Efficient synthesis of differentially protected (S,S)-2,7- diaminooctanedioic acid, the dicarba analogue of cystine

Article abstract of DOI:10.1002/(SICI)1522-2675(19981111)81:11<2053::AID-HLCA2053>3.0.CO;2-4

Convenient preparative syntheses of differentially protected forms of (S,S)-2,7-diaminooctanedioic acid (2), suitable for application in peptide chemistry, are described. The key compound, the di-Cbz-protected phenacyl monoester 7a (Cbz = [(benzyloxy)carb

Full text of DOI:10.1002/(SICI)1522-2675(19981111)81:11<2053::AID-HLCA2053>3.0.CO;2-4

Helvetica Chimica Acta ± Vol. 81 (1998)
2053
Efficient Synthesis of Differentially Protected (S,S)-2,7-
Diaminooctanedioic Acid, the Dicarba Analogue of Cystine
1
2
by Meinolf Lange ) and Peter M. Fischer )*
Â
Nycomed Imaging AS, Bioreg Research, Gaustadalleen 21, N-0371 Oslo
Convenient preparative syntheses of differentially protected forms of (S,S)-2,7-diaminooctanedioic acid
(2), suitable for application in peptide chemistry, are described. The key compound, the di-Cbz-protected
phenacyl monoester 7a (Cbz ˆ [(benzyloxy)carbonyl]), was obtained by means of Schöllkopf bis-lactim ether
methodology and optimized monoesterification procedures. Selective amino deprotection at the non-esterified
amino-acid function of 7a by dichloromethyl-methyl-ether-induced ꢀN-carboxyanhydrideꢁ formation, and
hydrolysis permitted access to the Boc/Cbz- and Fmoc/Cbz-protected monophenacyl esters 11a and 11b, as well
as to the fully orthogonally protected Fmoc/Boc monophenacyl ester 12 (Boc ˆ (tert-butoxy)carbonyl, Fmoc ˆ
(9H-fluoren-9-ylmethoxy)carbonyl).
Introduction. ± Disulfide bonding through cystine residues is a main feature of
peptide and protein architecture. For many applications of polypeptides it is desirable
to replace cystine 1 with its non-reducible isosteric dicarba analogue, i.e., (S,S)-2,7-
diaminooctanedioic acid ( ˆ l,l-2,7-diaminosuberic acid; 2) [1]. For symmetrical
polypeptides containing such a cystine replacement, this does not present any particular
synthetic problem as N-protected derivatives of 2 are available: mixtures of stereo-
isomers of 2 can be obtained by Gabriel synthesis from octanedioic acid [2], and a
number of related synthetic routes are known [3]. Separation of the stereoisomers has
been shown to be possible but is tedious [4]. For this reason, asymmetric-synthesis
methods have been developed. Electrochemical dimerization via Kolbe synthesis from
glutamic-acid derivatives [5] has been used preparatively for this purpose [6].
Furthermore, asymmetric-synthesis methods using various chiral auxiliaries have been
used for the preparation of optically pure derivatives of certain diamino dicarboxylic
acids [7]. The results we present here for the synthesis of derivatives of diaminoocta-
nedioic acid 2 are based on the use of one such method, viz. the Schöllkopf bis-lactim
ether technique [8].
For most applications where a cystine residue in a polypeptide is to be replaced with
the isoster 2, however, the resulting synthetic bridged intermediates will be unsym-
metrical, and differentially protected derivatives of 2 are required for unambiguous
and selective synthesis. Originally, a-(tert-butyl) Na-Boc-Na'-Cbz-l,l-2,7-diaminosub-
erate3) (ˆ 8-(tert-butyl) 1-hydrogen (S,S)-2-{[(benzyloxy)carbonyl]amino}-7-{[(tert-
1
)
)
)
Current address: ArQule, Inc., 200 Boston Avenue, Medford, MA 02155, USA.
Current address: Cyclacel Ltd., 5 Whitehall Crescent, Dundee DD1 4AR, Scotland, UK.
Cbz ˆ (Benzyloxy)carbonyl, Boc ˆ (tert-butoxy)carbonyl, Fmoc ˆ (9H-fluoren-9-ylmethoxy)carbonyl,
Pac ˆ phenacyl ˆ 2-oxo-2-phenylethyl.
2
3

2054
Helvetica Chimica Acta ± Vol. 81 (1998)
butoxy)carbonyl]amino}octanedioate) was obtained by mixed Kolbe electrolytic
decarboxylative dimerization of appropriate glutamic-acid derivatives [9], and this
approach has recently been extended to the synthesis of orthogonally protected
diamino dicarboxylic acids containing up to four different protecting groups [10]. The
disadvantage of electrolytic dimerization methods lies in the fact that statistically
controlled product mixtures are formed from which the desired derivatives have to be
isolated.
Differentially protected derivatives of diaminoheptanedioic acid (ˆ diaminopi-
melic acid) have also been synthesized via connection and elaboration of two different
chiral auxiliaries [11]; a similar approach was adopted for the preparation of
differentially protected 2,9-diaminodecanedioic acid [12]. Furthermore, it was shown
in principle that diaminooctanedioic-acid derivatives with two different amino-
protecting groups are accessible using the chiral auxiliary Boc-BMI of Seebach and
coworkers [13]. Starting from diaminoheptanedioic acid, selective introduction of
protecting groups with the aid of dimeric copper chelates has also been shown to be
feasible [14]. The most recent approach towards differentially protected diaminodi-
carbocylic acids uses Grubbꢁs ring closure olefin metathesis reactions on allylglycine-
derived templates [15].
The method we have developed for the differential protection of 2 is based on work
by Arendt et al. [16] who showed that using racemic di-Cbz-protected3) 4-nitrobenzyl
hydrogen 2,6-diaminopimelate (i.e., 4-nitrobenzyl hydrogen 2,6-bis{(benzyloxy)carbo-
nyl]amino}heptanedioate), it was possible to address selectively the a-amino-acid
function containing the free carboxylic acid rather than the ester function by formation
of an ꢀN-carboxyanhydrideꢁ (NCA). Using this approach, we show that enantiomeri-
cally pure derivatives of 2, in orthogonally protected forms directly applicable to
peptide synthesis, can be obtained.
Results and Discussion. ± The bis-lactim ether 3 (Scheme 1), synthesized from
cyclo(d-Val-Gly) [17], was lithiated and alkylated with 0.5 mol-equiv. of 1,4-dibromo-
butane to afford a mixture of diastereoisomers 4a and 4b in a ratio of ꢀ 4 : 1. The
desired stereoisomer 4a was obtained in enantiomerically pure form after column
chromatography (silica gel) and recrystallization. Controlled hydrolysis of the imino-
ether functions in 4a provided the diester 5 after removal of valine methyl ester by
high-vacuum distillation. Rather than hydrolysis of the methyl esters prior to the
introduction of the N-Cbz groups as is usually the practice, we chose to reverse the

Helvetica Chimica Acta ± Vol. 81 (1998)
2055
process. This is advantageous due to the problems attendant on N-protection of free
2,7-diaminooctanedioic acid [18]. Thus, the di-Cbz-protected diester 6 was obtained in
high yield from 5.
Scheme 1
Monoesterification of diaminodicarboxylic acids is most easily effected using
methods relying on alkylation of carboxylates, since the alkylating agent can be
employed in stoichiometric amounts. Thus, 4-nitrobenzyl esters have been used for this
purpose [14]. We chose the phenacyl (Pac3) ester [19], which can be introduced
similarly and has the additional advantage of being orthogonal with respect to both the
Cbz and Boc3) groups in terms of deprotection methods. Thus, it is possible to remove
the Pac ester from amino-acid derivatives as well as from peptides, e.g., with the aid of
Zn in AcOH or with Bu4NF [20]. Furthermore, its acid stability is such that both the
Boc and Cbz groups can be removed acidolytically without interference with the Pac
ester.

2056
Helvetica Chimica Acta ± Vol. 81 (1998)
In our early experiments, we achieved monoesterification by treatment of di-Cbz-
protected diaminooctanedioic acid (after acid hydrolysis of the methyl esters in 6;
Scheme 2) with 1 mol-equiv. of Pac-Br in DMF in the presence of Et3N. With this
procedure, monoester 7a and diester 7b were formed in a ratio of 2 :1 at best, and the
chemical yields were low. We subsequently found that much better yields were
achieved if 6 was hydrolysed carefully using LiOH in H2O/dioxane, followed directly
by treatment of the hydrolysate with Pac-Br in the same solvent mixture. Under
appropriate concentration conditions, the monoester 7a precipitated immediately upon
formation, and excess Pac-Br could be used to drive the reaction nearly to completion,
thus, only trace amount of diester 7b were observed.
Conversion of Cbz-amino acids to NCAs is usually achieved by preparation of the
corresponding acyl chlorides with PCl5 [14] or SOCl2 [21], followed by heating of the
reaction mixtures. Using these strategies, we were able to detect the formation of what
is likely to be the acyl chloride 8 by TLC, as well as the NCA 94). Unfortunately, the
transformations were not smooth, and several unidentified by-products were observed.
Thus, we took recourse to a method of NCA formation from Cbz-protected amino acids
using the reagent dichloromethyl methyl ether [22]. Analyses of reaction mixtures
obtained with this reagent showed clean and rapid formation of 9, and no additional
solvent was necessary. Hydrolysis of the NCA 9 with aqueous AcOH to give 10,
followed by introduction of a Boc or Fmoc3) protecting group, without purification of
the hydrolysed intermediate, finally afforded the triprotected target compounds 11a
and 11b, respectively, in excellent yield and purity.
In terms of the envisaged use of our triprotected target compounds in peptide
syntheses, the combination of protecting groups in 11a and 11b is not fully satisfactory
due to the fact that hydrogenolytic deblocking of the Cbz group is compatible with
neither the Pac nor the Fmoc [23] groups. We have demonstrated the compatibility of
the Fmoc and Pac groups [24] and thus, in principle, the three protecting groups in 11b
are fully orthogonal if the Cbz group is removed acidolytically rather than hydro-
genolytically. However, a further element of orthogonality is desirable with respect to
the amino-acid side-chain protecting groups present in prospective peptides incorpo-
rating derivatives of 2. These protecting groups are commonly of the benzyl type, and
the choice of Cbz as one of the amino-blocking groups in 2 is, therefore, not optimal.
Compound 12, on the other hand, is fully orthogonal when permanent benzyl-type
peptide protecting groups are to be used. This derivative was readily prepared by
removal of the Cbz groups from 11b with strong acid, followed by introduction of a Boc
group in the usual manner.
The absence of racemization during LiOH saponification of the methyl diester 6
and the following esterification step was verified by removal of the ester functions in
the diester 7b and comparison of the resulting product with authentic [18] (S,S)-2,7-
bis{[(benzyloxy)carbonyl]amino}octanedioic acid (13C-NMR and optical rotation).
Similarly, chiral integrity during the transformation of monoester 7a to amino acid 10
via NCA 9 was established by comparison of authentic material with the product
obtained after reprotection of 10 by Cbz and Pac deprotection.
4
)
The Rf value upon TLC (AcOEt/hexane/AcOH 50:45:5) was 0.57 for the methyl ester derived from acyl
chloride 8 (after quenching with MeOH), 0.47 for NCA 9, and 0.42 for the starting material 7a.

Helvetica Chimica Acta ± Vol. 81 (1998)
2057
Scheme 2

2058
Helvetica Chimica Acta ± Vol. 81 (1998)
Experimental Part
General. Reagents and solvents were of the highest commercial grades available and were used as supplied,
except THF, which was freshly distilled from sodium diphenylketyl. Column chromatography [25]: Merck silica
gel 60, 230 ± 400 mesh. TLC: Merck silica gel 60 F254 anal. plates; detection by UV light or by spraying with 2%
ninhydrin soln. or 5% phosphomolybdic acid in 95% EtOH and heating. Optical rotations: Perkin-Elmer-141
polarimeter, d ˆ 10 cm, c in g/100 ml. NMR Spectra: Varian Unity 300; d values in ppm rel. to SiMe4, coupling
constants J in Hz. MS: performed at the Laboratory for Mass Spectrometry, Chemical Institute, University of
Oslo, Norway (EI-MS) and by M-Scan Ltd., Ascot, Berkshire, England (FAB-MS). Elemental analyses were
carried out by Ilse Beetz Mikroanalytisches Laboratorium, Kronach, Germany.
2,2'-(Butane-1,4-diyl)bis[(2S,5R)-2,5'-dihydro-3,6-dimethoxy-5-isopropylpyrazine] (4a). A soln. of 1.60m
BuLi in hexane (26.0 ml, 41.6 mmol) was injected into a soln. of bis-lactim ether 3 (7.0 g, 37.8 mmol) and 1,3-
dimethylimidazolidin-2-one (8.3 ml, 75.6mmol) in THF (375 ml) at 788 while stirring. Stirring was continued
for 15 min to complete the formation of the aza-enolate. A soln. of 1,4-dibromobutane (2.3 ml, 4.1 g,
18.9 mmol) in THF (75 ml) was then added slowly, and stirring was continued for 12 h at 788 to 08. After
addition of phosphate buffer (pH 7), the mixture was allowed to warm up to r.t. and evaporated and the residue
partitioned between H2O (200 ml) and Et2O (300 ml). The aq. layer was extracted with Et2O (2 Â 100 ml), the
combined extract washed with brine, dried (MgSO4), and evaporated, and excess of 1,4-dibromobutane
removed by bulb-to-bulb distillation (508/0.2 Torr). The crude alkylation product (diastereoisomer ratio 4a/4b
4 : 1; d.e. 90% by capillary GLC) was chromatographed (silica gel (850 g), Et2O/hexane 1 : 20; TLC; Rf 0.28).
Pure (> 99% d.e.) 4a (5.03 g, 63%) was obtained after recrystallization from MeOH or MeCN. Colourless solid.
M.p. 848. IR (CH2Cl2): 1690s (CˆN). 1H-NMR (300 MHz, CDCl3): 0.67, 1.04 (2d, J ˆ 7.7, 2 Me2CH); 1.14 ± 1.34
(m, CH2(CH2)2CH2); 1.61 ± 1.84 (m, CH2(CH2)2CH2); 2.27 (dsept., J ˆ 7.7, 3.6, 2 MeCH); 3.67, 3.68
(2s, 4 MeO); 3.91 (dd, J ˆ 4.0, 3.6, 2 H,
H
C(5)); 4.00 (ddd, J ˆ 4.3, 4.0, 2 H,
H
C(2)). 13C-NMR
(75.4 MHz, CDCl3): 16.54, 19.07 (Me2CH); 24.47, 34.12 ((CH2)4); 28.60 (C(2)); 31.63 (Me2CH); 52.26, 52.29
‡
(MeO); 55.44 (C(2)); 60.97 (C(5)); 163.40, 163.89 (CˆN). HR-EI-MS: 422.2893 (C22H38N4O4, M ;
calc. 422.2893). Anal. calc. for C22H38N4O4 (422.56): C 62.53, H 9.06, N 13.26; found: C 62.51, H 9.08, N 13.28.
(S,S)-2,7-Diaminooctanedioic Acid Dimethyl Ester (5). To a stirred soln. of 4a (6.0 g, 14.2 mmol) in MeCN
(40 ml), 0.5m aq. HCl (170.4 ml, 85.2 mmol) was added dropwise. Stirring was continued overnight at r.t. The
mixture was extracted with Et2O (50 ml), the aq. phase adjusted to pH 11 with NH4OH soln. and extracted with
CHCl3 (3 Â 50 ml), and the CHCl3 phase dried (MgSO4) and evaporated. The valine methyl ester was removed
from the residue by bulb-to-bulb distillation (358/0.05 Torr) to afford 5 (3.07 g, 93%) which was used in the next
1
step without further purification. Colourless solid. IR (CH2Cl2): 1732s (CˆO). H-NMR (300 MHz, CDCl3):
1.23 ± 1.41 (m, 2 NH2); 1.42 ± 1.59 (m, CH2(CH2)2CH2); 1.60 ± 1.71 (m, CH2(CH2)2CH2); 3.26 ± 3.46 (m, 2 CH);
3.65 (s, 2 MeO). 13C-NMR (75.4 MHz, CDCl3): 25.19, 34.51 ((CH2)4); 51.81; 51.71 (MeO); 54.11 (CH); 176.34
(CˆO).
(S,S)-2,7-Bis{[(benzyloxy)carbonyl]amino}octanedioic Acid Dimethyl Ester (6). Amino ester 5 (3.0 g,
12.9 mmol) was dissolved in dioxane (30 ml) and the soln. cooled (08). Benzyl carbonochloridate Cbz-Cl;
(6.4 ml, 45.2 mmol) and 1m aq. NaHCO3 (45.2 ml, 45.2 mmol) were added simultaneously and gradually with
stirring. After the addition, the mixture was further stirred for 3 h at 08 and for 5 h at r.t. Dioxane was removed
by evaporation and the residue extracted with CHCl3 (3 Â 30 ml). The combined extract was washed with brine,
dried (MgSO4), and evaporated and the residue chromatographed (silica gel (170 g), hexane/AcOEt 2 : 1, TLC:
Rf 0.25): 6 (5.8 g, 89%). Colourless solid. M.p. 888. [a]2D0 ˆ‡ 14.2 (c ˆ 1.0, CHCl3). IR (CH2Cl2): 1743s, 1718s,
1700s (CˆO). 1H-NMR (300 MHz, CDCl3): 1.21 ± 1.46 (m, CH2(CH2)2CH2); 1.55 ± 1.62, 1.63 ± 1.89
(2m, CH2(CH2)2CH2); 3.73 (s, 2 MeO); 4.35 (ddd, J ˆ 4, 2 CH); 5.10 (s, 2 PhCH2); 5.31 (d, J ˆ 8.7, 2 NHCˆO);
7.33 (s, 2 PhCH2). 13C-NMR (75.4 MHz, CDCl3): 24.71, 32.45 ((CH2)4); 52.37 (MeO); 53.68 (CH); 67.02
(PhCH2); 128.12, 128.19, 128.52, 136.23 (PhCH2); 155.82 (NHCˆO); 172.81 (CˆO). HR-EI-MS: 500.2199
‡
(C26H32N2O8, M ; calc. 500.2199). Anal. calc. for C26H32N2O8 (500.54): C 62.39, H 6.44, N 5.60; found: C 62.38,
H 6.42, N 5.65.
(S,S)-2,7-Bis{[(benzyloxy)carbonyl]amino}octanoic Acid 2-Oxo-2-phenylethyl Ester (7a) and (S,S)-2,7-
Bis{[(benzyloxy)carbonyl]amino}octanoic Acid Bis(2-oxo-2-phenylethyl) Ester (7b). To a soln. of 6 (2.5 g,
5.0 mmol) in dioxane (25 ml) and H2O (12.5 ml), 1m aq. LiOH (10 ml, 10 mmol) was added. The mixture was
stirred overnight at r.t. When methyl ester hydrolysis was complete (TLC), 2-bromo-1-phenylethan-1-one
( ˆ phenacyl bromide, Pac-Br; 1.1 g, 5.5 mmol) in dioxane (1 ml) was added. Onset of precipitation could
normally be observed soon after addition was complete. If this was not the case, H2O (1 ± 5 ml) was added until
the mixture became turbid. Stirring was continued for 2 d, when more Pac-Br (0.22 g, 1.10 mmol) was added.

Helvetica Chimica Acta ± Vol. 81 (1998)
2059
After a further 2 d stirring, the solvents were evaporated, and the residue was chromatographed (200 g of silica
gel). The by-product 7b was eluted with CHCl3 and 7a (1.86 g, 63%) with hexane/AcOEt 1 : 2 containing 2%
AcOH.
Data of 7a: Amorphous powder. [a]D20
ˆ
3.4 (c ˆ 1.0, DMF). IR (CH2Cl2): 1747s, 1714s (CˆO). 1H-NMR
(300 MHz, CDCl3): 1.28 ± 1.58 (m, CH2(CH2)2CH2); 1.60 ± 1.92, 1.93 ± 2.12 (2m, CH2(CH2)2)CH2); 4.31 ± 4.43,
4.48 ± 4.61 (2m, 2 CH); 5.10, 5.12 (2s, 2 PhCH2); 5.27, 5.50 (2dd, J ˆ 18.3, 2.3, CH2COPh); 5.36 (d, J ˆ 16.3,
NHCˆO); 5.41 (d, J ˆ 17.0, NHCˆO); 7.34 (s, 2 PhCH2); 7.47 (ddd, J ˆ 15.0, 6.6, 1.3, 2 H, CH2COPh); 7.56 ±
7.65 (m, 1 H, CH2COPh); 7.88 (dd, J ˆ 8.3, 1.3, 2 H, CH2COPh). 13C-NMR (75.4 MHz, CDCl3): 24.63, 24.69,
32.38, 32.39 ((CH2)4); 52.31, 53.73 (CH); 66.42, 66.97 (PhCH2, CH2COPh); 127.71, 128.08, 128.14, 128.48,
128.85, 128.89, 133.92, 133.98, 136.98 (PhCH2, CH2COPh); 155.82 (NHCˆO); 171.88, 171.96, 172.86 (CˆO);
‡
191.36 (COOH). HR-FAB-MS: 591.2348 (C32H35N2O9, [M ‡ H] ; calc. 591.2343). Anal. calc. for C32H34N2O9
(590.62): C 65.07, H 5.80, N 4.74; found: C 64.97, H 5.85, N 4.88.
Data of 7b: Colourless solid. M.p. 161 ± 1648. [a]2D0 ˆ‡ 14.3 (c ˆ 1.0, CHCl3). IR (CH2Cl2): 1753s, 1698s
(CˆO). 1H-NMR (300 MHz, CDCl3): 1.48 ± 1.59 (m, CH2(CH2)2CH2); 1.78 ± 1.93, 1.98 ± 2.12 (2m, CH2(CH2)2-
CH2); 4.51 ± 4.61 (m, 2 CH); 5.12 (2s, 2 PhCH2); 5.27, 5.50 (2d, J ˆ 18.3, 2 CH2COPh); 5.43 (d, J ˆ 9.3,
2 NHCˆO); 7.34 (s, 2 PhCH2); 7.46 (dd, J ˆ 8.3, 4 H, CH2COPh); 7.60 (dd, J ˆ 8.3, 2 H, CH2COPh); 7.88
(d, J ˆ 8.3, 4 H, CH2COPh). 13C-NMR (75.4 MHz, CDCl3): 24.72, 32.34 ((CH2)4); 53.80 (CH); 66.46, 67.01
(PhCH2, CH2COPh); 127.77, 128.10, 128.13, 128.51, 128.89, 133.97, 134.04, 136.30 (PhCH2); 155.42 (NHCˆO);
‡
171.98 (CˆO); 191.44 (COOH). HR-FAB-MS: 709.2753 (C41H41N2O10, [M ‡ H] ; calc. 709.2761). Anal. calc.
for C40H40N2O10 (708.75): C 67.78, H 5.69, N 3.95; found: C 67.88, H 5.74, N 3.98.
Phenacyl Ester Deprotection of 7b. A sample of 7b (0.10 g, 0.14 mmol) in AcOH (25 ml) was treated with
Zn powder (0.18 g, 2.8 mmol). The suspension was stirred at 608 for 2 h. More Zn (0.18 g, 2.8 mmol) was added
and stirring continued at r.t. for 4 h. The mixture was filtered through a pad of Celite and the filtrate evaporated.
The residue was partitioned between H2O (10 ml) and CH2Cl2 (25 ml). The aq. phase was extracted twice more
with CH2Cl2 (10 ml), the combined extract washed with brine, dried (MgSO4), and evaporated, and the reside
crystallized from MeCN/Et2O: (S,S)-2,7-bis{[(benzyloxy)carbonyl]amino}octanedioic acid (62 mg, 94%).
[a]2D0
13C-NMR: in accordance with [6].
ˆ
10.5 Æ 0.5 (c ˆ 1.0, DMF); [18]: [a]D
ˆ
10.8 (c ˆ 1, DMF); [6]: [a]D
ˆ
9.8 (c ˆ 5, DMF).
(S,S)-7-Amino-2-{[(benzyloxy)carbonyl]amino}octanedioic Acid 1-(2-Oxo-2-phenylethyl) Ester 7-(N-Car-
boxyanhydride) (ˆ(aS,4S)-a-{[(Benzyloxy)carbonyl]amino}-2,5-dioxooxazolidine-4-hexanoic Acid 2-Oxo-2-
phenylethyl Ester; 9). A soln. of 7a (1.50 g, 2.54mmol) in dichloromethyl methyl ether (20.0 g, 174 mmol) was
stirred at 608 for 30 min and then for 2 h at r.t. The mixture was evaporated: 9 (1.23 g, 100%) which was not
purified (semistable compound). 1H-NMR (300 MHz, CDCl3): 1.38 ± 1.62 (m, CH2(CH2)2CH2); 1.64 ± 1.92,
1.94 ± 2.09 (2m, CH2(CH2)2CH2); 3.93 ± 4.05, 4.48 ± 4.59 (2m, 2 CH); 5.06, 5.15 (2d, J ˆ 13.7, PhCH2); 5.29, 5.50
(2d, J ˆ 18.3, CH2COPh); 5.67, 7.15 (2s, 2NHCˆO); 7.33 (s, PhCH2); 7.48 (dd, J ˆ 8.3, 2 H, CH2COPh); 7.61
(dd, J ˆ 8.3, 1 H, CH2COPh); 7.87 (d, J ˆ 8.3, 2 H, CH2COPh). 13C-NMR (75.4 MHz, CDCl3): 23.44, 23.68,
31.36, 32.34 ((CH2)4); 53.05, 56.78 (CH); 66.66, 67.20 (PhCH2, CH2COPh); 127.82, 128.04, 128.09, 128.60,
128.93, 129.05, 133.75, 134.31, 136.36 (PhCH2, CH2COPh); 152.35, 156.34 (NHCˆO); 170.37, 171.88, 191.86
(CˆO).
(S,S)-2-Amino-7-{[(benzyloxy)carbonyl]amino}octanedioic Acid 8-(2-Oxo-2-phenylethyl) Ester (10). A
soln. of 9 (1.20 g, 2.49 mmol) in AcOH/H2O 3 :1 (50 ml) was stirred at r.t. for 12 h. After evaporation, the
residue was suspended in CHCl3, evaporated again, and dried: acetate salt of 10 (1.29 g, 100%) which was not
purified. IR (CH2Cl2): 1740s (CˆO). 1H-NMR (300 MHz, (D6)DMSO): 1.22 ± 1.52 (m, CH2(CH2)2CH2); 1.62 ±
1.89 (m, CH2(CH2)2CH2, NH2); 3.55 ± 3.68, 4.10 ± 4.23 (2m, 2 CH); 5.03 (s, 2 PhCH2); 5.46, 5.58 (2d, J ˆ 18.3,
CH2COPh); 7.36 (s, PhCH2); 7.54 (dd, J ˆ 8.3, 2 H, CH2COPh); 7.67 (dd, J ˆ 8.3, 1 H, CH2COPh); 7.82 (d, J ˆ
8.7, NHCˆO); 7.95 (d, J ˆ 8.3, 2 H, CH2COPh). 13C-NMR (75.4 MHz, (D6)DMSO): 24.72, 25.61, 30.63, 31.21
((CH2)4); 53.25, 54.34 (CH); 66.05, 67.25 (PhCH2, CH2COPh); 128.23, 128.30, 128.34, 128.52, 128.87, 129.43,
134.51, 137.41 (PhCH2, CH2COPh); 155.70 (NHCˆO); 172.57 (CˆO); 193.09 (COOH).
(S,S)-2-{[(Benzyloxy)carbonyl]amino}-7-{[(tert-butoxy)carbonyl]amino}octanedioic Acid 1-(2-Oxo-2-
phenylethyl) Ester (11a). To a cooled (08) soln. of 10 (0.40 g, 0.77 mmol) in CH2Cl2 (30 ml), Boc2O (0.34 g,
1.55 mmol) and Et3N (0.32 ml, 2.32 mmol) were added gradually with stirring. The mixture was stirred for
further 3 h at 08 and 5 h at r.t. After evaporation, the residue was suspended in H2O and extracted with CHCl3
(3 Â 30 ml), the combined extract washed with brine, dried (MgSO4), and evaporated, and the crude product
chromatographed (silica gel (170 g), hexane/AcOEt 5 : 1; TLC; Rf 0.28): 11a (0.41 g, 95%). Amorphous
powder. [a]2D0
ˆ
2.8 (c ˆ 1.0, DMF). IR (CH2Cl2): 1748s, 1694s (CˆO). 1H-NMR (300 MHz, CDCl3): 1.42
(s, t-Bu); 1.59 ± 1.81 (m, CH2(CH2)2CH2); 1.85 ± 2.38 (m, CH2(CH2)2CH2); 4.37 ± 4.46, 4.48 ± 4.58 (2m, 2 CH);

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Helvetica Chimica Acta ± Vol. 81 (1998)
5.11 (s, PhCH2); 5.21 (s, NHCˆO); 5.28, 5.48 (2d, J ˆ 18.3, CH2COPh); 5.62 (s, NHCˆO); 7.34 (s, PhCH2);
7.47 (dd, J ˆ 8.3, 2 H, CH2COPh); 7.60 (dd, J ˆ 8.3, 1 H, CH2COPh); 7.88 (d, J ˆ 8.3, 2 H, CH2COPh).
13C-NMR (75.4 MHz, CDCl3): 24.68, 24.86, 32.22 ((CH2)4); 28.26 (Me3); 53.74 (CH); 66.46, 67.04 (PhCH2,
CH2COPh); 79.96 (Me3C); 127.74, 128.08, 128.12, 128.47, 128.87, 133.94, 133.98, 136.16 (PhCH2, CH2COPh);
‡
‡
156.06 (NHCˆO); 172.03 (CˆO); 191.53 (COOH). HR-FAB-MS: 557.2459 (C29H37N2O9 , [M ‡ H] ; calc.
557.2499). Anal. calc. for C29H36N2O9 (556.60): C 62.58, H 6.52, N 5.03; found: C 62.49, H 6.54, N 5.00.
(S,S)-2-{[(Benzyloxy)carbonyl]amino}-7-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}octanedioic Acid 1-
(2-Oxo-2-phenylethyl) Ester (11b). To a cooled (08) soln. of 10 (0.75 g, 1.45 mmol) in dioxane (20 ml), Fmoc-Cl
(0.75 g, 2.90 mmol) and 1m NaHCO3 (5.8 ml, 5.8 mmol) were added gradually with stirring. The mixture was
stirred for further 3 h at 08 and for 9 h at r.t. The reaction was quenched by adding AcOH (2 ml), and the
solvents were evaporated. The residue was suspended in H2O and extracted with CHCl3 (3 Â 30 ml), the
combined extract washed with brine, dried (MgSO4), and evaporated and the crude product chromatographed
(silica gel (170 g), hexane/AcOEt 2 : 1; TLC: Rf 0.31): 11b (0.76 g, 77%). Amorphous powder. [a]D20
ˆ
4.2
(c ˆ 1.0, DMF). IR (CH2Cl2): 1746s, 1693s (CˆO). 1H-NMR (300 MHz, (D6)DMSO): 1.25 ± 1.34
(m, CH2(CH2)2CH2); 1.52 ± 1.78 (m, CH2(CH2)2CH2); 3.84 ± 3.92 (m, CH); 4.10 ± 4.28 (m, 3 H, CH,
C13H9CH2); 5.03 (s, PhCH2); 5.42, 5.59 (2d, J ˆ 18.7, CH2COPh); 7.23 ± 7.41 (m, 10 H, NHCO, PhCH2,
CH2COPh, C13H9CH2); 7.50 ± 7.58, 7.61 ± 7.73 (2m, 6 H, CH2COPh, C13H9CH2); 7.81 (d, J ˆ 7.6, NHCˆO);
7.86, 7.95 (2d, J ˆ 8.3, 4 H, CH2COPh, C13H9CH2). 13C-NMR (75.4 MHz, (D6)DMSO): 24.79, 32.11 ((CH2)4);
52.36, 53.74 (CH); 66.49, 66.75, 67.08 (PhCH2, CH2COPh, C13H9CH2); 120.02, 124.92, 127.08, 127.56, 127.67,
127.92, 128.10, 128.15, 128.50, 128.87, 128.99 (PhCH2, CH2COPh, C13H9CH2); 133.92, 134.01 (CH2COPh);
136.20 (PhCH2); 141.25, 143.95 (C13H9CH2); 156.24 (NHCˆO); 171.33, 172.19 (CˆO); 191.69 (COOH). HR-
‡
‡
FAB-MS: 679.2658 (C39H39N2O9 , [M ‡ H] ; calc. 679.2656). Anal. calc. for C39H38N2O9 (678.73): C 69.01,
H 5.64, N 4.13; found: C 69.10, H 5.59, N 4.02.
(S,S)-2-{[(tert-Butoxy)carbonyl]amino}-7-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}octanedioic Acid
1-(2-Oxo-2-phenylethyl) Ester (12). A soln. of 11b (0.70 g, 1.03 mmol) in 47% HBr/AcOH (7 ml) at 08 was
stirred for 2 h at r.t. After evaporation, the residue was suspended in CHCl3 and dried under high vacuum for
24 h: HBr salt (0.64 g, 99%) of Cbz-deprotected 11b which was not purified. IR (CH2Cl2): 1747s, 1703s (CˆO).
‡
1H-NMR (300 MHz, (D6)DMSO): 1.24 ± 1.82 (m, CH2(CH2)2CH2); 1.89 (s, NH3 ); 3.90 ± 3.98 (m, CH); 4.18 ±
4.32 (m, CH, C13H9CH2); 5.63, 5.73 (2d, J ˆ 19.0, CH2COPh); 7.30 (dd, J ˆ 8.6, 2 H, C13H9CH2); 7.40 (dd, J ˆ
8.3, 2 H, C13H9CH2); 7.56 (dd, J ˆ 8.6, 3 H, CH2COPh, C13H9CH2); 7.65 (d, J ˆ 9.0, NHCˆO); 7.70 (dd, J ˆ 8.6,
3 H, CH2COPh, C13H9CH2); 7.88, 7.97 (2d, J ˆ 8.3, 4 H, CH2COPh, C13H9CH2); 8.43 (s, COOH). 13C-NMR
(75.4 MHz, (D6)DMSO): 24.18, 25.64, 30.57, 30.95 ((CH2)4); 52.32, 58.14 (CH); 66.10, 68.13 (CH2COPh,
C13H9CH2); 120.63, 125.77, 127.58, 128.16, 128.40, 128.82, 129.18 (CH2COPh, C13H9CH2); 134.04, 134.75
(CH2COPh); 141.22, 144.28 (C13H9CH2); 156.06, 169.88 (CˆO); 192.47 (COOH).
An aliquot of this material (0.50 g, 0.80 mmol) in CH2Cl2 (20 ml) was cooled (08) and treated gradually with
Boc2O (0.53 g, 2.40 mmol) and Et3N (0.39 ml, 2.80 mmol). The mixture was stirred for 3 h at 08 and 5 h at r.t.
After evaporation, the residue was suspended in H2O and extracted with CHCl3 (3 Â 30 ml), the combined
extract washed with brine, dried (MgSO4), and evaporated, and the crude product chromatographed (silica gel
(170 g), hexane/AcOEt 1 : 1 (2% AcOH); TLC: Rf 0.30): 12 (475 mg, 92%). Amorphous powder. [a]D20
ˆ
1.5
(c ˆ 1.0, DMF). IR (CH2Cl2): 1749s, 1703s (CˆO). 1H-NMR (300 MHz, (D6)DMSO): 1.17 ± 1.39
(m, CH2(CH2)2CH2); 1.35 (s, 9 H, Me3C); 1.44 ± 1.83 (m, CH2(CH2)2CH2); 3.66 ± 3.84, 3.98 ± 4.11 (2m, 2 CH);
4.13 ± 4.32 (m, 2 H, C13H9CH2); 5.41, 5.56 (2d, J ˆ 19.0, CH2COPh); 7.02 (d, J ˆ 7.3, NHCˆO); 7.30 (dd, J ˆ 8.6,
4 H, CH2COPh, C13H9CH2); 7.39 (dd, J ˆ 8.3, 2 H, CH2COPh, C13H9CH2); 7.52 (dd, J ˆ 8.6, 2 H, CH2COPh,
C13H9CH2); 7.62 (d, J ˆ 7.3, NHCˆO); 7.69 (dd, J ˆ 8.6, 2 H, CH2COPh, C13H9CH2); 7.86, 7.94 (2d, J ˆ 8.3, 4 H,
CH2COPh, C13H9CH2). 13C-NMR (75.4 MHz, (D6)DMSO): 25.58, 26.01, 31.45, 32.33 ((CH2)4); 28.70 (Me3C);
54.06, 55.12 (CH); 65.84, 67.08 (C13H9CH2, CH2COPh); 78.68 (Me3C); 120.52, 121.87, 125.77, 127.56, 128.08,
128.40, 129.39, 134.37 (C13H9CH2, CH2COPh); 137.93, 139.92 (C13H9CH2); 143.08, 156.06, 172.86 (CˆO);
‡
193.11 (COOH). HR-FAB-MS: 645.2811 (C36H41N2O9, [M ‡ H] ; calc. 645.2812). Anal. calc. for C36H40N2O9
(644.71): C 67.07, H 6.25, N 4.35; found: C 67.34, H 6.17, N 4.19.

Helvetica Chimica Acta ± Vol. 81 (1998)
2061
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Received July 27, 1998