Scientific paper
Synthesis of Bis-Aminoazirines and their Application in Peptide Synthesis
Peter Köttgen, Anthony Linden and Heinz Heimgartner*
Institute of Organic Chemistry, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland
* Corresponding author: E-mail: heimgart@oci.uzh.ch
Received: 29-09-2008
Dedicated to Professor Branko Stanovnik on the occasion of his 70th birthday
Abstract
The 'cyclohexane-bridged' bis-(3-amino-2H-azirines) cis- and trans-^,^'-dimethyl-^,^'-diphenyl-1,7-diazadispi-ro[2.2.2.2]deca-1,7-diene-2,8-diamine (cis-21 and trans-21) were synthesized from the corresponding bis-thioamide 20 by consecutive treatment with COCl2, 1,4-diazabicyclo[2.2.2]octane (DABCO) and NaN3. The reaction of these bis-azi-rines with different natural a-amino acids gave peptide amides 23. In addition, hydrolysis of the C-terminal amide groups of 23c and subsequent coupling with the Aib synthon 2, i.e., 2,2,^-trimethyl-^-phenyl-2H-azirin-3-amine, showed the applicability of building blocks 21 for peptide synthesis and peptide chain ligation.
Keywords: 2H-azirin-3-amines, a,a-disubstituted a-amino acids, aminoisobutyric acid (Aib), peptide synthesis
1. Introduction
Tools for the reduction of the conformational freedom of a protein and, therefore, for controlling its three-dimensional structure are of considerable interest in organic, medicinal and biochemistry.1,2 Conformational constraints can be achieved by incorporation of non-protein amino acids into the polypeptide chain.3 Substitution of
the H-atom at C(a) of a natural amino acid is one of the widest spread strategies for backbone modification leading to an enhanced tendency to form secondary structures such as ß-turns or helices.4-12 The most well-known a,a-disubstituted a-amino acid (2,2-disubstituted glyci-ne) is aminoisobutyric acid (Aib), which is ubiquitous in natural peptaibols and responsible for the helical conformation of these oligopeptides with antibiotic properties.13
Figure 1. Different types of 2H-azirin-3-amines.
The preferred formation of the 310-helical conformation has been demonstrated for several Aib-containing oligopeptides.14-20
A useful strategy for the introduction of these disub-stituted amino acids into peptides is the 'azirine/oxazolo-ne method',18-23 in which 2H-azirin-3-amines 1 are used as amino acid synthons. The reaction of the latter, e.g., the Aib synthon 2, with amino or peptide acids leads to pepti-de amides, the terminal amide groups of which can be hydrolyzed selectively.
Based on the first synthesis of Rens and Ghosez,24 numerous 2H-azirin-3-amines have been prepared,22,25 27 including enantiomerically pure chiral compounds,28,29 spirocyclic8a,30 and heterospirocyclic compounds (e.g. 3),31,32 as well as dipeptide synthons of type 4 and 518,33-35 (Figure 1). These molecules have been applied successfully in the synthesis of peptaibols,18-20,23,36 endothiopep-
tides,37-40 cyclic peptides,41-43 and cyclic depsipepti-des.44-47 Furthermore, the crystal structure of the 1,4-bis(2,2dimethyl-2H-azirin-3-yl)piperazine (6) has been published,48 without disclosing its synthesis and reactions.
A very general and effective way of introducing a global constraint into a peptide chain is the formation of a covalent bond between distant parts in the sequence, e.g., by formation of disulfide bridges between cysteine resi-dues.49 Because this might possibly also be achieved between two carboxylic acid functions by using a bis-(3-ami-no-2H-azirine) of type 7 (Scheme 1), it was of interest to develop a synthesis of such molecules by using similar methods as described for the monomeric analogues.24,50-52
In the present paper we report the synthesis of the bis-(3-amino-2H-azirines) cis-21 and trans-21, representing the first examples of this new class of molecules. Reactions with several ^-protected a-amino acids confirmed that these new building blocks are suitable for pepti-de synthesis.
2. Results and Discussion
In preliminary studies we elucidated the appropriateness of bis-(N-methyl-N-phenylamides) for azirine synthesis and attempted the preparation of compounds with two (3-amino-2H-azirine) structures linked together by an alkyl chain. The preparation of 2,4-dimethylpenta-nedioic acid bis-(methylphenylamide) (9) and 2,7-di-methyloctanedioic acid bis-(methylphenylamide) (10) was achieved via a-alkylation of two equivalents of N-methyl-^-phenylpropanamide (8) with dibromomethane or 1,4-dibromobutane, respectively (Scheme 2). Since the conversion of these compounds into the corresponding bis-azirines according to the procedure of Villalgordo and Heimgartner^^ was not successful, 9 and 10 were converted into the corresponding thioamides 11 and 12 by reaction with P2S5 and hexamethyldisiloxane (HMDO).53 These are the required starting materials for a synthesis analogous to the procedure described earlier.50,51 In the case of 11, consecutive treatment with COCl2 in CH2Cl2, evaporation of the solvent, dissolution of the residue in THF, addition of 1,4-diazabicyclo[2.2.2]octane (DABCO), filtration, and reaction of the filtrate with NaN3 gave 4H-
vw ^
H
R
"HN.
O O
N
.N(Me)Ph
.N(Me)Ph
R
wv»[Sj
H
\ H
H
O
o
R
O
N(Me)Ph
,N(Me)Ph
O
R
H
\ H
o o
O
H
H
O
O
I
Me
1. LDA ,Ph 2. Br-(CH2)n-Br
Ph-
I
Me
Scheme 2

11 n= 1 12n = 4
-N' Me
-Rh
1.	COCI2
2.	DABCO
3.	NaNg
O
Rh,
N'
i
Me
.(CH2)4.
14
N
Me I
■Rh
thiopyran-2,6-diamine 13 in quantitative yield, instead of the desired bis-azirine.
A plausible mechanism for the formation of 13 is shown in Scheme 3. The key step is the cyclization of the mono-chloriminium salt 16 via nucleophilic attack of the neighboring thioamide group to give the bis-iminium salt 17, which stabilizes by twofold deprotonation. It is likely that this side reaction is favored in the case of the formation of a six-membered ring.
After the treatment of 12 under the same conditions, no product could be isolated due to decomposition of the crude material during the purification process. However, analysis of the crude product by mass spectrometry showed the absence of a nine-membered cyclization product analogous to 13, but indicated the presence of a mixture of
Rh'
Me
I
Y
N
(CH2)4
15
Tvr
/ N
Me
i
~Rh
the corresponding mono-azirine 14 and the bis-azirine 15. In addition, an IR-absorption at 1750 cm-1, which is characteristic for 2H-azirin-3-amines, was observed. Although the desired compound could not be isolated, it seemed possible that bis-(3-amino-2H-azirines) could be synthesized by this method using phosgene.
Me
\
N
Rh^
11
Me
/
N
>h
COCl2,CH2Cl2
-COS
16
Me
Me
CI
Rh
,e
^	Rh

„0
DABCO
-2 HCl
Rh'
Me I
Me I
~Rh
17
13
CO2H
1.	SOCI2
2.	Ph(Me)NH/Et3N
CO2H
18 {cis/trans)
1.	COCI2
2.	DABCO
3.	NaNg
Me-N
Ph
Me
P2S5, (Me3Si)20
O^ N
I
Me
19 (cis/trans) Ph
N-Me
' + /
'N	/
Me-N
Ph
Me
Me
20 (cis/trans)
N-Me
Scheme 4
trans-21 (39%)
Ph^ c/s-21 (31%)
Since in the case of 15 the formation of different stereoisomers is possible, which probably would be difficult to separate because of the flexibility of the molecule, we decided to use a cyclohexane ring as a symmetric and less flexible connection between the two three-membered hete-rocycles. The preparation of the required starting material 20 for the azirine synthesis was performed starting with the commercially available cis/trans-1,4-cyclohexanedicar-boxylic acid 18. Conversion to the bis-(^-methyl-^-phenyl-amide) 19 (cis/trans mixture) via the bis-acyl chloride and subsequent thionation with P2S5/HMDO gave cyclohexane-1,4-dicarboxylic acid bis-(^-methyl-^-phenylthioamide) 20.* Treatment of the latter with COa2, DABCO and NaN3
as described above yielded a mixture of cis-21 and trans-21. These isomers were separated by means of column chromatography and were obtained as solid products in 31% and 39% yield, respectively (Scheme 4).
Recrystallization of cis-21 from a mixture of CH2Cl2, acetone, hexane, and Et2O by slow evaporation of the solvent gave crystals that were sufficiently adequate for a crude X-ray crystal structure determination, which allowed the essential conformation of the molecule to be established. The molecular structure of cis-21 is shown in Figure 2.
The cyclohexane ring shows the chair conformation with the unsubstituted N(9) and N(16) atoms of the aziri-
Figure 2. ORTEP Plot54 of the molecular structure of cis-21 (50% probability ellipsoids, arbitrary numbering of the atoms).
* NMR spectroscopy did not give any indication for the presence of two diastereoisomers. As the reaction pathway for the synthesis of 3-amino-2ff-azi-rines involves intermediates with a sp2-C(a) atom of the thioamide,22 the stereochemical properties of the starting material are irrelevant.
ne rings cis to one another and in pseudo-equatorial and pseudo-axial orientations, respectively.
To examine the reactivity of the new bis-(3-amino-2H-azirines) cis-21 and trans-2\, they were reacted with Z-protected L-alanine (22a)** in CHCl, which lead to the corresponding peptides 23a (Scheme 5). Compound trans-21 was chosen for reactions with other natural a-amino acids, which bear different protecting groups, i.e., Z-Phe-OH (22b), Fmoc-Leu-OH (22c), and Z-Lys(Boc)-OH (22d), leading to peptides 23b-d. In an analogous manner, the reaction of trans-21 with acetic acid gave the corresponding trans-1,4-bis(acetylamino)cyclohexane-1,4-dicarboxamide 24 (not shown in Scheme 5). All reactions proceeded smoothly at room temperature to give the products in high to very high yields (81-97%) without the formation of any side products.
The terminal amide groups of peptide 23c were selectively hydrolyzed using the standard procedure (3N
HCl in water/THF) to give the peptide 25c with unprotected C-termini. The latter was subsequently reacted with the Aib synthon 2 to yield the extended peptide 26c, showing that bis-azirine trans-21 (as a representative of both diastereoisomers) is a convenient building block for the 'azirine/oxazolone method' (Scheme 6).
3. Conclusions
The first examples of bis-(2H-azirin-3-amines) connected via the C(2)-atoms, i.e., cis- and trans-21, were prepared in good yields from the corresponding bis-(thio-amide) by using the 'phosgene methodology'. Furthermore, it has been shown that the reactivity of these bis-aziri-nes is similar to that of known 2H-azirin-3-amines and, therefore, they can be used as synthons of bis-(amino acids) in the preparation of ligated peptide chains.

N-
Me
I

N' I
Me
.Ph
c/s-21
22a
c/s-23a
N K ^Ph
H // N R	O I
Me
trans-2A
Scheme 5
22a-d a R = Me, PG = Z	trans-23a-d
bR = CH2-Ph, PG = Z c R = CH2-CHMe2, PG = Fmoc d R = (CH2)4-NH-Boc, PG = Z
Abbreviations: Z = benzyloxycarbonyl, Boc = tert-butyloxycarbonyl, Fmoc = (9H-fluoren-9-yl)methoxycarbonyl
Fmoc'
H
-N,
O
O

Fmoc
3N HCl, H2O/THF
H J N(Me)Ph
trans-23c
Fmoc
Fmoc
N(Me)Ph
Scheme 6
trans-26c
4. Experimental
4. 1. General Procedures
Melting points were determined on a Büchi 540 apparatus; they are uncorrected. IR spectra were recorded on a Perkin-Elmer spectrum one spectrophotometer; absorption bands in cm-1. 1H NMR (300 MHz) and 13C NMR (75.5 MHz) spectra were obtained on a Bruker ARX-300 instrument at room temperature (r.t.); TMS was used as internal standard, 5 in ppm. Mass spectra (MS) were recorded on a Finnigan SSQ-700 spectrometer for chemical ionization (CI, with NH3) and electrospray ionization (ESI, in MeOH + NaI), and on a Finnigan MAT95 spectrometer for HR-MS (CI). Thin-layer chromatography (TLC) was performed on Merck TLC aluminium sheets, silica gel 60 F254, and flash chromatography (CC) on Ueti-kon-Chemie 'Chromatographiegel C-560'. The Aib syn-thon 2 was prepared according to ref. 52, and amide 8 was prepared according to ref. 30. All other products used were commercially available.
4. 1. 1. General Procedure 1 (GP1)
To a solution of diisopropylamine (2.16 ml, 15.3 mmol) in THF (30 ml) at -80 °C was added butyllithium (8.8 ml, 1.6M in hexane, 14.1 mmol), and the mixture was stirred for 30 min. Then, amide 8 (2.1 g, 12.8 mmol) in THF (5 ml) was added, and after 30 min stirring at -80 °C, the corresponding dibromoalkane (6.4 mmol) was added. The
reaction mixture was allowed to warm up to r.t. slowly. A saturated aqueous NH4Cl-solution (40 ml) was added and the aqueous layer extracted with EtjO (2 x 30 ml). The combined organic fractions were washed with water (40 ml) and brine (20 ml). After drying over MgSO4, filtration, and evaporation of the solvent, the residue was purified by CC.
4. 1. 2. General Procedure 2 (GP2)
A mixture of the corresponding amide, P2S5 and he-xamethyldisiloxane (HMDO) in CHCl3 was heated under reflux until complete conversion of the amide was observed by TLC. After cooling to r.t., the solvent was evaporated and the residue purified by CC.
4. 1. 3. General Procedure 3 (GP3)
To a solution of the corresponding thioamide (3.81 mmol) and 3 drops of DMF in CH2Cl2 (20 ml) at 0 °C was added phosgene*** (20% solution in toluene, 9 ml, 18.1 mmol). The mixture was stirred at r.t. for 60 min and was then concentrated under reduced pressure. THF (40 ml) and DABCO (852 mg, 7.62 mmol) were added and the mixture was stirred for 30 min at r.t. The formed precipitation was filtered under nitrogen, and after the addition of
*** Warning: Phosgene is a highly toxic gas (b.p. 8 °C), which has to be handled with extreme caution. Its solution in toluene is more safe and was used in a closed system.
DMF (30 ml) and NaN3 (2.0 g, 30.8 mmol), the suspension was stirred for 60 h at ambient temperature. Filtration and evaporation of the solvent gave the crude product that was purified as indicated.
4. 1. 4. General Procedure 4 (GP4)
A solution of azirine cis-21 or trans-21 and the corresponding carboxylic acid in CHCl3 was stirred at r.t. until complete conversion of the starting materials was observed (TLC). The mixture was then washed three times with saturated aqueous NaHCO3-solution and dried over MgSO4. Evaporation of the solvent gave the crude product that was purified as indicated.
4. 2. Attempted Synthesis of Bis-azirines of Type 15
2,4,^,^'-Tetramethyl-^,^'-diphenylpentanedioic Acid Diamide (9). Prepared according to GP1; with dibromo-methane (1.11 g, 0.45 ml, 6.4 mmol), CC (SiO2, hexa-ne/AcOEt 1:1, then 2:3). Yield: 950 mg (44%) of amide 9. White powder; mp 83 °C. IR (KBr): vmax 3291w, 3052w, 2970s, 2931m, 2871w, 1651s, 1594max 1494s, 1455s, 1425s, 1387s, 1329m, 1273s, 1113s, 1026m, 773m, 703s cm-1. 1H NMR (CDCl3): 5 0.79 (6H, d, J = 6.7 Hz, 2 MeCH); 1.59-1.64 (2^, m, CH2); 2.37-2.45 (2H, m, 2 MeC^); 3.28 (6H, s, 2 MeN); 7.19-7.47 (10H, m, 2 Ph). 13C NMR (CDCl3): 5 16.9 (q, 2 MeCH); 33.6 (d, 2 MeCH); 37.3 (q, 2 MeN); 37.8 (t, CH2); 127.3, 127.6,
129.6	(3d, 10 arom. CH); 143.9 (s, 2 arom. CN); 176.0 (s, 2 CO). ESI-MS (m/z): 361 (100, [M + Na]+).
2,7,^,^'-Tetramethyl-^,^'-diphenyloctanedioic Acid Diamide (10). Prepared according to GP1; with 1,4-di-bromobutane (1.38 g, 0.76 ml, 6.4 mmol), CC (SiO2, he-xane/AcOEt 3:2). Yield: 1.92 g (79%) of amide 10. WVhite powder; mp 55-56 °C. IR (KBr): vmax 3041w, 2930m, 2861w, 1650s, 1593s, 1493s, 1464m, 1386m, 1263m, 1117m, 1031m, 776m, 702s cm-1. 1H NMR (CDCl3): 5 0.98 (6H, d, J = 6.7 Hz, 2 MeCH); 0.98-1.29, 1.50-1.69 (6H + 2H, 2m, 4 CH2); 2.26-2.38 (2H, m, 2 MeC^); 3.25 (6H, s, 2 MeN); 7.12-7.15, 7.28-7.43 (4H + 6H, 2m, 2 Ph). 13C NMR (CDCl3): 5 18.1 (q, 2 MeCH); 27.5, 34.4 (2t, 4 CH2); 36.3 (d, ^ MeCH); 37.2 (q, 2 MeN); 127.3, 127.5, 129.6 (3d, 10 arom. CH); 144.1 (s, 2 arom. CN);
176.7	(s, 2 CO). ESI-MS (m/z): 403 (100, [M + Na]+).
992s, 777m, 771m, 699s cm-1. 1H NMR (CDCl3): 5 0.74 (6H, d, J = 6.4 Hz, 2 MeCH); 1.83 (2H, t, J ^ 6.9 Hz, CH2); 2.73-2.80 (2H, m, 2 MeC^); 3.69 (6H, s, 2 MeN); 7.344-7.49 (10H, m, 2 Ph). 13C NMR (CDCl3): 5 20.2 (q, 2 MeCH); 40.5 (t, CH2); 44.9 (d, 2 MeCH); 435.4 (q, 2 MeN); 125.5, 128.4, 1^9.9 (3d, 10 arom. CH); 145.3 (s, 2 arom. CN); 211.1 (s, 2 CS). ESI-MS (m/z): 393 (100, [M + Na]+).
2,7,^,^'-Tetramethyl-^,^'-diphenyloctane-bis-thioic Acid Diamide (12). Prepared according to GP2; with amide 10 (1.14 g, 3.0 mmol), P^Sj (1.28 g, 5.7 mmol) and HMDO (3.97g, 5.2 ml, 24.4 mmol) in CHCl3 (20 ml), 14 h, CC (SiO2, hexane/AcOEt 10:1). Yield: 408 mg (33%) of thioamide 12. Yellowish powder; mp 109 °C. IR (KBr): vmax 3427w, 2957m, 2925m, 2858m, 1694m, 1492s, 14(:,1s, 1443s, 1383s, 1272m, 1110m, 1033s, 773m, 700s cm-1. 1H NMR (CDCl3): 5 1.08 (6H, d, J = 6.6 Hz, 2 MeCH): 0.89-1.09, 1.2^3-1.39, 1.60-1.82 (4H + 2H + 2H, 3m, 4 CH2); 2.56-2.70 (2H, m, 2 MeC^); 3.72 (6H, s, 2 MeN); 7.07-7.14, 7.36-7.48 (4H + 6H, 2m, 2 Ph). 13C NMR (CDCl3): 5 22.1 (q, 2 MeCH); 27.6, 38.1 (2t, 4 CH2); 43.9 (d, 2 MeCH); 45.4 (q, 2 MeN); 125.4, 128.3, 129.9 (3d, 10 arom. CH); 145.62 (s, 2 arom. CN); 212.1 (s, 2 CS). ESI-MS (m/z): 435 (100%, [M + Na]+).
3,5,^,^'-Tetramethyl-^,^'-diphenyl-4H-thiopyran-2,6-diamine (13). Prepared according to GP3; with thioamide 11 (1.41 g), CC (SiO2, hexane/AcOEt 4:1). Yield: 1.23 g (96%) of 13. Orangej oil. IR (film): vmax 3026w, 2985w, 2913w, 2903m, 2810w, 1598s, 1577m, 1499s, 1451m, 1336s, 1297m, 1236m, 1108m, 1034m, 1001m, 909m, 748s cm-1. 1H NMR (CDCl3): 5 1.69 (6H, s, 2 Me); 3.05 (6H, s, 2 MeN); 6.72-6.80, 7.15-7.22 (6H + 4H, 2m, 2 Ph). 13C NMR (CDCl3): 5 18.7 (q, 2 Me); 37.4 (q, 2 MeN); 40.0 (t, CH2); 113.0, 117.6, 128.9 (3d, 10 arom. CH); 125.1, 133.8, 147.0 (3s, 2 C=C + 2 arom. CN). CI-MS (m/z): 337 (100, [M + H]+), 91 (5%).
Attempted synthesis of 15. Acoording to GP3, thioamide 12 (1.57 g), was treated with COCl2, DABCO, and NaN3; attempted isolation led to decomposition. IR (film, crude mixture): vmax 3398m, 2933m, 2857w, 2222w, 2155m, 2029w, 1750s, 1654m, 1599s, 1501s, 1460w, 1113m, 755m cm-1. ESI-MS of the crude mixture (m/z): 403 (84, [M(10) + Na]+), 400 (100, [M(14) + Na]+), 397 (34, M(15) + Na]+).
2,4,^,^'-Tetramethyl-^,^'-diphenylpentane-bis-thi-oic Acid Diamide (11). Prepared according to GP2; with amide 9 (1.01 g, 3.0 mmol), P^Sj (1.28 g, 5.7 mmol) and HMDO (3.97g, 5.2 ml, 24.4 mmol) in CHCl3 (20 ml), 14 h, CC (SiO2, hexane/AcOEt 6:1). Yield: 930 mg (84%) of thioamide 11. Yellowish powder; mp 110-112 °C. IR (KBr): vmas 3043w, 2973m, 2925m, 2861w, 1592m, 1492s, 14K53s, 1379s, 1343m, 1268m, 1106s, 1069m,
4. 3. Synthesis of cis- and trans-N,N'-
Dimethyl-N,N'-diphenylyclohexane-1,7-diazadispiro[2.2.2.2]deca-1,7-diene-2,8-diamine (cis-21 and trans-21) N,N'-Dimethyl-N,N'-diphenylclohexane-1,4-dicarbox-amide (19). A solution of 1,4-cyclohexanedicarboxylic acid (cis/trans mixture, 10.0 g, 58 mmol) in SOCl2 (100 ml)
was heated to reflux for 6 h. The mixture was concentrated under reduced pressure and the residue dried in high vacuum. Ethyl acetate (300 ml) and, after cooling to 0 °C, triethylamine (13.1 g, 18.2 ml, 130 mmol) and N-methy-lanilin (13.9 g, 14.1 ml, 130 mmol) were added slowly. The mixture was stirred at r.t. for 16 h; then water (200 ml) was added. The organic layer was extracted with 1N aqueous HCl-solution (3 x 100 ml), 1N aqueous NaOH-solution (3 x 100 ml), and brine (50 ml). Evaporation of the solvent and crystallization from toluene gave bis-ami-de 19 (mixture of 2 diastereoisomers, ratio ca. 1:1). White powder; mp 156-158 °C. IR (KBr): vmax 2946m, 2920m, 2858w, 1644s, 1592m, 1495m, 1451w, 1417w, 1388m, 1270m, 1117m, 779w, 702s cm-1. 1H NMR (CDCl3): 5 1.18-1.41, 1.54-2.03, 2.10-2.41 (4H + 4H + 2H, 3m, 4 CH2 + 2 CH); 3.19, 3.22 (6H, 2s, 2 MeN); 7.10-7.46 (10H, m, 2 Ph). 13C NMR (CDCl3): 5 26.0, 28.2 (2t, 4 CH2); 37.4, 37.6 (2q, 2 MeN); 40.2 (d, 2 CH); 126.2, 127.1, 127.3, 127.9, 129.6, 129.8 (6d, 10 arom. CH); 144.0, 144.6 (2s, 2 arom. CN); 175.7, 175.9 (2s, 2 CO). ESI-MS (m/z): 373 (100, [M + Na]+). Anal. Calcd. for C22H26N2O2 (350.20): C, 75.40; H, 7.48; N, 7.99. Found: C, 75.31; H, 7.22; N, 7.78.
7ra«s-^,^-Dimethyl-^,^-diphenylcyclohexane-1,4-biscarbothioamide (20). Prepared according to GP2; with amide 19 (2.1g, 6.0 mmol), P^Sj (1.28 g, 5.7 mmol), and HMDO (3.97g, 5.2 ml, 24.4 mmol) in CHCl3 (20 ml), 3 h, CC (SiO2, CH2Cl2). Yield: 1.93 g (84%) of thioamide 20. Yellowisli pow;der; mp 249-250 °C. IR (KBr): vmax 3043w, 2947m, 2923m, 2856m, 1593w, 1586w, 1492sx 1467s, 1444s, 1380s, 1358m, 1269m, 1179m, 1101m, 1050m, 766m, 751m, 694s cm-1. 1H NMR (CDCl3): 5 1.53-1.64 (8H, m, 4 CH2); 2.53-2.61 (2H, m, 2 CH); 3.65 (6H, s, 2 MeN); 7.08-7.12, 7.40-7.53 (4H + 6H, 2m, 2 Ph). 13C NMR (CDCl3): 5 32.5 (t, 4 CH2); 45.6 (d, 2 CH); 47.9 (q, 2 MeN); 125.1, 128.7, 130.0 (^d, 10 arom. CH); 145.4 (s, 2 arom. CN); 210.3 (s, 2 CS). CI-MS (m/z): 383 (100, [M + H]+). Anal. Calcd. for C22H2gN2S2 (382.59): C, 69.07; H, 6.85; N, 7.32; S, 16.76; Found: C, 69.02; H, 6.48; N, 7.21; S, 16.79.
Cis- and ^ra«s-^,^'-Dimethyl-^,^'-diphenyl-1,7-di-azadispiro[2.2.2.2]deca-1,7-diene-2,8-diamine (cis-21 and trans-21). Prepared according to GP3; with thioamide 20 (1.45 g), CC (SiO2, hexane/AcOEt 1:2, then 1:5). Yields: 405 mg (31%) of cis-21 and 510 mg (39%) of trans-21.
Data of cis-21: Yellowish powder; mp 110-112 °C. IR (KBr): vmax 3399w, 2944m, 2909m, 2212w, 1759s, 1647m, 1599s, 1500s, 1354m, 1279s, 1112m, 1009m, 949m, 890w, 759s, 693m cm-1. 1H NMR (CDCl3): 5 1.62-2.21 (8H, m, 4 CH2); 3.51 (6H, s, 2 MeN); 7.02-7.50 (10H, m, 2 Ph). 13C NMR (CDCl3): 5 34.2 (t, 4 CH2); 117.4, 123.6, 129.2 (3d, 10 arom. CU); 142.7 (s, 2 aroim. CN); 167.9 (s, 2 C=N); MeN and spiro-C could not
be detected. CI-MS (m/z): 345 (100, [M + H]+), 238 (19), 108 (41). HR-CIMS (m/z): 345.2082 ([M + H]+); calcd. for C22H25N4: 345.2079 (5 = 0.8 ppm).
Crystals for an X-ray crystal-structure determination were grown from a solution of cis-21 in a mixture of CH2Cl2, acetone, hexane, and Et2O by slow evaporation of the solvent.
Data of trans-21: Yellowish powder; mp 98-99 °C. IR (KBr): v9ax 3433,w 2950m, 2929m, 2905m, 2827m, 1744s, 1596s, 1503s, 1320m, 1235m, 1189m, 1101s, 1034w, 752s, 690m cm-1. 1H NMR (CDCl3): 5 1.31-1.39, 2.54-2.63 (4H + 4H, 2m, 4 CH2); 3.49 ((5H, s, 2 MeN); 7.11-7.21, 7.39-7.48 (4H + 6H, 2m, 2 Ph). 13C NMR (CDCl3): 5 33.2 (t, 4 CH2); 115.8, 123.1, 129.4 (3d, 10 arom. CH); 142.2 (s, 2 arom. CN); 176.8 (s, 2 C=N); MeN and spiro-C could not be detected. CI-MS (m/z): 345 (100, [M + H]+), 238 (33), 108 (10). HR-CIMS (m/z): 345.2088 ([M + H]+); calcd. for C22H25N4: 345.2079 (5 = 2.6 ppm).
4. 4. Reactions of cis-21 and trans-21 with Amino Acids
Benzyl Cis-[(S)-1-(4-{(S)-2-[(Benzyloxycarbonyl)ami-no]propanoylamino}-1,4-bis(^-methyl-^-phenylcar-bamoyl)cyclohexylcarbamoyl)ethyl]carbamate (cis-23a). Prepared according to GP4; with cis-21 (30 mg, 0.087 mmol) and Z-Ala-OH (42 mg, 0.19 mmol) in CHCl3 (5 ml), 2 h, CC (SiO2, hexane/AcOEt 1:3). Yield: 63 m^ (91%) of cis-23a. Yellowish powder; mp 177-178 °C. IR (KBr): V9ax 3295s, 3061m, 2975m, 2936m, 1722s, 1674s, 1630s, 15^2m, 1493s, 1453m, 1386m, 1239s, 1098m, 1069m, 738m, 698s cm-1. 1H NMR (dg-DMSO): 5 1.20 (6H, d, J = 6.9 Hz, 2 MeCH); 1.60-1.90, 2.05-2.30 (4H + 4H, 2m, 4 CH2); 3.17 (6H, s, 2 MeN); 3.95-4.08 (2H, m, 2 MeCH); 5.01, 5.05 (4H, 2d, J = 12.7 Hz, 2 CH2O); 7.06-7.41 (22H, m, 4 Ph + 2 NH); 7.88 (2H, br, 2 NNH). 13C NMR (dg-DMSO): 5 18.7 (q, 2 MeCH); 28.6, 29.1 (2t, 4 CH2); 39.8 (q, 2 MeN); 49.5 (d, 2 MeCH); 57.8 (s, 2 (CH2);;C); 65.1 (t, 2 CH2O); 126.4, 127.3, 127.5, 127.6, 128.2, 128.7 (6d, 20 arom. CH); 137.1 (s, 2 arom. C); 145.4 (s, 2 arom. CN); 155.5 (s, 2 O-CO-N); 171.2, 171.5 (2s, 4 CO). ESI-MS (m/z): 813 (100, [M + Na]+). [a]25D: -7.7 (c 0.5, MeOH). Anal. Calcd. for C44H50NgO8■0.5 H2O (799.92): C, 66.07; H, 6.42; N, 10.51. I^ound: C, 66.11; H, 6.40; N, 10.31.
Benzyl Trans- [(S)-1-(4-{(S)-2-[(Benzyloxycarbonyl) amino]propanoylamino}-1,4-bis(^-methyl-^-phenyl-carbamoyl)cyclohexylcarbamoyl)ethyl]carbamate (trans-23a). Prepared according to GP4; with trans-21 (30 mg, 0.087 mmol) and Z-Ala-OH (43 mg, 0.19 mmol) in CHCl3 (5 ml), 2 h, CC (SiO2, hexane/AcOEt 1:3). Yield: 67 mg (97%) of trans-23a. White powder; mp 195 °C. IR (KBr): V9ax 3329s, 3033m, 2937m, 1717s, 1674s, 1627s, 1593m, 1528s, 1493s, 1453s, 1378m, 1248s,
1070m, 739m, 700s cm-1. 1H NMR (CDCl3): 5 1.25 (6H, d, J = 6.7 Hz, 2 MeCH); 1.89-2.24 (8H, m, 4 CH2); 3.14 (6H, s, 2 MeN); 3.53 (2H, br, 2 NH); 5.11-5.30 (6H, m, 2 CH20 + 2 MeCH); 6.02 (2H, br, 2 NH); 7.07-7.48 (20H, m, 4 Ph). 13C NMR (CDCl3): 5 17.3 (q, 2 MeCH); 27.1, 28.0 (2t, 4 CH2); 41.1 (q, 2 IVIeN); 49.9 (d, 2 MeCH); 58.9 (s, 2 (CH2)2C); 67.4 (t, 2 CH2O); 126.8, 127.6, 128.1, 128.3, 12^.42, 129.4 (6d, 20 arom. CH); 136.2 (s, 2 arom. C); 144.9 (s, 2 arom. CN); 156.1 (s, 2 O-CO-N); 171.1, 171.8 (2s, 4 CO). ESI-MS (m/z): 813 (100, [M + Na]+). [a]25D: -9.6 (c 0.6, MeOH). Anal. Calcd. for C44H50Ng08-0.5 H20 (799.92): C, 66.07; H, 6.42; N, 10.51. Found: C, 6^.17; H, 6.39; N, 10.42.
Benzyl 7rans-[(S)-1-(4-{(S)-2-[(Benzyloxycarbonyl) amino]-3-phenylpropanoylamino}-1,4-bis-(^-methyl-^-phenylcarbamoyl)cyclohexylcarbamoyl)-2-phenyl-ethyl]carbamate (trans-23b). Prepared according to GP4; with trans-21 (100 mg, 0.29 mmol) and Z-Phe-OH (190 mg, 0.64 mmol) in CHCl3 (10 ml), 2 h, CC (SiO2, CH2Cl2/MeOH 25:1). Yield: 220 mg (81%) of trans-23^. White powder; mp 177-178 °C. IR (KBr): vmax 3305s, 3061m, 3030m, 2948m, 1954m, 1717s, 1661;^, 1627s, 1592m, 1493s, 1454s 1381m, 1255s, 1148m, 1032m, 748m, 698s cm-1. 1H NMR (dg-DMSO): 5 1.71-2.02 (8H, m, 4 CH2); 2.63 (2H, t, J = 13.1 Hz, 2 PhCH); 2.89 (2H, dd, J1 = 3.6 Hz, J2 = 13.1 Hz, 2 PhCH); 3.03 (s, 6H, 2 MeN); 41.11-4.21 (2H, m, 2 CHN); 4.79, 4,87 (4H, 2d, J = 12.7 Hz, 2 CH2O); 6.96-7.27 (m, 30H, 6 Ph); 7.39 (2H, d, J = 8.7 Hz, 2 NH); 7.86 (2H, s, 2 NH). 13C NMR (dg-DMSO): 5 26.5, 26.9 (2t, 4 CH2); 37.2 (t, 2 PhCH2); 39.7 (q, 2 MeN); 57.8 (s, 2 (CH2)2C); 65.2 (t, 2 PhCH2O); 126.1, 126.3, 127.1, 127.3, 1^7.5, 127.9, 128.1, 128.8, 129.1 (9d, 30 arom. CH); 136.9, 137.9 (2s, 4 arom. C); 145.5 (s, 2 arom. C); 155.9 (s, 2 O-CO-N); 170.7, 172.1 (2s, 2 CO). ESI-MS (m/z): 981 (8, [M + K]+), 965 (100, [M + Na]+). [a]25D: -12.1 (c 0.6, MeOH). Anal. Calcd. for C5gH58NgO8-0.5 H2O (952.12): C, 70.64; H, 6.25; N, 8.83. Found: C:, ^0.45; H, 6.19; N, 8.61.
(9H-Fluoren-9-yl)methyl rrans-((S)-1-{4-[(S)-2-{[(9H-Fluoren-9-yl)methoxy]carbonylamino}-4-(methylpen-tanoyl)amino]-1,4-bis(^-methyl-^-phenylcarba-moyl)cyclohexylcarbamoyl}-3-methylbutyl)carbama-te (trans-23c). Prepared according to GP4; with trans-21 (100 mg, 0.29 mmol) and Fmoc-Leu-OH (226 mg, 0.64 mmol) in CHCl3 (10 ml), 2 h, CC (SiO2, CH2Cl2/MeOH 33:1). Yield: 268 mg (88%) of trans-23c. White powder; mp 218 °C. IR (KBr): vmax 3304s, 3064m, 3039m, 2955s, 2868m, 1724s, 1653s,mT629s, 1593m, 1524s, 1493s, 1449s, 1383m, 1331m, 1252s, 1033m, 758m, 738s, 700m cm-1. 1H NMR (dg-DMSO): 5 1.04, 1.07 (12H, 2d, J = 6.6Hz, 2 Me2CH); 1.52-1.89, 2.04-2.30 (6H + 8H, 2m, 2 Me2CHCH2 + 2 CH2C); 3.32 (6H, s, 2 MeN); 4.20-4.57 (8^, m, 2 CCHCH2O + 2 NCHCO); 7.20-8.11 (30H, m, 2 fluorenyl + 2 Ph + 4 NH). 13C NMR (dg-DMSO): 5 21.3,
23.0 (2q, 2 Me2CH); 24.1 (d, 2 Me2CH); 26.5, 26.6 (2t, 2 (CH2)2C); 39.6 (q, 2 MeN); 40.4 (t, Me2CHCH2); 46.5 (d, 2 CHCH2O); 52.7 (d, 2 NCHCO); 57.6 (s, 2 (CH2)2C); 65.5 (t, 2 CH2O); 119.9, 120.1, 125.1, 125.2, 126.3, 127.0,
127.4,	127.6, 128.7, 128.8 (10d, 26 arom. CH); 140.6,
143.5,	143.9, 145.7 (4s, 6 arom. C + 2 arom. CN); 155.9 (s, 2 O-CO-N); 171.5, 172.1 (2s, 4 CON). ESI-MS (m/z): 1089 (9, [M + K]+), 1073 (100, [M + Na]+). [a]25D: -18.9 (c 1.0, CHCl3). Anal. Calcd. for Cg4H70NgO80.5 H2O (1060.30): C, 72.50; H, 6.75; N, 7.93. Found: C, 72.63; H, 6.53; N, 7.75.
Benzyl rrans-[(S)-1-(4-{(S)-2-[(Benzyloxycarbonyl) amino]-6-(tert-butylamino)hexanoylamino}-1,4-bis(^-methyl-^-phenylcarbamoyl)cyclohexylcarbamoyl)-5-(tert-butylamino)pentyl]carbamate (trans-23d). Prepared according to GP4; with trans-21 (100 mg, 0.29 mmol) and Z-Lys(Boc)-OH (243 mg, 0.64 mmol) in CHCl3 (10 ml), 2 h, CC (SiO2, CH2Cl2/MeOH 15:1). Yield: 295 mg (92%) of trans-23d. Wliite powder; mp 190-191 °C. IR (KBr): vmax 3327s, 3034w, 2959m, 2932m, 2865w, 1717s, 1659s, 16^6s, 1593m, 1525s, 1454s, 1365s, 1250s, 1170s, 1101m, 1034m, 750m, 699s cm-1. 1H NMR (dg-DMSO): 5 1.37 (18H, s, 2 Me3C); 1.24-1.70, 1.94-2.10 (12H + 8H, 2m, 4 CH2 + 2 CHCH2CH2CH2); 2.85-2.94 (4H, m, 2 CH2N); 3.^3-4.01 (2H, m, 2 CH(C H2); 5.00, 5.08 (4H, 2d, J = 12.5 Hz, 2 CH2O); 6.72 (2H, br, 2 NH); 7.11-7.38 (22H, 4 Ph + 2 NH); 7.79 (2H, br, 2 NH). 13C NMR (dg-DMSO): 5 22.7, 26.3, 26.8, 29.1, 31.5, 39.6 (6t, 2 (CH2)2C + 2 (CH2)4); 28.1 (q, 2 Me3Cy; 39.8 (q, 2 MeN); 54.2 (d, 2 NCHCO); 65.3 (t, 2 CH20); 57.7 (s, (CH2)2C); 77.2 (s, 2 Me3C); 126.3, 127.0, 1^7.5, 128.1, 128.^ (5d, 20 arom. CH); 136.9 (s, 2 arom. C); 145.5 (s, arom. 2 CN); 155.4, 156.0 (2s, 4 O-CO-N); 171.2, 172.1 (2s, 4 CON). ESI-MS (m/z): 1143 (8, [M + K]+), 1127 (100, [M + Na]+). [a]25D: -6.7 (c 0.5, MeOH). Anal. Calcd. for Cg0H80N8O120.5 H2O (1114.35): C, 64.67; H, 7.33; N, 10.06. Found: C, 6.1.47; H, 7.04; N, 9.80.
7'rans-1,4-Bis(acetylamino)cyclohexane-1,4-dicar-boxylic Acid bis(^-methyl-^-phenylamide) (trans-24).
Prepared according to GP4; with trans-21 (100 mg, 0.29 mmol) and acetic acid (52 mg, 50 ml, 0.87 mmol) in CHCl3 (5 ml), 2 h, washing with MeOH. Yield: 127 mg (94%) of trans-24. White powder (only soluble in CF3CO2H); mp 144-145 °C. IR (KBr): vmax 3263m, 30^3m, 2972w, 2926w, 1648s, 1592m, 1549s, 1493m, 1367s, 1297m, 1274w, 1083w, 1073w, 1025w, 771w, 706m cm1. 1H NMR (CF3CO2D): 5 2.21-3.13 (14H, m, 2 MeCO + 2 CH2); 3.30 (6H, s, 2 MeN); 7.40-7.72 (10H, m, 2 Ph). ESI-MdS (m/z): 503 (14, [M + K]+), 487 (100, [M + Na]+).
rrans-1,4-Bis((S)-2-{[(9H-fluoren-9-yl)methoxy]car-bonylamino}-4-methylpentanoylamino)cyclohexane-1,4-dicarboxylic acid (trans-25c). To a solution of trans-
23c (80 mg, 0.076 mmol) in THF (15 ml) was slowly added an aqueous HCl-solution (32%, 6.4 ml), and the mixture was stirred at r.t. for 24 h. Then, aqueous HCl (2M solution, 10 ml) was added and the mixture was extracted with Et2O (3 X 10 ml). After drying over MgSO4 and evaporation of the solvent, the residue was recrystallized from acetone/hexane, yielding trans-25c (64 mg, 96%). White powder; mp 171-173 °C. IR (KBr): vmax 3325s, 3065m, 2956s, 1719s, 1662s, 1525s, 1540m, 1338m, 1262s, 1108m, 1050m, 758m, 739m cm-1. 1H NMR (CD30D): 5 0.92, 0.95 (12H, 2d, J = 6.5 Hz, 2 Me^CH); 1.45-1.78, 1.91-2.20 (6H + 8H, 2m, 2 Me2CHC^2 + 2 (CH2)2C); 4.18-4.29, 4.40 (8H, m + d, J = 6.6 H z, 2 CHC:H20 + 2 NCHCO); 7.25-7.41, 7.60-7.83 (16H, 2m, 2 fluorenyl). 13C NMR (CD3OD): 5 22.0, 23.3 (2q, 2 Me2CH); 25.8 (d, 2 Me2CH); 27.7, 28.0 (2t, 2 (CH2)2C);
41.8	(t, 2 MeCHCH2); 48.4, 54.8 (2d, 2 CHCH2O + 2 NCHCO); 59.1 (s, 2 (CH2)2C); 67.8 (t, 2 CH2O); 120.8, 126.1, 126.2, 128.1, 128.^ (5d, 16 arom. CH); 142.5, 145.1, 145.4 (3s, 8 arom. C); 158.3 (s, 2 O-CO-N); 175.4, 176.9 (2s, 2 CON + 2 CO2). ESI-MS (m/z): 895 (100, [M + Na]+). [a]25D: -16.4 (c 1.0, acetone).
(9H-Fluoren-9-yl)methyl Trans-{(S)-1-[4-((S)-2-{i(9H-Fluoren-9-yl)methoxy]carbonylamino}-4-methylpen-tanoylamino)-1,4-bis[1-methyl-1-(N-methyl-N -phenylcarbamoyl)ethylcarbamoyl]cyclohexylcarba-moyl]-3-methylbutyl}carbamate (trans-26c). A solution of trans-25c (150 mg, 0.17 mmol) and aminoazirine 2 (63 mg, 0.36 mmol) in THF (10 ml) was stirred at r.t. for 48 h. Then, the solvent was evaporated and the residue was purified by CC (SiO2, CH2Cl2/MeOH 20:1), yielding 149 mg (71%) of trans-26c. W^hite powder; mp 203-204 °C. IR (KBr): vmax 3314s, 2953m, 2868w, 1718s, 1665s, 1654s, 1631s, 1593m, 1513s, 1494s, 1449m, 1393m, 1364m, 1259m, 1203m, 1088m, 1025m, 780w, 734m, 706m cm-1. 1H NMR (CD3OD): 5 0.99, 1.04 (12H, 2d, J = 6.4 Hz, 2 Me2CH); 1.39, 1.46 (12H, 2s, 2 Me2C); 1.55-2.10, 2.31-2.48 (12H + 2H, 2m, 2 MeCHCH2 + 2 (CH2)2C); 3.24 (6H, s, 2 MeN); 4.10-4.51 (8H, m, 2 CHC2H2O + 2 NCHCO); 7.17-8.10 (32H, m, 2 Ph + 2 fluorenyl +6 NH). 13C NMR (CD3OD): 5 22.3, 23.1, 26.2, 26.4 (4q, 4 Me2C + 2 Me2CH); 25.9 (d, 2 Me2CH); 27.9,
27.9	(2t, 2 (CH2)2C); 41.1 (q, 2 MeN); 41.4 (t, Me2CHCH2); 48.3 (d, 2 CHCH2O); 55.7 (d, 2 CHN); 68.0 (t, 2 CH(O); 120.9, 126.0, 126.2, 128.1, 128.2, 128.3, 128.8, 130.2 (8d, 26 arom. CH); 142.5, 145.1, 145.2 (3s, 8 arom. C + 2 arom. CN); 158.0 (s, 2 N-CO-O); 175.2, 175.4, 176.0 (3s, 6 CON). ESI-MS (m/z): 1243 (100, [M + Na]+). [a]25.: -13.5 (c 1.0, acetone).
nochromated Mo^a radiation (/10.71073 À) and an Oxford Cryosystems Cryostream 700 cooler. Data reduction was performed with HKL Denzo and Scalepack.56 The intensities were corrected for Lorentz and polarization effects, but not for absorption. Equivalent reflections were merged. Data collection and refinement parameters are given below, and a view of the molecule is shown in Figure 2. The structure of cis-21 was solved by direct methods using SIR92,57 which revealed the positions of all non-hydrogen atoms. The non-hydrogen atoms were refined anisotropically. All of the H-atoms were placed in geometrically calculated positions and refined using a riding model where each H-atom was assigned a fixed isotropic displacement parameter with a value equal to 1.2 ^eq of its parent C-atom (1.5^eq for the Me groups). Refinement of the structure was carried out on F2 using full-matrix least-squares procedures, which minimized the function 2'w(Fo(-Fc()(. A correction for secondary extinction was applied. Although the conformation of the molecule is clearly defined, the refinement results are poor. This appears to be because of the nature of the crystals. Either they are twinned or are intergrown, as the diffraction images show evidence of interleaving lattices with many reflections overlapping. Data recorded from several crystals yielded similar results. An attempt to extract twinned data was unsuccessful. The imprecise nature of the geometric parameters means that it is inadvisable to attempt to draw any far-reaching conclusions from a detailed analysis of the geometrical parameters. Neutral atom scattering factors for non-H-atoms were taken from ref.,58a and the scattering factors for H-atoms were taken from ref.59 Anomalous dispersion effects were included in Fc;60 the values for f ' and f " were those of ref.58b The values of the mass attenuation coefficients are those of ref.58c All calculations were performed using the SHELXL9761 program.
Crystal data for cis-21: C((H(4N4, M = 344.46, colorless, prism, crystal dimensions 0.20 X 0.30 X 0.35 mm, monoclinic, space group P21/c, Z = 4, a = 19.4051(6) À, h = 9.7739(3) À, c = 10.1001(2) À, ß = 98.747(2)°, V = 1893.34(9) À3, T = 160 K, Dx = 1.208 gcm3, ^(Mo^a) = 0.0732 mm-1, scan type 0 and a, 20(max) = 60°, total reflections measured 48208, symmetry independent reflections 5539, reflections with I > 2ö(/) 4123, reflections used in refinement 5539, parameters refined 239, R(F) [I > 20(1) reflections] = 0.1315, wR(F() [all data] = 0.4719 (w = [o((Fo() + (0.2P)2]-1, where P = (F^2 + 2Fc()/3), goodness of fit 1.923, secondary extinction coefficient 0.17(4), final A /o0.001, (max; min) = 0.50; -0.45 e À-3.
4. 5. X-Ray Crystal-Structure Determination of cis-21 (see Figure 2).****
All measurements were made on a Nonius Kappa-CCD area detector diffractometer55 using graphite-mo-
.... CCDC-704899 contains the supplementary crystallographic data for this paper. These data can he ohtained free of charge from the Camhridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/ data_request/cif.
4. References
1.	R. Kaul, P. Balaram, Bioorg. Med. Chem. 1999, 7, 105-117.
2.	S. Aravinda, N. Shamala, R. S. Rao, P. Balaram, Proc. Ind. Acad. Sci. (Chem. Sci.) 2003, 115, 373-400.
3.	J. M. Humphrey, A. R. Chamberlin, Chem. Rev. 1997, 97, 2243-2266.
4.	B. V. Prasad, P. Balaram, CRC Crit. Rev. Biochem. 1984, 16, 307-348.
5.	C. Toniolo, M. Crisma, F. Formaggio, G. Valle, G. Cavic-chioni, G. Precigoux, A. Aubry, J. Kamphuis, Biopolymers 1993, 33, 1061-1072.
6.	S. Vijayalakshmi, R. Balaji Rao, I. L. Karle, P. Balaram, Biopolymers 2000, 53, 84-98.
7.	M. Sahebi, P. Wipf, H. Heimgartner, Tetrahedron 1989, 45, 2999-3010.
8.	a) P. Wipf, H. Heimgartner, Helv. Chim. Acta 1988, 71, 258-267. b) P. Wipf, R. W. Kunz, R. Prewo, H. Heimgartner, Helv. Chim. Acta 1988, 71, 268-273.
9.	M. Narita, K. Ishikawa, H. Sugasawa, M. Doi, Bull. Chem. Soc. Jpn. 1985, 58, 1731-1737.
10.	C. Toniolo, E. Benedetti, Trends Biochem. Sci. 1991, 16, 350-353.
11.	V. Pavone, B. Di Blasio, A. Santini, E. Benedetti, C. Pedone, C. Toniolo, M. Crisma, J. Mol. Biol. 1990, 214, 633-635.
12.	B. Di Blasio, V. Pavone, A. Lombardi, C. Pedone, E. Benedetti, Biopolymers 1993, 33, 1037-1049.
13.	C. Toniolo, H. Brückner (Eds.), Peptaibiotics, Topical Issue of Chem. Biodivers. 2007, 4, 1021-1412.
14.	M. Iqbal, P. Balaram, J. Am. Chem. Soc. 1981, 103, 5548-5552.
15.	R. Bosch, G. Jung, W. Winter, Acta Crystallogr., Sect. C. 1983, 39, 776-778.
16.	R. Gessmann, H. Brückner, M. Kokkinidis, Acta Crystallogr., Sect. B 1998, 54, 300-307.
17.	M. Oba, M. Tanaka, H. Suemune, Y. Sato, M. Kurihara, in 'Peptide Science 2003', Ed. M. Ueki, Japanese Peptide Society, Osaka, 2004, pp. 397-398.
18.	R. Luykx, C. B. Bucher, A. Linden, H. Heimgartner, Helv. Chim. Acta 1996, 79, 527-540.
19.	R. T. N. Luykx, A. Linden, H. Heimgartner, Helv. Chim. Acta 2003, 86, 4093-4111.
20.	N. Pradeille, O. Zerbe, K. Möhle, A. Linden, H. Heimgartner, Chem. Biodivers. 2005, 2, 1127-1152.
21.	P. Wipf, H. Heimgartner, Helv. Chim. Acta 1986, 69, 1153-1162.
22.	H. Heimgartner, Angew. Chem. Int. Ed. Engl. 1991, 30, 238-264.
23.	W. Altherr, A. Linden, H. Heimgartner, Chem. Biodivers. 2007, 4, 1170-1182.
24.	M. Rens, L. Ghosez, Tetrahedron Lett. 1970, 3765-3768.
25.	F. Hilty, K. A. Brun, H. Heimgartner, Helv. Chim. Acta 2004, 87, 2539-2548.
26.	K. A. Brun, H. Heimgartner, Helv. Chim. Acta 2005, 88, 2951-2959.
27.	J. L. Räber, K. A. Brun, H. Heimgartner, Heterocycles 2007, 74, 397-409.
28.	C. Bucher, A. Linden, H. Heimgartner, Helv. Chim. Acta 1995, 78, 935-946.
29.	K. A. Brun, A. Linden, H. Heimgartner, Helv. Chim. Acta
2002,	85, 3422-3443.
30.	J. M. Villalgordo, A. Enderli, A. Linden, H. Heimgartner, Helv. Chim. Acta 1995, 78, 1983-1998.
31.	C. Strässler, A. Linden, H. Heimgartner, Helv. Chim Acta
1997,	80, 1528-1554.
32.	S. Stamm, A. Linden, H. Heimgartner, Helv. Chim. Acta
2003,	86, 1371-1396.
33.	G. Suter, S. A. Stoykova, A. Linden, H. Heimgartner, Helv. Chim. Acta 2000, 83, 2961-2974.
34.	R. A. Breitenmoser, A. Linden, H. Heimgartner, Helv. Chim. Acta 2002, 85, 885-912.
35.	S. Stamm, H. Heimgartner, Helv. Chim. Acta 2006, 89, 1841-1855.
36.	P. Wipf, H. Heimgartner, Helv. Chim. Acta 1990, 73, 13-24.
36.	N. Pradeille, H. Heimgartner, J. Pept. Sci. 2003, 9, 827-837.
37.	a) J. Lehmann, A. Linden, H. Heimgartner, Tetrahedron
1998,	54, 8721-8736. b) J. Lehmann, A. Linden, H. Heim-gartner, Tetrahedron 1999, 55, 5359-5376. c) J. Lehmann, A. Linden, H. Heimgartner, Helv. Chim. Acta. 1999, 82, 888- 908. d) J. Lehmann, A. Linden, H. Heimgartner, Helv. Chim. Acta. 1999, 82, 1899-1915.
38.	a) R. A. Breitenmoser, H. Heimgartner, Helv. Chim. Acta 2001, 84, 786-796. b) R. A. Breitenmoser, A. Linden, H. Heimgartner, Helv. Chim. Acta 2002, 85, 990-1018.
39.	A. Bärtsch, B. Bischof, H. Heimgartner, Pol. J. Chem. 2009, 83, 195-206
40.	A. Budzowski, A. Linden, H. Heimgartner, Helv. Chim. Acta 2008, 91, 1471-1488.
41.	I. Dannecker-Dörig, A. Linden, H. Heimgartner, Collect. Czech. Chem. Commun. 2009, 74, 901-925.
42.	F. S. Arnhold, Ph.D. thesis, Universität Zürich, 1997.
43.	a) T. Jeremic, A. Linden, K. Moehle, H. Heimgartner, Tetrahedron 2005, 61, 1871-1883. b) T. Jeremic, A. Linden, H. Heimgartner, Helv. Chim. Acta 2004, 87, 3056-3079. c) T. Jeremic, A. Linden, H. Heimgartner, Chem. Biodivers. 2004, 1, 1730-1761. d) T. Jeremic, A. Linden, H. Heimgartner, J. Pept. Sci. 2008, 14, 1051-1061.
44.	D. Obrecht, H. Heimgartner, Helv. Chim. Acta 1987, 70, 329-338.
45.	a) K. N. Koch, H. Heimgartner, Helv. Chim. Acta 2000, 83, 1881-1900. b) K. N. Koch, A. Linden, H. Heimgartner, Tetrahedron 2001, 57, 2311-2326. c) K. N. Koch, A. Linden, H. Heimgartner, Helv. Chim. Acta 2000, 83, 233-257. d) K. N. Koch, G. Hopp, A. Linden, K. Moehle, H. Heimgartner, Helv. Chim. Acta 2001, 84, 502-512.
46.	a) B. Iliev, A. Linden, H. Heimgartner, Helv. Chim. Acta 2003, 86, 3215-3234. b) B. Iliev, A. Linden, R. W. Kunz, H. Heimgartner, Tetrahedron 2006, 62, 1079-1094.
47.	P. Köttgen, A. Linden, H. Heimgartner, Helv. Chim. Acta 2006, 89, 731-746.
48.	J. Galloy, J.-P. Declercq, M. Van Meerssche, Cryst. Struct. Commun. 1980, 9, 151-156.
49.	J. Rizo, L. M. Gierasch, Annu. Rev. Biochem. 1992, 61,
387-418.
50.	K. Dietliker, H. Heimgartner, Helv. Chim. Acta 1983, 66, 262-295.
51.	P. Wipf, Ph.D. thesis, Universität Zürich, 1987.
52.	a) J. M. Villalgordo, H. Heimgartner, Tetrahedron 1993, 49, 7215-7222. b) J. M. Villalgordo, H. Heimgartner, Helv. Chim. Acta 1993, 76, 2830-2837.
53.	T. J. Curphey, J. Org. Chem. 2002, 67, 6461-6473.
54.	C. K. Johnson, ORTEP II, Report ORNL-5138, Oak Ridge National Laboratory, Oak Ridge, Tenessee, 1976.
55.	R. Hooft, KappaCCD Collect Software, Nonius BV, Delft, The Netherlands, 1999.
56.	Z. Otwinowski, W. Minor, in 'Methods in Enzymology', Vol. 276, 'Macromolecular Crystallography', Part A, Eds. C. W. Carter, Jr., R. M. Sweet, Academic Press, New York, 1997, pp. 307-326.
57.	A. Altomare, G. Cascarano, C. Giacovazzo, A. Guagliardi, M. C. Burla, G. Polidori, M. Camalli, SIR92, J. Appl. Cry-
stallogr., 1994, 27, 435-436.
58.	a) E. N. Maslen, A. G. Fox, M. A. O'Keefe, in 'International Tables for Crystallography', Ed. A. J. C. Wilson, Kluwer Academic Publishers, Dordrecht, 1992, Vol. C, Table 6.1.1.1, pp. 477-486. b) D. C. Creagh, W. J. McAuley, in 'International Tables for Crystallography', Ed. A. J. C. Wilson, Kluwer Academic Publishers, Dordrecht, 1992, Vol. C, Table 4.2.6.8, pp. 219-222. c) D. C. Creagh, J. H. Hubbell, in 'International Tables for Crystallography', Ed. A. J. C. Wilson, Kluwer Academic Publishers, Dordrecht, 1992, Vol. C, Table 4.2.4.3, p. 200-206.
59.	R. F. Stewart, E. R. Davidson, W. T. Simpson, J. Chem. Phys., 1965, 42, 3175-3187.
60.	J. A. Ibers, W. C. Hamilton, Acta Crystallogr., 1964, 17, 781-782.
61.	G. M. Sheldrick, SHELXL97, Program for the Refinement of Crystal Structures, University of Göttingen, Germany, 1997.
Povzetek
Opisana je sinteza žcikloheksan-premostenih' bis-(3-amino-2H-azirinov), cis- in trans-W,W-di9etil-W,W-difenil-1,7-diazadispiro[2.2.2.2]deka-1,7-dien-2,8-diaminov iz ustreznih bis-thioamidov 20 z zaporednimi pretvorbami s COCl2, 1,4-diazabiciklo[2.2.2]oktanom in NaN3. Reakcije teh bis-azirinov z različnimi naravnimi a-amino kislinami so vodile do peptid amidov 23. Hidroliza C-terminalnih amidnih skupin spojin 23c s slewdeči9 pripajanjem na Aib sinton 2, 2,2,N-trimetil-N-fenil-2H-azirin-3-amin, je pokazala uporabnost gradnikov 21 v sintezi peptidov.