 174.8, 176.0. IR (nujol): ν˜ ϭ
BnO-mAib-Gly-OH (3): KMnO4 (105 mmol, 16.6 g) was added
portionwise at 0 °C to a stirred solution of 2 (30 mmol, 8 g) in
acetone (175 mL), water (25 mL) and acetic acid (14 mL). The mix-
ture was stirred for 1 h, and Na2SO3 (3 g) was then added until the
purple colour disappeared. Acetone was removed under reduced
pressure, and HCl (1 ) was added to pH ഠ 2. Ethyl acetate
(200 mL) was added, and the organic layer was separated, washed
with brine (100 mL) and water (100 mL), dried over sodium sulfate
and concentrated under reduced pressure. Compound 3 was ob-
tained pure as a solid in 70% yield (5.86 g); m.p. 69Ϫ71 °C. 1H
NMR (200 MHz, CDCl3): δ ϭ 1.50 (s, 6 H, 2 ϫ CH3), 4.03 [d,
3JH,H ϭ 5.4 Hz; 2 H, NCH2CO), 5.18 (s, 2 H, OCH2Ph), 7.07 [br.
3336 (NϪH), 1699, 1652 (CϭO) cmϪ1
.
Boc(-gGly-mAib)2-OBn (7): 65% yield; m.p. 155Ϫ156 °C. 1H NMR
(400 MHz, CDCl3): δ ϭ 1.37 (s, 6 H, 2 ϫ CH3), 1.43 (s, 6 H, 2 ϫ
3
CH3), 1.44 (s, 9 H, tBu), 4.48 (t, JH,H ϭ 6.4 Hz; 2 H, NCH2N),
3
4.59 (t, JH,H ϭ 6.3 Hz; 2 H, NCH2N), 5.15 (s, 2 H, OCH2Ph),
3
5.61 [br. t, JH,H ϭ 6.4 Hz; 1 H, NH], 7.24 [br. t, 3J (H, H) ϭ
6.3 Hz; 1 H, NH], 7.30Ϫ7.41 (m, 6 H, Ph ϩ NH), 7.46 [br. t,
3JH,H ϭ 6.4 Hz; 1 H, NH]. 13C NMR (100 MHz, CDCl3): δ ϭ 23.5,
23.7, 28.5, 45.5, 46.6, 49.6, 50.3, 67.5, 80.4, 128.2, 128.6, 128.9,
135.6, 156.2, 173.4, 174.1, 174.5, 174.7. IR (nujol): ν˜ ϭ 3340, 3318
3
t, JH,H ϭ 5.4 Hz; 1 H, NH], 7.31Ϫ7.38 (m, 5 H, Ph). 13C NMR
(75 MHz, CDCl3): δ ϭ 23.8, 41.9, 50.2, 67.6, 128.3, 128.7, 128.9,
135.6, 173.0, 173.5, 174.5 ppm. IR (nujol): ν˜ ϭ 3380 (NϪH), 1733,
(NϪH), 1746, 1695, 1645 (CϭO) cmϪ1
.
1653 (CϭO) cmϪ1
.
Boc(-gGly-mAib)2-OH (8): Quantitative yield; m.p. 205Ϫ207 °C.
1H NMR (300 MHz, CDCl3): δ ϭ 1.41 (s, 6 H, 2 ϫ CH3), 1.44 (s,
Boc-gGly-mAib-OBn (4): A solution of 3 (3.6 mmol, 1.0 g), DPPA
(0.36 mmol, 0.78 mL) and triethylamine (3.6 mmol, 0.5 mL) in tert-
butyl alcohol (20 mL) was stirred at reflux for 4 h. The solvent was
then removed and replaced with ethyl acetate (20 mL). The re-
sulting solution was washed with HCl (1 , 15 mL), water (15 mL),
5% aqueous NaHCO3 (15 mL) and brine (15 mL), dried over so-
dium sulfate and concentrated in vacuo. Compound 4 was obtained
pure as a liquid in 50% yield (0.63 g) after silica gel chromatogra-
phy (cyclohexane/ethyl acetate, 8:2); m.p. 79Ϫ81 °C. 1H NMR
(300 MHz, CDCl3): δ ϭ 1.43 (s, 9 H, tBu), 1.45 (s, 6 H, 2 ϫ CH3),
4.48 [t, 3JH,H ϭ 6.3 Hz; 2 H, NCH2N], 5.17 (s, 2 H, OCH2Ph), 5.50
(br. s, 1 H, NH), 7.08 (br. s, 1 H, NH), 7.26Ϫ7.39 (m, 5 H, Ph).
13C NMR (50 MHz, CDCl3): δ ϭ 23.2, 28.2, 46.3, 49.9, 67.0, 79.8,
127.7, 128.1, 128.4, 135.3, 155.8, 172.7, 173.5. IR (nujol): ν˜ ϭ 3317
3
6 H, 2 ϫ CH3), 1.47 (s, 9 H, tBu), 4.50 [t, JH,H ϭ 5.7 Hz; 2 H,
3
NCH2N], 4.64 [t, JH,H ϭ 5.7 Hz; 2 H, NCH2N], 5.87 (br. s, 1 H,
NH), 7.30 (br. s, 1 H, NH), 7.53 (br. s, 1 H, NH), 7.69 (br. s, 1 H,
NH). 13C NMR (75 MHz, CD3OD): δ ϭ 23.7, 23.8, 28.7, 46.2,
47.2, 51.1, 51.2, 80.7, 175.9, 176.0, 176.3, 176.9. IR (nujol): ν˜ ϭ
3326 (NϪH), 1737, 1635 (CϭO) cmϪ1
.
Boc(-gGly-mAib)3-OBn (9): 62% yield; m.p. 214Ϫ215 °C. 1H NMR
(300 MHz, CDCl3): δ ϭ 1.37 (s, 6 H, 2 ϫ CH3), 1.41 (s, 6 H, 2 ϫ
3
CH3), 1.44 (s, 6 H, 2 ϫ CH3), 1.46 (s, 9 H, tBu), 4.51 [t, JH,H
ϭ
3
6.3 Hz; 2 H, NCH2N], 4.59 [t, JH,H ϭ 6 Hz; 2 H, NCH2N], 4.60
3
[t, JH,H ϭ 6 Hz; 2 H, NCH2N], 5.17 (s, 2 H, OCH2Ph), 5.90 (br.
s, 1 H, NH), 7.21Ϫ7.48 (m, 10 H, Ph ϩ5 ϫ NH). 13C NMR
(75 MHz, CDCl3): δ ϭ 23.2, 23.4, 28.3, 45.4, 46.5, 49.7, 50.1, 67.1,
79.9, 127.8, 128.3, 128.6, 135.5, 155.0, 173.2, 173.8, 174.4. IR (nu-
(NϪH), 1739, 1699, 1646 (CϭO) cmϪ1
.
TFA·؉H2-gGly-mAib-OBn (5): Boc-gGly-mAib-OBn (4, 1.6 mmol,
0.55 mg) in dry dichloromethane (10 mL) and TFA (28.8 mmol,
2.2 mL) was stirred for 4 h at room temp., and the volatiles were
removed in vacuo. Compound 5 was obtained in quantitative yield
jol): ν˜ ϭ 3410, 3337 (NϪH), 1711, 1649 (CϭO) cmϪ1
.
Boc(-gGly-mAib)3-OH (10): Quantitative yield; m.p. 220Ϫ223 °C
1
1
(dec.). H NMR (300 MHz, CDCl3): δ ϭ 1.40 (s, 6 H, 2 ϫ CH3),
and was used without any further purification; m.p. 88Ϫ90 °C. H
1.43 (s, 6 H, 2 ϫ CH3), 1.45 (s, 6 H, 2 ϫ CH3), 1.48 (s, 9 H, tBu),
NMR (300 MHz, CDCl3): δ ϭ 1.45 (s, 6 H, 2 ϫ CH3), 4.52 (d,
3
3JH,H ϭ 5.4 Hz; 2 H,), 5.14 (s, 2 H, OCH2Ph), 7.23Ϫ7.39 (m, 5 H,
4.55 [t, JH,H ϭ 6.3 Hz; 2 H, NCH2N], 4.60Ϫ4.66 [m, 4 H, 2 ϫ
3
NCH2N], 5.94 (br. s, 1 H, NH), 7.27Ϫ7.32 (br. s, 2 H, 2 ϫ NH),
7.57 (br. s, 3 H, 3 ϫ NH). 13C NMR (75 MHz, CD3OD): δ ϭ 23.7,
23.8, 28.7, 46.2, 46.4, 47.3, 51.1, 51.2, 51.3, 66.5, 80.7, 158.1, 175.9,
Ph), 8.19 (br. s, 3 H, NH3), 8.46 [br. t, JH,H ϭ 5.4 Hz; 1 H, NH].
13C NMR (75 MHz, CDCl3): δ ϭ 22.8, 46.7, 50.2, 67.7, 127.9,
128.1, 128.6, 135.0, 161.3, 173.7, 175.4. IR (nujol): ν˜ ϭ 3357
(NϪH), 1719, 1672 (CϭO) cmϪ1
.
176.3, 177.0. IR (nujol): ν ϭ 3324 (NϪH), 1733, 1714, 1704, 1652
˜
(CϭO) cmϪ1
.
General Method for the Hydrogenolysis of the Benzyl Esters: Pal-
ladium on charcoal (10%, 20 mg) was added to a solution of Boc(-
gGly-mAib)n-OBn (1 mmol) in methanol (20 mL), and the mixture
was stirred under H2 (ഠ3 atm) for 12 h. The catalyst was then fil-
tered through a celite pad and the mixture was concentrated. The
corresponding carboxylic acid was obtained pure as a solid in
quantitative yield without any further purification.
Boc(-gGly-mAib)4-OBn (11): 40% yield; m.p. 235Ϫ237 °C. 1H
NMR (400 MHz, [D6]DMSO): δ ϭ 1.21 (s, 6 H, 2 ϫ CH3), 1.23
(s, 12 H, 4 ϫ CH3), 1.31 (s, 6 H, 2 ϫ CH3), 1.36 (s, 9 H, tBu), 4.29
3
[t, JH,H ϭ 4.0 Hz; 2 H, NCH2N], 4.39Ϫ4.44 (m, 6 H, 3 ϫ
NCH2N), 5.08 (s, 2 H, OCH2Ph), 7.09 (br. s, 1 H, NH), 7.29Ϫ7.37
(m, 5 H, Ph), 7.95 (br. s, 6 H, NH), 8.14 (br. s, 1 H, NH). 13C
NMR (75 MHz, [D6]DMSO): δ ϭ 22.7, 23.2, 28.0, 30.5, 44.8, 49.1,
49.7, 65.8, 127.2, 127.7, 128.2, 136.0, 159.2, 171.9, 172.8, 172.9,
General Method for Coupling: A solution of TFA·ϩH2-gGly-mAib-
OBn (5, 2.6 mmol, 0.95 g) and triethylamine (9.1 mmol, 1.3 mL) in
acetonitrile (20 mL) was stirred for 30 min at room temp. Separ-
ately, Boc(-gGly-mAib)n-OH (2.6 mmol) and HCTU (2.6 mmol) in
173.0. IR (nujol): ν˜ ϭ 3403, 3337 (NϪH), 1712, 1649 (CϭO) cmϪ1
.
Boc(-gGly-mAib)4-OH (12): Quantitative yield; m.p. 255Ϫ260 °C
dry acetonitrile (20 mL) were stirred for 10 min at room temp. The (dec.). 1H NMR (600 MHz, [D6]DMSO): δ ϭ 1.20 (s, 6 H, 2 ϫ
Eur. J. Org. Chem. 2004, 4188Ϫ4196
www.eurjoc.org  2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 4193

C. Tomasini et al.
FULL PAPER
CH3), 1.22 (s, 18 H, 6 ϫ CH3), 1.34 (s, 9 H, tBu), 4.28 [t, JH,H
3
ϭ
mixture was stirred at room temp. in a closed flask for 240 h. The
5.8 Hz; 2 H, NCH2N], 4.38Ϫ4.43 (m, 6 H, 3 ϫ NCH2N), 7.08 (br. mixture was washed with aqueous NaHCO3 (5%, 2 ϫ 250 mL)
3
s, 1 H, NH), 7.91 [t, 1 H, JH,H ϭ 5.4 Hz; NH], 7.92Ϫ8.20 (m, 5 and water (150 mL), dried over sodium sulfate and concentrated in
H, 5 ϫ NH), 8.10 (br. s, 1 H, NH). 13C NMR (150 MHz,
vacuo. Di-tert-butyl dimethylmalonate was obtained pure as a
[D6]DMSO): δ ϭ 23.8, 23.9, 24.0, 28.8, 31.4, 45.4, 45.6, 49.9, 50.0, liquid in 95% yield (52.6 g). 1H NMR (250 MHz, CDCl3): δ ϭ 1.34
173.8, 173.9. IR (nujol): ν˜ ϭ 3330 (NϪH), 1720, 1708, 1688, 1653 (s, 6 H, 2 ϫ CH3), 1.45 (s, 18 H, 2 ϫ tBu). IR (KBr): ν˜ ϭ 1726
(CϭO) cmϪ1
(CϭO) cmϪ1
BnO-mAib-
.
.
Di-tert-butyl dimethylmalonate (24.5 mmol, 6 g) in dry dichloro-
methane (40 mL) and TFA (196 mmol, 15 mL) was stirred for
20 min at 0 °C. The volatiles were then removed in vacuo, and
dichloromethane (50 mL) and water (50 mL) were added. The
aqueous layer was separated and solid KHSO4 was added to pH
2. CH2Cl2 (4 ϫ 200 mL) was then added and the layers were sepa-
rated. The combined organic layers were dried over sodium sulfate
and concentrated in vacuo. Compound 16 was obtained pure as a
white solid in 35% yield (1.61 g); m.p. 70Ϫ72 °C. 1H NMR
(250 MHz, CDCl3): 1.43 (s, 6 H, 2 ϫ CH3), 1.46 (s, 9 H, tBu). 13C
NMR (50 MHz, CDCl3): δ ϭ 22.5, 27.6, 50.5, 81.7, 171.6, 179.3.
L
-Ala-OtBu (13): A solution of HCl·H--Ala-OtBu
(24 mmol, 4.36 g) and triethylamine (72 mmol, 10 mL) in aceto-
nitrile (50 mL) was stirred for 30 min at room temp. Separately, a
solution of benzyl dimethylmalonate (1, 24 mmol, 5.29 g) and
HCTU (24 mmol, 9.93 g) in acetonitrile (50 mL) was stirred at
room temp. for 5 min. The first solution was then added dropwise
at room temp., and the resulting mixture was stirred for an ad-
ditional 20 min. The acetonitrile was then removed under reduced
pressure and replaced with dichloromethane (20 mL). The mixture
was washed with aqueous HCl (1 , 3 ϫ 30 mL) and aqueous
NaHCO3 (5%, 1 ϫ 30 mL), dried with sodium sulfate and concen-
trated in vacuo. The product was obtained pure as a liquid after
silica gel chromatography (cyclohexane/ethyl acetate, 8:2) in 83%
yield (6.95 g). [α]2D0 ϭ Ϫ1.1 (c ϭ 1.0, CH2Cl2). 1H NMR (200 MHz,
IR (KBr): ν˜ ϭ 1745, 1698 (CϭO) cmϪ1
.
tBuO-mAib- -Ala-NH2 (17): EDC·HCl (58.3 mmol, 7.2 g) was ad-
L
ded at 0 °C to a stirred solution of mono tert-butyl dimethylmalon-
ate (16, 53 mmol, 10 g) and HOAt (53 mmol, 7.2 g) in dichloro-
methane (100 mL). The mixture was stirred at room temp. until the
solution became clear. H--Ala-NH2 (63.6 mmol, 14.1 g) and
NMM (63.6 mmol, 14.1 g) were then added, and the mixture was
stirred for 48 h at room temp. Dichloromethane was removed in
vacuo and replaced with ethyl acetate (100 mL). The organic sol-
vent was washed with aqueous KHSO4 (10%, 50 mL), water
(50 mL), aqueous NaHCO3 (5%, 50 mL) and water (50 mL), dried
over sodium sulfate and concentrated in vacuo. Compound 17 was
obtained pure as a solid in 80% yield (10.9 g) after recrystallization
from ethyl acetate/petroleum ether; m.p. 119Ϫ120 °C. [α]2D0 ϭ Ϫ9.2
(c ϭ 0.5, MeOH). 1H NMR (250 MHz, CDCl3): δ ϭ 1.37 [d,
3JH,H ϭ 6.0 Hz; 3 H, AlaϪCH3], 1.41 (s, 6 H, 2 ϫ CH3), 1.46 (s,
9 H, tBu), 4.54 (m, 1 H, Ala CH), 5.86 (s, 1 H, NHH), 6.94 (s, 1
3
CDCl3): δ ϭ 1.28 [d, JH,H ϭ 7.2 Hz; 3 H, AlaϪCH3], 1.45 (s, 9
H, tBu), 1.47 (s, 3 H, CH3), 1.48 (s, 3 H, CH3), 4.38 [q, JH,H
3
ϭ
3
7.2 Hz; 1 H, Ala-CH), 5.18 (s, 2 H, OCH2Ph), 6.83 [d, JH,H
ϭ
6.8 Hz; 1 H, NH], 7.34 (m, 5 H, Ph). 13C NMR (75 MHz, CDCl3):
δ ϭ 18.0, 23.2, 23.3, 27.8, 48.7, 49.8, 66.9, 81.7, 127.9, 128.1, 128.4,
135.4, 165.6, 170.9, 171.8, 174.0. IR (film): ν˜ ϭ 3350 (NϪH), 1739,
1666 (CϭO) cmϪ1
.
BnO-mAib-L-Ala-OH (14): BnO-mAib--Ala-OtBu (13, 20 mmol,
6.9 g) in dry dichloromethane (50 mL) and TFA (180 mmol,
14 mL) was stirred for 4 h at room temp. The volatiles were then
removed in vacuo, and the product 14 was obtained as a thick
liquid in quantitative yield and was used without any further purifi-
cation. [α]2D0 ϭ Ϫ18.2 (c ϭ 1.1, CH2Cl2). 1H NMR (200 MHz,
3
H, NHH), 6.97 [d, JH,H ϭ 7.8 Hz; 1 H, Ala NH]. 13C NMR
3
CDCl3): δ ϭ 1.38 [d, JH,H ϭ 7.0 Hz; 3 H, AlaϪCH3], 1.49 (s, 6
H, tBu), 4.52 [dq, JH,H ϭ 7.0 Hz; 1 H, AlaϪCH], 5.19 (s, 2 H,
3
(100 MHz, CDCl3): δ ϭ 18.2, 23.2, 23.3, 27.7, 48.6, 50.6, 82.1,
172.4, 173.3, 174.6. IR (KBr): ν˜ ϭ 3389, 3277 (NϪH), 1731, 1641,
3
OCH2Ph), 6.97 [d, JH,H ϭ 6.6 Hz; 1 H, NH], 7.32Ϫ7.39 (m, 5 H,
Ph), 7.80 (s, 1 H, NH). 13C NMR (75 MHz, CDCl3): δ ϭ 17.5,
1620 (CϭO) cmϪ1
.
23.2, 23.4, 48.4, 49.8, 67.3, 128.0, 128.3, 128.5, 135.2, 172.3, 174.0,
TFA·؉H2-gAla-mAib-OtBu (18): A solution of tBuO-mAib--Ala-
NH2 (17, 5.8 mmol, 1.5 g) and TIB (8.1 mmol, 3.4 mmol) in aceto-
nitrile (60 mL) and water (40 mL) was stirred for 6 h at room temp.
under an inert atmosphere. The solvents were then removed under
an inert atmosphere. Compound 18 was obtained pure as a solid
in 61% yield (1.22 g) after repeated crystallization from diethyl
ether; m.p. 108Ϫ109 °C. 1H NMR (200 MHz, DMSO): δ ϭ 1.26
(s, 3 H, CH3), 1.28 (s, 3 H, CH3), 1.36 (br. s, 12 H, OtBu and
AlaϪCH3), 5.05 (m, 1 H, CHN), 8.42 (s, 3 H, ϩNH3), 8.61 [d,
3JH,H ϭ 7.2 Hz, 1 H, NH]. 13C NMR (50 MHz, DMSO): δ ϭ 18.4,
176.5. IR (film): ν˜ ϭ 3364 (NϪH), 1733, 1716, 1652 (CϭO) cmϪ1
.
Boc-gAla-mAib-OBn (15): A solution of 14 (4.7 mmol, 1.38 g),
DPPA (4.7 mmol, 1.02 mL) and triethylamine (5.2 mmol, 0.72 mL)
in tert-butyl alcohol (25 mL) was stirred at reflux for 5 h. The sol-
vent was then removed and replaced with ethyl acetate (20 mL).
The resulting solution was washed with HCl (1 , 15 mL), water
(15 mL), aqueous NaHCO3 (5%, 15 mL) and brine (15 mL), dried
over sodium sulfate and concentrated in vacuo. Compound 15 was
obtained pure as a liquid in 43% yield (0.74 g), after silica gel chro-
1
2
matography (cyclohexane/ethyl acetate, 8:2); m.p. 150Ϫ153 °C. H
NMR (300 MHz, CDCl3): δ ϭ 1.44 [d, JH,H ϭ 6.6 Hz; 3 H,
22.5, 22.7, 27.5, 50.3, 54.3, 80.6, 117.0 [q, JC,F ϭ 296 Hz, CF3],
3
3
159.4 [q, JC,F ϭ 36.8 Hz, CF3CO], 171.9, 172.4. IR (KBr): ν˜ ϭ
3394 (NϪH), 1717, 1677 (CϭO) cmϪ1
.
AlaϪCH3], 1.46 (s, 6 H, 2 ϫ CH3), 1.49 (s, 9 H, tBu), 5.18 [AB,
2JH,H ϭ 12.4 Hz; 2 H, OCH2Ph], 5.49 (m, 1 H, Ala-CH), 6.98 [d,
3JH,H ϭ 6.9 Hz; 1 H, NH], 7.30Ϫ7.40 (m, 5 H, Ph), 8.20 (d, 3JH,H ϭ
7.8 Hz; 1 H, NH). 13C NMR (75 MHz, CDCl3): δ ϭ 20.6, 23.2,
23.3, 28.0, 50.0, 54.5, 67.0, 82.8, 127.9, 128.2, 128.5, 135.5, 152.6,
152.8, 170.8, 174.2. IR (film): ν˜ ϭ 3344, 3304 (NϪH), 1759, 1738,
Z-gAla-mAib-OtBu (19): NMM (11.6 mmol, 1.28 mL) was added
at 0 °C to a stirred solution of TFA·ϩH2-gAla-mAib-OtBu (18,
5.8 mmol, 2.0 g) and ZϪOSu (7.5 mmol, 1.9 g) in dichloromethane
(25 mL). The mixture was stirred for 48 at room temp., and the
dichloromethane was removed in vacuo and replaced with ethyl
acetate (40 mL). The organic solution was washed with aqueous
KHSO4 (10%, 20 mL), water (20 mL), aqueous NaHCO3 (5%,
1690, 1639 (CϭO) cmϪ1
.
tert-Butyl Dimethylmalonate (16): A solution of dimethylmalonic
acid (227 mmol, 30 g) in dichloromethane (750 mL) was stirred at 20 mL) and water (20 mL), dried over sodium sulfate and concen-
Ϫ60 °C, and gaseous isobutene (8 mol, 450 g) was bubbled
through. Sulfuric acid (56 mmol, 3 mL) was then added, and the
trated in vacuo. Compound 19 was obtained pure as a solid in 80%
yield (0.99 g) after silica gel chromatography (dichloromethane/
4194
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2004, 4188Ϫ4196

Total Synthesis of Sequential Retro-Peptide Oligomers
FULL PAPER
1
ethanol, 98:2); m.p. 78Ϫ81 °C. H NMR (400 MHz, CDCl3): δ ϭ FT-IR Absorption: The FT-IR absorption spectra were recorded
1.35 (s, 6 H, 2 ϫ CH3), 1.43 (s, 9 H, tBu), 1.51 [d, 3JH,H ϭ 5.6 Hz;
with a PerkinϪElmer 1720X spectrophotometer, nitrogen flushed,
3 H, CH3], 5.08 (s, 2 H, OCH2Ph), 5.30 (m, 1 H, CHN), 5.76 (br. s, equipped with a sample-shuttle device, at 2 cmϪ1 nominal resolu-
1 H, NH), 7.04 (br. s, NH), 7.33 (m, 5 H, Ph). 13C NMR (100 MHz, tion, averaging 100 scans. Solvent (base-line) spectra were recorded
CDCl3): δ ϭ 20.5, 23.2, 23.4, 27.7, 50.5, 55.6, 66.7, 81.8, 128.0, under the same conditions. Cells with path lengths of 0.1, 1.0 and
128.1, 128.5, 136.2, 155.3, 172.0, 173.5. IR (KBr): ν˜ ϭ 3340 10 mm (with CaF2 windows) were used. Spectrograde CDCl3
(NϪH), 1728, 1681, 1662 (CϭO) cmϪ1
.
(99.8% D) was purchased from Fluka.
1H NMR: The H NMR spectra were recorded with a Bruker AM
400 spectrometer. Measurements were carried in deuteriochloro-
form (99.96% D; Aldrich) with tetramethylsilane as the internal
standard.
1
Z-gAla-mAib-OH (20): Z-gAla-mAib-OtBu (4.1 mmol, 1.50 g) in
dry dichloromethane (20 mL) and TFA (73.8 mmol, 5.6 mL) was
stirred for 35 min at 0 °C. The volatiles were then removed in va-
cuo, and dichloromethane (50 mL) and water (50 mL) were added.
The aqueous layer was separated and solid KHSO4 was added to
pH 2. Dichloromethane (4 ϫ 200 mL) was added and the layers
were separated. The organic layer was dried over sodium sulfate
and concentrated in vacuo. Compound 20 was obtained pure as a
X-ray Crystallography: Formula C13H23F3N2O5. Crystal (by slow
evaporation of a diethyl ether solution) dimensions 0.4 ϫ 0.3 ϫ
˚
˚
0.2 mm. Monoclinic, P21; a ϭ 9.160(2) A, b ϭ 10.946(3) A, c ϭ
3
9.282(2) A, β ϭ 98.93(2)°; V ϭ 919.4(4) A ; ρcalcd. ϭ 1.244 Mg·mϪ3
;
˚
˚
1
˚
solid in 21% yield (0.14 g); m.p. 148Ϫ150 °C. H NMR (400 MHz,
[D6]DMSO): δ ϭ 1.25 (s, 9 H, 3 ϫ CH3), 5.00 (br. s, 2 H, OCH2Ph),
2θmax. ϭ 120°; Cu-Kα radiation (λ ϭ 1.54178 A), θϪ2θ scan mode,
T ϭ 293 K; 1521 collected reflections, 1435 of which independent;
hkl limits: Ϫ10 Յ h Յ 10, 0 Յ k Յ 12, 0 Յ l Յ 10. Data collection
was performed on a Philips PW1100 four-circle diffractometer.
Intensities were corrected for Lorentz and polarization effects, but
not for absorption (µ ϭ 0.988 mmϪ1). The structure was solved by
direct methods (SHELXS 97 program[33]) and refined by full-ma-
trix block least-squares on F2, using all data, by application of the
SHELXL 97 program,[34] with all non-H atoms anisotropic, and
allowing the positional parameters and anisotropic displacement
parameters of the non-H atoms to refine at alternate cycles. The
ϪCF3 moiety of the trifluoroacetate anion shows rotational dis-
order. It was refined with the three fluorine atoms in two sets of
positions with population parameters of 0.60 and 0.40, respectively.
Restraints were applied to the CϪF bond lengths and to the aniso-
tropic displacement parameters of the fluorine atoms, the latter to
approach isotropic behaviour. Data/restraints/parameters: 1435/43/
5.31 (m, 1 H, CHN), 6.45 (br. s, 1 H, NH), 7.32 (m, 5 H, Ph), 7.68
3
[d, JH,H ϭ 6.8 Hz; 1 H, NH], 10.09 (br. s, 1 H, OH). 13C NMR
(100 MHz, [D6]DMSO): δ ϭ 20.9, 22.9, 23.0, 49.3, 54.6, 65.3,
127.7, 127.8, 128.3, 136.9 154.8, 171.1, 175.0. IR (KBr): ν˜ ϭ 3419,
3328 (NϪH), 1726, 1693, 1645 (CϭO) cmϪ1
.
Z-(gAla-mAib)2-OtBu (21): EDC·HCl (1.53 mmol, 0.29 g) was ad-
ded at 0 °C to a stirred solution of Z-gAla-mAib-OH (1.39 mmol,
0.43 mg) and HOAt (1.39 mmol, 0.19 g) in dichloromethane
(30 mL). The mixture was stirred at room temp. until the solution
became clear. Separately, tBuO-mAib-gAla-H·TFA (1.53 mmol,
0.53 g) and NMM (2.78 mmol, 0.31 mL) were stirred in dry di-
chloromethane (15 mL) for 5 min at room temp. The two solutions
were then mixed, and the resulting mixture was stirred for 48 h.
The volatiles were removed in vacuo and the product 21 was ob-
tained pure as a solid in 80% yield (0.58 g) after silica gel chroma-
tography (dichloromethane/ethanol, 98:2); m.p. 115Ϫ117 °C. 1H
NMR (400 MHz, CDCl3): δ ϭ 1.35Ϫ1.60 (m, 27 H, 9 ϫ CH3).
5.09 (m, 2 H, OCH2Ph), 5.35Ϫ5.38 (m, 2 H, 2 ϫ NCHN), 5.60 [br.
ϩ
242. The positions of the H-atoms of the terminal ϪNH3 group
were recovered from a difference Fourier map, while the remaining
H-atoms were calculated at the idealized positions. All H-atoms
were refined as riding. The refinement converged to R1 ϭ 0.067 [on
F ϭ 4σ(F)]; wR2 ϭ 0.180 (on F2, all data). Goodness of fit on F2 ϭ
1.071; max. and min. residual electron density peaks ϩ0.238 and
3
d, JH,H ϭ 13.1 Hz; 1 H, NH], 7.15Ϫ6.95 (m, 3 H, 3 ϫ NH),
7.32Ϫ7.35 (s, 5 H, Ph). 13C NMR (100 MHz, CDCl3, mixture of
diastereoisomers): δ ϭ 20.1, 20.7, 23.2, 23.3, 23.5, 27.7, 27.8, 49.5,
50.5, 54.5, 54.6, 55.5, 66.6, 81.9, 128.0, 128.1, 128.4, 136.2, 155.2,
172.0, 172.1, 172.2, 172.6, 172.7, 173.2, 173.3, 173.6. IR (KBr): ν˜ ϭ
3
˚
Ϫ0.292 e·A .
CCDC-234290 contains the supplementary crystallographic data
for this paper. These data can be obtained online free of charge [or
from the Cambridge Crystallographic Data Centre, 12 Union
Road, Cambridge CB2 1EZ, UK; fax (ϩ44)-1223-336-033; or
deposit@ccdc.cam.ac.uk].
3322 (NϪH), 1718, 1665 (CϭO) cmϪ1
.
Ac-(gAla-mAib)2-OtBu (22): Palladium on charcoal (10%, 5 mg)
was added to a solution of Z-(gAla-mAib)2-OtBu (21, 0.13 mmol,
68 mg) in methanol (10 mL) and the mixture was stirred under H2
(ca. 3 atm) for 12 h. The catalyst was then filtered through a celite
pad and the mixture was concentrated. The corresponding amine
tBuO-(mAib-gAla)2-H, obtained pure as a solid in quantitative
yield, was dissolved in dichloromethane (10 mL). Acetic anhydride
(10.6 mmol, 1 mL) was added, and the mixture was stirred for 24 h
at room temp. The volatiles were removed in vacuo. Compound 22
was obtained as a solid in 84% overall yield (47 mg) after recrystal-
lization from diethyl ether; m.p. 162Ϫ164 °C. 1H NMR (400 MHz,
CDCl3, mixture of diastereoisomers): δ ϭ 1.30Ϫ1.10 (m, 18 H, 6
ϫ CH3); 1.52Ϫ1.44 (m, 15 H, 5 ϫ CH3), 1.97 (s, 3 H, CH3), 5.35
(m, 1 H, NCHN), 5.49 (m, 1 H, NCHN), 6.52 and 6.60 (2d, J ϭ
6.9 Hz and 5.6 Hz; 1 H, NH) 7.11 (m, 3 H, 3 ϫ NH). 13C NMR
(50 MHz, CDCl3, mixture of diastereoisomers): δ ϭ 20.0 and 20.1
ppm, 20.4 and 20.5, 23.1, 23.2, 23.3, 23.4, 23.5, 27.8, 49.7 and 49.8,
50.6 and 50.7, 53.9 and 54.2, 54.5 and 54.7, 169.8 and 169.9, 172.3
and 172.4, 173.0, 173.1 and 173.2, 173.5. IR (KBr): ν˜ ϭ 3320
Acknowledgments
C. T., S. C. and G. L. thank the MURST Cofin 2002 (‘‘Sintesi di
peptidi ciclici o lineari contenenti ammino acidi inusuali analoghi
di arginina, glicina o acido aspartico (RGD) come nuovi inbitori di
integrine’’), FIRB 2001 (‘‘Eterocicli azotati come potenziali inbitori
enzimatici’’) and 60% (Ricerca Fondamentale Orientata), and the
University of Bologna (funds for selected topics ‘‘Sintesi e Caratte-
rizzazione di Biomolecole per la Salute Umana’’) for financial sup-
port.
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Received April 10, 2004
4196
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