Chemoenzymatic synthesis of Fmoc-protected (2S,3S)-2-hydroxy-3-amino acids and their application in the synthesis of an α-hydroxylated β-hexapeptide

Article abstract of DOI:10.1016/S0957-4166(01)00193-8

A chemoenzymatic and stereoselective synthesis of Fmoc-protected (2S,3S)-2-hydroxy-3-amino acids from 2-furaldehyde is described as well as their application, without prior hydroxyl protection, in the solid-phase synthesis of a novel completely α-hydroxylated β-hexapeptide.

Full text of DOI:10.1016/S0957-4166(01)00193-8

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 12 (2001) 1109–1112
Chemoenzymatic synthesis of Fmoc-protected
2S,3S)-2-hydroxy-3-amino acids and their application in the
synthesis of an a-hydroxylated b-hexapeptide
(
Reynier A. Tromp, Michael van der Hoeven, Alessia Amore, Johannes Brussee,* Mark Overhand,
Gijs A. van der Marel and Arne van der Gen
Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, PO Box 9502, 2300 RA Leiden, The Netherlands
Received 2 April 2001; accepted 27 April 2001
Abstract—A chemoenzymatic and stereoselective synthesis of Fmoc-protected (2S,3S)-2-hydroxy-3-amino acids from 2-furalde-
hyde is described as well as their application, without prior hydroxyl protection, in the solid-phase synthesis of a novel completely
a-hydroxylated b-hexapeptide. © 2001 Elsevier Science Ltd. All rights reserved.
1
. Introduction
Recently, Motorina and co-workers showed that a
hexapeptide composed of (2R,3S)-2-silyloxy-3-amino
17
Over the last decade considerable attention has been
acids formed stable b-strands. Finally, Wen et al. have
directed toward the enantioselective synthesis of b-
reported the synthesis of b-peptides containing guani-
1
–3
18
amino acids.
These compounds not only occur in
dino side groups. Our interest is focused on the
influence of an unprotected a-hydroxyl function on the
formation and stability of the secondary structures of
b-peptides. In this paper we present a chemoenzymatic
route towards a novel completely a-hydroxylated b-
various natural products, which exhibit interesting
pharmaceutical properties, including paclitaxel and
4,5
6
bestatin, they also function as precursors for the syn-
7
,8
thesis of b-lactams.
Apart from this, it has been
demonstrated that oligomers composed of b-amino
acids can fold in stable secondary structures similar to
hexapeptide. For structural comparison, the a-hydroxyl-
19
ated analogue of the 3 helix forming b-hexapeptide
9–11
14
2,3
those of a-peptides, i.e. helices, sheets and turns.
2,3
2,3
H-(b -Val(a-Me)-b -Ala(a-Me)-b -Leu(a-Me)) -
2
Most intriguing was the discovery that only six b-amino
20
OH, was synthesised.
acid residues are necessary for the formation of a stable
1
2,13
helix,
whereas an a-amino acid oligomer normally
requires 15–20 residues. Formation of the helical struc-
ture of b-peptides was strongly influenced by the nature
and stereochemistry of the amino acid side chains at
2
. Results and discussion
Several routes towards (2S,3S)-2-hydroxy-3-amino
1
4
both the a- and the b-position. It is important that the
substituents of the b-amino acid residues are placed
equatorial with respect to the helical axis to minimise
steric hindrance with the adjacent turn.
2
acids have been described in the literature. An earlier
21
route developed in our laboratories proved to give
low diastereoselectivity for hindered side groups (d.e.
22
5
20%). Herein, we present a short chemoenzymatic
route towards Fmoc-protected a-hydroxy-b-amino
acids, starting from 2-furaldehyde, thereby increasing
the d.e. and simultaneously decreasing the number of
So far, the effect of polar a-side chains on the stability
of the secondary structures of b-peptides has scarcely
been investigated. Seebach et al. synthesised helix form-
23
2,3
steps (Scheme 1).
ing oligopeptides containing two (2R,3R)-b -amino
1
5
16
acids with two serine or cysteine side chains.
Enantioselective addition of HCN to 2-furaldehyde
catalysed by R-oxynitrilase (E.C. 4.1.2.10) (as present
24
in defatted almond meal) afforded the cyanohydrin
with high enantiomeric excess (e.e.=98%). Subsequent
silyl protection using TBS-Cl and imidazole in DMF
*
0
Corresponding author. E-mail: brussee@chem.leidenuniv.nl
957-4166/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(01)00193-8

1
110
R. A. Tromp et al. / Tetrahedron: Asymmetry 12 (2001) 1109–1112
Scheme 1. (a) R-Oxynitrilase, HCN, MTBE, pH 5.5, 4°C; (b) TBS-Cl (1.2 equiv.), imidazole (2.4 equiv.), DMF, 0°C to rt; (c) 1.
RMgX (1.2 equiv.), Et O, reflux; 2. MeOH, −20°C; 3. NaBH (2 equiv.), −70°C to rt; (d) Fmoc-Cl (1.4 equiv.), CH Cl , NaHCO
3
2
4
2
2
(
aq.), 0°C to rt; (e) 1. O , MeOH, −70°C; 2. THF/MeCN/H O, 40°C, 3 h; (f) Rink amide MBHA, HOBt, BOP, DIPEA and NMP.
3 2
2
5
gave 2 in high yield (88% from 1). Next, the amino
acid side chains for the required a-hydroxylated b-ala-
nine, b-valine and b-leucine were introduced by Grig-
nard reaction. The silyl protected cyanohydrin 2 was
dissolved in diethyl ether and a solution of the Grig-
nard reagent RMgX (R=Me, iso-Pr, iso-Bu; X=I, Cl)
was added dropwise. After 1 h of reflux, the mixture
was cooled to −20°C. Dry methanol was added to
destroy excess Grignard reagent and liberate the free
imine. The solution was further cooled to −70°C, after
which the amino function was obtained by reduction
with sodium borohydride, which occurred with good
to stir for 3 h in THF/MeCN/H O (3/1/1) at 40°C. The
2
required amino acids 4a–4c could be obtained in pure
form and with high d.e. by crystallisation from ethyl
acetate and pentane in 61, 52 and 61% yield, respec-
tively. After one recrystallisation, no diastereomeric
1
impurities were visible in the H NMR spectrum.
The N-protected a-hydroxy-b-amino acids 4a–4c were
used in the construction of a novel a-hydroxylated
b-hexapeptide by solid-phase synthesis using Rink
amide MBHA as resin, without prior protection of the
hydroxyl function. Conventional procedures for solid-
3
2
2
6,27
phase peptide synthesis were applied (Scheme 1).
selectivity (Table 1).
The crude amino alcohols were
Remarkably, after lyophilisation, the newly formed b-
hexapeptide proved to be slightly soluble in most sol-
vents, including water, methanol, acetonitrile,
chloroform, ethyl acetate, 2,2,2-trifluoroethanol and
hexafluoro-acetone. It could be dissolved in pyridine
and TFA. So, impurities could be easily removed by
washing the peptide with water/acetonitrile (80/20)
yielding the pure b-hexapeptide (36%), as confirmed by
LCMS and NMR spectroscopy.
then protected with 9-fluorenylmethyloxocarbonyl
(
3
Fmoc) to give the fully protected amino alcohols 3a–
c. Valine analogue 3b was initially obtained in only
2
0–25% (from 2). The yield was greatly improved (48%)
by addition of a catalytic amount of Cu(I)Br, using
2
8
THF as solvent, and by using 2 equiv. of Fmoc-Cl
and 1 equiv. of DIPEA in dry dichloromethane for
2
9
protection of the amino group.
Finally, the Fmoc-protected a-hydroxy-b-amino acids
were obtained by a one-pot oxidation–deprotection
procedure. Upon ozonolysis of the furan ring of com-
1
H NMR spectra (in pyridine-d ) showed all methyl
5
groups resolved. Also, the protons of the methylene
group of each leucine residue were found to have
different chemical shifts, indicating restricted mobility.
30
pounds 3a–3c at low temperature, the TBS group
partly migrated to the newly formed carboxylic acid,
due to the presence of the amino group at the b-posi-
3
1
tion. To force the deprotection to completion, the
solvent was evaporated and the crude product was left
Table 2. Chemical shifts and coupling constants of the
amide and H protons of hexapeptide 5
a
b
Table 1. Diastereoselectivity of the Grignard addition–
l (NH)a
J(NH,Hb)b
l (Ha)a
J(H ,H )
a
b
reduction sequence
Val1
5.10
4.76
4.81
4.70
4.82
4.81
4.4
3.7
3.0
4.4
3.0
3.0
a
2
R
(2S,3S)/(2S,3R)-3 (%)
Ala
8.85
8.42
8.32
8.45
8.39
8.6
9.4
9.7
8.7
9.3
3
Leu
4
3a
3b
3c
Me
iso-Pr
iso-Bu
19/1 (80)
9/1 (48)
5/1 (61)
Val
Ala
5
6
Leu
a
a
(
2S,3S)/(2S,3R)-Ratio determined by NMR.
Chemical shift (l) in ppm.
Coupling constant J in Hz.
b
Yields (in parenthesis) after purification from 2.

R. A. Tromp et al. / Tetrahedron: Asymmetry 12 (2001) 1109–1112
6,17,33,34
1111
1
As has been found by other groups,
NH peaks
Acknowledgements
all contained coupling constants ranging between 8.6
and 9.7 Hz (Table 2). The coupling constant between
Ha,i and Hb,i proved to be in the range of 3–4.4 Hz,
unlike those reported for the helix forming b-peptides,
We thank Nico Meeuwenoord, Hans van der Elst and
Cees Erkelens for their assistance with solid-phase syn-
thesis, LCMS and NMR spectroscopy.
2
,3
including the a-methylated b -hexapeptide by Seebach
2
0
et al. (J=9.3–11.3 Hz). Except for the NH protons at
2
the C-terminus, no interactions between amide protons
were observed by NOESY experiments (Fig. 1). Fur-
thermore, NOESY experiments indicated that no long-
range interactions between residues i and i+2 or i+3
were present, as has been reported for folded b-pep-
References
1. Abele, S.; Seebach, D. Eur. J. Org. Chem. 2000, 1–15.
2. Andruszkiewicz, R. Pol. J. Chem. 1998, 72, 1–48.
3. Juaristi, E. Enantioselective Synthesis of i-Amino Acids;
Wiley-VCH: New York, 1997.
1
6,20,33–35
tides, including helices.
Thus, although the stereochemistry of the residues was
chosen to enable helix formation, first findings indicate
that in pyridine a stable secondary structure is formed,
which is probably not helical in character. Currently we
are investigating the nature of the secondary structure
using extensive NMR techniques (e.g. NOESY and
ROESY) supported by calculations.
4. Rowinsky, E. K. Annu. Rev. Med. 1997, 48, 353–374.
5. Nicolaou, K. C.; Dai, W.-M.; Guy, R. K. Angew. Chem.,
Int. Ed. Engl. 1994, 33, 15–44.
6. Umezawa, H.; Aoyagi, T.; Suda, H.; Hamada, M.;
Takeuchi, T. J. Antibiot. 1976, 29, 97–99.
7. Kise, N.; Ueda, N. J. Org. Chem. 1999, 64, 7511–7514.
8. Murayama, T.; Kobayashi, T.; Miura, T. Tetrahedron
Lett. 1995, 36, 3703–3706.
9. DeGrado, W. F.; Schneider, J. P.; Hamuro, Y. J. Peptide
Res. 1999, 54, 206–217.
0. Gademann, K.; Hintermann, T.; Schreiber, J. V. Curr.
3. Conclusions
1
Med. Chem. 1999, 6, 605–925.
In summary, a new straightforward and stereoselective
route towards Fmoc-protected (2S,3S)-2-hydroxy-3-
amino acids has been developed, starting from 2-
furaldehyde. It has been demonstrated that these
a-hydroxy-b-amino acids can be used for solid-phase
synthesis of a-hydroxylated b-oligopeptides without
protection at the hydroxyl function. NMR studies on
the resulting a-hydroxylated b-hexapeptide gave strong
indications that in pyridine no helical structure was
formed. Thus, the presence of unprotected a-hydroxyl
groups have a great influence on the formation of the
secondary structure.
1
1
1. Gellman, S. H. Acc. Chem. Res. 1998, 31, 173–180.
2. Seebach, D.; Overhand, M.; K u¨ hnle, F. N. M.; Marti-
noni, B.; Oberer, L.; Hommel, U.; Widmer, H. Helv.
Chim. Acta 1996, 79, 913–941.
1
3. Appella, D. H.; Christianson, L. A.; Karle, I. L.; Powell,
D. R.; Gellman, S. H. J. Am. Chem. Soc. 1996, 118,
13071–13072.
1
4. Seebach, D.; Ciceri, P. E.; Overhand, M.; Jaun, B.; Rigo,
D.; Oberer, L.; Hommel, U.; Amstutz, R.; Widmer, H.
Helv. Chim. Acta 1996, 79, 2043–2066.
2,3
1
1
5. b : b-Amino acid carrying a side chain at both the two
and the three position.
6. Seebach, D.; Jacobi, A.; Rueping, M.; Gademann, K.;
Ernst, M.; Juan, B. Helv. Chim. Acta 2001, 83, 2115–
2140.
17. Motorina, I. A.; Huel, C.; Quiniou, E.; Mispelter, J.;
Adjadj, E.; Grierson, D. S. J. Am. Chem. Soc. 2001, 123,
8–17.
1
1
2
8. Wen, K.; Han, H.; Hoffman, T. Z.; Janda, K. D.; Orgel,
L. E. Bioorg. Med. Chem. Lett. 2001, 11, 689–691.
9. Three residues per turn, the hydrogen bonds closing
14-membered rings.
0. Seebach, D.; Abele, S.; Gademann, K.; Giuchard, G.;
Hintermann, T.; Juan, B.; Matthews, J. L.; Schreiber, J.
V.; Oberer, L.; Hommel, U.; Widmer, H. Helv. Chim.
Acta 1998, 81, 932–982.
21. Warmerdam, E. G. J. C.; van Rijn, R. D.; Brussee, J.;
Kruse, C. G.; van der Gen, A. Tetrahedron: Asymmetry
1996, 7, 1723–1732.
2
2
2. Unpublished results.
3. Compounds were characterised by H, C NMR, IR and
1
13
mass spectroscopy. Hexapeptide 5 was characterised by
1
LCMS and H NMR.
24. Zandbergen, P.; van der Linden, J.; Brussee, J.; van der
Gen, A. Synth. Commun. 1991, 21, 1387–1391.
Figure 1. NH region of the NOESY spectrum (600 MHz) of
hexapeptide 5 in pyridine-d5.
25. Brussee, J.; Roos, E. C.; van der Gen, A. Tetrahedron
Lett. 1988, 29, 4485–4488.

1
112
R. A. Tromp et al. / Tetrahedron: Asymmetry 12 (2001) 1109–1112
2
2
6. Effenberger, F.; Eichhorn, J. Tetrahedron: Asymmetry
997, 8, 469–476.
32. Rink amide MBHA (50 mmol) was used as the resin and
1
HOBt, BOP and DIPEA as coupling reagents with NMP
as solvent. The Fmoc group was cleaved by piperidine. Per
coupling step 4 equiv. of amino acid were used with respect
to the resin. The a-hydroxylated b-hexapeptide was cleaved
7. Formation of the (2S,3S)-diastereoisomer was established
by synthesis of an oxazolidinone from the Boc-protected
analogue of 3b (R=iso-Pr), via desilylation and NaH
assisted ring closure. The coupling constant (7.7 Hz) of the
vicinal protons was in accordance with literature values.
8. Weiberth, F. J.; Hall, S. S. J. Org. Chem. 1987, 52,
from the solid support by stirring the resin in TFA/H O
2
(9/1) for 1 h.
2
2
3
3
33. Gung, B. W.; Zou, D.; Miyahara, Y. Tetrahedron 2000, 56,
9739–9746 and references cited therein.
34. Chung, Y. J.; Huck, B. R.; Christianson, L. A.; Stanger,
H. E.; Krauth a¨ user, S.; Powell, D. R.; Gelmann, S. H. J.
Am. Chem. Soc. 2000, 122, 3995–4004 and references cited
therein.
3
901–3904.
9. Pfeifer, M. E.; Linden, A.; Robinson, J. A. Helv. Chim.
Acta 1997, 80, 1513–1527.
0. Bednarski, M.; Danishefsky, S. J. Am. Chem. Soc. 1986,
1
08, 7060–7067.
1. Greco, M. N.; Zhong, H. M.; Maryanoff, B. E. Tetra-
hedron Lett. 1998, 39, 4959–4962.
35. Wang, X.; Espinosa, J. F.; Gelmann, S. H. J. Am. Chem.
Soc. 2000, 12, 4821–4822 and references cited therein.
.