Orthogonally protected, carboxy-activated L-homoisoserine, 2-methyl-L-homoisoserine, and homoisocysteine derivatives. New building blocks for peptide and depsipeptide modification

Article abstract of DOI:10.1007/s00706-005-0291-1

Starting from L-malic, L-citramalic, and rac. thiomalic acids routes to L-homoisoserine, 2-methyl-L-homoisoserine and rac. homoisocysteine have been developed. The new orthogonally protected and carboxy-activated building blocks are GABA as well as α-hydr

Full text of DOI:10.1007/s00706-005-0291-1

Monatshefte f€ur Chemie 136, 763–776 (2005)
DOI 10.1007/s00706-005-0291-1
Orthogonally Protected, Carboxy-Activated
L-Homoisoserine, 2-Methyl-L-homoisoserine,
and Homoisocysteine Derivatives.
New Building Blocks for Peptide
and Depsipeptide Modification#
Klaus Burger1;ꢀ, Gabor Radics1, Lothar Hennig1,
¨
Christoph Bottcher , Jan Spengler , and Fernando Albericio
1
2
2
1
Department of Organic Chemistry, University of Leipzig, D-04103 Leipzig, Germany
2
ꢀ
Institut de Recerca Biomedica de Barcelona, Parc Cientific Barcelona,
08028 Barcelona, Spain
Received August 23, 2004; accepted (revised) October 18, 2004
Published online April 22, 2005 # Springer-Verlag 2005
Summary. Starting from L-malic, L-citramalic, and rac. thiomalic acids routes to L-homoisoserine, 2-
methyl-L-homoisoserine and rac. homoisocysteine have been developed. The new orthogonally pro-
tected and carboxy-activated building blocks are GABA as well as ꢀ-hydroxy and ꢀ-mercapto acid
derivatives, suitable for the construction of peptide and depsipeptide surrogates.
Keywords. Malic acid; Citramalic acid; Thiomalic acid; Hexafluoroacetone; ꢁ-Amino acids;
ꢀ-Hydroxy acids; ꢀ-Mercapto acids; Wolff rearrangement; Curtius rearrangement; Arndt-Eistert
reaction; Peptide modification; Depsipeptide modification; Azapeptides; Hydroxamates.
Introduction
The number of reports dealing with peptidomimetics built from two or more dif-
ferent types of monomers is rapidly growing [1]. The application of !-peptides (ꢂ-
peptides and ꢁ-peptides) as drug candidates and of !-amino acids as building
blocks for the incorporation into strategical positions of biologically active pep-
tides is a newly emerging area of current research [2]. This is mainly due to the
ability of these amino acids to modify the geometry of the peptide backbone [2d],
providing proteolytically stable bioactive domains. Insertion of one or more extra
carbon atoms into intramolecular hydrogen bound, folded structures leads to the
ꢀ
Corresponding author. E-mail: burger@organik.chemie.uni-leipzig.de
Dedicated to Prof. Dr. Manfred M€uhlst€adt on the occassion of his 75th birthday
#

764
K. Burger et al.
creation of unusual turns and novel helical folds [3], which are often responsible
for biological activity [4].
Helix stability increases on homologation of monomers. Incorporation of ꢀ-
methylated !-amino acids further stabilizes helical structures, provided that the spatial
orientation of the ꢀ-placed group is compatible with the main backbone [5]. Structural
diversity can be achieved by incorporation of additional functional groups, like –OH
and –SH, into different positions of the backbone, suitable for further transformations.
Prominent members of this class of amino acids are GABA (4-aminobutyrate)
and homoisoserine (2-hydroxy-4-aminobutyrate). The latter was isolated from anti-
biotics [6] and phytosiderophores [7]. Homoisoserine is an inhibitor of GABA
uptake and exhibits antitumor activities [8]. Because of these and other interesting
properties, several routes to homoisoserine have been developed [9].
However, to the best of our knowledge, 2-methylhomoisoserine has not been
found in nature. More surprisingly, this amino acid seems to be unknown. Like-
wise, homoisocysteine (4-amino-2-mercaptobutyrate) has not been found in nature
so far. Although, homoisocysteine was first mentioned in a Japanese patent in 1961,
there exists nearly no information about its chemistry [10].
In a series of papers we disclosed that a new protection=activation concept
developed for the site-selective derivatization of multifunctional amino acids, like
iminodiacetic acid [11], aspartic acid [12], and homologues [13] can also be
applied to multifunctional ꢀ-hydroxy (malic and citramalic acid) [14] and ꢀ-mer-
capto acids (thiomalic acid) [15].
Results and Discussion
We now report on the syntheses of L-homoisoserine, 2-methyl-L-homoisoserine
and rac. homoisocysteine and their incorporation into peptides using HFA (hexa-
fluoroacetone) as protecting and activating reagent [16]. Malic (1a), citramalic
(1b), and thiomalic acid (2) react readily with HFA in solvents like DMSO
(dimethylsulphoxide) or DMF (dimethylformamide) at room temperature to give
2,2-bis(trifluoromethyl)-1,3-dioxolan-4-ones (3) [17] and 2,2-bis(trifluoromethyl)-
1,3-oxathiolan-5-ones (4) [18] in excellent yields (86–87%). A second equivalent
of HFA is necessary to trap the water which is eliminated during lactone formation.
In a tandem process, protection of the ꢀ-placed group (–OH, –SH) and the adja-
cent carboxy group are achieved. Noteworthy, the ꢀ-carboxy group is protected
and activated, while the !-carboxy group remains unaffected and can be selectively
derivatized after separate activation (Scheme 1). Compared to conventional strate-
Scheme 1. i) HFA, DMSO, rt; ii) SOCl2, reflux; iii) CH2N2, Et2O, ꢁ78!0ꢂC

New Building Blocks for Peptide and Depsipeptide Modification
765
gies, this concept saves steps, as demonstrated by a two step Aspartame synthesis
with an overall yield of 72% [19].
On heating compounds 3=4 with an excess of thionyl chloride, acid chlorides
5=6 are formed (86–87%). Compounds 5=6 are preparatively useful members of a
new class of dielectrophiles, because their two electrophilic centers are different in
reactivity, which allows site-selective derivatizations. With diazomethane (>3
equivalents) the acid chloride moiety reacts to give the diazoketones 7=8. Best yields
(90–93%) were obtained when the acid chlorides were slowly dropped into a solu-
tion of diazomethane in ether with stirring at ꢁ78 ! 0ꢂC. An excess of diazo-
methane is necessary to trap the HCl formed during the reaction to suppress the
formation of chloroketones, which are difficult to separate from diazoketones 7=8.
Arndt-Eistert homologation can be achieved via photo-Wolff rearrangement
[20] of diazoketones 7=8 in aqueous dioxane. The corresponding ꢀ-hydroxy and
ꢀ-mercapto glutaric acid derivatives can be readily transformed into acid chlorides
on treatment with thionyl chloride.
Improved yields are obtained when a silver benzoate catalyzed decomposition
[21] of the diazoketones 7=8 is performed in tert-butanol to trap the ketenes as tert-
butylesters 9=10. Deprotection of the tert-butylester and transformation into the
acid chlorides 11=12 are achieved in excellent yields on heating with an excess of
thionyl chloride. The acid chlorides 11=12 can be readily purified by distillation
under reduced pressure (Scheme 2).
t
Scheme 2. i) BuOH, PhCO2Ag, reflux; ii) SOCl2, reflux; iii) (CH3)3SiN3, toluene, 80–90ꢂC
Scheme 3. i) tBuOH, CHCl3, reflux; ii) PhCH2OH, CHCl3, reflux; iii) 9-fluorenylmethanol, CHCl3,
reflux

766
K. Burger et al.
Introduction of the isocyanato group into the !-position requires two steps,
namely Curtius rearrangement of the azides [22] obtained from 11=12 and tri-
methylsilyl azide. Compounds 13=14 are members of a class of dielectrophiles
with high synthetic potential. Urethane protection of the ꢁ-amino group (e.g. Boc-
NH, Z-NH, Fmoc-NH) can be achieved in high yields on heating the isocyanates
13=14 with 1 equivalent of the corresponding alcohol to give the carboxy-acti-
vated orthogonally protected derivatives of homoisoserine (15a), 2-methyl-
homoisoserine (15b), and homoisocysteine (16), respectively (Scheme 3). Further
highly useful synthetical applications of compounds 13=14 are i.a. N-glycosyla-
tion [23], N-lipidation [24], and isopeptide formation [25]. Compounds 13=14
can be stored in a fridge on exclusion of moisture for several weeks without
decomposition.
Likewise, urethane-protected carboxy-activated species 15=16 are valuable
starting materials for the generation of libraries of homoisoserine, 2-methylhomo-
Scheme 4. i) CH3OH, reflux; ii) HClꢃNH2OH, propene oxide, DMF, rt
Scheme 5. i) HCl ꢃ H-Phe-OPg2, DIPEA, DMF, rt; ii) H2NNHCO2CH3, DMF, rt

New Building Blocks for Peptide and Depsipeptide Modification
767
isoserine and homoisocysteine derivatives. Since they represent GABA derivatives
they are interesting per se or as building blocks for the synthesis of drug candidates.
Esters, e.g. 17, are readily obtainable on heating with an excess of corresponding
alcohols, hydroxamates, e.g. 18, are obtainable on cleavage of the lactone moiety
with the reagent pair hydroxylamine hydrochloride=propene oxide (Scheme 4).
Hydroxamates exhibit strong inhibitory effects on metal-containing enzymes (metal-
lozymes) [26] and meet growing interest since a hydroxamate subunit is the phar-
macophor of several HDAC inhibitors [27].
Small peptides [28] and peptide surrogates (depsipeptides 19, 21–23 and aza-
peptides 24–27) become readily available using 15 and 16 as acyl transfer reagents.
Nucleophilic cleavage of the lactone ring is always coupled with a deprotection of
the ꢀ-placed functional group (Scheme 5). Consequently, chain elongation can be
continued without the need of any extra deprotection step. Depending on the reac-
tion conditions applied, up to 10% of N-protected 3-hydroxypyrrolidin-2-ones
represented by structure 20 are formed via an intramolecular cyclocondensation
process [29]. N-Unprotected 3-hydroxypyrrolidin-2-ones were obtained from com-
pounds 15 in good yields on selective cleavage of the urethane protecting group
and intramolecular ring closure [20c, 30].
For L-malic acid we proved that all reaction steps studied so far proceed stereo-
conservatively (NMR analysis). In the case of thiomalic acid only the racemate
was commercially available, therefore all reactions described are starting from
rac thiomalic acid. On further aspects of the synthetic potential of the HFA
protection=activation concept we report elsewhere.
Experimental
General
Solvents were purified and dried prior to use. Reagents were used as purchased. TLC was performed
on Silica Gel 60 F254 (Merck) with detection by UV light or phosphomolybdic acid=ceric sulphate in
5% aqueous sulfuric acid followed by heating. Melting points (uncorrected) were determined on a
Boetius heating table. Optical rotation indices were measured using a Schmidt & Haensch Polartronic-
D polarimeter in a 5 cm cell. Mass spectra were recorded on a VG 12-250 and a MAT 212 (Masslab)
electron ionization spectrometer (EI-MS, EI¼ 70eV), on a Bruker Daltonics APEX II ESI-FT-ICR
spectrometer or on a Finnigan ZAB-HSQ spectrometer (FAB-matrix: 3-NBA). IR spectra were
obtained with a FTIR spectrometer (Genesis ATI Mattson=Unicam). 1H (400MHz, 600 MHz,
700 MHz), 13C (101 MHz, 151 MHz), and 19F (376MHz) NMR were recorded on Bruker DRX-
1
AVANCE spectrometers. TMS was used for H and 13C NMR spectra, and CCl3F for 19F NMR
spectra as internal references. Flash chromatography was performed using silica gel (32–63ꢃm) with
solvent systems given in the text. Elemental analyses were performed with a CHNO-S Rapid apparatus
(Fa. Heraeus); their results agreed with calculated values.
Wolff Rearrangement of HFA-Protected Diazoketones (General Procedure)
A stirred solution of diazoketones 7=8 (20 mmol) in dry tert-butanol (50 cm3) was heated (oil bath
temperature 85ꢂC). Then silver benzoate (200mg) was added in two portions. When no gas evolution
was observed even on further addition of silver benzoate the solvent was evaporated in vacuo and the
residue purified by column chromatography (eluent: dichloromethane (DCM)).

768
K. Burger et al.
tert-Butyl 3-[(5S)-2,2-bis(trifluoromethyl)-4-oxo-1,3-dioxolan-5-yl]propionate
(9a, C12H14F6O5)
Yield 66%; colorless oil; [ꢀ]D ¼ ꢁ17.4ꢂ cm3 gꢁ1 dmꢁ1 (c ¼ 1.0, DCM); IR (film): ꢄꢁ¼ 2981, 2937,
1
1851, 1730 cmꢁ1; H NMR (600MHz, CDCl3): ꢅ ¼ 1.43 (s, 9H), 2.03–2.09 (m, 1H), 2.21–2.27 (m,
1H), 2.45 (m, 2H), 4.76 (dd, J ¼ 8.3, 5.1 Hz, 1H) ppm; 13C NMR (151MHz, CDCl3): ꢅ ¼ 26.9, 27.9,
29.7, 74.1, 81.3, 97.6 (sept, J ¼ 36.0Hz), 118.8 (q, J ¼ 287.2Hz), 119.6 (q, J ¼ 289.8 Hz), 167.5,
19
170.7ppm; F NMR (376 MHz, CDCl3): ꢅ ¼ ꢁ81.5 (m, 6F) ppm; MS (EI): m=z (%) ¼ 352 [M]þ
(1), 336 (29), 295 [Mꢁ C4H9]þ (14), 278 (100), 85 (18), 59 (90), 42 [C2H2O]þ (24).
tert-Butyl 3-[(5S)-2,2-bis(trifluoromethyl)-5-methyl-4-oxo-1,3-dioxolan-5-yl]propionate
(9b, C13H16F6O5)
Yield 77%; colorless oil; [ꢀ]D ¼ ꢁ6.6ꢂ cm3 gꢁ1 dmꢁ1 (c ¼ 1.21, DCM); IR (film): ꢄꢁ¼ 1844,
1
1732 cmꢁ1; H NMR (400 MHz, CDCl3): ꢅ ¼ 1.45 (s, 9H), 1.59 (s, 3H), 2.18 (m, 2H), 2.43 (m,
2H) ppm; 13C NMR (101 MHz, CDCl3): ꢅ ¼ 22.0, 28.1, 29.2, 32.8, 81.3, 97.0 (sept, J ¼ 35.7Hz),
119.2 (q, J ¼ 288.4 Hz), 119.3 (q, J ¼ 289.0 Hz), 170.1, 170.9 ppm; 19F NMR (376MHz, CDCl3):
ꢅ ¼ ꢁ81.1 (q, J ¼ 8.3 Hz, 3F), ꢁ81.3 (q, J ¼ 8.3 Hz, 3F) ppm; MS (EI): m=z (%) ¼ 366 [M]þ (1),
351 (1), 293 [Mꢁ C4H9O]þ (25), 147 (8), 99 (100), 69 (55), 57 (98).
rac. tert-Butyl 3-[2,2-bis(trifluoromethyl)-5-oxo-1,3-oxathiolan-4-yl]propionate
(10, C12H14F6O4S)
1
Yield 55%; slightly yellow oil; IR (film): ꢄꢁ¼ 2980, 2933, 1817, 1726cmꢁ1; H NMR (600 MHz,
CDCl3): ꢅ ¼ 1.42 (s, 9 H), 2.09–2.17 (m, 1H), 2.35–2.45 (m, 3H), 4.34 (dd, J ¼ 7.8, 3.1 Hz, 1H) ppm;
13C NMR (151 MHz, CDCl3): ꢅ ¼ 27.9, 28.2, 32.0, 45.1, 81.4, 82.9 (sept, J ¼ 35.0Hz), 120.8 (q,
J ¼ 283.5 Hz), 121.3 (q, J ¼ 284.5Hz), 170.5, 170.8ppm; 19F NMR (376MHz, CDCl3): ꢅ ¼ ꢁ76.4
(q, J ¼ 9.2 Hz, 3F), ꢁ77.5 (q, J ¼ 9.2 Hz, 3F) ppm; MS (EI): m=z (%) ¼ 368 [M]þ (1), 352 (29), 311
(39), 294 (49), 101 (42), 57 (100), 41 (34).
Synthesis of Propionyl Chlorides 11, 12 (General Procedure)
Thionyl chloride (30 cm3) was added to compounds 9=10 (20 mmol) with stirring. Then the reaction
mixture was heated under reflux for 12h. After removal of the excess of thionyl chloride, the residue
was distilled in vacuo.
3-[(5S)-2,2-Bis(trifluoromethyl)-4-oxo-1,3-dioxolan-5-yl]propionyl chloride
(11a, C8H5ClF6O4)
Yield 80%; colorless oil; bp 47–48ꢂC=106.7Pa; [ꢀ]D ¼ ꢁ17.1ꢂ cm3 gꢁ1 dmꢁ1 (c ¼ 1.05, DCM); IR
1
(film): ꢄꢁ¼ 1851, 1793cmꢁ1; H NMR (400MHz, CDCl3): ꢅ ¼ 2.20 (ddt, J ¼ 14.9, 8.4, 7.0 Hz, 1H),
2.39 (ddt, J ¼ 14.9, 7.5, 5.0 Hz, 1H), 3.18 (t, J ¼ 7.0 Hz, 2H), 4.75 (dd, J ¼ 8.4, 5.0 Hz, 1H) ppm;
13C NMR (101MHz, CDCl3): ꢅ ¼ 26.9, 41.5, 73.3, 97.8 (sept, J ¼ 36.4Hz), 118.9 (q, J ¼ 286.8Hz),
119.6 (q, J ¼ 288.8 Hz), 167.0, 172.7 ppm; 19F NMR (376 MHz, CDCl3): ꢅ ¼ ꢁ81.4 (m, 6F) ppm; MS
(EI): m=z (%) ¼ 316=314 [M]þ (1), 293 (33), 279 [Mꢁ Cl]þ (20), 247=245 [Mꢁ CF3]þ (6), 217 (19),
203 (11), 97 (17), 85 (76), 57 (44), 42 [C2H2O]þ (100).
3-[(5S)-2,2-Bis(trifluoromethyl)-5-methyl-4-oxo-1,3-dioxolan-5-yl]propionyl chloride
(11b, C9H7ClF6O4)
Yield 78%; yellow oil; bp 56–60ꢂC=53.3Pa; [ꢀ]D ¼ ꢁ14.3ꢂcm3 gꢁ1 dmꢁ1 (c ¼ 1.26, DCM); IR (film):
ꢄꢁ¼ 1842, 1797 cmꢁ1; 1H NMR (700 MHz, CDCl3): ꢅ ¼ 1.62 (s, 3H), 2.28 (m, 2H), 3.08 (m, 1H), 3.17

New Building Blocks for Peptide and Depsipeptide Modification
769
(m, 1H) ppm; 13C NMR (176 MHz, CDCl3): ꢅ ¼ 22.0, 32.5, 40.7, 80.4, 97.1 (sept, J ¼ 35.9Hz), 119.0
(q, J ¼ 288.5Hz), 119.1 (q, J ¼ 289.0Hz), 169.5, 172.4 ppm; 19F NMR (376MHz, CDCl3): ꢅ ¼ ꢁ81.2
(m, 6F) ppm; MS (EI): m=z (%) ¼ 328 [M]þ (2), 293 [Mꢁ Cl]þ (37), 259 (3), 231 (6), 195 (4), 167 (5),
117 (8), 99 (51), 89 (15), 69 (20).
rac. 3-[2,2-Bis(trifluoromethyl)-5-oxo-1,3-oxathiolan-4-yl]propionyl chloride
(12, C8H5ClF6O3S)
Yield 84%; yellow oil; bp 52ꢂC=240 Pa; IR (film): ꢄꢁ¼ 1813, 1735cmꢁ1; 1H NMR (600MHz, CDCl3):
ꢅ ¼ 2.29–2.35 (m, 1H), 2.47–2.53 (m, 1H), 3.17 (m, 2H), 4.35 (t, J ¼ 6.6Hz, 1H) ppm; 13C NMR
(151 MHz, CDCl3): ꢅ ¼ 27.9, 43.2, 44.1, 83.0 (sept, J ¼ 35.0Hz), 120.6 (q, J ¼ 284.0Hz), 121.2 (q,
J ¼ 284.5 Hz), 169.9, 172.7ppm; 19F NMR (376MHz, CDCl3): ꢅ ¼ ꢁ76.4 (q, J ¼ 9.2 Hz, 3F), ꢁ77.4
(q, J ¼ 9.2Hz, 3F) ppm; MS (EI): m=z (%) ¼ 330 [M]þ (1), 294 [Mꢁ Cl]þ (100), 266 (31), 252 (26),
101 (69), 73 (29).
Synthesis of Isocyanates 13=14 (General Procedure)
Trimethylsilyl azide (21 mmol) was added to a stirred solution of propionyl chloride 11=12 (20 mmol)
in dry toluene (30 cm3) at room temperature. After 12h the reaction mixture was heated to 80–90ꢂC
(bath temperature). When the reaction was complete (19F NMR analysis, ca. 12h) the solvent was
evaporated under reduced pressure and the residue was purified by distillation in vacuo or by column
chromatography (eluent: DCM).
(5S)-5-(2-Isocyanatoethyl)-2,2-bis(trifluoromethyl)-1,3-dioxolan-4-one (13a, C8H5F6NO4)
Yield 88%; yellow oil; bp 65ꢂC=70.7Pa; [ꢀ]D ¼ ꢁ15.3ꢂ cm3 gꢁ1 dmꢁ1 (c ¼ 1.1, CHCl3); IR (film):
1
ꢄꢁ¼ 2281, 1853 cmꢁ1; H NMR (400MHz, CDCl3): ꢅ ¼ 2.07 (dddd, J ¼ 14.6, 9.0, 5.9, 5.4 Hz, 1H),
2.22 (dddd, J ¼ 14.6, 8.4, 5.8, 4.2 Hz, 1H), 3.61 (ddd, J ¼ 13.4, 8.4, 5.4 Hz, 1H), 3.67 (ddd, J ¼ 13.4,
5.9, 5.8 Hz, 1H), 4.80 (dd, J ¼ 9.0, 4.2 Hz, 1H) ppm; 13C NMR (101MHz, CDCl3): ꢅ ¼ 33.0, 38.5,
72.5, 98.0 (sept, J ¼ 36.0 Hz), 119.0 (q, J ¼ 287.2 Hz), 119.8 (q, J ¼ 289.1Hz), 123.16, 167.56 ppm;
19F NMR (376MHz, CDCl3): ꢅ ¼ ꢁ81.5 (m, 6F) ppm; MS (EI): m=z (%) ¼ 293 [M]þ (2), 224
[Mꢁ CF3]þ (10), 250 (10), 196 (11), 155 (15), 99 (23), 69 [CF3]þ (50), 56 [CH2NO]þ (100).
(5S)-5-(2-Isocyanatoethyl)-5-methyl-2,2-bis(trifluoromethyl)-1,3-dioxolan-4-one
(13b, C9H7F6NO4)
Yield 70%; yellow oil; bp 34–38ꢂC=45.3Pa; [ꢀ]D ¼ ꢁ6.8ꢂ cm3 gꢁ1 dmꢁ1 (c ¼ 1.33, DCM); IR (film):
ꢄꢁ¼ 2276, 1844 cmꢁ1; 1H NMR (400 MHz, CDCl3): ꢅ ¼ 1.66 (s, 3H), 2.17 (t br, J ¼ 7.0 Hz, 2H), 3.59
(m, 2H) ppm; 13C NMR (101 MHz, CDCl3): ꢅ ¼ 22.3, 37.4, 38.5, 80.6, 97.4 (sept, J ¼ 35.9Hz), 119.3
(q, J ¼ 288.0 Hz), 122.6, 169.9ppm; 19F NMR (376MHz, CDCl3): ꢅ ¼ ꢁ81.1 (m, 6F) ppm; MS (EI):
m=z (%) ¼ 307 [M]þ (3), 238 (28), 169 (14), 72 (66), 56 [CH2NCO]þ (100).
rac. 4-(2-Isocyanatoethyl)-2,2-bis(trifluoromethyl)-1,3-oxathiolan-5-one (14, C8H5F6NO3S)
Yield 82%; light yellow oil; bp 36–38ꢂC=24.0 Pa; IR (film): ꢄꢁ¼ 2279, 1815 cmꢁ1
;
1H NMR
(600 MHz, CDCl3): ꢅ ¼ 2.11 (m, 1H), 2.48 (m, 1H), 3.58–3.62 (m, 1H), 3.66–3.71 (m, 1H), 4.32
(m, 1H) ppm; 13C NMR (151 MHz, CDCl3): ꢅ ¼ 34.3, 40.7, 43.4, 83.3 (sept, J ¼ 35.0Hz), 120.7 (q,
J ¼ 284.0 Hz), 121.3 (q, J ¼ 284.5 Hz), 123.1, 170.4 ppm; 19F NMR (376MHz, CDCl3): ꢅ ¼ ꢁ76.4 (q,
J ¼ 9.2 Hz, 3F), ꢁ77.4 (q, J ¼ 9.2 Hz, 3F) ppm; MS (EI): m=z (%) ¼ 309 [M]þ (2), 281 [Mꢁ CO]þ

770
K. Burger et al.
(35), 266 (50), 253 [Mꢁ NCO]þ (18), 236 (20), 171 (23), 113 (29), 82 (39), 72 (63), 69 [CF3]þ (30),
56 [CH2NCO]þ (28).
Introduction of Boc-, Z-, and Fmoc Protection (General Procedure)
A solution of equimolar amounts (10 mmol) of the isocyanate (13, 14) and the corresponding alcohol
was heated in dry CHCl3 (30 cm3) for 12h under reflux (TLC control). After evaporation of the
solvent, the residue was purified by column chromatography (eluent: DCM) or crystallization.
(5S)-5-[2-(tert-Butyloxycarbonylamino)ethyl]-2,2-bis(trifluoromethyl)-1,3-dioxolan-4-one
(15a=1, C12H15F6NO5)
Yield 61%; mp 43ꢂC; [ꢀ]D ¼ ꢁ8.0ꢂ cm3 gꢁ1 dmꢁ1 (c ¼ 0.75, DCM); IR (KBr): ꢄꢁ¼ 3620–3035, 1854,
1
1782, 1700 cmꢁ1; H NMR (600MHz, CDCl3): ꢅ ¼ 1.40 (s, 9H), 1.91–2.21 (m, 2H), 3.26–3.35 (m,
2H), 4.45 (m, 1H, NH), 4.69 (dd, J ¼ 8.0, 4.0 Hz, 1H) ppm; 13C NMR (151MHz, CDCl3): ꢅ ¼ 27.8,
31.7, 33.9, 72.9, 79.4, 97.2 (sept, J ¼ 36.0Hz), 118.4 (q, J ¼ 286.0 Hz), 119.1 (q, J ¼ 286.0 Hz), 155.4,
167.4ppm; 19F NMR (376 MHz, CDCl3): ꢅ ¼ ꢁ81.5 (m, 6F) ppm; MS (EI): m=e (%) ¼ 352
[Mꢁ CH3] (1.5), 69 [CF3]þ (41), 56 [C4H9]þ (100).
(5S)-5-[2-(Benzyloxycarbonylamino)ethyl]-2,2-bis(trifluoromethyl)-1,3-dioxolan-4-one
(15a=2, C15H13F6NO5)
Yield 51%; colorless crystals; mp 55ꢂC (CHCl3=hexanes); [ꢀ]D ¼ ꢁ13.4ꢂ cm3 gꢁ1 dmꢁ1 (c ¼ 1.05,
1
DCM); IR (KBr): ꢄꢁ¼ 3500–3300, 1830, 1689, 1539 cmꢁ1; H NMR (600 MHz, CDCl3): ꢅ ¼ 1.92–
2.20 (m, 2H), 3.32–3.41 (m, 2H), 4.69 (dd, J ¼ 8.0, 4.0 Hz, 1H), 5.06 (s, 2H), 5.23 (m, 1H, NH), 7.32
(m, 5H) ppm; 13C NMR (151 MHz, CDCl3): ꢅ ¼ 31.2, 36.1, 66.3, 72.5, 97.0 (sept, J ¼ 35.0Hz), 118.1 (q,
J ¼ 288.0Hz), 118.8 (q, J ¼ 289.0Hz), 127.5, 127.6, 127.9, 135.5, 155.8, 167.1ppm; 19F NMR
(376 MHz, CDCl3): ꢅ ¼ ꢁ81.5 (m, 6F) ppm; MS (EI): m=z (%) ¼ 401 [M]þ (22), 108 [C6H5CH2OH]þ
(100), 92 [C7H8]þ (31).
(5S)-5-[2-(9-Fluorenylmethoxycarbonylamino)ethyl]-2,2-bis(trifluoromethyl)-
1,3-dioxolan-4-one (15a=3, C22H17F6NO5)
Yield 66%; mp 119ꢂC (CHCl3=hexanes); [ꢀ]D ¼ ꢁ7.9ꢂ cm3 gꢁ1 dmꢁ1 (c ¼ 1.14, DCM); IR (KBr):
1
ꢄꢁ¼ 3600–3300, 1834, 1697, 1535 cmꢁ1; H NMR (600MHz, CDCl3): ꢅ ¼ 1.98 (m, 1H), 2.18 (m,
1H), 3.32–3.41 (m, 2H), 4.16 (m, 1H), 4.44 (m, 2H), 4.63 (dd, J ¼ 8.0, 4.0Hz, 1H), 4.95 (m, 1H, NH),
7.26–7.30 (m, 2H), 7.35–7.39 (m, 2H), 7.53–7.55 (m, 2H), 7.72–7.74 (m, 2H) ppm; 13C NMR
(151 MHz, CHCl3): ꢅ ¼ 32.3, 37.1, 47.7, 67.1, 73.6, 98.1 (sept, J ¼ 36.0Hz), 119.3 (q, J ¼ 288.0Hz),
120.1 (q, J ¼ 289.0Hz), 120.5, 125.4, 127.6, 128.3, 141.9, 144.3, 157.0, 168.3ppm; 19F NMR
(376 MHz, CDCl3): ꢅ ¼ ꢁ81.5 (m, 6F) ppm; MS (EI): m=z (%) ¼ 489 [M]þ (14), 323 [Mꢁ C3F6O]þ
(14), 179 [C14H11]þ (100), 166 (54), 69 (41), 55 (21).
þ
(5S)-5-[2-(tert-Butyloxycarbonylamino)ethyl]-2,2-bis(trifluoromethyl)-
5-methyl-1,3-dioxolan-4-one (15b=1, C13H17F6NO5)
Yield 70%; colorless crystals; Rf ¼ 0.22 (eluent: DCM); mp 49–51ꢂC; [ꢀ]D ¼ ꢁ4.0ꢂ cm3 gꢁ1 dmꢁ1
1
(c ¼ 1.0, DCM); IR (KBr): ꢄꢁ¼ 3340, 1842, 1687, 1543 cmꢁ1; H NMR (600 MHz, CDCl3): ꢅ ¼ 1.43
(s, 9H), 1.65 (s, 3H), 2.07 (m, 2H), 3.29 (m, 1H), 3.44 (m, 1H), 4.70 (s br, 1H, NH) ppm; 13C NMR
(151 MHz, CDCl3): ꢅ ¼ 22.0, 28.4, 35.1, 37.5, 79.8, 81.1, 97.1 (sept, J ¼ 35.7Hz), 119.2 (q,
J ¼ 288.5 Hz), 155.7, 170.2ppm; 19F NMR (376MHz, CDCl3): ꢅ ¼ ꢁ81.2 (m, 6F) ppm; MS (EI):
m=z (%) ¼ 381 [M]þ (2), 367 (5), 328 (16), 282 (14), 59 (93), 57 (100).

New Building Blocks for Peptide and Depsipeptide Modification
771
(5S)-5-[2-(Benzyloxycarbonylamino)ethyl]-2,2-bis(trifluoromethyl)-
5-methyl-1,3-dioxolan-4-one (15b=2, C16H15F6NO5)
Yield 74%; colorless oil; Rf ¼ 0.32 (eluent: DCM); [ꢀ]D ¼ ꢁ4.0ꢂ cm3 gꢁ1 dmꢁ1 (c ¼ 1.0, DCM); IR
1
(film): ꢄꢁ¼ 3500–3200, 1842, 1720, 1699, 1527cmꢁ1; H NMR (700 MHz, CDCl3): ꢅ ¼ 1.63 (s br,
3H); 2.09 (m, 2H); 3.35 (m, 1H), 3.48 (m, 1H), 5.01 (t br, 1H, NH), 5.09 (s br, 2H), 7.33 (m, 5H) ppm;
13C NMR (176 MHz, CDCl3): ꢅ ¼ 22.0, 35.6, 37.3, 67.0, 81.0, 97.1 (sept, J ¼ 35.7Hz), 119.2 (q,
J ¼ 288.6 Hz), 128.2, 128.3, 128.6, 136.4, 156.3, 170.1ppm; 19F NMR (376MHz, CDCl3):
ꢅ ¼ ꢁ81.2 (m, 6F) ppm; MS (FAB, 3-NBA): m=z ¼ 416 [Mþ H]þ.
(5S)-5-[2-(9-Fluorenymethyloxycarbonylamino)ethyl]-2,2-bis(trifluoromethyl)-
5-methyl-1,3-dioxolan-4-one (15b=3, C23H19F6NO5)
Yield 76%; mp 75–78ꢂC; Rf ¼ 0.30 (eluent: DCM); [ꢀ]D ¼ ꢁ5.5ꢂ cm3 gꢁ1 dmꢁ1 (c ¼ 1.09, DCM); IR
(KBr): ꢄꢁ¼ 3500–3300, 1838, 1720, 1693, 1541cmꢁ1; 1H NMR (700MHz, CDCl3): ꢅ ¼ 1.64 (s, 3H),
2.09 (t br, J ¼ 7.0 Hz, 2H), 3.36 (m, 1H), 3.49 (m, 1H), 4.20 (t br, J ¼ 7.0 Hz, 1H), 4.42 (d br,
J ¼ 7.0 Hz, 2H), 4.90 (t br, 1H, NH), 7.30 (m, 2H), 7.40 (m, 2H), 7.57 (m, 2H), 7.76 (m, 2H) ppm;
13C NMR (176 MHz, CDCl3): ꢅ ¼ 22.0, 35.5, 37.3, 47.3, 66.8, 81.0, 97.3 (sept, J ¼ 35.0 Hz), 119.1 (q,
J ¼ 288.3 Hz), 120.0, 125.0, 127.1, 127.8, 141.2, 143.8, 156.2, 170.1 ppm; 19F NMR (376MHz,
CDCl3): ꢅ ¼ ꢁ81.2 (m, 6F) ppm; MS (EI): m=z (%) ¼ 503 [M]þ (2), 178 (100), 165 (13).
rac. 4-[2-(tert-Butyloxycarbonylamino)ethyl]-2,2-bis(trifluoromethyl)-
1,3-oxathiolan-5-one (16=1, C12H15F6NO4S)
1
Yield 64%; colorless oil; IR (film): ꢄꢁ¼ 3600–3200, 1815, 1685, 1525 cmꢁ1; H NMR (400 MHz,
CDCl3): ꢅ ¼ 1.45 (s, 9H), 2.06 (m, 1H), 2.41 (m, 1H), 3.33 (m, 2H), 4.25 (dd, J ¼ 9.8, 4.1 Hz, 1H),
4.73 (t br, 1H, NH) ppm; 13C NMR (101 MHz, CDCl3): ꢅ ¼ 28.3, 34.6, 38.4, 44.0, 80.2, 83.3 (sept,
J ¼ 34.9Hz), 120.9 (q, J ¼ 283.5Hz), 121.4 (q, J ¼ 284.3Hz), 156.1, 171.1ppm; 19F NMR (376 MHz,
CDCl3): ꢅ ¼ ꢁ76.5 (q, J ¼ 9.1 Hz, 3F), ꢁ77.4 (q, J ¼ 9.1Hz, 3F) ppm; MS (EI): m=z (%) ¼ 383 [M]þ
(1), 327 (69), 282 (100), 243 (17), 178 (12), 117 (71); MS (FAB, 3-NBA): m=z ¼ 384 [Mþ H]þ.
rac. 4-[2-(Benzyloxycarbonylamino)ethyl]-2,2-bis(trifluoromethyl)-
1,3-oxathiolan-5-one (16=2, C15H13F6NO4S)
Yield 76%; light yellow oil; IR (film): ꢄꢁ¼ 3500–3200, 1815, 1699 cmꢁ1; 1H NMR (400MHz, CDCl3):
ꢅ ¼ 2.06 (m, 1H), 2.38 (m, 1H), 3.23–3.50 (m, 2H), 4.22 (m, 1H), 5.06 (t br, J ¼ 6.0 Hz, 1H, NH), 5.10
(s br, 2H), 7.29–7.39 (m, 5H) ppm; 13C NMR (101MHz, CDCl3): ꢅ ¼ 34.4, 38.8, 43.9, 67.2, 83.3 (sept,
J ¼ 34.8Hz), 120.9 (q, J ¼ 283.3 Hz), 121.3 (q, J ¼ 284.3 Hz), 128.2, 128.4, 128.6, 136.2, 156.8,
171.0ppm; 19F NMR (376 MHz, CDCl3): ꢅ ¼ ꢁ76.5 (q, J ¼ 9.2 Hz, 3F), ꢁ77.3 (q, J ¼ 9.2 Hz, 3F)
ppm; MS (EI): m=z (%) ¼ 417 [M]þ (34), 147 (35) 100 (88), 92 (100), 69 [CF3]þ (84).
rac. 4-[2-(9-Fluorenylmethyloxycarbonylamino)ethyl]-2,2-bis(trifluoromethyl)-
1,3-oxathiolan-5-one (16=3, C22H17F6NO4S)
1
Yield 76%; viscous oil; mp 88ꢂC; IR (film): ꢄꢁ¼ 3600–3300, 1817, 1695cmꢁ1; H NMR (400 MHz,
CDCl3): ꢅ ¼ 2.02 (m, 1H), 2.32 (m, 1H), 3.29 (m, 1H), 3.38 (m, 1H), 4.11 (m, 1H), 4.18 (dd, J ¼ 10.0,
4.0 Hz, 1H), 4.48 (m, 2H), 4.88 (m, 1H, NH), 7.30 (m, 2H), 7.39 (m, 2H), 7.56 (m, 2H), 7.75 (m, 2H)
ppm; 13C NMR (101 MHz, CDCl3): ꢅ ¼ 34.4, 38.7, 43.9, 47.3, 66.7, 83.3 (sept, J ¼ 34.8Hz), 120.1,
120.8 (q, J ¼ 283.9 Hz), 121.3 (q, J ¼ 284.6 Hz), 124.9, 127.1, 127.8, 141.4, 143.7, 156.7, 171.0ppm;
19F NMR (376MHz, CDCl3): ꢅ ¼ ꢁ76.6 (q, J ¼ 9.0 Hz, 3F), ꢁ77.2 (q, J ¼ 9.0 Hz, 3F) ppm; MS (EI):
m=z (%) ¼ 505 [M]þ (19), 178 (100), 165 (34).

772
K. Burger et al.
Methyl (2S)-4-tert-butyloxycarbonylamino-2-hydroxy-2-methylbutyrate (17, C11H21NO5)
A solution of 15b=1 (3.81 g, 10 mmol) in dry methanol (30 cm3) was heated under reflux for 72h (TLC
control). Then the solvent was evaporated under reduced pressure and the residue was purified by
column chromatography (eluent: CHCl3=methanol, 20=1, Rf ¼ 0.32). Yield 49%; colorless oil;
[ꢀ]D ¼ ꢁ4.2ꢂ cm3 gꢁ1 dmꢁ1 (c ¼ 0.48, methanol); IR (film): ꢄꢁ¼ 3600–3200, 2978, 2933, 1782,
1
1712, 1696 cmꢁ1; H NMR (400 MHz, CDCl3): ꢅ ¼ 1.35 (s, 3H), 1.36 (s, 9H), 1.74 (m, 1H), 2.01
(m, 1H), 2.97 (s, 1H, OH), 3.10 (m, 1H), 3.20 (m, 1H), 3.72 (s, 3H), 4.83 (s br, 1H, NH) ppm;
13C NMR (101MHz, CDCl3): ꢅ ¼ 26.6, 28.4, 36.2, 39.2, 52.9, 74.8, 83.4, 156.0, 177.5ppm; MS (ESI):
m=z ¼ [Mþ Na]þ calcd 270.13119, found 270.13144.
tert-Butyl (S)-3-hydroxycarbamoyl-3-hydroxybutylcarbamate (18, C10H20N2O5)
To a stirred solution of 15b=1 (3.81 g, 10mmol) in DMF (30 cm3) hydroxylamine hydrochloride
(0.70 g, 10.0mmol) and propylene oxide (0.60 g, 10 mmol) were added. The reaction mixture was
stirred for 24h at room temperature (TLC control). Then the solvent was evaporated under reduced
pressure and the residue was purified by column chromatography (eluent: CHCl3=CH3OH), 10=1,
Rf ¼ 0.11). 18 was isolated as a hygroscopic white solid after lyophilization. Yield 67%;
[ꢀ]D ¼ ꢁ3.0ꢂ cm3 gꢁ1 dmꢁ1 (c ¼ 0.50, CH3OH); IR (KBr): ꢄꢁ¼ 3600–3100, 2976, 2929, 1689cmꢁ1
;
1H NMR (400 MHz, CDCl3): ꢅ ¼ 1.22 (s, 3H), 1.35 (s, 9H), 1.56 (m, 1H), 1.75 (m, 1H), 2.83 (m, 1H),
3.01 (m, 1H), 5.14 (s, 1H, OH), 6.59 (t br, 1H, NH), 8.59 (s br, 1H), 10.29 (s br, 1H) ppm; 13C NMR
(101 MHz, CDCl3): ꢅ ¼ 26.4, 28.2, 35.7, 39.9, 73.2, 77.3, 155.5, 171.7 ppm; MS (ESI):
m=z ¼ [Mþ Na]þ calcd 271.12644, found 271.12670, [2M þ Na]þ calcd 519.26366, found 519.26389.
Aminolysis of 15 and 16 (General procedure)
To a stirred solution of equimolar amounts of DIPEA (1.29 g, 10.0mmol) in DMF (20 cm3) a solution
of L-Phe-OMeꢄHCl (2.15 g, 10.0 mmol) in DMF was added. After 5 min 15=16 (10.0 mmol) was
added. When the reaction was complete (TLC control) DMF was evaporated in vacuo and the residue
was purified by column chromatography.
Methyl [(2S)-4-(benzyloxycarbonylamino)-2-hydroxybutyryl]phenylalaninate
(19a, C22H26N2O6)
Yield 62%; yellow oil; crystallizing on standing, mp 69–71ꢂC; Rf ¼ 0.38 (eluent: ethyl acetate=
petroleum ether, 1=3); [ꢀ]D ¼ ꢁ5.6ꢂ cm3 gꢁ1 dmꢁ1 (c ¼ 1.0, CH3OH); IR (KBr): ꢄꢁ¼ 3700–3200,
1
1725, 1711, 1660 cmꢁ1; H NMR (600 MHz, CDCl3): ꢅ ¼ 1.56 (m, 1H), 1.91 (m, 1H), 3.03 (dd,
J ¼ 14.0, 7.2Hz, 1H), 3.14 (dd, J ¼ 14.0, 5.7 Hz, 2H), 3.37 (m, 1H), 3.68 (s, 3H), 4.06 (m, 1H),
4.54 (s br, 1H, OH), 4.84 (dt br, J ¼ 7.8, 5.9 Hz, 1H), 5.04 (d, J ¼ 12.3Hz, 1H), 5.07 (d, J ¼ 12.3Hz,
Hz, 1H), 5.38 (t, J ¼ 6.2 Hz, 1H, NH), 7.10–7.33 (m, 10H), 7.40 (d, J ¼ 8.1 Hz, 1H; NH) ppm; 13C
NMR (151MHz, CDCl3): ꢅ ¼ 34.9, 37.0, 38.1, 52.3, 52.8, 67.0, 69.1, 127.1, 128.1, 128.2, 128.5, 128.6,
129.2, 135.9, 136.3, 157.8, 171.9, 173.5ppm; MS (ESI): m=z ¼ [Mþ Na]þ calcd 399.10015, found
399.10077, [Mþ H]þ calcd 377.11820, found 377.11871.
Methyl [(2S)-4-(benzyloxycarbonylamino)-2-hydroxy-2-methylbutyryl]phenylalaninate
(19b, C23H28N2O6)
Yield 84%; colorless oil; Rf ¼ 0.48 (eluent: CHCl3=CH3OH, 10=1); [ꢀ]D ¼ ꢁ5.9ꢂ cm3 gꢁ1 dmꢁ1
1
(c ¼ 1.0, CH3OH); IR (film): ꢄꢁ¼ 3600–3150, 3100–2850, 1736, 1662 cmꢁ1; H NMR (400MHz,
DMSO-d6): ꢅ ¼ 1.19 (s, 3H), 1.54 (m, 1H), 1.75 (m, 1H), 2.71 (m, 1H), 3.00 (m, 1H), 3.06 (d br,
J ¼ 7.0 Hz, 2H), 3.62 (s, 3H), 4.52 (dt, J ¼ 8.1, 7.1 Hz, 1H), 4.99 (s br, 2H), 5.44 (s, 1H, OH), 6.97 (t,

New Building Blocks for Peptide and Depsipeptide Modification
773
J ¼ 5.2 Hz, 1H, NH), 7.12–7.38 (m, 10H), 7.77 (d, J ¼ 8.1 Hz, 1H, NH) ppm; 13C NMR (101 MHz,
DMSO-d6): ꢅ ¼ 26.3, 35.9, 36.3, 40.6, 51.8, 52.7, 65.0, 73.2, 126.4–137.2, 155.7, 171.6, 175.0 ppm;
MS (ESI): m=z ¼ [Mþ H]þ calcd 429.20201, found 429.20238, [Mþ Na]þ calcd 451.18396, found
451.18448.
(3S)-1-Benzyloxycarbonylamino-3-hydroxypyrrolidin-2-one (20, C12H13NO4)
A solution of 15a=2 (5mmol) in DMF (25 cm3) was heated in a microwave oven under reflux for 30–
60min (TLC control). Then the solvent was evaporated in vacuo and the residue was purified by
column chromatography (eluent: DCM). Yield 64%; colorless oil; Rf ¼ 0.21 (eluent: DCM);
[ꢀ]D ¼ ꢁ17.8ꢂ cm3 gꢁ1 dmꢁ1 (c ¼ 0.45, DCM); IR (film): ꢄꢁ¼ 3600–3000, 1721, 1702, 1691 cmꢁ1
;
1H NMR (400MHz, CDCl3): ꢅ ¼ 1.95 (m, 1H), 2.40 (m, 1H), 3.54 (dt br, J ¼ 10.0, 7.0 Hz, 1H),
3.86 (t br, J ¼ 10.0Hz, 1H), 4.37 (t, J ¼ 10.0Hz, 1H), 4.68 (s br, 1H, OH), 5.24 (s br, 2H), 7.26–
7.41 (m, 5H) ppm; 13C NMR (101MHz, CDCl3): ꢅ ¼ 26.8, 42.1, 68.2, 70.3, 128.1, 128.4, 128.5, 134.9,
151.1, 174.8 ppm; MS (EI): m=z (%) ¼ 235 [M]þ (9), 209 (5), 197 (5), 167 (8), 147 (62), 115 (24), 18
(91), 95 (28), 91 (100), 79 (19), 69 (46).
Methyl [4-(benzyloxycarbonylamino)-2-mercaptobutyryl]phenylalaninate (21, C22H26N2O5S)
Yield 44%; yellow oil; Rf ¼ 0.55 (eluent: CHCl3=CH3OH, 10=1, mixture of 2 diastereomers); IR
(film): ꢄꢁ¼ 3450–3300, 3064, 3032, 2951, 1745, 1651 cmꢁ1
;
1H NMR (400 MHz, DMSO-d6):
ꢅ ¼ 1.62 (m, 1H), 1.84 (m, 1H), 2.66=2.71 (2d, J ¼ 9.2, 8.5 Hz, 1H, SH), 2.90 (m, 2H), 3.04 (m,
2H), 3.37 (m, 1H), 3.59=3.61 (s, 3H), 4.47 (m, 1H), 5.01 (s br, 2H), 7.10–7.40 (m, 10H arom, 1NH),
8.48=8.50 (2d, J ¼ 5.9, 6.3 Hz, 1H, NH) ppm; 13C NMR (101 MHz, DMSO-d6): ꢅ ¼ 35.8, 36.5=36.7,
38.0=38.1, 38.5=38.7, 51.7=51.8, 53.5=53.6, 65.1=65.2, 126.4=126.5, 128.1=128.2, 128.3,
129.0=129.0, 136.9=137.0, 137.0=137.1, 155.9=156.0, 171.5=171.6, 171.7=171.9ppm; MS (ESI):
m=z ¼ [Mþ H]þ calcd 431.16352, found 431.16364, [Mþ Na]þ calcd 453.14546, found 453.14598,
[2M þ Na]þ calcd 883.30171, found 883.30230.
tert-Butyl [(2S)-4-(9-fluorenylmethyloxycarbonylamino)-2-hydroxybutyryl]phenylalaninate
(22a, C32H36N2O6)
Yield 62%; mp 126ꢂC; [ꢀ]D ¼ 6.7ꢂ cm3 gꢁ1 dmꢁ1 (c ¼ 1.00, DCM); IR (KBr): ꢄꢁ¼ 3360–3290, 1720,
1
1685, 1630, 1530 cmꢁ1; H NMR (101MHz, CDCl3): ꢅ ¼ 1.37 (s, 9H), 1.57 (m, 1H), 1.91 (m, 1H),
3.01 (dd, J ¼ 14.0, 7.0 Hz, 1H), 3.08 (dd, J ¼ 14.0, 7.0 Hz, 1H), 3.15 (m, 1H), 3.38 (m, 1H), 3.96 (m,
1H), 4.12 (m, 1H), 4.36 (m, 2H), 4.63 (s br, 1H, OH), 4.74 (m, 1H), 5.51 (m, 1H, NH), 7.13–7.17 (m,
2H), 7.18–7.27 (m, 5H), 7.32–7.36 (m, 2H), 7.44 (d, J ¼ 7.0 Hz, 1H, NH), 7.51–7.54 (m, 2H), 7.69–
7.71 (m, 2H) ppm; 13C NMR (101 MHz, CDCl3): ꢅ ¼ 27.4, 34.4, 36.4, 37.8, 46.8, 52.7, 66.3, 68.3,
81.8, 119.5, 124.5, 126.3, 126.6, 128.0, 129.0, 140.8, 140.8, 143.2, 143.3, 157.4, 170.1, 172.9ppm; MS
(EI): m=z (%) ¼ 500 [Mꢁ CO2]þ (0.7), 178 (100).
tert-Butyl [(2S)-4-(9-fluorenylmethyloxycarbonylamino)-2-hydroxy-2-
methylbutyryl]phenylalaninate (22b, C33H38N2O6)
Yield 56%; mp 129ꢂC; Rf ¼ 0.36 (eluent: CHCl3=CH3OH, 10=1); [ꢀ]D ¼ ꢁ4.0ꢂ cm3 gꢁ1 dmꢁ1
1
(c ¼ 0.75, CH3OH); IR (KBr): ꢄꢁ¼ 3600–3200, 2975, 2929, 1725, 1654 cmꢁ1; H NMR (400 MHz,
DMSO-d6): ꢅ ¼ 1.30 (s, 3H), 1.34 (s, 9H), 1.58 (m, 1H), 1.79 (m, 1H), 2.77 (m, 1H), 3.02 (m, 3H), 4.20
(t, J ¼ 6.8 Hz, 1H), 4.26 (d, J ¼ 6.8 Hz, 2H), 4.41 (dt, J ¼ 7.2, 7.1 Hz, 1H), 5.46 (s br, 1H, OH), 7.07 (t
br, J ¼ 5.0 Hz, 1H, NH), 7.14–7.29 (m, 5H), 7.64 (d, J ¼ 9.0 Hz, 1H, NH), 7.32 (m, 2H), 7.40 (m, 2H),
7.67 (m, 2H), 7.88 (m, 2H) ppm; 13C NMR (101MHz, DMSO-d6): ꢅ ¼ 27.4, 27.5, 35.9, 36.7, 39.7,

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K. Burger et al.
46.6, 53.2, 65.1, 79.7, 80.8, 120.0, 125.0, 126.4, 126.9, 127.5, 128.0, 129.1, 136.9, 140.6, 143.8, 155.8,
170.1, 174.8 ppm; MS (ESI): m=z ¼ [Mþ H]þ calcd 559.28026, found 559.28079, [Mþ Na]þ calcd
581.26221, found 581.26252.
tert-Butyl [4-(9-fluorenylmethyloxycarbonylamino)-2-mercaptobutyryl]phenylalaninate
(23, C32H36N2O5S)
Yield 47%; yellow oil; Rf ¼ 0.45 (eluent: CHCl3=CH3OH, 10=1, mixture of diastereomers); IR (film):
ꢄꢁ¼ 3600–3200, 2992, 2948, 1740, 1655 cmꢁ1; 1H NMR (400MHz, DMSO-d6): ꢅ ¼ 1.28=1.30 (s, 9H),
1.81 (m, 2H), 2.79–3.11 (m, 5H), 3.54 (m, 1H), 4.19 (t br, 1H), 4.28 (d br, 2H), 4.41 (m, 1H), 7.12–
7.91 (m, 14H), 8.37=8.46 (d br, 1H, NH) ppm; 13C NMR (101MHz, DMSO-d6): ꢅ ¼ 27.4, 31.4, 36.9,
37.9, 46.7, 50.6, 54.6, 65.3, 80.8, 120.0, 125.0, 126.4, 127.0, 127.5, 128.1, 129.1, 136.8, 140.7, 143.8,
155.9, 156.0, 170.1ppm; MS (ESI): m=z ¼ [Mþ H]þ calcd 561.24187, found 561.24177.
Incorporation of Azaglycine (General Procedure)
15 (10.0 mol) was added to a stirred solution of NH2NHCO2Me (0.90 g, 10.0 mol) in DMF (15 cm3).
When the reaction was complete (TLC control) DMF was evaporated and the residue was purified by
column chromatography (eluent: CHCl3=CH3OH, 10=1). Reactions with 16 were performed under
argon.
Methyl [(2S)-4-benzyloxycarbonylamino-2-hydroxybutyryl]azaglycinate (24a, C14H19N3O6)
Yield 74%; hygroscopic brownish crystals; mp 103–105ꢂC; [ꢀ]D ¼ ꢁ21.6ꢂ cm3 gꢁ1 dmꢁ1 (c ¼ 0.51,
CH3OH); IR (KBr): ꢄꢁ¼ 3600–3200, 1732, 1697, 1653 cmꢁ1
;
1H NMR (600MHz, DMSO-d6):
ꢅ ¼ 1.63 (m, 1H), 1.80 (m, 1H), 3.12 (s br, 2H), 3.58 (s, 3H), 3.97 (s br, 1H), 5.01 (s br, 2H), 5.70
(s br, 1H, OH), 7.20 (s br, 1H, NH), 7.31–7.36 (m, 5H), 8.98 (s br, 1H, NH), 9.57 (s br, 1H, NH) ppm;
13C NMR (151 MHz, DMSO-d6): ꢅ ¼ 34.6, 36.8, 51.7, 65.1, 68.4, 127.6, 128.2, 137.2, 156.0, 156.4,
173.1ppm; MS (ESI): m=z ¼ [Mþ H]þ calcd 326.13466, found 326.13420, [Mþ Na]þ calcd
348.11661, found 348.11595.
Methyl [(2S)-4-benzyloxycarbonylamino-2-hydroxy-2-methylbutyryl)azaglycinate
(24b, C15H21N3O6)
Yield 82%; oil; Rf ¼ 0.21 (eluent: CHCl3=CH3OH, 10=1); [ꢀ]D ¼ ꢁ2.7ꢂ cm3 gꢁ1 dmꢁ1 (c ¼ 0.75,
CH3OH); IR (film): ꢄꢁ¼ 3700–3100, 3100–2800, 1698brcmꢁ1
;
1H NMR (400 MHz, DMSO-d6):
ꢅ ¼ 1.25 (s, 3H), 1.61 (m, 1H), 1.82 (m, 1H), 2.98 (m, 1H), 3.18 (m, 1H), 3.56 (s, 3H), 4.99 (s br,
2H), 5.38 (s br, 1H, OH), 7.07 (t br, J ¼ 5.0 Hz, 1H, NH), 7.31 (m, 5H), 8.94 (s br, 1H, NH), 9.44 (s br,
1H, NH) ppm; 13C NMR (101MHz, DMSO-d6): ꢅ ¼ 26.4, 35.9, 40.4, 51.4, 65.0, 73.4, 127.6, 128.2,
137.2, 155.8, 156.4, 174.8 ppm; MS (ESI): m=z ¼ [Mþ H]þ calcd 340.15031, found 340.15065,
[Mþ Na]þ calcd 362.13226, found 362.13268.
Methyl [4-benzyloxycarbonylamino-2-mercaptobutyryl]azaglycinate (25, C14H19N3O5S)
Yield 71%; hygroscopic white solid; Rf ¼ 0.27 (eluent: CHCl3=CH3OH, 10=1); IR (KBr): ꢄꢁ¼ 3500–
3200, 3032, 1745, 1716, 1670 cmꢁ1; 1H NMR (600MHz, DMSO-d6): ꢅ ¼ 1.71 (m, 1H), 1.92 (m, 1H),
2.86 (d, J ¼ 8.4 Hz, 1H, SH), 3.09 (m, 2H), 3.33 (m, 1H), 3.59 (s, 3H), 5.01 (s br, 2H), 7.26 (t br,
J ¼ 5.0 Hz, 1H, NH), 7.29–7.39 (m, 5H), 9.18 (s br, 1H, NH), 9.86 (s br 1H, NH) ppm; 13C NMR
(101 MHz, DMSO-d6): ꢅ ¼ 35.9, 37.0, 38.0, 52.0, 65.3, 127.7, 128.3, 137.1, 156.1, 156.5, 171.7ppm;
MS (ESI): m=z ¼ [Mþ H]þ calcd 342.11182, found 342.11135.

New Building Blocks for Peptide and Depsipeptide Modification
775
Methyl [(2S)-4-(9-fluorenylmethyloxycarbonylamino)-2-hydroxybutyryl]azaglycinate
(26a, C21H23N3O6)
Yield 67%; white solid; mp 144–145ꢂC; [ꢀ]D ¼ ꢁ13.3ꢂ cm3 gꢁ1 dmꢁ1 (c ¼ 0.60, CH3OH), IR (KBr):
ꢄꢁ¼ 3500–3200, 1695 cmꢁ1; 1H NMR (600MHz, DMSO-d6): ꢅ ¼ 1.63 (m, 1H), 1.81 (m, 1H), 3.11 (m,
2H), 3.58 (s, 3H), 3.97 (m, 1H), 4.21 (t, J ¼ 7.0 Hz, 1H), 4.27 (d, J ¼ 7.0 Hz, 2H), 5.54 (s br, 1H, OH),
7.26 (t br, J ¼ 5.0 Hz, 1H, NH), 7.33 (m, 2H), 7.41 (m, 2H), 7.69 (m, 2H), 7.89 (m, 2H), 8.99 (s br, 1H,
NH), 9.57 (s br, 1H, NH) ppm; 13C NMR (151MHz, DMSO-d6): ꢅ ¼ 34.5, 36.7, 46.6, 51.7, 65.2, 68.4,
120.0, 125.1, 127.0, 127.5, 140.6, 143.8, 156.0, 156.4, 173.1ppm; MS (ESI): m=z ¼ [Mþ H]þ calcd
414.16596, found 414.16553.
Methyl [(2S)-4-(9-fluorenylmethyloxycarbonylamino)-2-hydroxy-2-methylbutyryl]aza-
glycinate (26b, C22H25N3O6)
Yield 87%; white solid; mp 75–77ꢂC; Rf ¼ 0.38 (eluent: CHCl3=CH3OH, 10=1);
[ꢀ]D ¼ ꢁ3ꢂ cm3 gꢁ1 dmꢁ1 (c ¼ 1.0, CH3OH); IR (KBr): ꢄꢁ¼ 3600–3100, 3100–2900, 1811, 1737,
1
1697 cmꢁ1; H NMR (400MHz, DMSO-d6): ꢅ ¼ 1.26 (s, 3H), 1.63 (m, 1H), 1.84 (m, 1H), 2.99 (m,
1H), 3.19 (m, 1H), 3.57 (s, 3H), 4.21 (m, 3H), 5.48 (s br, 1H, NH), 7.16 (s br, 1H, NH), 7.32 (m, 2H),
7.40 (m, 2H), 7.67 (m, 2H), 7.87 (m, 2H), 8.95 (s br, 1H, NH), 9.46 (s br, 1H, NH) ppm; 13C NMR
(101 MHz, DMSO-d6): ꢅ ¼ 26.4, 35.8, 40.1, 46.6, 51.7, 65.2, 73.3, 119.9, 125.1, 126.9, 127.5, 140.6,
143.8, 155.9, 156.5, 174.8 ppm; MS (ESI): m=z ¼ [Mþ Na]þ calcd 450.16356, found 450.16395.
Methyl [4-(9-fluorenylmethyloxycarbonylamino)-2-mercaptobutyryl]azaglycinate
(27, C21H23N3O5S)
Yield 62%; hygroscopic solid; Rf ¼ 0.21 (eluent: CHCl3=CH3OH, 10=1); IR (KBr): ꢄꢁ¼ 3500–3300,
1
2920, 2852, 1736, 1658 cmꢁ1; H NMR (400 MHz, DMSO-d6): ꢅ ¼ 1.72 (m, 1H), 1.94 (m, 1H), 2.85
(d, J ¼ 9.7 Hz, 1H, SH), 3.10 (m, 2H), 3.32 (m, 1H), 3.59 (s, 3H), 4.21 (t, J ¼ 6.7 Hz, 1H), 4.31 (d,
J ¼ 6.7 Hz, 2H), 7.33 (m, 3H), 7.41 (m, 2H), 7.69 (m, 2H), 7.89 (m, 2H), 9.18 (s br, 1H, NH), 9.87 (s
br, 1H, NH) ppm; 13C NMR (101 MHz, DMSO-d6): ꢅ ¼ 35.9, 37.1, 38.0, 46.7, 51.9, 65.3, 120.1, 125.1,
127.0, 127.6, 140.7, 143.9, 156.1, 156.6, 171.7ppm; MS (ESI): m=z ¼ [2M þ Na]þ calcd 881.26090,
found 881.26006.
Acknowledgements
The authors are grateful to Stiftung Volkswagenwerk, Hannover, to DFG, Bonn, and FCI, Frankfurt, for
financial support.
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