A practical synthesis of Nα-Fmoc protected L-threo-β-hydroxyaspartic acid derivatives for coupling via α- Or β-carboxylic group

Article abstract of DOI:10.1007/s00726-010-0806-x

A simple and practical general synthetic protocol towards orthogonally protected tHyAsp derivatives fully compatible with Fmoc solid-phase peptide synthetic methodology is reported. Our approach includes enantioresolution of commercially available D,L-tHyAsp racemic mixture by co-crystallization with L-Lys, followed by ion exchange chromatography yielding enantiomerically pure L-tHyAsp and D-tHyAsp, and their selective orthogonal protection. In this way N^α-Fmoc protected tHyAsp derivatives were prepared ready for couplings via either α- or β-carboxylic group onto the resins or the growing peptide chain. In addition, coupling of tHyAsp via β-carboxylic group onto amino resins allows preparation of peptides containing tHyAsn sequences, further increasing the synthetic utility of prepared tHyAsp derivatives. Springer-Verlag 2010.

Full text of DOI:10.1007/s00726-010-0806-x

Amino Acids (2012) 42:285–293
DOI 10.1007/s00726-010-0806-x
ORIGINAL ARTICLE
A practical synthesis of Na-Fmoc protected
L-threo-b-hydroxyaspartic acid derivatives for coupling
via a- or b-carboxylic group
•
Nina Bionda Mare Cudic Lidija Barisic
•
•
´
Maciej Stawikowski Roma Stawikowska
•
•
•
Diego Binetti Predrag Cudic
Received: 31 August 2010 / Accepted: 1 November 2010 / Published online: 17 November 2010
Ó Springer-Verlag 2010
Abstract A simple and practical general synthetic pro-
tocol towards orthogonally protected tHyAsp derivatives
fully compatible with Fmoc solid-phase peptide synthetic
methodology is reported. Our approach includes enantio-
resolution of commercially available D,L-tHyAsp racemic
mixture by co-crystallization with L-Lys, followed by ion
exchange chromatography yielding enantiomerically pure
L-tHyAsp and D-tHyAsp, and their selective orthogonal
protection. In this way Na-Fmoc protected tHyAsp deriv-
atives were prepared ready for couplings via either a- or
b-carboxylic group onto the resins or the growing peptide
chain. In addition, coupling of tHyAsp via b-carboxylic
group onto amino resins allows preparation of peptides
containing tHyAsn sequences, further increasing the syn-
thetic utility of prepared tHyAsp derivatives.
Abbreviations
ACN
Acetonitrile
Bn–OH
DBU
Benzyl alcohol
1,8-Diazabicyclo[5.4.0]undec-7-ene
Dichloromethane
DCM
DHP
Dihydropyran
DIC
Diisopropylcarbodiimide
Dmab–OH 4-{N-[1-(4,4-Dimethyl-2,6-
dioxocyclohexylidene)-3-methylbutyl]-
amino} benzyl alcohol
Dimethylaminopyridine
Dimethyl sulfoxide
Ethyl acetate
DMAP
DMSO
EtOAc
EtOH
Ethanol
Fmoc-OSu N-(9-Fluorenylmethoxycarbonyloxy)
succinimide
Keywords Hydroxyaspartic acid ꢀ Enantioresolution ꢀ
Orthogonal protection ꢀ Fmoc solid-phase peptide synthesis
HOAc
IPA
Acetic acid
Isopropanol
MeOH
PPTS
TBAF
Methanol
Pyridinium p-toluenesulfonate
Tetrabutylammonium fluoride
Electronic supplementary material The online version of this
article (doi:10.1007/s00726-010-0806-x) contains supplementary
material, which is available to authorized users.
TBDMS-Cl tert-Butyldimethylsilyl chloride
TFA
THF
Trifluoroacetic acid
Tetrahydrofurane
N. Bionda ꢀ M. Cudic ꢀ L. Barisic ꢀ M. Stawikowski ꢀ
R. Stawikowska ꢀ D. Binetti ꢀ P. Cudic
Department of Chemistry and Biochemistry,
Florida Atlantic University, Boca Raton, FL 33431, USA
Present Address:
N. Bionda ꢀ M. Cudic ꢀ P. Cudic (&)
Torrey Pines Institute for Molecular Studies,
11350 SW Village Parkway, Port St. Lucie, FL 34987, USA
e-mail: pcudic@tpims.org
Introduction
Stereoisomers of b-hydroxyaspartic acid (HyAsp) as well
as its analog b-hydroxyasparagine (HyAsn) are found in
both free form and as peptide constituents in many natural
products. L-threo-b-hydroxyaspartic acid (L-tHyAsp) alone
inhibits growth of various fungi (Ishiyama et al. 1975), and
Present Address:
L. Barisic
Faculty of Food Technology and Biotechnology,
Pierottijeva 6, 10000 Zagreb, Croatia
123

286
N. Bionda et al.
its derivatives exhibit a series of biological activities such
as inhibition of glutamate/aspartate transporters (Lebrun
et al. 1997; Shimamoto et al. 2000), inhibition of tumor cell
growth (Thomasset et al. 1991; Tournaire et al. 1994), and
antiretroviral activity (Malley et al. 1994). HyAsp and
HyAsn were found in sequences of antibacterial peptides
such as ramoplanin (Cavalleri et al. 1984; Pallanza et al.
1984), katanosins (O’Sullivan et al. 1988; Kato et al.
1988), lanthiopeptin (Naruse et al. 1989), alterobactins
(Reid et al. 1993), plusbacins (Shoji et al. 1992; Maki et al.
2001), and cormycin A (Scaloni et al. 2004). Among them,
ramoplanin shows most promising clinical potential and is
currently in Phase III trials for treatment of Clostridium
difficile-associated disease (CDAD) and for the prevention
of vancomycin-resistant enterococci bloodstream infec-
tions (Montecalvo 2003; Rogers and Leach 2004). Other
examples of naturally occurring peptides containing
HyAsp/HyAsn are theonellamide (Matsunaga et al. 1989)
and cepacidine (Lee et al. 1994; Lim et al. 1994), exhib-
iting strong antifungal activity, and microviridins (Reshef
and Carmeli 2006) that act as protease inhibitors.
protected L-tHyAsp. All reported Na-Boc-HyAsp derivatives
possess a free a-carboxylic group, limiting somewhat their
synthetic applicability.
We report here a practical and efficient synthetic
approach towards orthogonally protected D- and L-tHyAsp
fully compatible with the standard Fmoc SPPS methodol-
ogy. Besides complete orthogonal protection, our approach
allows synthetic exploitation of both HyAsp carboxylic
groups expanding the versatility of these peptide building
blocks.
Materials and methods
General methods
Unless otherwise specified, all chemicals and solvents were
purchased from Fisher Scientific (Atlanta, GA) and
were analytical reagent grade or better. All solvents used
˚
were dried over 4 A molecular sieves prior to use, unless
specified differently. D,L-tHyAsp and Marfey’s reagent were
purchased from TCI America (Portland, OR, USA). Thin-
layer chromatography (TLC) used for monitoring reactions
was performed on Whatman precoated silica gel F-254
plates (Whatman Inc., Piscataway, NJ, USA) and visualized
by ultraviolet light and/or staining with ninhydrin solution.
Silica gel (Dynamic Adsorbents Inc., Atlanta, GA;
32–63 lm) was used for flash chromatography. The 1H and
13C NMR spectra were obtained on Varian 400 MHz NMR
spectrometer (Varian Inc., Palo Alto, CA, USA). Chemical
shifts (d) are reported in units of parts per million (ppm)
downfield from tetramethylsilyl chloride. High resolution
mass spectroscopy (HRMS) was performed on MALDI–
TOF Voyager-DETM STR (Applied Biosystems, Foster
City, CA, USA). Enantiomeric purity of resolved D- and
L-tHyAsp was determined by derivatization with Marfey’s
reagent and RP-HPLC analyses of corresponding deriva-
tives on a Thermo Electron Corporation SpectraSYSTEM
(Thermo Fischer Scientific Inc., Waltham, MA, USA) liquid
chromatography system. For RP-HPLC analysis, a C18
monomeric column (Grace Vydac, 250 9 4.6 mm, 5 lm,
However, limited synthetic access to these peptides and
particularly their analogs hampered complete exploitation of
their biological potential and utilization as lead compounds
for drug development. Since Fmoc solid-phase peptide
synthesis (Fmoc SPPS) represents a standard approach for
the routine peptide synthesis, access to orthogonally pro-
tected HyAsp and HyAsn fully compatible with this meth-
odology is of practical importance. Asymmetric syntheses of
L-tHyAsn bearing standard protecting groups for Fmoc-
solution or solid-phase peptide synthesis have been reported
´
´
in the literature (Boger et al. 2000; Guzman-Martınez and
VanNieuwenhze 2007; Spengler et al. 2010). In contrast, a
few synthetic protocols are reported for preparation of
HyAsp, but none of the reported derivatives are compatible
with the standard Fmoc SPPS methodology. Hanessian and
Vanasse (1993) reported the synthesis of L-threo-HyAsp
(L-tHyAsp) and L-erythro-HyAsp (L-eHyAsp) mixtures of
diesters by hydroxylation of L-aspartic acid (L-Asp) diesters.
Starting with trans-ethyl cinnamate, Sharpless asymmetric
aminohydroxylation was used by Khalaf and Datta (2008) to
prepare D-tHyAsp. In all cases reported, HyAsp derivatives
possess unprotected a-amino and a-carboxylic groups. Boc-
protecting strategy was chosen to synthesize Na-protected
HyAsp derivatives. Hansson and Kihlberg (1986) used
tartaric acid to prepare protected L-eHyAsp, whereas
Na-Boc-L-Ser was used by Wagner and Tilley (1990) to
prepare protected L-tHyAsp fully compatible with Boc
peptide synthetic methodology. Na-Boc-L-tHyAsp was also
prepared by Deng et al. (1995) starting from methyl cinna-
mate and using Sharpless dihydroxylation reaction. In this
case, synthesized L-tHyAsp derivative underwent additional
deprotection and reprotection to obtain the desired Na-Boc
˚
120 A), 1 mL/min flow rate, and elution method 100% A for
5 min followed by linear gradient of 0–100% B over
40 min, where A was 0.1% TFA in H2O and B was 0.1%
TFA in ACN, was used. Eluting products were detected by
UV at 340 nm. Optical rotations were measured on Autopol
III RA 7214 automatic polarimeter (Rudolph Research, NJ,
USA) at 25°C, and 589 nm (Na-D).
Enantioresolution of D,L-tHyAsp
D,L-tHyAsp racemic mixture 1 was successfully separated
by co-crystallization with L-Lys (equimolar ratio) followed
123

Synthesis of orthogonally protected L-tHyAsp
287
by ion exchange chromatography (Okai et al. 1967),
Scheme 1. Enantiomerically pure L-Lys ꢀ L-tHyAsp salt
crystallized from the H2O:MeOH solvent mixture over-
night at 4°C, whereas L-Lys ꢀ D-tHyAsp remained in the
mother liquor (Supporting information). In both cases
L-Lys was separated by ion exchange chromatography, and
pure L- and D-tHyAsp, 1a and 1b, were obtained in 75%
yields.
completion, the pH was adjusted to 6 with NH4OH, and
cold EtOH was added to precipitate the product. Precipitate
was recrystallized from hot water, and pure (2S,3S)-2-
amino-4-(benzyloxy)-3-hydroxy-4-oxobutanoic acid (2)
(1.4 g, 5.86 mmol, 87%) was obtained as white solid.
1H NMR (400 MHz, DMSO-d6) d 7.33–7.43 (m, 5H, Bn–
ArH), 5.11 (dd, 2H, Bn–CH2), 4.52 (d, 1H, Hb), 3.47 (d,
1H, Ha) ppm; 13C NMR (100 MHz, DMSO-d6) d 171.7,
167.7, 136.0, 128.4, 128.0, 69.7, 66.1, 55.9 ppm.
RP-HPLC analysis of D- and L-tHyAsp after derivati-
zation with Marfey’s reagent (Bhushan and Bru¨ckner
2004), Fig. 1, shows that in both cases enantiomeric purity
was[99%. Specific optical rotations ([a]D) for the obtained
D- and L-tHyAsp are in very good agreement with the
literature data (Okai et al. 1967), further confirming the
enantiomeric resolution efficiency, Table 1.
(2S,3S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-
(benzyloxy)-3-hydroxy-4-oxobutanoic acid (3)
A solution of Fmoc-OSu (0.141 g, 0.42 mmol) in acetone
was added dropwise to a solution of compound 2 (0.1 g,
0.42 mmol) and NaHCO3 (0.07 g, 0.84 mmol) in
water:acetone mixture (1:2, 15 mL). The reaction was
completed within 2 h, as determined by TLC. The reaction
mixture was concentrated via rotary evaporation, and the
residual water was acidified with 2 N HCl until pH 4 and
extracted two times with EtOAc. EtOAc was evaporated
and the crude residue was loaded on a silica gel column
(solvent system toluene:EtOAc:HOAc, 5:5:1) yielding the
product (0.185 g, 0.4 mmol, 96%) as a white solid. HRMS
calculated for C26H23NO7 m/z (M ? Na)? 484.1372; found
Compound synthesis and characterization
(2S,3S)-2-amino-4-(benzyloxy)-3-hydroxy-4-oxobutanoic
acid (2)
L-tHyAsp 1a (1 g, 6.71 mmol) was added to benzyl alcohol
(10 mL) and concentrated HCl (1.2 mL), and the resulting
mixture was stirred at 70°C for 4.5 h. Upon reaction
1
484.4560. H NMR (400 MHz, CD3OD) d 7.12–7.82 (m,
NH2
NH2
O
13H, Fmoc–ArH, Bn–ArH), 5.16 (dd, 2H, Bn–CH2), 4.83
(d, 1H, Ha), 4.76 (d, 1H, Hb), 4.27 (m, 2H, Fmoc–CH2),
4.19 (t, 1H, Fmoc–CH) ppm; 13C NMR (100 MHz,
CD3OD) d 173.0, 172.8, 158.6, 145.4, 145.3, 142.7, 137.1,
129.6, 129.6, 129.5, 128.9, 128.4, 128.3, 126.53, 126.49,
121.1, 72.5, 68.5, 68.5, 58.5 ppm.
HO
HO
+
NH2
OH
O
O
OH
D
,
L
-threo-HyAsp
L-Lys
1
1) Co-crystallization
water/MeOH 1:1
2) Filtration
(2S,3S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-
(benzyloxy)-4-oxo-3-(tetrahydro-2H-pyran-2-
yloxy)butanoic acid (4)
Crystals
Mother liquor
PPTS was added (0.008 g, 0.0325 mmol) to a solution of 3
(0.15 g, 0.325 mmol) in dry DCM (15 mL).To the stirred
reaction mixture, DHP was added dropwise over 10 min
(0.045 mL, 0.487 mmol). Stirred was continued at room
temperature for 72 h with occasional addition of PPTS and
DHP (total 0.3 equiv. PPTS and 2.5 equiv. DHP). Reaction
mixture was then extracted with water. DCM was evapo-
rated and crude product purified using silica gel chroma-
tography (solvent system toluene:EtOAc:HOAc, 10:1:1)
yielding 0.115 g of product (0.211 mmol, 65%). HRMS
calculated for C31H31NO8 m/z (M ? Na)? 568.1947; found
L
-threo-HyAsp
L-Lys
D-threo-HyAsp L-Lys
Ion-exchange
chromatography
NH2
O
NH2
O
HO
HO
OH
OH
O
OH
1
O
OH
568.4458. H NMR (400 MHz, CDCl3) d 7.28–7.77 (m,
13H, Fmoc–ArH, Bn–ArH), 5.69 (d, 1H, Ha), 5.17 (s, 2H,
Bn–CH2), 5.01 (m, 1H, Hb), 4.86 (t, 1H, THP), 4.33 (m,
2H, Fmoc–CH2), 4.22 (t, 1H, Fmoc–CH), 3.49–3.75 (m,
2H, THP), 1.50–1.77 (m, 6H, THP) ppm; 13C NMR
L
-threo-HyAsp
D
-threo-HyAsp
1a
1b
Scheme 1 General scheme of D,L-tHyAsp enantioresolution
123

288
N. Bionda et al.
2200
2000
1800
1600
1400
1200
1000
800
2200
2000
1800
1600
1400
1200
1000
800
UV2000-340n
DHyAsp
m
2200
2000
1800
1600
1400
1200
1000
800
UV2000-340n
L-HyAsp run2-1 120908
m
(c)
(a)
(b)
Marfey’s
reagent
Rt-21.6 min
Rt-21.2 min
600
600
600
400
400
400
200
200
200
0
0
0
0
2
4
6
8
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
Minutes
0
2
4
6
8
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
Minutes
0
2
4
6
8
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
Minutes
Fig. 1 RP-HPLC traces after derivatization with Marfey’s reagent a D,L-tHyAsp, b D-tHyAsp, c L-tHyAsp
67.7, 67.7, 67.5, 66.8, 66.8, 61.9, 56.4, 47.2, 30.2, 29.9,
25.3, 25.2, 19.0, 18.4 ppm.
Table 1 Specific optical rotations for resolved tHyAsp enantiomers
[a]D
Literature valuesa
Observed valuesb
5 N HCl
H2O
5 N HCl
H2O
(2S,3S)-2-amino-4-(benzyloxy)-3-(tert-
butyldimethylsilyloxy)-4-oxobutanoic acid (7)
L-tHyAsp (1a)
D-tHyAsp (1b)
?6.4°
-6.5°
-8.5°
?8.9°
?6.0°
-6.0°
-8.2°
?8.4°
To a stirred solution of 2 (0.56 g, 2.33 mmol) in dry ACN
(300 mL), DBU was added (0.351 mL, 2.33 mmol) to
obtain pH approximately 9 after which TBDMS-Cl was
added in half equimolar portions (0.175 g, 1.17 mmol).
Occasionally DBU was added to correct the pH of the
solution. Reaction mixture was refluxed at 50°C. Total
2 equiv. of TBDMS-Cl and 3.5 equiv. of DBU were used.
The reaction was monitored by TLC and was completed
within 3–5 h. The solvent was evaporated; residue redis-
solved in water, acidified with 2 N HCl until pH 4 and
extracted two times with EtOAc. Crude product (yellow
oil, 0.674 g, 82% yield) was used in the next step without
further purification.
a
Okai et al. 1967
b
For the detailed method see Supporting Information
(100 MHz, CDCl3) d 173.0, 169.1, 156.5, 143.9, 141.4,
135.2, 128.8, 128.7, 128.6, 127.9, 127.3, 125.4, 120.2,
97.6, 73.5, 67.8, 67.7, 62.6, 56.2, 47.1, 30.0, 25.2,
18.9 ppm.
(2S,3S)-1-allyl 4-benzyl 2-(((9H-fluoren-9-
yl)methoxy)carbonylamino)-3-(tetrahydro-2H-pyran-2-
yloxy)succinate (5)
Aliquat 336 (0.084 mL, 0.183 mmol) and allyl–Br
(0.017 mL, 0.183 mmol) were dissolved in DCM (15 mL).
This mixture was added to a stirred solution of 4 (0.1 g,
0.183 mmol) and NaHCO3 (0.016 g, 0.183 mmol) in water
(15 mL). Reaction was stirred at room temperature for 72 h
with occasional addition of 0.5 equiv. of allyl–Br (total
added was 2.5 equiv.). DCM was separated from the
aqueous layer and extracted one more time with water,
evaporated, and crude product was purified on a silica
column (solvent system toluene:EtOAc:HOAc, 10:1:1)
yielding 0.109 g (85%) of pure product 5. HRMS calcu-
lated for C34H35NO8 m/z (M ? Na)? 608.2260; found
(2S,3S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-
(benzyloxy)-3-(tert-butyldimethylsilyloxy)-4-oxobutanoic
acid (8)
To a solution of 7 (0.674 g, 1.91 mmol) and NaHCO3
(0.321 g, 3.82 mmol) in water:acetone mixture (1:2,
100 mL), a solution of Fmoc-OSu (0.644 g, 1.91 mmol) in
acetone was added dropwise. The reaction was monitored
by TLC and was completed within 2 h. Reaction mixture
was concentrated, residual water acidified with 2 N HCl
until pH 4 and extracted two times with EtOAc. EtOAc was
evaporated and crude residue was loaded on a silica gel
column and purified (solvent system toluene:EtOAc:
HOAc, 10:1:1) yielding pure product 8 (1.06 g, 1.84 mmol,
96% yield) as a white solid. HRMS calculated for
C32H37NO7Si m/z (M ? Na)? 598.2237; found 598.2075.
1H NMR (400 MHz, CDCl3) d 7.3–7.76 (m, 13H, Fmoc–
ArH, Bn–ArH), 5.58 (d, 1H, Ha), 5.16 (dd, 2H, Bn–CH2),
4.93 (d, 1H, Hb), 4.33 (m,2H, Fmoc–CH2), 4,23 (t, 1H,
Fmoc–CH), 0.89 (s, 9H, TBDMS-tBu), 0.11 (s, 3H,
TBDMS–CH3), 0.03 (s, 3H, TBDMS–CH3) ppm; 13C NMR
1
608.6071. H NMR (400 MHz, CDCl3) d 7.25–7.77 (m,
13H, Fmoc–ArH, Bn–ArH), 5.91 (m, 1H, Allyl), 5.65 (d,
1H, Ha), 5.36 (dd, 1H, Allyl), 5.27 (dd, 1H, Allyl), 5.16 (s,
2H, Bn–CH2), 5.02 (m, 1H, Hb), 4.87 (t, 1H, THP), 4.67
(m, 2H, Allyl–CH2), 4.33 (m, 2H, Fmoc–CH2), 4.22 (t, 1H,
Fmoc–CH), 3.43–3.62 (m, 2H, THP), 1.46–1.79 (m, 6H,
THP) ppm; 13C NMR (100 MHz, CDCl3) d 169.2, 169.1,
156.3, 144.0, 143.9, 141.4, 135.2, 131.5, 128.8, 128.7,
128.7, 127.9, 127.3, 125.5, 120.2, 119. 5, 101.0, 96.9, 73.2,
123

Synthesis of orthogonally protected L-tHyAsp
289
(100 MHz, CDCl3) d 174.3, 169.9, 156.1, 143.7, 141.2,
141.2, 135.0, 128.57, 128.55, 128.50, 127.7, 127.1, 125.2,
119.9, 72.2, 67.6, 67.5, 57.3, 46.9, 25.5, 25.4, 18.7, -4.9,
-5.8 ppm.
CH), 2.49 (s, 2H, Dmab–CH2), 2.41 (s, 2H, Dmab–CH2),
2.06 (d, 2H, Dmab–CH2 isopropyl), 1.81 (m, 1H, Dmab–
CH isopropyl), 1.06 (d, 6H, 2 Dmab–CH3), 0.90 (s, 9H,
TBDMS–tBu), 0.75 (s, 3H, Dmab–CH3 isopropyl), 0.73 (s,
3H, Dmab–CH3 isopropyl), 0.14 (s, 3H, TBDMS–CH3),
0.01 (s, 3H, TBDMS–CH3) ppm; 13C NMR (100 MHz,
CDCl3) d 203.2, 197.0, 176.8, 176.5, 173.3, 169.2, 167.4,
156.3, 144.3, 143.7, 143.6, 141.5, 141.2, 136.9, 134.4,
129.2, 127.7, 127.6, 127.1, 126.7, 125.2, 125.2, 124.7,
120.0, 107.6, 71.9, 67.7, 66.8, 61.4, 57.6, 53.5, 49.3, 46. 9,
39.0, 38.5, 30.0, 29.6, 28.2, 25.6, 22.7, 22.6, 20.7, 18.2,
-4.8, -5.7 ppm.
(2S,3S)-1-(4-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-
3-methylbutylamino)benzyl) 4-benzyl 2-(((9H-fluoren-9-
yl)methoxy)carbonylamino)-3-(tert-
butyldimethylsilyloxy)succinate (9)
To a solution of 8 (0.3 g, 0.52 mmol) in DCM (30 mL),
DMAP (0.013 g, 0.1 mmol) and Dmab–OH (0.205 g,
0.624 mmol) were added. DIC (0.097 mL, 0.624 mmol)
was added dropwise over 15 min. Reaction was stirred at
room temperature for 4–6 h with monitoring using TLC.
The solvent was removed and silica gel chromatography
(solvent system toluene:EtOAc:HOAc, 10:5:1) afforded
product 9 (0.319 g, 0.36 mmol, 70%) as a yellowish solid.
HRMS calculated for C52H62N2O9Si m/z (M ? Na)?
Results and Discussion
The goal of this work was to develop a practical and effi-
cient synthesis of tHyAsp derivatives suitable for Fmoc
SPPS. Selection of orthogonal protecting groups allows
activation and subsequent coupling of Na-Fmoc protected
derivatives via either a- or b-carboxylic group onto the free
hydroxyl or amine of the resin linker or of the growing
peptide chain. Synthesis commences with enantioresolu-
tion of commercially available D,L-tHyAsp racemic mix-
ture, Scheme 1, thus avoiding the multi-step process of
enantioselective synthesis. Selective orthogonal protections
of the resulting enantiomers 1a and 1b, Scheme 2, make
these amino acid derivatives versatile building blocks in
Fmoc solid phase peptide synthesis.
1
909.4122; found 909.1248. H NMR (400 MHz, CDCl3) d
7.07–7.78 (m, 17H, Fmoc–ArH, Bn–ArH, Dmab–ArH),
5.63 (d, 1H, Ha), 5.10–5.15 (m, 4 H, Bn–CH2,Dmab–CH2),
4.97 (d, 1H, Hb), 4.31 (m, 2H, Fmoc–CH2), 4.23 (t, 1H,
Fmoc–CH), 2.49 (s, 2H, Dmab–CH2), 2.40 (s, 2H, Dmab–
CH2), 2.09 (d, 2H, Dmab–CH2 isopropyl), 1.82 (m, 1H,
Dmab–CH isopropyl), 1.08 (d, 6H, 2 Dmab–CH3), 0.88 (s,
9H, TBDMS-tBu), 0.76 (s, 3H, Dmab–CH3 isopropyl),
0.74 (s, 3H, Dmab–CH3 isopropyl), 0.09 (s, 3H, TBDMS–
CH3), -0.04 (s, 3H, TBDMS–CH3) ppm; 13C NMR
(100 MHz, CDCl3) d 200.2, 196.5, 176.5, 170.0, 169.8,
169.2, 169.5, 156.0, 155.5, 143.74, 143.71, 143.6, 141.2,
137.1, 136. 9, 135.1, 135.1, 134.4, 134.3, 129.2, 129.2,
128.64, 128.58, 128.53, 127.7, 127.1, 126.7, 126. 6, 120.0,
107.8, 72.4, 67.6, 67.4, 67.0, 66.8, 66.8, 57.7, 53.7, 52.3,
47.0, 46.9, 38.4, 30.0, 29.6, 28.3, 25.6, 22.6, 18.2, -4.8,
-5.8 ppm.
Enantioresolution of D,L-tHyAsp
The enantioselective enzyme-catalyzed hydrolysis of
Na-acyl amino acids had been a widely used method in the
resolution of amino acid racemic mixtures. In particular,
acylase I (N-acetylamino-acid amidohydrolase) was shown
to be very efficient in the preparation of large quantities of
ˇ
enantiopure L-amino acids (Svedas and Galaev 1983;
(2S,3S)-4-(4-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-
3-methylbutylamino)benzyloxy)-3-(((9H-fluoren-9-
yl)methoxy)carbonylamino)-2-(tert-butyldimethylsilyloxy)-
4-oxobutanoic acid (10)
Bommarius et al. 1992; Sato and Tosa 1993). However,
several studies demonstrated that acyl derivatives of
aspartic acid are not substrates for acylase I due to unfa-
vorable charge interactions (Chenault et al. 1989; Liljeblad
et al. 2001), thereby eliminating this method as an option
for enantioresolution of D,L-tHyAsp. Instead, we have
focused on their chemical resolution. Attempts to separate
the D,L-mixture of compound 3 via co-crystallization with
(-)-ephedrine hydrochloride (Oki et al. 1970; Wong and
Wang 1978), a method traditionally used for resolution of
Na-benzyloxycarbonyl-D,L-amino acids, were unsuccessful.
Limited solubility of D,L-3 in a variety of solvents including
CHCl3, MeOH, EtOH, ACN, acetone, THF, EtOAc,
EtOAc:MeOH 1:1, EtOAc:petroleum ether 2:3, EtOAc:
diethyl ether 1:1, EtOAc:CHCl3 1:1, EtOAc:DCM 2:3,
Fully protected amino acid 9 (0.27 g, 0.3 mmol) was dis-
solved in EtOAc, and 5% Pd/C (270 mg) was added. The
reaction vessel was set up at 1 atm of H2 and shaken at
room temperature for 50 min. Reaction mixture was fil-
tered through Celite and evaporated. Product was precipi-
tated with petroleum ether yielding pure 10 (0.184 g,
0.23 mmol, 75%). HRMS calculated for C45H56N2O9Si m/z
(M)? 795.3677; found 795.6108. 1H NMR (400 MHz,
CDCl3) d 7.04–7.76 (m, 12H, Fmoc–ArH, Dmab–ArH),
5.71 (d, 1H, Ha), 5.10–5.13 (dd, 2 H, Dmab–CH2), 4.98 (d,
1H, Hb), 4.33 (m, 2H, Fmoc–CH2), 4.23 (t, 1H, Fmoc–
123

290
N. Bionda et al.
Bn-OH, HCl,
4 h, 87%
NH2
O
NH2 O
a- or b-carboxylic group, combination of the Fmoc, Dmab,
TBDMS and Bn protecting groups gave the best results. The
general protection scheme for L-tHyAsp 1a is shown in
Scheme 2. Selective protection of b-carboxylic group was
achieved by Fischer-type esterification, resulting in com-
pound 2 (87% yield). Due to the presence of acid catalyst,
protonation of the a-amino group determined the esterifi-
cation selectivity (Okonya et al. 1995). In order to prepare
tHyAsp derivative with a free a-carboxylic group, we have
devised two protecting approaches. One includes Fmoc
protection of the a-amino group resulting in compound 3,
96% yield (Cudic et al. 2005), followed by b-hydroxyl group
protection with THP (Miyashita et al. 1977) with a 65%
yield to obtain compound 4.
HO
HO
OH
OBn
O
OH
1a
O
OH
2
Fmoc-OSu (1.1 eq),
NaHCO3 (2 eq),
acetone/H2O, 2 h, 96%
TBDMS-Cl (2 eq),
DBU (3.5 eq),
ACN, 3-5 h, 82%
Fmoc
NH
O
NH2
O
HO
HO
OBn
OBn
O
OH
O
OTBDMS
3
7
DHP (2.5 eq),
PPTS (0.3 eq),
DCM, 72 h, 65%
Fmoc-OSu (1.1 eq),
NaHCO3 (2 eq),
acetone/H2O, 2 h, 96%
Fmoc
NH
Fmoc
NH
O
O
In another approach, due to the basic reaction conditions
requirements for TBDMS introduction, order of protections
was reversed. The b-hydroxyl group was protected first
with TBDMS (Orsini et al. 1989) (7, 82% yield), followed
by a-amino group protection with Fmoc (8, 96% yield).
Although both THP and TBDMS protecting groups fulfill
the requirement for orthogonal protection, we gave pref-
erence to the TBDMS due to better reaction yields, the lack
of need for chromatographic purification, less complex
reaction monitoring and final product characterization.
Introduction of the THP group added an additional
stereocenter to the tHyAsp molecule, resulting in two
diastereomers of 4 with distinguishable Rf values on the
silica-gel TLC plates (0.35, 0.33; toluene:EtOAc:AcOH
10:1:1), and complex NMR spectra (Green and Wuts
1999). On the other hand, preparation of the orthogonally
protected L-tHyAsp derivate with a free b-carboxyl group
turned out to be more challenging. After successful intro-
duction of an allyl protecting group (5, 85% yield, Fried-
rich-Bochnitschek et al. 1989), selective removal of benzyl
ester using various approaches was unsuccessful. We have
tested several reaction conditions including mild base,
Lewis acids, catalytic hydrogenation, neutral reagents, or
lipase C from Candida Antarctica, and in all cases either
no Bn removal or more that one protecting group removal
was observed. The reaction conditions used for benzyl ester
removal are summarized in Table 2.
HO
HO
OBn
OBn
O
OTHP
O
OTBDMS
4
8
NaHCO3 (1 eq),
Allyl-Br (2.5 eq),
Aliquat 336 (1 eq),
DCM, 72 h, 85%
Dmab-OH (1.2 eq),
DIC (1.2 eq),
DMAP (0.2 eq),
DCM, 4-6 h, 70%
Fmoc
NH
Fmoc
NH
O
O
AllylO
DmabO
OBn
OBn
O
OTHP
O
OTBDMS
5
9
H2, 5% Pd/C, EtOAc,
50 min, 75%
see Table 2
Fmoc
NH
Fmoc
NH
O
O
AllylO
DmabO
OH
OTHP
OH
OTBDMS
O
O
6
10
Scheme 2 General protection scheme for HyAsp
EtOAc:ACN 1:3 resulted in poor resolution of the corre-
sponding diastereomeric ephedrine salts. Here, D,L-tHyAsp
was successfully enantioresolved by modified method
previously described by Okai et al. (1967) via co-crystal-
lization with L-Lys followed by ion-exchange chromato-
graphy. Both enantiomers were obtained with satisfactory
yield (75%) and enantiomeric purity (e.e. [99%).
Design of a protection scheme
Appropriate selection of orthogonal protecting groups and
their selective manipulation is an essential requirement in
peptide and amino acid synthesis. In order to find the optimal
combination of orthogonal protecting groups for tHyAsp,
we have tested benzyl (Bn), allyl (Allyl), and (4{N-[1-(4,4-
dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl]-amino}
benzyl (Dmab) for protection of carboxyl groups, 9-fluor-
enylmethylmethoxycarbonyl (Fmoc) for protection of
amino, and tert-butyldimethylsilyl (TBDMS) and tetrahy-
dropyranyl (THP) for protection of the hydroxyl group.
Considering this reaction conditions for the standard Fmoc
SPPS and the requirement for the tHyAsp coupling via either
Replacement of the allyl protecting group with the
Dmab, compound 9, allowed preparation of the desired
L-tHyAsp derivative 10 over five reaction steps in a satis-
factory yield of 36%. Among several previously published
methods for coupling of Dmab-OH to amino acid’s car-
boxyl group, the most satisfactory results were obtained
using DIC/DMAP coupling reagents (Berthelot et al.
2006). However, this method had to be modified for opti-
mal Dmab-OH coupling to compound 8. If catalytic
amounts of DMAP were to be used, the required reaction
time is significantly prolonged resulting in a poor yield for
compound 9. On the other hand, use of equimolar amount
123

Synthesis of orthogonally protected L-tHyAsp
291
Table 2 Tested conditions for selective removal of benzyl protecting group from 5
Fmoc
Fmoc
NH O
NH
O
AllylO
AllylO
OBn
OTHP
OH
OTHP
O
O
5
6
Conditions
Observeda
20% K2CO3/H2O, EtOH, 1 h (Verbeure et al. 2002)
15% K2CO3/H2O, THF, 4 h (Huffman et al. 1978)
Removal of Fmoc, Bn and Allyl
No deprotection
4 mM NaOH, 0.5 M CaCl2, IPA/H20 (7:3), 2 h (Pascal and Sola 1998)
1 N LiOH, THF, 2 h (Chauhan et al. 2007)
Removal of Fmoc and Bn
Removal of Fmoc and Bn
Removal of THP first
1 N BCl3, DCM, -10°C to r.t., 1 h (Schmidt et al. 1991)
Pd(OAc)2, Et3SiH, Et3N, DCM, 1 h-overnight
(Coleman and Shah 1999)
Short time-no deprotection, longer time-all
protecting groups removed except THP
H2, 10% Pd/C, EtOAc, 1 h (Broddefalk et al. 1998)
TBAF, THF, 1 h (Namikoshi et al. 1991)
Removal of Bn and Allyl or Allyl reduction
Removal of Fmoc first
HCOONH4/Mg, MeOH, 3 h (Channe Gowda et al. 2002)
Enzymatic removal, 24 h (Barbayianni et al. 2005)
Removal of Bn and Allyl
No deprotection
a
1
Based on H NMR analysis and TLC comparison of reaction mixtures with compounds 1–5
of DMAP causes Fmoc deprotection. The best results were
obtained with 0.2 equiv. of DMAP. In this case compound
9 was obtained in 70% yield, with no detectable epimer-
ization as indicated by RP-HPLC and NMR analysis (see
Supporting Information). Final benzyl protecting group
removal was successfully achieved by hydrogenation of
compound 9 with 5% Pd/C in ethyl-acetate (Broddefalk
et al. 1998). Yield for this final step was 75%. The outlined
protection scheme is also expected to be useful for the
preparation of orthogonally protected D-tHyAsp, as well as
D- and L-eHyAsp derivatives.
activities, the described synthesis of Na-Fmoc protected
tHyAsp building blocks is of particular practical value
allowing multiple possibilities for their incorporation into a
peptide backbone.
Acknowledgments We gratefully acknowledge the financial sup-
port of the National Institutes of Health (1S06-GM073621-01), the
American Heart Association (0630175 N) to P.C. L.B. was supported
by Unity through Knowledge Fund, Croatia (29/08, 7320-HR).
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