Synthesis and evaluation of enantiomeric purity of protected α-amino and peptide aldehydes

Article abstract of DOI:10.1002/(SICI)1522-2675(19991110)82:11<1960::AID-HLCA1960>3.0.CO;2-2

The synthesis of enantiomerically pure Ac-Tyr-Val-Ala-Asp(O'Bu)-H dimethyl acetal ((S)-1) is reported, a protected tetrapeptide C-terminal aldehyde belonging to a class of potent, reversible inhibitors of cysteine proteases (e.g., interleukin-1β-convertin

Full text of DOI:10.1002/(SICI)1522-2675(19991110)82:11<1960::AID-HLCA1960>3.0.CO;2-2

1960
Helvetica Chimica Acta ± Vol. 82 (1999)
Synthesis and Evaluation of Enantiomeric Purity of Protected a-Amino
and Peptide Aldehydes1)
by Thomas Mindt and Urs Michel*
University of Applied Sciences, Department of Chemistry, Technikumstrasse 9, CH-8401 Winterthur
and F. Dick*
Bachem AG, Hauptstrasse 144, CH-4416 Bubendorf
The synthesis of enantiomerically pure Ac-Tyr-Val-Ala-Asp(OtBu)-H dimethyl acetal ((S)-1) is reported, a
protected tetrapeptide C-terminal aldehyde belonging to a class of potent, reversible inhibitors of cysteine
proteases (e.g., interleukin-1b-converting enzyme (ICE), also called caspase-1). The coupling of the precursors
Ac-Tyr-Val-Ala-OH ((S)-8) and H-Asp(OtBu)-H dimethyl acetal ((S)-6) gave (S)-1 in a yield of 85%, with
epimerization of < 2% at the alanine and aspartic-acid residue. (S)-6 itself was synthesized in four steps in an
overall yield of 83% with an ee >98%.
Introduction. ± a-Amino and peptide aldehydes are useful synthetic intermediates
[1], and some of them are potent inhibitors of proteases [2]. A range of interleukin-1b-
converting enzyme (ICE) inhibitors is represented by small peptides with a modified
C-terminus. Aspartic acid frequently appears as its a-semialdehyde at the C-terminus
in such inhibitors. Especially the acetylated tetrapeptide aldehyde Ac-Tyr-Val-Ala-
Asp-H has been reported to be a potent reversible inhibitor of ICE [2 ± 4]. Since a few
years ago, it is known that ICE is involved in apoptosis (programmed cell death), which
is among the putative initiating factors in chronic and acute inflammatory diseases [5].
As a consequence, there is an increasing demand for such inhibitors, and, therefore,
easy synthetic access is of some importance. Unprotected chiral a-amino and peptide
aldehydes have the tendency to racemize (epimerize) at the C(a)-atom next to the
aldehyde group [1]. The racemization (epimerization) rate depends on structure,
temperature, and pH. Under the conditions of flash chromatography (silica gel), such
aldehydes are especially prone to racemization (epimerization) [6]. Consequently,
viable strategies of synthesis entail a suitably protected form of a given peptide
aldehyde which can be transformed easily into the biologically active free form while
keeping the stereogenic center at the C-terminal residue intact [2 ± 4]. This is very
important since the activity of such inhibitors depends on their enantiomeric purity [5].
Nevertheless, there is only little information available concerning the enantiomeric and
diastereoisomeric purity of intermediates and end products, especially with respect to
the aspartic acid C(a)-atom [2 ± 4].
We now describe the synthesis of the protected, enantiomerically pure tetrapeptide
aldehyde Ac-Tyr-Val-Ala-Asp(OtBu)-H dimethyl acetal ((S)-1), with the main focus
1
)
Diploma Thesis of Thomas Mindt. Present address: Brown University, Providence RI 02912-1867, USA.

Helvetica Chimica Acta ± Vol. 82 (1999)
1961
on the following: i) Preparation of the key building block (S)-6 and determination of its
19
enantiomeric purity by NMR spectroscopy (1H- and F-NMR of its MTPA-amides2)
[9]), and ii) coupling of Ac-Tyr-Val-Ala-OH ((S)-8) and (S)-6 and determination of
1
the extent of epimerization at the aspartic-acid and alanine residue by H-NMR
spectroscopy, including full analytical characterization of (S)-1.
Results and Discussion. ± When planning the synthesis of the target compound (S)-
1, we realized that preparation of the key building block H-Asp(OtBu)-H dimethyl
acetal ((S)-6) would be a critical step. In particular, no reliable information about
conservation/loss of the chiral integrity during the acetalization could be found. Based
on theoretical considerations [7] as well as literature [8], we assumed a high risk of
racemization at C(a) during the conversion of protected aspart-1-al in the correspond-
ing dimethyl acetal. Therefore, we decided to synthesize H-(R)-Asp(OtBu)-H dimethyl
acetal ((R)-6) and H-(RS)-Asp(OtBu)-H dimethyl acetal ((RS)-6) beside (S)-6, and
to convert all three to the MTPA-amides (S)-MTPA-Asp(OtBu)-H dimethyl acetal
((S)-7), (S)-MTPA-(R)-Asp(OtBu)-H dimethyl acetal ((R)-7), and (S)-MTPA-(RS)-
Asp(OtBu)-H dimethyl acetal ((RS)-7), in order to establish a methodology for the
determination of the enantiomeric purity by means of chromatography and/or
spectroscopy [9].
Thus, commercially available Z-Asp(OtBu)-OH ((S)-2) was activated to the mixed
anhydride with isobutyl choroformate (ˆ isobutyl carbonochloridate) and N-methyl-
morpholine and reduced to the corresponding alcohol Z Asp(OtBu) ol ((S)-3;
purity >95%, yield 95%) [10][11] (Scheme 1). (S)-3 was oxidized by mild Swern
oxidation [12] to Z Asp(OtBu)-H ((S)-4; purity > 95%, yield 95%), which was
immediately converted with trimethyl orthoformate/MeOH/TsOH to the stable
Z-Asp(OtBu)-H dimethyl acetal ((S)-5; purity >95%, yield 83%) [13]. The (benzyl-
oxy)carbonyl(Z) protecting group was finally removed by hydrogenolysis with 10%
Pd/C in MeOH to give (S)-6 (purity > 95%, yield 91%) [14]. According to the same
procedure and with comparable yields and purities, (R)- and (RS)-6 were synthesized.
Samples of (S)-, (R)-, and (RS)-6 were subsequently reacted with ( )-(2R)-3,3,3-
trifluoro-2-methoxy-2-phenylpropanoyl chloride ((R)-MTPA-Cl) to furnish the dia-
2
)
MTPA ˆ 2-Methoxy-2-(trifluoromethyl)-2-phenylacetyl (ˆ 3,3,3-trifluoro-2-methoxy-2-phenylpropanoyl).

1962
Helvetica Chimica Acta ± Vol. 82 (1999)
Scheme 1
a) Isobutyl chloroformate/N-methylmorpholine/THF, NaBH4,
788. b) (COCl)2/DMSO/CH2Cl2,
458,
iPr2EtN. c) MeOH/TsOH/CH(OMe3)3, r.t. d) H2, 10% Pd/C, MeOH, 48 h, r.t.
stereoisomeric MTPA-amides as individual compounds (S)-MTPA-Asp(OtBu)-H
dimethyl acetal ((S)-7), (S)-MTPA-(R)-Asp(OtBu)-H dimethyl acetal ((R)-7) and
as diastereomer mixture (S)-MTPA-(RS)-Asp(OtBu)-H dimethyl acetal ((RS)-7)
(Scheme 2).
Scheme 2
a) (R)-MTPA-Cl, pyridine/CCl4, 30 min, r.t., N,N-dimethylpropane-1,3-diamine [9].

Helvetica Chimica Acta ± Vol. 82 (1999)
1963
Attempts to separate the diastereoisomers by TLC or HPLC were unsuccessful.
Careful spectroscopic analyses, however, allowed us to unambiguously distinguish
between (S)- and (R)-7. Determination of the (R/S) ratio with respect to aspart-1-al
1
was accomplished by comparing selected H- and 19F-NMR data (see Table 1) of
compounds (S)-, (R)-, and (RS)-7 with corresponding data of artificial mixtures of (S)-
and (R)-7 (90 :10, 99 :1; data not shown). The results can be summarized as follows: i)
The method of choice for checking enantiomeric purity is 1H-NMR spectroscopy using
the s of the Boc group (19F-NMR is slightly less sensitive but suitable as well). ii) The
detection limit of (S)-7 in (R)-7 (and vice versa) is 1% or better. iii) The prepared (S)-
and (R)-7 have enantiomeric purities of > 99% (i.e., ee >98%). These results suggest
that acetalization of an a-amino aldehyde ± at least in the case of aspartic acid ± is
possible without concomitant racemization. The behavior of other a-amino aldehydes
derived from amino acids is currently under investigation.
1
Table 1. Relevant 19F- and H-NMR Signals (CDCl3) of (S)-MTPA-Asp(OtBu)-H Dimethyl Acetals
d(F) [ppm]
s of CF3
d(H) [ppm]
s of Asp (OttBu)
(S)-7
69.45
1.39
(R)-7
(RS)-7
69.35
69.35, 69.45
1.45
1.45, 1.39
After having verified the quality of the key building block (S)-6, we concentrated
on the final coupling to the target compound (S)-1. Thus, (S)-8 and (S)-6 were coupled
in CH2Cl2/DMF with EDCl3)/N-methylmorpholine and 1-hydroxybenzotriazole
(HOBt) as additive, conditions which cleanly afforded (S)-1 (purity >95%, yield
85%; Scheme 3).
Since one of the most serious problems in peptide synthesis remains epimerization
of the C-terminal residue of the carboxy component during fragment couplings [15] ±
and the reaction in discussion represents such a coupling ± possible epimerization at the
alanyl residue in (S)-8 had to be kept in mind. To quantify the extent of epimerization,
a similar approach as described for compound (S)-6 was chosen, i.e., diastereoisomers
with opposite or mixed configuration at the stereogenic centers in Asp and Ala were
prepared and analyzed by 1H-NMR spectroscopy (Scheme 3).
Table 2 lists characteristic NMR data of Ac-Tyr-Val-Ala-Asp(OtBu)-H dimethyl
acetal ((S)-1), Ac-Tyr-Val-Ala-(R)-Asp(OtBu)-H dimethyl acetal ((R)-1), Ac-Tyr-Val-
Ala-(RS)-Asp(OtBu)-H dimethyl acetal ((RS)-1), and Ac-Tyr-Val-(R)-Ala-Asp-
(OtBu)-H dimethyl acetal (1a), which were prepared analogously to (S)-1 (for details,
t
see Exper. Part). Again, differences in chemical shifts (s of BuO in Asp and/or d of
Me(3) in Ala) enabled us to differentiate between individual diastereoisomers. The
results of this study are as follows: i) The chiral integrity of the Asp(OtBu)-H dimethyl
acetal part is not affected at all, as expected. ii) The detection limit of 1a in (S)-1, which
is the most important in this context, is 2% or better. iii) Our preparation of (S)-1
contains less than 2% of 1a.
3
)
EDCI ˆ 1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide.

1964
Helvetica Chimica Acta ± Vol. 82 (1999)
Scheme 3
a) Ac-Tyr-Val-Ala-OH ((S)-8), HOBt, N-methylmorpholine, EDCl, CH2Cl2/DMF. b) Ac-Tyr-Val-(R)-Ala-OH
((R)-8), HOBt, N-methylmorpholine, EDCl, CH2Cl2/DMF; reaction carried out with (S)-6 only.
Table 2. Relevant 1H-NMR Signals ((D6)DMSO) of the Diastereoisomeric Tetrapeptides
d(H) [ppm]
d of Me(3) (Ala)
s of tBuO (Asp)
(S)-1
(R)-1
(RS)-1
1a
1.19
1.17
1.17, 1.19
1.15
1.35
1.36
1.35, 1.36
1.36
In parallel, further trials to separate diastereoisomers by anal. HPLC were carried
out with varying degrees of success (data not shown), i.e., the mixtures (S)-1/1a and
Ac-Tyr-Val-Ala-Asp-H/Ac-Tyr-Val-(R)-Ala-Asp-H were separable, whereas Ac-Tyr-
Val-Ala-Asp-H/Ac-Tyr-Val-Ala-(R)-Asp-H was not.
The Figure illustrates the 2D-COSY spectrum of compound (S)-1, which is fully
consistent with expectation and thus confirms the correct structure, together with the
13C-NMR and FAB-MS data.
In summary, we have developed a five-step synthesis of the protected peptide
aldehyde (S)-1 (overall yield ca. 70%) which may be of general practicability. In
particular, racemization- and epimerization-endangered key steps ± especially the

Helvetica Chimica Acta ± Vol. 82 (1999)
1965
Figure. 2D-COSY(45) of Ac-Tyr-Val-Ala-Asp(OtBu)-H dimethyl acetal ((S)-1) in (D6)DMSO at 300 MHz
acetalization of the protected amino aldehyde (S)-4 ± were carefully looked at and
shown to proceed with almost complete conservation of the chiral integrity at the
critical stereocenters.
We thank F. Nydegger for MS, F. Fehr for NMR spectra, and A. Ruf and M. Vock for skillful technical
assistance.

1966
Helvetica Chimica Acta ± Vol. 82 (1999)
Experimental Part
General. Reagents were reagent-grade commercials and used without further purification. Amino-acid
derivatives and the tripeptides Ac-Tyr-Val-Ala-OH ((S)-8) and Ac-Tyr-Val-(R)-Ala-OH ((R)-8) were from
Bachem AG, CH-4416 Bubendorf. Pyridine was distilled over KOH. Other solvents were dried over activated
molecular sieves for at least 12 h. Evaporation was done at water-aspirator pressure. Flash chromatography
(FC): SiO2 60 (230 ± 400 mesh, 0.040 ± 0.063 mm) from Bachem and Fluka. TLC: precoated plates SiO2- 60 F254
from Fluka; visualization by UV light. M.p. Büchi Smp-20; uncorrected. [a]D: Perkin-Elmer-241 polarimeter. IR
Spectra (cm 1): Perkin-Elmer FT-IR 16 PC. NMR Spectra: Bruker Avance DPX-300, DRX-500; d in ppm rel. to
internal SiMe4 (1H) or CFCl3 (19F; unless mentioned otherwise); J in Hz. MS: VG Micromass 70/70e; FAB (fast-
atom bombardment): Ar atoms and 3-nitrobenzyl alcohol matrix.
(S)-4-Hydroxy-3-{[(phenylmethoxy)carbonyl]amino}butanoic Acid 1,1-Dimethylethyl Ester ((S)-3; Z-
Asp(OtBu)-ol). At 0 ± 58, N-methylmorpholine (2.80 g, 27.5 mmol) and isobutyl carbonochloridate (3.60 g,
26.25 mmol) were added to a soln. of (S)-2 (8.10 g, 25 mmol) in THF (150 ml). After 15 min, the white
suspension was added dropwise at 788 to a suspension of NaBH4 (3.8 g, 50 mmol) in THF/MeOH 3 : 1
(200 ml) (immediate gas production). After 20 min at 788, the mixture was quenched with 10% AcOH/H2O
(100 ml), most of the solvent evaporated, the residue extracted with AcOEt (3 Â 200 ml), the org. layer washed
with 5% NaHCO3 soln. (2 Â 200 ml) and H2O (100 ml), dried (Na2SO4), and evaporated, and the residue
(9.5 g) purified by FC (SiO2 (300 g), CH2Cl2/MeOH 20 : 1): (S)-3 (7.7 g, 95%; purity >95% by 1H-NMR).
Slightly yellow oil. [a]D20
ˆ
9.7 (c ˆ 1.2 ´ 10 2, MeOH). IR (1% in CCl4): 3450m (OH, NH), 2991m (CH),
1733vs (CˆO), 1513m (amide II), 1375m (Me), 1261m (C O), 1068m. 1H-NMR (300 MHz, CDCl3): 1.40
(s, tBu); 2.40 ± 2.55 (m, 2 H C(2)); 3.52 (br. s, OH); 3.58 ± 3.70 (m, 2 H C(4)); 3.98 ± 4.11 (m, H C(3)); 5.02,
5.08 (2d, 2J ˆ 12.2, PhCH2); 5.82 (d, 3J ˆ 8.6, NH); 7.35 ± 7.23 (m, 5 arom. H). 13C-NMR (DEPT; 75 MHz,
CDCl3): 27.7 (Me); 37.0, 63.8, 66.5 (CH2); 49.8, 127.8, 128.2, 128.22 (CH); 80.9, 136.2, 156.1, 170.8 (C).
(S)-4-Oxo-3-{[(phenylmethoxy)carbonyl]amino}butanoic Acid 1,1-Dimethylethyl Ester ((S)-4; Z-Asp-
(OtBu)-H). Dry DMSO (3.95 g, 50.5 mmol) was dissolved under Ar at 458 in CH2Cl2 (60 ml), and oxalyl
chloride (3.5 g, 27.5 mmol) was added dropwise (immediate gas production). After 5 min, a soln. of (S)-3 (7.1 g,
22.9 mmol) in CH2Cl2 (35 ml) was added dropwise at 458 and the white suspension stirred 30 min at this temp.
Then iPr2EtN (9.2 g, 71 mmol) was added, the mixture allowed to warm up to 208, and stirring continued for
30 min at 208. The soln. was diluted with CH2Cl2 (200 ml) and extracted with H2O (50 ml), 1n NaHSO4
(50 ml), and again with H2O (3 Â 50 ml). The org. phase was dried (Na2SO4) and evaporated: (S)-4 (7.0 g, 95%;
purity >95% by 1H-NMR). White crystals. M.p. 63 ± 648. [a]2D0
ˆ
23.4 (c ˆ 1.3 ´ 10 2, MeOH). IR (1% in CCl4):
3455m (NH), 2996w (C H), 2837w and 2717w (C H, aldehyde), 1736vs (CˆO), 1511s (amide II), 1381m
(Me), 1247m (C O), 1172m, 1062m, 848w. 1H-NMR (300 MHz, CDCl3): 1.41 (s, tBu); 2.75 (dd, 2J ˆ 17.1, 3J ˆ
5.0, 1 H C(2)); 2.89 (dd, 2J ˆ 17.1, 3J ˆ 5.0, 1 H C(2)); 4.37 (ddd, 3J ˆ 8.1, 5.0, 5.0, H C(3)); 5.12 (s, PhCH2);
6.09 (d, 3J ˆ 8.1, NH); 7.39 ± 7.26 (m, 5 arom. H); 9.60 (s, H C(4)). 13C-NMR (DEPT; 75 MHz, CDCl3): 27.8
(Me); 35.4, 67.0 (CH2); 56.4, 128.0, 128.1, 128.37, 198.8 (CH); 81.8, 135.9, 156.0, 169.9 (C).
(S)-4,4-Dimethoxy-3-{[(phenylmethoxy)carbonyl]amino}butanoic Acid 1,1-Dimethylethyl Ester ((S)-5; Z-
Asp(OtBu)-H dimethyl acetal). At r.t., trimethyl orthoformate (11.2 g, 105 mmol) and TsOH (100 mg) were
added to a soln. of (S)-4 (6.50 g, 21.1 mmol) in MeOH (30 ml). After 30 min, the mixture was evaporated, the
residue dissolved in CH2Cl2 (200 ml), and the soln. extracted with 5% NaHCO3 soln. (50 ml) and H2O (50 ml).
The extraction of the aq. layer with CH2Cl2 (200 ml) was repeated. The combined org. phase was dried (Na2SO4)
and evaporated and the residue purified by FC (SiO2 (300 g), AcOEt/hexane 1:4 with 0.5% Et3N): (S)-5 (6.5 g,
83%; purity >95% by 1H-NMR). Yellow oil. [a]2D0
ˆ
17.7 (c ˆ 10 2, MeOH). IR (1% in CCl4): 3465m (NH),
2996m and 2947m (C H), 2847m (acetal); 1740vs (CˆO), 1516s (amide II), 1381m (Me), 1237m (C O),
1
t
3
1173m, 1093m, 933m. H-NMR (300 MHz, CDCl3): 1.42 (s, Bu); 2.43 (dd, 2J ˆ 15.6, J ˆ 6.9, 1 H C(2)); 2.53
(dd, 2J ˆ 15.6, 3J ˆ 5.7, 1 H C(2)); 4.28 ± 4.13 (m, H C(3)); 3.39 (s, (MeO)2CH); 4.33 (d, 3J ˆ 4.0,
(MeO)2CH); 5.12, 5.06 (2d, 2J ˆ 12.3, PhCH2); 5.40 (d, 3J ˆ 9.2, NH); 7.38 ± 7.25 (m, 5 arom. H). 13C-NMR
(DEPT; 75 MHz, CDCl3): 27.8 (Me); 35.5, 66.5 (CH2); 49.5, 127.9, 128.30 (CH); 80.7, 136.4, 155.8, 170.0 (C).
(S)-3-Amino-4,4-dimethoxybutanoic Acid 1,1-Dimethylethyl Ester ((S)-6; H-Asp(OtBu)-H dimethyl
acetal). A suspension of (S)-5 (5.90 g, 17 mmol) and 10% Pd/C (300 mg) in MeOH (30 ml) was stirred for
48 h at r.t. under 1 atm H2. The black suspension was filtered over Hyflo, the filtrate evaporated, and the residue
triturated with hexane. The insoluble components were filtered off, and the filtrate was evaporated to give (S)-6
(3.5 g, 91%; purity >95% by 1H-NMR). Slightly yellow liquid. B.p. 160 ± 1658. [a]2D0
ˆ
20.3 (c ˆ 10 2, MeOH).
IR (1% in CCl4): 3406w (N H), 2996m (C H), 2779m (C O, acetal), 1735s (CˆO), 1461w, 1381m (Me),
1167s (C O), 1127m, 1087m, 933w, 863w. 1H-NMR (300 MHz, CDCl3): 1.46 (s, tBu); 1.63 (br. s, NH2); 2.23

Helvetica Chimica Acta ± Vol. 82 (1999)
1967
(dd, 2J ˆ 16.1, 3J ˆ 8.8, 1 H C(2)); 2.52 (dd, 2J ˆ 16.1, 3J ˆ 3.9, 1 H C(2)); 3.22 ± 3.31 (m, H C(3)); 3.40, 3.42
(2s, (MeO)2CH); 4.15 (d, 3J ˆ 5.8, H C(4)). 13C-NMR (DEPT; 75 MHz, CDCl3): 27.9, 54.4, 54.8 (Me); 38.1
(CH2); 49.5, 106.9 (CH); 80.0, 171.3 (C).
(R)-3-Amino-4,4-dimethoxybutanoic Acid 1,1-Dimethylethyl Ester ((R)-6; H-(R)-Asp(OtBu)-H dimethyl
acetal). As described for (S)-6, from (R)-2 (10.0 g, 30.9 mmol). Overall yield 52%. HPLC: purity >95%.
(RS)-3-Amino-4,4-dimethoxybutanoic Acid 1,1-Dimethylethyl Ester ((RS)-6; H-(RS)-Asp(OtBu)-H di-
methyl acetal). As described for (S)-6. Overall yield 68%. Purity >95% by 1H-NMR. 1H-NMR (300 MHz,
CDCl3): 1.46 (s, tBu); 1.97 (br. s, NH2); 2.25 (dd, 2J ˆ 16.2, 3J ˆ 8.8, 1 H C(2)); 2.53 (dd, 2J ˆ 16.2, 3J ˆ 4.0,
1 H C(2)); 3.27 (ddd, 3J ˆ 8.8, 5.8, 4.0, H C(3)); 3.41, 3.43 (2s, (MeO)2CH); 4.15 (d, 3J ˆ 5.8, H C(4)).&
(3S)-4,4-Dimethoxy-3-{[(2S)-3,3,3-trifluoro-2-methoxy-2-phenylpropanoyl]amino}butanoic Acid 1,1-Di-
methylethyl Ester ((S)-7; (S)-MTPA-Asp(OtBu)-H dimethyl acetal). Under Ar at r.t., ( )-(2R)-3,3,3-trifluoro-
2-methoxy-2-phenylpropanoyl chloride (( )-(R)-MTPA-Cl) (140 mg, 0.48 mmol) was dissolved in dry pyridine
(1.2 ml) and diluted with CCl4 (1 ml). Subsequently, (S)-6 (90 mg, 0.4 mmol) was added to the white suspension,
the mixture stirred for 30 min at r.t., and then an excess of N,N-dimethylpropane-1,3-diamine (82 mg, 0.8 mmol)
added. After 5 min, the soln. was diluted with Et2O (50 ml) and extracted with 10% aq. citric acid (50 ml), 5%
Na2CO3 (50 ml), and sat. NaCl soln. (2 Â 20 ml). The org. phase was dried (Na2SO4) and evaporated: (S)-7
(0.17 g, >90%; purity >90% by 1H-NMR ). Yellow oil. 1H-NMR (300 MHz, CDCl3): 1.39 (s, tBu); 2.47
3
3
(dd, 2J ˆ 15.9, J ˆ 6.4, 1 H C(2)); 2.53 (dd, 2J ˆ 15.9, J ˆ 5.4, 1 H C(2)); 3.41, 3.43 (2s, (MeO)2CH); 3.42 ±
3.46 (q, 5J(H,F) ˆ 1.5 MeO (MTPA)); 4.40 ± 4.53 (m, H C(3), H C(4)); 7.25 (d, 3J ˆ 10.1, NH); 7.30 ± 7.60
(m, 5 arom. H). 19F-NMR (282 MHz, CDCl3): 69.45 (s, CF3); ee > 98%.
(3R)-4,4-Dimethoxy-3-{[(2S)-3,3,3-trifluoro-2-methoxy-2-phenylpropanoyl]amino}butanoic Acid 1,1-Di-
methylethyl Ester ((R)-7; (S)-MTPA-(R)-Asp(OtBu)-H dimethyl acetal). From (R)-6, as described for (S)-7.
1
Yellow oil (0.17 g, >90%; purity >90% by H-NMR/HPLC). 1H-NMR (500 MHz, CDCl3): 1.45 (s, tBu); 2.46
(dd, 2J ˆ 16.0, 3J ˆ 6.5, 1 H C(2)); 2.51 (dd, 2J ˆ 15.9, 3J ˆ 5.4, 1 H C(2)); 3.24, 3.28 (2s, (MeO)2CH); 3.34
(q, 5J(H,F) ˆ 1.5, MeO(MTPA)); 4.31 (d, H C(4)); 4.40 (m, H C(3)); 7.23 (d, 3J ˆ 9.1, NH); 7.3 ± 7.5
(m, 5 arom. H). 19F-NMR (282 MHz, CDCl3): 69.35 (s, CF3); ee > 98%.
(3R)- and (3S)-4,4-Dimethoxy-3-{[(2S)-3,3,3-trifluoro-2-methoxy-2-phenylpropanoyl]amino}butanoic
Acid 1,1-Dimethylethyl Ester ((RS)-7; (S)-MTPA-(RS)-Asp(OtBu)-H dimethyl acetal). From (RS)-6, as described
for (S)-7. Yellow oil (0.17 g, >90%; purity >90% by 1H-NMR). 1H-NMR (300 MHz, CDCl3): 1.39 (s, tBu ((S)-
7)); 1.45 (s, tBu ((R)-7)); 2.42 ± 2.63 (m, 2 H C(2)); 3.30, 3.35 (2s, (MeO)2CH ((R)-7)); 3.41, 3.42 (2s,
(MeO)2CH ((S)-7)); 3.4 ± 3.45 (m, MeO (MTPA)); 4.37 ± 4.53 (m, H C(3), H C(4)); 7.15 ± 7.25 (m, NH);
7.20 ± 7.60 (m, 5 arom. H). 19F-NMR (282 MHz, CDCl3): 69.45 (s, CF3 ((S)-7)); 69.35 (s, CF3 ((R)-7)).
Ac-Tyr-Val-Ala-Asp(OtBu)-H Dimethyl Acetal ((S)-1). At 0 ± 58, 1-hydroxybenzotriazole monohydrate
(1.53 g, 10 mmol), N-methylmorpholine (0.91 g, 9 mmol), (S)-6 (1.1 g, 5 mmol), and 1-[3-(dimethylamino)-
propyl]-3-ethylcarbodiimide hydrochloride (1.16 g, 6.05 mmol) were added to a soln. of Ac-Tyr-Val-Ala-OH
((S)-8; 2.16 g, 5.5 mmol) in CH2Cl2/DMF 1:1 (25 ml). The reaction mixture was stirred for 1 h at 0 ± 58 and
additionally 18 h at r.t. After dilution with AcOEt (300 ml), the org. phase was extracted with 10% aq. citric acid
(200 ml) and 5% NaHCO3 soln. (200 ml), dried (Na2SO4), and evaporated and the residue purified by FC (SiO2
1
(250 g), CH2Cl2/MeOH 20 :1 with 0.5% Et3N): (S)-1 (2.6 g, 85%; purity >96% by H-NMR). White crystals.
M.p. 212 ± 2148 (dec.). [a]D20
ˆ
28.9 (c ˆ 5.1 ´ 10 3, MeOH). ee > 98% (by comparison of the 1H-NMR with that
of the diastereoisomeric analogs (R)-1 and 1a). IR (1% in KBr): 3423m (N H), 3092w (C H, arom.), 2982w
and 2936w (C H), 2854w (C O, acetal), 1743m (CˆO, ester), 1642s (CˆO, amide), 1554m and 1527m (amide
II), 1458w, 1380w (Me), 1242w (C O), 1169m, 1095w, 857w. 1H-NMR (300 MHz, (D6)DMSO): 0.82 (d, 3J ˆ
6.8, Me(4) or Me(4') (Val)); 0.85 (d, 3J ˆ 7.2, Me(4) or Me(4') (Val)); 1.19 (d, 3J ˆ 7.1, Me(3) (Ala)); 1.35
(s, tBu (Asp)); 1.76 (s, Ac); 1.87 ± 2.04 (m, H C(3) (Val)); 2.22 (dd, 2J ˆ 15.0, 3J ˆ 7.5, 1 H C(2) (Asp)); 2.43
(dd, 2J ˆ 15.0, 3J ˆ 4.3, 1 H C(2) (Asp)); 2.61 (dd, 2J ˆ 13.8, 3J ˆ 10.2, 1 H C(3) (Tyr)); 2.87 (dd, 2J ˆ 13.8,
3J ˆ 3.8, 1 H C(3) (Tyr)); 3.27, 3.30 (2s, each (MeO)2CH); 4.13 ± 4.25 (m, H C(2) (Val), H C(3) (Asp),
H
C(4) (Asp)); 4.25 ± 4.36 (m, H C(2) (Ala)); 4.41 ± 4.53 (m, H C(2) (Tyr)); 6.63 (d, 3J ˆ 8.3, 2 arom. H
(Tyr)); 7.03 (d, 3J ˆ 8.4, 2 arom. H (Tyr)); 7.74 ± 7.90 (m, NH (Val), NH (Asp)); 7.93 ± 8.11 (m, NH (Tyr), NH
(Ala)); 9.17 (s, OH (Tyr)). 13C-NMR (DEPT; 75 MHz, (D6)DMSO): 18.0, 18.7, 19.9, 22.5, 27.7, 54.2, 55.8
(Me); 35.8, 36.5 (CH2); 30.6, 47.8, 48.0, 54.3, 57.4, 104.8, 114.8, 130.1 (CH); 79.1, 128.1, 155.7, 169.2, 169.8, 170.2,
‡
‡
171.5, 171.6 (C). FAB-MS: 595.3 ([M ‡ H] ), 617.3 ([M ‡ Na] ).
Ac-Tyr-Val-Ala-(R)-Asp(OtBu)-H Dimethyl Acetal ((R)-1). As described for (S)-1, from (R)-6 (1.39 g,
6.35 mmol): (R)-1 (1.37 g, 35%; purity >95% by HPLC). M.p. 226 ± 2278. White crystals. [a]D20
ˆ
13.7 (c ˆ 5.1 ´
10 3, MeOH). 1H-NMR (500 MHz, (D6)DMSO): 0.83 (d, 3J ˆ 6.9, Me(4) or Me(4') (Val)); 0.85 (d, 3J ˆ 6.7,
Me(4) or Me(4') (Val)); 1.17 (d, 3J ˆ 7.0, Me(3) (Ala)); 1.36 (s, tBu (Asp)); 1.75 (s, Ac); 1.94 ± 2.00 (m, H C(3)

1968
Helvetica Chimica Acta ± Vol. 82 (1999)
(Val)); 2.20 (dd, 2J ˆ 15.3, 3J ˆ 8.6, 1 H C(2) (Asp)); 2.46 (dd, 2J ˆ 15.3, 3J ˆ 4.1, 1 H C(2) (Asp)); 2.61
(dd, 2J ˆ 13.9, 3J ˆ 10.1, 1 H C(3) (Tyr)); 2.87 (dd, 2J ˆ 14.1, 3J ˆ 3.9, 1 H C(3) (Tyr)); 3.25, 3.31 (2s,
(MeO)2CH); 4.15 ± 4.24 (m, H C(2) (Val), H C(3) (Asp), H C(4) (Asp)); 4.25 ± 4.33 (m, H C(2) (Ala));
4.42 ± 4.50 (m, H C(2) (Tyr)); 6.62 (d, 3J ˆ 8.6, 2 arom. H (Tyr)); 7.03 (d, 3J ˆ 8.6, 2 arom. H (Tyr)); 7.83
(d, 3J ˆ 8.9, NH (Val or Asp)); 7.87 (d, 3J ˆ 8.3, NH (Val or Asp)); 7.98 (d, 3J ˆ 7.4, NH (Ala or Tyr)); 8.5
(d, 3J ˆ 7.4, NH (Ala or Tyr)); 9.13 (s, OH (Tyr)).
Ac-Tyr-Val-Ala-(RS)-Asp(OtBu)-H Dimethyl Acetal ((RS)-1). As described for (S)-1, from (RS)-6
(220 mg, 1.0 mmol) and (S)-8 (433 mg, 1.1 mmol): (RS)-1 (0.2 g, 40%; purity >95% by 1H-NMR). White
crystals. 1H-NMR (300 MHz, (D6)DMSO): 0.825 (d, 3J ˆ 5.1, Me(4) or Me(4') (Val)); 0.85 (d, 3J ˆ 6.5, Me(4)
or Me(4') (Val)); 1.17, 1.19 (2d, 3J ˆ 6.7, 6.85, Me(3) (Ala, both diastereoisomers)); 1.348, 1.364 (2s, tBu (Asp,
both diastereoisomers)); 1.75 (s, Ac); 1.91 ± 2.03 (m, H C(3), (Val)); 2.14 ± 2.33 (m, 1 H C(2) (Asp)); 2.38 ±
2.48 (m, 1 H C(2) (Asp)); 2.61 (dd, 2J ˆ 13.7, 3J ˆ 10.0, 1 H C(3) (Tyr)); 2.87 (dd, 2J ˆ 13.7, 3J ˆ 3.5,
1 H C(3) (Tyr)); 3.27, 3.30 (2s, (MeO)2CH ((S)-Asp diastereoisomer)); 3.25, 3.31 (2s, (MeO)2CH ((R)-
Asp diastereoisomer)); 4.07 ± 4.33 (m, H C(2) (Ala), H C(2) (Val), H C(3) (Asp), H C(4) (Asp));
4.41 ± 4.53 (m, H C(2) (Tyr)); 6.63 (d, 3J ˆ 8.3, 2 arom. H (Tyr)); 7.03 (d, 3J ˆ 7.4, 2 arom. H (Tyr)); 7.62 ± 8.37
(m, NH (Val), NH (Asp), NH (Tyr), NH (Ala)); 9.11 (s, OH (Tyr)).
Ac-Tyr-Val-(R)-Ala-Asp(OtBu)-H Dimethyl Acetal (1a). As described for (S)-1, from (S)-6 (110 mg,
0.5 mmol) and Ac-Tyr-Val-(R)-Ala-OH ((R)-8; 217 mg, 0.6 mmol): 1a (0.25 g, 70%; purity >95% by
1H-NMR). ee > 98% (by comparison with the diastereoisomeric analogs). White crystals. 1H-NMR
(300 MHz, (D6)DMSO): 0.83 (d, 3J ˆ 6.7, Me(4) (Val), Me(4') (Val)); 1.15 (d, 3J ˆ 7.0, Me(3) (Ala)); 1.36
(s, tBu (Asp)); 1.75 (s, Ac); 1.87 ± 2.01 (m, H C(3) (Val)); 2.22 (dd, 2J ˆ 15.2, 3J ˆ 8.5, 1 H C(2) (Asp)); 2.43
(dd, 2J ˆ 15.5, 3J ˆ 4.0, 1 H C(2) (Asp)); 2.61 (dd, 2J ˆ 13.9, 3J ˆ 10.1, 1 H C(3) (Tyr)); 2.87 (dd, 2J ˆ 13.9,
3J ˆ 3.9, 1 H C(3) (Tyr)); 3.26, 3.31 (2s, (MeO)2CH); 4.10 ± 4.37 (m, H C(2) (Val), H C(2) (Ala), H C(3)
(Asp), H C(4) (Asp)); 4.40 ± 4.52 (m, 1 H C(2) (Tyr)); 6.63 (d, 3J ˆ 8.4, 2 arom. H (Tyr)); 7.02 (d, 3J ˆ 8.4,
2 arom. H (Tyr)); 7.80 ± 8.15 (4d, 3J ˆ 7.7, 8.3, 8.4, 8.7, NH (Tyr), NH (Ala), NH (Val), NH (Asp)); 9.16 (s, OH
(Tyr)).
REFERENCES
[1] J. Jurczak, A. Golebiowski, Chem. Rev. 1989, 68, 14; M. Reetz, Angew. Chem. 1991, 12, 1559.
[2] K. Chapman, Bioorg. Med. Chem. Lett. 1992, 6, 613.
[3] T. D. Graybill, R. E. Dolle, C. T. Helaszek, R. E. Miller, M. A. Ator, Int. J. Pept. Protein Res. 1994, 44, 173.
[4] M. D. Mullican, D. J. Lauffer, R. J. Gillespie, S. S. Matharau, D. Kay, G. M. Porritt, L. Evans, J. M. C. Golec,
M. A. Murcko, Y. P. Luong, S. A. Raybuck, D. J. Livingston, Biorg. Med. Chem. Lett. 1994, 4, 2359.
[5] A. Thornberry, Nature (London) 1992, 356, 768; F. G. Evan, Cell (Cambridge, Mass.) 1996, 86, 781; S.
Nagata, ibid. 1997, 88, 355; N. A. Thornberry, Y. Lazebnik, Science (Washington, D.C.) 1998, 281, 1312.
[6] A. Ito, R. Takahashi, Y. Baba, Chem. Pharm. Bull. 1975, 23, 3081.
[7] J. March, ꢀAdvanced Organic Chemistryꢁ, John Wiley & Sons, 4th Edition, New York ± Chichester ±
Brisbane ± Toronto ± Singapore 1992, 890.
[8] T. Saino, T. Someno, H. Myazaki, S. Ishii, Chem. Pharm. Bull. 1982, 30, 2319.
[9] J. A. Dale, D. Dull, H. S. Mosher, J. Org. Chem. 1969, 9, 2543; J. A. Dale, H. A. Mosher, J. Am. Chem. Soc.
1973, 95, 512.
[10] G. Kokotos, J. Org. Chem. 1990, 4, 299.
[11] A. El Marini, M. L. Roumestant, Ph. Viallefont, D. Razafindramboa, M. Bonato, M. Follet, Synthesis 1992,
1104.
[12] A. Manusco, S. Huang, D. Swern, J. Org. Chem. 1978, 12, 2480.
[13] ꢀ Houben-Weyl, Methoden der organischen Chemieꢁ, Georg Thieme Verlag, Stuttgart, 1954, Vol. VII/1.
[14] J. Jones, ꢀThe Chemical Synthesis of Peptidesꢁ, Clarendon Press, Oxford, 1991; P. Bailey, ꢀAn Introduction
to Peptide Chemistryꢁ, Salle
& Sauerländer, Aarau ± Frankfurt ± Salzburg, 1992; M. Bodanski, A.
Bodansky, ꢀThe Practice of Peptide Synthesisꢁ, Springer, Berlin, 1994, pp. 17, 28, and 199.
[15] N. L. Benoiton, Biopolymers (Pept. Sci.) 1996, 40, 245 and refs. cit. therein.
Received July 14, 1999