Synthesis of alanine and proline amino acids with amino or guanidinium substitution on the side chain

Article abstract of DOI:10.1016/S0040-4020(00)00123-X

Competitive binding of peptides containing basic amino acids to disrupt or prevent the Tat-TAR interaction could result in diminished transcription as well as translation and hence constitutes an alternative way of controlling HIV replication. Therefore, we synthesized guanidinium and amino containing amino acids, based on a proline or an alanine scaffold. The introduction of the guanidinium moiety was best accomplished using 1H- pyrazole-1-carboxamidine hydrochloride, with Pmc used for its protection. The absence of racemization, maintained throughout the whole synthesis, was confirmed by chiral purity determination. These building blocks were smoothly incorporated into oligopeptides, which proved their suitability for use in a combinatorial approach for selecting TAR binding ligands. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4020(00)00123-X

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 2513–2522
Synthesis of Alanine and Proline Amino Acids with Amino or
Guanidinium Substitution on the Side Chain
Zhenyu Zhang,a Arthur Van Aerschot,a Chris Hendrix,a Roger Busson,a Frank David,b Pat Sandrab
and Piet Herdewijna,
*
aLaboratory of Medicinal Chemistry, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10,
B-3000 Leuven, Belgium
bResearch Institute for Chromatography, Kennedypark 20, B-8500 Kortrijk, Belgium
Received 2 November 1999; revised 24 January 2000; accepted 10 February 2000
Abstract—Competitive binding of peptides containing basic amino acids to disrupt or prevent the Tat–TAR interaction could result in
diminished transcription as well as translation and hence constitutes an alternative way of controlling HIV replication. Therefore, we
synthesized guanidinium and amino containing amino acids, based on a proline or an alanine scaffold. The introduction of the guanidinium
moiety was best accomplished using 1H-pyrazole-1-carboxamidine hydrochloride, with Pmc used for its protection. The absence of
racemization, maintained throughout the whole synthesis, was confirmed by chiral purity determination. These building blocks were
smoothly incorporated into oligopeptides, which proved their suitability for use in a combinatorial approach for selecting TAR binding
ligands. ᭧ 2000 Elsevier Science Ltd. All rights reserved.
Introduction
transcriptional elongation by binding to a region near
the start of transcription in the viral long terminal repeat
(LTR) called the trans-activation response element.4,5 The
regulatory protein is one of the important viral proteins,
which have scarcely been used as targets. A lower rate of
resistance against TAR-binding compounds can be
expected because the drugs would act at the level of gene
expression and Tat–TAR interactions are highly
conserved.4 Moreover, mutations in the protein-binding
site of the RNA will (most probably) result in replication
of incompetent viruses.
In the past decade, remarkable clinical success in the field of
target-directed AIDS treatment has been achieved with
drugs against two important enzymes in the HIV-1 life
cycle, the reverse transcriptase and the HIV protease.1,2
However, the rapid development of viral resistance against
existing drugs has stimulated the exploration of alternative
targets.3 One of these targets is the viral trans-activator
protein, Tat. This regulatory protein is required early in
the viral life cycle to increase the efficiency of
Figure 1. The basic region of Tat protein induces a local conformational rearrangement, which enhances the accessibility of unfunctional groups on TAR RNA.
Keywords: guanidinium substitution; Tat–TAR interaction; HIV replication.
* Corresponding author. Tel.: ϩ32-16-33-73-87; fax: ϩ32-16-33-73-40; e-mail: piet.herdewijn@rega.kuleuven.ac.be
0040–4020/00/$ - see front matter ᭧ 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00123-X

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Z. Zhang et al. / Tetrahedron 56 (2000) 2513–2522
Figure 2. Orthogonal protected basic amino acids using proline 1, 2 or alanine 3a, 4a, 3b, 4b as scaffold.
During past years, the interaction of Tat protein with TAR
RNA has been studied in more detail. NMR studies showed
that the accessibility of functional groups on TAR RNA to
be recognized by Tat protein is enhanced by a local confor-
mational rearrangement.6–8 This folding process is induced
by the basic region of Tat, which comprises a series of eight
positively charged residues located within a stretch of nine
amino acids4 (Fig. 1). Following this observation, it has
been suggested8 that ligands carrying the basic guanidinium
group may bind to TAR RNA and induce folding. As a
result the ligand may block the regulatory function of the
Tat–TAR complex.
In a first step we plan to develop libraries of unnatural
peptides containing one or more positively charged
residues. Therefore we synthesized orthogonal protected
amino acid analogues containing a basic functionality
(amino or guanidine group) on the side chain. These
amino acids are structurally based on a proline (1, 2) or
alanine (3a, 3b, 4a, 4b) scaffold (Fig. 2).
All building blocks were functionalized to be incorporated
into peptides following the Fmoc-strategy. During the total
synthesis the enantiomeric purity of these residues was
properly retained as confirmed by chiral purity analysis.
To evaluate the potential use of these modified a-amino
acids as components in an oligopeptide library, the modified
amino acids were incorporated in a peptide sequence.
Following deprotection and cleavage from the solid support,
the unnatural peptide was analyzed for its purity and correct
structure, and the yield of incorporation was determined.
This work lets us conclude that the newly described
amino acids may be useful building blocks in a potential
anti-Tat library.
Because of the complex interaction pattern involved in RNA
structure organization and folding, de novo design of
RNA-binding ligands is very difficult. Combinatorial
chemistry might prove to be an alternative.9–12 This
approach allows the simultaneous synthesis of a large
number of compounds that can be used for screening for
TAR-binding properties.
Since the library components are envisaged to compete with
Tat for binding to TAR RNA, it seems reasonable to intro-
duce functional groups interacting with TAR RNA via a
similar mechanism. The basic region of Tat protein is
extremely important for Tat–TAR RNA sequence specific
interaction.4,8 In this region eight positively charged
amino acids are found, incorporating two lysine and six
arginine residues. Therefore, a first library approach to
select TAR binding compounds may be based on a selection
of various positively charged and structurally more
constrained residues that can be assembled in a peptide-
like structure.
Results and Discussion
The synthesis of 4-amino-N-benzyloxycarbonyl-l-cis-
proline, (5) has been previously reported.13 Since peptide
assembly using the Fmoc-strategy was envisaged, the Boc
group was used to protect the primary amino group of 5.
Therefore, 5 was reacted with ditertbutyl dicarbonate in the
presence of 10% Na2CO3, affording 6 in 93% yield.
Removal of the Z group by catalytic hydrogenation enabled
the final protection of the a-amine with Fmoc-Cl under
Scheme 1. (i) 1.2 equiv. (Boc)2O in 10% Na2CO3 and dioxane; 93%, (ii) a. H2, Pd/C in MeOH and H2O, 45 psi; b. 1.2 equiv. Fmoc-Cl in 10% Na2CO3 and
dioxane; 81%.

Z. Zhang et al. / Tetrahedron 56 (2000) 2513–2522
2515
Scheme 2. (i) 1.2 equiv. Z-Cl in 10% Na2CO3 and dioxane; 84%, (ii) 1.5 equiv. [bis(trifluroacetoxy)iodo]-benzene in DMF/H2O (1:1) and pyridine; 47%, (iii)
1.2 equiv. (Boc)2O in 10% Na2CO3 and dioxane; 91%, (iv) a. H2, Pd/C in MeOH and H2O, 45 psi; b. 1.2 equiv. Fmoc-Cl in 10% Na2CO3 and dioxane; 81%.
Schotten–Baumann conditions. The desired product 1 was
obtained in 81% yield (Scheme 1).
material from ether. The b-amino group was protected with
a Boc group to yield 10. The Z group was cleaved and
replaced with Fmoc to afford 3a in 80% yield.
For the synthesis of the amino analogue 3, we first started
from serine. However, mesylation of its b-alcohol group
easily led to an elimination reaction. Therefore we investi-
gated opening of serine-b-lactone with ammonia.14 This led
predominantly to formation of the amide of serine instead of
the desired b-amino alanine derivative. Finally, we utilized
a modified Hoffmann rearrangement as reported by Waki
et al.,15 starting from l-asparagine (7) (Scheme 2). Here also
the a-amino function of asparagine was protected with a
benzyloxycarbonyl group to give 8 in a yield of 84%.
This derivative was converted into the desired diamino-
propionic acid by addition of a solution of [bis(trifluro-
acetoxy)iodo]-benzene in a mixture of DMF-water (1:1)
and pyridine and 9 was obtained in 47% yield as crystalline
Several approaches for the conversion of an amino group
into a guanidine function have been reported in literature.16
Guanylation via the formation of a carbodiimide inter-
mediate, prepared in situ by reaction of di-Boc protected
thiourea with Mukaiyama’s reagent16,17 or with HgCl2,18
has been widely used. More recently, triflated di-Boc
guanidine was reported19 to effectively carry out guanidine
formation starting from an amine. However, both
approaches suffer from an unpleasant and complicated
manipulation. Moreover a di-Boc protected approach was
not very attractive to us as it rendered the guanidine moiety
less stable during subsequent reactions (often the guanidine
group was degraded to yield the starting amino analogue).20
Scheme 3. (i) 1 equiv. 1H-pyrazole-1-carboxamidine hydrochloride in 1.0 M Na2CO3; 40%, (ii) 1.8 equiv. Pmc-Cl in 3.2 M NaOH and acetone at 0ЊC; 50%,
(iii) a. H2, Pd/C in MeOH and H2O, 45 psi; b. 1.2 equiv. Fmoc-Cl in 10% Na2CO3 and dioxane; 81%.
Scheme 4. (i) 1 equiv. 1H-pyrazole-1-carboxamidine hydrochloride in 1.0 M Na2CO3; 40%, (ii) 1.8 equiv. Pmc-Cl in 3.2 M NaOH and acetone at 0ЊC; 52%,
(iii) a. H2, Pd/C in MeOH and H2O, 45 psi; b. 1.2 equiv. Fmoc-Cl in 10% Na2CO3 and dioxane; 81%.

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Z. Zhang et al. / Tetrahedron 56 (2000) 2513–2522
Figure 3. Analysis of the enantiomeric purity of 4a(l) and 4b(d) by using HPLC (Chirobiotic T-5 mm column).
The reaction could be successfully carried out using 1H-
pyrazole-1-carboxamidine hydrochloride as transferable
amidine moiety.21 After 3 h reaction of 5 with the pyrazole
reagent in 1.0 M Na2CO3 at room temperature, 11 was
obtained in 40% yield (Scheme 3). In a similar way, 9
was converted to 12 (Scheme 4).
with these groups, such as the 4-methoxy-2,3,6-trimethyl-
benzenesulfonyl group (Mtr), have shown a slow coupling
and cleavage rate in solid phase synthesis.24 More recently
the pentamethylchroman sulfonyl group (Pmc) was
developed.25 It inherits the advantage of the aryl sulfonyl
group with respect to the suppression of side reactions and
demonstrates facile cleavage under mild acidic conditions.
Therefore, we introduced the Pmc group to the guanidine
moieties of 11 and 12 under strongly alkaline conditions
(in 3.2 M NaOH) to afford 13 and 14, respectively, in
50% yield (Schemes 3 and 4). Following removal of the Z
group, the a-amine was protected with Fmoc to yield 2 and
4a, respectively, in 80%.
For the protection of guanidine moieties, three types of
protecting groups are available: a nitro group, a urethane
group or an arylsulfonyl group. The nitro-protected arginine
is prone to numerous side reactions during synthesis, as well
as acylation and cleavage in solid phase synthesis,22
whereas urethane-protected arginine suffers from side
chain acylation and subsequent ornithine formation under
piperidine treatment in Fmoc-strategy-based solid phase
synthesis.23 Because of these limitations, arylsulfonyl
groups have been used widely since they can suppress
side reactions to a minimum. However, arginines protected
In order to evaluate whether introduction of the Pmc group
under strongly basic conditions does not induce racemiza-
tion, the chiral purity of the final compounds was verified.
Hence, both l and d enantiomers of the alanine derivative
Figure 4. Structure of synthetic peptide consisting of amino–alanine, amino–proline, guanidino–alanine, guanidino–proline, phenylalanine, tyrosine, isoni-
pecotic acid and b-alanine monomers.

Z. Zhang et al. / Tetrahedron 56 (2000) 2513–2522
Table 1. Coupling yields of the amino acids in solid phase peptide synthesis
2517
Peptide 1
Coupling yield (%)
Peptide 2
Gly
90
Gly
90
b-Ala
97.2
b-Ala
97.2
Inp
103.8
Inp
l-Tyr
102
l-Tyr
102
l-Pro-NH2 (1)
95.3
d-Phe
105
d-Phe
105
l-Ala-NH2 (3a)
97
l-Pro-Gua (2)
l-Ala-Gua (4a)
Coupling yield (%)
103.8
77a/103
77a/93
a First figure is the yield from a single coupling step using HOAt/DIC activation, second figure represents the yield after a second coupling cycle.
(4a, 4b) were prepared and subjected to chiral purity
determination by liquid chromatography. In both cases,
the spectra showed a single peak, while two well-separated
peaks for the mixture of both isomers were obtained (Fig. 3).
It is therefore clear that the enantiomeric purity of those
compounds was sufficiently retained during the Pmc
protection step as well as throughout the whole synthetic
process.
Yield was determined using UV absorption of the piper-
idine-dibenzofulvene adduct. For most of the amino acids
used, one coupling step was enough to obtain a yield of ca.
90%. The arginine analogues required a second coupling
cycle to attain a yield of ca. 95% (Table 1). The yield
over 100% is most probably due to the errors in the weight
of small amounts of loaded beads. A capping and Fmoc
deprotection reaction completed each coupling cycle.
Since the newly synthesized amino acids are to be used for
the assembly of peptide libraries, their incorporation and
deprotection efficiencies need to be controlled. Therefore,
1, 2, 3a and 4a were incorporated into two test peptides (Fig.
4), along with other amino acids, intended for use in our
library. Assembly on solid support afforded good yield
using HOAt/DIC activation.
Upon 90 min of treatment with the TFA-based cleavage
cocktail containing thioanisole as scavenger, the protecting
groups (Pmc, Boc, and tBu) were removed and the peptides
were released from the resin. The HPLC profile of the
crude reaction mixture indicated that one major
compound was formed (Fig. 5) (the peak which comes out
with 76% B corresponds to thioanisole as verified by mass
Figure 5. HPLC profiles of the peptides released from resin using a linear gradient of A: 5% CH3CN in H2Oϩ0.1% TFA; and B: 80% CH3CN in H2Oϩ0.1%
TFA; flow rate: 1 ml/min.
Figure 6. Mass spectra analysis of the major peaks isolated from HPLC purification proved the correct constitution of the peptides.

2518
Z. Zhang et al. / Tetrahedron 56 (2000) 2513–2522
measurement). Subsequent analysis by mass spectrometry
proved this major compound corresponded to the desired
peptide sequence (Fig. 6), indicating successful incorpora-
tion and deprotection of the new amino and guanidinium
containing amino acids.
N-Fmoc-isonipecotic acid was synthesized from isoni-
pecotic acid, which was purchased from ACROS (Geel,
Belgium).
4-N-tert-Butyloxycarbonyl-N-benzyloxycarbonyl-l-cis-
proline (6). To a solution of 5.02 g (19 mmol) of 5 (13) in
29 ml of 10% Na2CO3 were added 19 ml of 1,4-dioxane and
4.97 g (23 mmol) of di-tert-butyldicarbonate at 0ЊC. The
reaction mixture was stirred overnight at room temperature
and was poured into 120 ml of H2O after which the mixture
was washed three times with diethyl ether. The aqueous
layer was acidified with 2N HCl and the white suspension
was extracted three times with ethyl acetate. The combined
ethyl acetate layer was dried on anhydrous MgSO4, filtered
and evaporated to dryness. The crude product was crystal-
lized from diethyl ether to afford compound 6 (6.5 g,
17.8 mmol, 93%). Mp 136ЊC; nmax 3328, 2976 (NH, OH),
1694 (CvO), 1530, 1365, 12453, 738, 697 cmϪ1. 1H NMR
(CDCl3) d 1.41 (s, 9H, Boc-CH3), 1.90–2.65 (m, 2H, 3-H),
3.29–3.90 (m, 2H, 5-H), 4.10–4.60 (m, 2H, 2-H, 4-H),
5.00–5.29 (m, 2H, Z-CH2), 7.29, 7.34 (two s, 5H,
Z-aromatic) ppm. 13C NMR (CDCl3) d 175.00 (COOH),
155.45 (2×OCON), 136.39 (Z-ar-Ci), 128.59, 128.14,
127.74 (Z-ar-Co,Cm,Cp), 79.82 (Boc-C), 68.10, 67.22
(Z-CH2), 58.60 (C-2), 53.26 (C-5), 49.86 (C-4), 36.90,
34.38 (C-3), 28.25 (Boc-CH3) ppm. Exact mass (LSIMS,
diethanolamine) calcd for C18H23N2O6 [MϪH]Ϫ
363.15560; found 363.15506; elem. anal. for C18H24N2O6
calcd C 59.33, H 6.64, N, 7.69; found C 59.22, H 6.73, N
7.50.
Conclusion
A set of new chirally pure, orthogonally protected, basic
amino acid analogues was synthesized. Their successful
incorporation in a peptide was demonstrated. These new
building blocks will be used in a potential anti-Tat
library.
Experimental
1
The H and 13C NMR spectra were recorded with a Varian
1
Gemini 200 spectrometer in the solvent indicated. For H
spectra tetramethylsilane (TMS) (for DMSO-d6 the solvent
signal at 2.5 ppm) was used as reference. For the 13C spectra
the center peak of the solvent multiplet, set at 77.00 ppm for
CDCl3 and at 39.60 ppm for DMSO-d6, was used. In the
case of Na-protected proline derivatives, some peaks are
double due to the presence of amide rotamers as a result
of restricted rotation. Exact mass measurements by liquid
secondary ion mass spectrometry (LSIMS) were obtained
using a Kratos Concept 1H mass spectrometer, except for
peptide 1 and peptide 2, which were analyzed on a PE
SCIEX QSTAR௣ Hybrid Quadrupole TOF System. Optical
Rotation was measured with a Thorn NPL Automatic
Polarimeter Type 243 at a temperature of 25ЊC in MeOH-
dichloromethane (90/10) and MeOH for 3a/3b and 4a/4b,
respectively. Infrared resonance was measured with a
Spectrum௣ RX I FT-IR System in reflectance mode. Melt-
ing point was measured with a Bu¨chi SMP-20. Precoated
Machery-Nagel Alugram᭨ SilG/UV 254 plates were used
for TLC and spots were examined with UV light, sulfuric
acid/anisaldehyde spray, ninhydrine spray or Sakaguchi
reagent. Column chromatography was performed on Acros
silica gel (0.06–200 nm). Anhydrous solvents were
obtained as follows: Pyridine and N,N-diisopropylethyl-
amine (DIEA) were refluxed overnight over potassium
hydroxide and distilled. N,N-dimethylformamide (DMF)
was stored on activated molecular sieves for 3 days and
was tested for the absence of dimethylamine by the bromo-
phenol test prior to use. CH3CN for HPLC was purchased
from Rathburn (grade S) and water for HPLC purification
was distilled twice. Tentagel-NH2 was obtained from
RAPP-Polymere (Tubingen, Germany). Dichloromethane
(DCM), N,N-dimethylformamide, acetic anhydride (Ac2O)
and pyridine were obtained from BDH (Poole, England).
1-Hydroxy-7-azabenzotriazole (HOAt), Fmoc-b-alanine
(b-Ala), Fmoc-glycine (Gly), Fmoc-d-phenylalanine
(d-Phe), Fmoc-O-t-butyl-l-tyrosine (l-Tyr) were purchased
from Advanced ChemTech (Louisville, Kentucky).
Asparagine (l or d), di-tert-butyldicarbonate [(Boc)2O],
[bis(trifluoro-acetoxy)iodo]benzene, benzyl chloroformate,
9-fluorenylmethyloxycarbonyl chloride, 1H-pyrazole-1-
carboxamidine, piperidine, trifluoroacetic acid (TFA),
diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole
(HOBt) were supplied by ACROS (Geel, Belgium). The
4-N-tert-Butyloxycarbonyl-N-9-fluorenylmethyloxy-
carbonyl-l-cis-proline (1). Nitrogen was bubbled for
10 min through a solution of 5.00 g (13.7 mmol) of 6 in
45 ml of methanol and 30 ml of H2O. To this solution was
added 1 g of palladium on charcoal (10%). The suspension
was hydrogenated overnight at 45 psi. After filtration, the
obtained solution was evaporated to dryness. The residue
was dissolved in 30 ml of 10% Na2CO3 in H2O. To the
solution was added 18 ml of 1,4-dioxane and 3.87 g
(15 mmol) of 9-fluorenylmethyloxycarbonyl chloride at
0ЊC. The reaction mixture was stirred overnight and then
poured into 100 ml of H2O and the solution was extracted
three times with diethyl ether. The aqueous layer was acid-
ified with a 2N HCl in water and the white suspension was
extracted three times with ethyl acetate. The combined
organic layer was dried on anhydrous MgSO4, filtered and
evaporated to afford compound 1 (5.02 g, 11.1 mmol, 81%).
Mp 104ЊC; nmax 3331, 2974 (NH, OH), 1702 (CvO), 1522,
1
1364, 1249, 738 cmϪ1. H NMR (CDCl3) d 1.43 (s, 9H,
Boc-CH3), 1.90–2.65 (m, 2H, 3-H), 3.30–3.90 (m, 2H,
5-H), 4.08–4.61 (m, 5H, 2-H, 4-H, Fmoc-CH2, Fmoc-90-
H), 7.20–7.80 (m, 8H, Fmoc-aromatic) ppm. 13C NMR
(CDCl3) d 174.97 (COOH), 157.02, 156.33, 155.45,
154.48 (OCON), 143.74 (Fmoc-100), 141.28 (Fmoc-110),
127.77 (Fmoc-30), 127.10 (Fmoc-20), 125.13 (Fmoc-10),
119.97 (Fmoc-40), 79.85 (Boc-C), 68.32, 67.83 (Fmoc-
CH2), 58.45 (C-2), 53.60, 53.26 (C-5), 49.95 (C-4), 46.95
(Fmoc-90), 37.18, 34.69 (C-3), 28.25 (Boc-CH3) ppm. Exact
mass (LSIMS, diethanolamine) calcd for C25H27N2O6
[MϪH]Ϫ 451.18690; found 451.18742; elem. anal. for
C25H28N2O6·C4H8O2 (1,4-dioxane) calcd C 64.43, H 6.71,
N 5.18; found C 64.75, H 6.85, N 4.90.

Z. Zhang et al. / Tetrahedron 56 (2000) 2513–2522
2519
Na-Benzyloxycarbonyl-l-asparagine (8). To a solution of
6.05 g (40 mmol) of l-asparagine 7 in 100 ml of 10%
Na2CO3 were added 56 ml of 1,4-dioxane and 6.9 ml
(48 mmol) of benzyl chloroformate at 0ЊC. The reaction
mixture was stirred overnight at room temperature and
was poured into 300 ml of H2O. The mixture was washed
three times with diethyl ether. The aqueous layer was acid-
ified with 2N HCl in water and a white precipitate fell out.
The precipitate was filtered off and washed extensively with
diethyl ether to afford compound 8 (9 g, 33.8 mmol, 84%).
Mp 164ЊC; nmax 3402, 3333 (NH), 1695 (CvO), 1638,
9H, Boc-CH3), 3.25–3.75 (br, 2H, b-H), 4.25–4.50 (br,
1H, a-H), 5.11 (s, 2H, Z-CH2), 7.32 (s, 5H, Z-aromatic)
ppm.
13C
NMR
(CDCl3)
d
173.57(COOH),
157.09(OCONH), 136.06(Z-ar-Ci), 128.53, 128.14(Z-ar-
Co,Cm,Cp), 82.03, 80.39 (Boc-C), 67.14 (Z-CH2), 55.08,
54.29 (C-a), 42.97, 42.06 (C-b), 28.07 (Boc-CH3) ppm.
Exact mass (LSIMS, glycerol) calcd for C16H21N2O6
[MϪH]Ϫ 337.13995; found 337.13980; elem. anal. for
C16H22N2O6·0.5 H2O calcd C 55.32, H 6.67, N 8.06; found
C 55.59, H 6.49, N 7.94.
1534, 735, 694 cmϪ1. H NMR (DMSO-d6) d 2.36–2.68
Na-(9-Fluorenylmethyloxycarbonyl)-Nb-tert-butyloxy-
carbonyl-l-a-2,3-diaminopropionic acid (3a). Nitrogen
was bubbled for 10 min through a solution of 6.14 g
(18.2 mmol) of 10 in 50 ml of methanol and 20 ml of
H2O. To this solution was added 1.2 g of palladium on
charcoal (10%). The suspension was hydrogenated over-
night at 45 psi. After filtration, the obtained solution was
evaporated to dryness. The residue was dissolved in 45 ml
of 10% Na2CO3 in H2O and 25 ml of 1,4-dioxane and 5.17 g
(20.0 mmol) of 9-fluorenylmethyloxycarbonyl chloride
were added at 0ЊC. The reaction mixture was stirred over-
night and then poured into 100 ml of H2O and the solution
was extracted three times with diethyl ether. The aqueous
layer was acidified with a 2N HCl and the white suspension
was extracted three times with ethyl acetate. The combined
organic layer was dried on anhydrous MgSO4, filtered and
evaporated to afford compound 3a (6.2 g, 14.6 mmol, 80%).
Mp 129ЊC; nmax 3334, 2977 (NH, OH), 1698 (CvO), 1518,
1366, 1251 738 cmϪ1. 1H NMR (CDCl3) d 1.41 (s, 9H, Boc-
CH3), 3.30–3.62 (br, 2H, b-H), 4.00–4.60 (br, 4H, a-H,
Fmoc-CH2, Fmoc-90-H), 7.18-7.80 (m, 8H, aromatic)
ppm. 13C NMR (CDCl3) d 173.85 (COOH), 157.18,
156.76 (2×OCONH), 143.86 (Fmoc-100), 141.25 (Fmoc-
110), 127.68 (Fmoc-30), 127.08 (Fmoc-20), 125.16 (Fmoc-
10), 119.91 (Fmoc-40), 80.33 (Boc-C), 67.22 (Fmoc-CH2),
55.36 (C-a), 46.92 (Fmoc-90), 42.03 (C-b), 28.10 (Boc-
CH3) ppm. Exact mass (LSIMS, glycerol) calcd for
C23H25N2O6 [MϪH]Ϫ 425.17125; found 425.16973; elem.
anal. for C23H26N2O6·0.5 C4H8O2 (1,4-dioxane) calcd C
63.82, H 6.43, N 5.95; found C 63.70, H 6.38, N 5.63;
[a]2D5ˆϪ6.20 [cˆ0.72 in MeOH/DCM (90/10)]. Compound
3b with [a]2D5ˆϩ5.78 [cˆ0.70 in MeOH/DCM (90/10)].was
prepared from d-asparagine in exactly the same way as
described for 3a.
1
(dAB, 2H, Jˆ6.0 and 15.4 Hz, b-H), 4.30–4.50 (m, 1H,
a-H), 5.04 (s, 2H, Z-CH2), 6.97 (s, 1H, NH), 7.35–7.60
(m, 7H, aromatic and 2×NH’s) ppm. 13C NMR (DMSO-
d6) d 173.45 (COOH), 171.59 (C-g), 156.15 (OCONH),
137.21 (Z-ar-Ci), 128.62, 128.07, 127.95 (Z-ar-Co,Cm,Cp),
65.64 (Z-CH2), 50.80 (C-a), 36.87 (C-b) ppm. Exact mass
(LSIMS, thioglycerol:NaOAc) calcd for C12H14N2O5Na
[MϩNa]ϩ 289.08006; found 289.07812; elem. anal. for
C12H14N2O5 0.4 H2O calcd C 52.71, H 5.46, N 10.24;
found C 52.84, H 5.09, N 10.05.
Na-Benzyloxycarbonyl-l-2,3-diaminopropionic acid (9).
To a stirred solution of 25.8 g (60 mmol) of [bis(trifluro-
acetoxy)iodo]-benzene in 320 ml (1/1, v/v) of dimethyl-
formamide/water was added 10.64 g (40 mmol) of 8 at
room temperature. After 15 min, 6.4 ml (80 mmol) of
pyridine was added and stirring was continued for 4.5 h.
The solvent was evaporated in vacuo and the residue was
dissolved in 400 ml of water. The solution was washed
extensively with diethyl ether and concentrated in vacuo.
The residue was precipitated from ethanol/ether and purified
by column chromatography with methanol in DCM to afford
compound 9 (4.5 g, 18.9 mmol, 47%). Mp 229ЊC; nmax
3296, 2909 (NH), 1690 (CvO), 1589 (COOϪ), 1537
1
(NH), 738, 694 cmϪ1. H NMR (D2O) d 2.98–3.42 (dAB,
2H, Jˆ5.0 and 13.6 Hz, b-H), 4.25–4.40 (m, 1H, a-H),
4.89 (s, 2H, Z-CH2), 7.18 (s, 5H, Z-aromatic) ppm. 13C
NMR (D2O) d 171.81 (COOH), 157.82 (OCONH), 135.78
(Z-ar-Ci), 128.65, 128.38, 127.68 (Z-ar-Co,Cm,Cp), 67.31
(Z-CH2), 51.26 (C-a), 39.51 (C-b) ppm. Exact mass
(LSIMS, thioglycerol) calcd for C11H15N2O4 [MϩH]ϩ
239.10317; found 239.10742; elem. anal. for
C11H14N2O4·0.1 CF3COOH calcd C 53.89, H 5.69, N
11.22; found C 53.67, H 5.63, N 10.99 (CF3COOH is used
to keep the free amino function in a protonated form).
4-Guanidino-Na-benzyloxycarbonyl-l-cis-proline (11).
To a solution of 5.28 g (20 mmol) of 5 in 20 ml of 1.0 M
Na2CO3 in H2O was added 2.93 g (20 mmol) of 1H-pyra-
zole-1-carboxamidine hydrochloride. The reaction mixture
was stirred overnight at room temperature and neutralized to
pH 5–6 with 1N HCl. The solution was evaporated to
dryness and the residue was adsorbed on silica gel and
purified by column chromatography with methanol in
DCM yielding 2.45 g (8.0 mmol, 40%) of compound 11 as
a foam. All attempts for crystallization failed. nmax 3356,
Na-Benzyloxycarbonyl-Nb-tert-butyloxycarbonyl-l-a-
2,3-diaminopropionic acid (10). To a solution of 4.5 g
(19 mmol) of 9 in 47 ml of 10% Na2CO3 were added
28 ml of 1,4-dioxane and 6.18 g (28.4 mmol) of di-tert-
butyl dicarbonate at 0ЊC. The reaction mixture was stirred
overnight at room temperature and was poured into 100 ml
of H2O after which the mixture was washed three times with
diethyl ether. The aqueous layer was acidified with 2N HCl
in water and the white suspension was extracted three times
with ethyl acetate. The combined ethyl acetate layer was
dried on anhydrous MgSO4, filtered and evaporated to
dryness. The crude product was crystallized from diethyl
ether to afford compound 10 (5.8 g, 17.2 mmol, 91%). Mp
144ЊC; nmax 3332, 2976 (NH, OH), 1693 (CvO), 1514,
3196, 2920 (NH), 1682 (CvO), 1650, 1541, 770, 697 cmϪ1
.
1H NMR (DMSO-d6) d 1.97–2.43 (m, 2H, 3-H), 3.20–3.67
(m, 2H, 5-H), 3.73–3.77 (m, 1H, 4-H), 3.98–4.08 (m, 1H, 2-
H), 5.04 (s, 2H, Z-CH2), 7.31, 7.35 (two s, 5H, Z-aromatic)
ppm. 13C NMR (DMSO-d6) d 175.21, 174.63 (COOH),
157.03 (Gua-C), 153.99 (OCON), 137.12 (Z-ar-Ci),
128.65, 127.74, 127.19 (Z-ar-Co,Cm,Cp), 68.18, 66.06
1
1365, 1250 738, 697 cmϪ1. H NMR (CDCl3) d 1.41 (s,

2520
Z. Zhang et al. / Tetrahedron 56 (2000) 2513–2522
(Z-CH2), 58.72, 58.51 (C-2), 51.64, 51.07 (C-5), 49.77,
49.10 (C-4), 36.20, 35.20 (C-3) ppm. Exact mass (LSIMS,
thioglycerol) calcd for C14H19N4O4 [MϩH]ϩ 307.14062;
found 307.13631.
Pmc 30-H, Pmc 40-H, 3-H), 3.43-3.68 (m, 2H, 5-H), 4.08–
4.44 (m, 5H, 4-H, 2-H, Fmoc CH, CH2), 6.00–6.50 (m, 3H,
guanidino NH’s), 7.20–7.76 (m, 8H, aromatic’s) ppm. 13C
NMR (CDCl3) d 175.29 (COOH), 155.67 (Gua-C), 155.23
(OCON), 153.82 (Pmc-90), 143.82–118.01 (aromatic C’s),
73.77 (Pmc-20) 68.25 (Fmoc-CH2), 58.36, 57.47 (C-2),
52.86 (C-5), 50.42, 49.55 (C-4), 47.04 (Fmoc-CH), 37.01,
35.08 (C-3), 32.73 (Pmc-40), 26.79 (2×CH3 on Pmc-20),
21.36 (Pmc-30), 18.52, 17.44 (2×CH3‘s o to –SO2Ϫ), 12.09
(CH3 m to –SO2Ϫ) ppm. Exact mass (LSIMS, thioglycerol:
TFA) calcd for C35H41N4O7S1 [MϩH]ϩ 661.26958; found
661.26696; elem. anal. for C35H40N4O7S1 calcd C 63.62, H
6.10, N 8.48; found C 63.33, H 6.15, N 8.49.
4-(2,2,5,7,8-Pentamethylchroman-6-sulfonyl)-guanidino-
Na-benzyloxycarbonyl-l-cis-proline (13). To a solution of
1.4 g (4.6 mmol) of 11 in a mixture of 5.4 ml of 3.2 M
NaOH and 15 ml of acetone were added 2.5 g (8.2 mmol)
of 2,2,5,7,8-pentamethylchroman-6-sulfonyl chloride in
10 ml of acetone at 0ЊC. The mixture was stirred at 0ЊC
for 2 h and another 2 h at room temperature. After acidifica-
tion to pH 6–7 with 1N HCl, the acetone was removed under
vacuo. The remaining solution was further acidified with 1N
HCl to pH 3, diluted with water and extracted with ethyl
acetate. The combined extract was dried on anhydrous
MgSO4, filtered and evaporated to dryness. The remaining
oil was adsorbed on silica gel and purified by column
chromatography with methanol in DCM yielding 1.3 g
(2.27 mmol, 49%) of compound 13. Mp 138ЊC; nmax
3434, 3336 2977 (NH, OH), 1682 (CvO), 1620, 1547
Na-Benzyloxycarbonyl-b-guanidino-l-a-alanine (12). To
a solution of 4.76 g (20 mmol) of 9 in 20 ml of 1.0 M
Na2CO3 in H2O was added 2.93 g (20 mmol) of 1H-pyra-
zole-1-carboxamidine hydrochloride. The reaction mixture
was stirred overnight at room temperature and neutralized to
pH 5–6 with 1N HCl. The solution was evaporated to
dryness and the residue was adsorbed on silica gel and puri-
fied by column chromatography with methanol in DCM.
The crude product was crystallized from methanol to yield
2.24 g (8.0 mmol, 40%) of compound 12. Mp 179ЊC; nmax
3414, 3315 2915 (NH), 1676 (CvO), 1654, 1527, 759,
(NH), 1297 (SvO), 1105, 742, 697 cmϪ1 1H NMR
.
(CDCl3) d 1.28 (s, 6H, 2×Pmc CH3‘s), 1.62–2.53 (m,
15H, 2×CH3‘s o to –SO2Ϫ, 1×CH3 m to –SO2Ϫ, Pmc 30-H,
Pmc 40-H, 3-H), 3.44-3.68 (m, 2H, 5-H), 4.32 (br, 2H, 4-H,
2-H), 4.95–5.15 (m, 2H, Z-CH2), 6.20–7.00 (br, 3H, guani-
dino NH’s), 7.25, 7.27 (m, 5H, Z-aromatic) ppm. 13C NMR
(CDCl3) d 175.88 (COOH), 155.48 (Gua-C), 154.72
(OCON), 153.88 (Pmc 90), 136.03 (Z-ar-Ci), 135.61 (Pmc-
60), 135.03 (Pmc-50), 132.87 (Pmc-70) 128.53, 128.22,
127.93 (Z-ar-Co,Cm,Cp), 124.19 (Pmc-100), 118.06 (Pmc-
80), 73.69 (Pmc-20) 67.65 (Z-CH2), 58.39, 57.67 (C-2),
52.50 (C-5), 50.20, 49.50 (C-4), 36.82, 35.23 (C-3), 32.68
(Pmc-40), 26.71 (2×CH3 on Pmc-C20), 21.28 (Pmc-30),
18.39, 17.33 (2×CH3‘s o to –SO2Ϫ), 11.99 (CH3 m to
–SO2Ϫ) ppm. Exact mass (LSIMS, thioglycerol:TFA)
calcd for C28H37N4O7S1 [MϩH]ϩ 573.23828; found
573.2386; elem. anal. for C28H36N4O7S1 · 1.5 H2O calcd C
56.08, H 6.55, N 9.34; found C 56.11, H 6.28, N 9.02.
699 cmϪ1 1H NMR (DMSO-d6) d 3.45 (m, 2H, b-H),
.
4.03–4.13 (m, 1H, a-H), 5.03 (s, 2H, Z-CH2), 7.34 (s, 5H,
Z-aromatic) ppm. 13C NMR (DMSO-d6) d 172.29 (COOH),
157.94 (Gua-C), 156.36 (OCONH), 137.18 (Z-ar-Ci),
128.62, 128.01, 127.89 (Z-ar-Co,Cm,Cp), 65.70 (Z-CH2),
54.29 (C-a), 42.39 (C-b) ppm. Exact mass (LSIMS, thio-
glycerol:NaOAc) calcd for C12H16N4O4Na [MϩNa]ϩ
303.10695; found 303.10774; elem. anal. for C12H16N4O4
calcd C 51.42, H 5.75, N 19.99; found C 51.27, H 5.56, N
19.36.
Na-Benzyloxycarbonyl-b-(2,2,5,7,8-pentamethylchroman-
6-sulfonyl)guanidino-l-a-alanine (14). To a solution of
1.4 g (5.0 mmol) of 12 in a mixture of 6 ml of 3.2 M
NaOH and 16 ml of acetone were added 2.72 g (9 mmol)
of 2,2,5,7,8-pentamethylchroman-6-sulfonyl chloride in
10 ml of acetone at 0ЊC. The mixture was stirred at 0ЊC
for 2 h and another 2 h at room temperature. After acidifica-
tion to pH 6–7 with 1N HCl, the acetone was removed under
vacuo. The remaining solution was further acidified with 1N
HCl to pH 3 and diluted with water, and was extracted with
ethyl acetate. The combined extracts were dried on
anhydrous MgSO4, filtered and evaporated to dryness. The
remaining oil was adsorbed on silica gel and purified by
column chromatography with methanol in DCM yielding
1.4 g (2.6 mmol, 52%) of compound 14. Mp 166ЊC; nmax
3445, 3347 2974 (NH, OH), 1713 (CvO), 1622, 1547
4-(2,2,5,7,8-Pentamethylchroman-6-sulfonyl)-guanidino-
Na-9-fluorenylmethyloxycarbonyl-l-cis-proline (2). Nitro-
gen was bubbled for 10 min through a solution of 1.5 g
(2.6 mmol) of 13 in 5 ml of methanol, 5 ml of dioxane
and 1 ml of acetic acid. To this solution was added 0.3 g
of palladium on charcoal (10%). The suspension was hydro-
genated overnight at 45 psi. After filtration, the obtained
solution was evaporated to dryness. The residue was
dissolved in 20 ml of 10% aqueous Na2CO3. To the solution
was added 12 ml of 1,4-dioxane and 0.8 g (3.1 mmol) of
9-fluorenylmethyloxycarbonyl chloride at 0ЊC. The reaction
mixture was stirred overnight and was poured into 20 ml of
H2O and the solution was extracted three times with
diethyl ether. The aqueous layer was acidified with a 2N
HCl and the white suspension was extracted three times
with ethyl acetate. The combined organic layer was dried
on anhydrous MgSO4, filtered and evaporated to dryness.
The crude product was crystallized from diethyl ether to
afford compound 2 (1.3 g, 2.0 mmol, 77%). Mp 199ЊC;
nmax 3448, 3345 2973 (NH, OH), 1702 (CvO), 1618,
(NH), 1298 (SvO), 1111, 743, 697 cmϪ1 1H NMR
.
(CDCl3) d 1.27 (s, 6H, 2×Pmc CH3‘s) 1.74 (t, 2H, Pmc
30-H), 2.05 (s, 3H, 1×CH3 m to –SO2Ϫ), 2.48–2.55 (m,
8H, 2×CH3‘s o to –SO2Ϫ, Pmc 40-H), 3.58 (br, 2H, b-H),
4.32 (br, 1H, a-H), 5.02 (s, 2H, Z-CH2), 6.57 (br, 3H, guani-
dino NH’s), 7.25 (s, 6H, aromatic, a-NH), 9.71 (br, 1H,
COOH) ppm. 13C NMR (CDCl3) d 173.48 (COOH),
156.70 (Gua-C, OCON), 154.09 (Pmc-90), 136.18 (Z-ar-
Ci), 135.85 (Pmc-60), 135.36 (Pmc-50), 132.33 (Pmc-70)
128.44, 127.86 (Z-ar-Co,Cm,Cp), 124.22 (Pmc-100),
118.06 (Pmc-80), 73.72 (Pmc-20) 66.98 (Z-CH2), 54.75
1535 (NH), 1298 (SvO), 1108, 740 cmϪ1
(CDCl3) d 1.25, 1.28 (two s, 6H, 2×Pmc CH3‘s), 1.68–
2.62 (m, 15H, 2×CH3‘s o to –SO2Ϫ, 1×CH3 m to –SO2Ϫ
.
1H NMR
,

Z. Zhang et al. / Tetrahedron 56 (2000) 2513–2522
2521
(C-a), 42.22 (C-b), 32.59 (Pmc-40), 26.65 (2×CH3 on Pmc
Solid phase synthesis of the heptapeptide
C-20), 21.22 (Pmc-30), 18.33, 17.30, 11.96 (2×CH3‘s o to
–SO2Ϫ, 1×CH3 m to –SO2Ϫ) ppm. Exact mass (LSIMS,
thioglycerol:NaOAc) calcd for C26H34N4O7SNa [MϩNa]ϩ
569.20460; found 569.20257; elem. anal. for C26H34N4O7S
0.25 H2O calcd C 56.66, H 6.31, N 10.17; found C 56.68, H
6.32, N 10.02.
A. Solid phase peptide synthesis. After 15 min swelling of
39 mg of Rink amide MBHA resin (21.6 mmol) in CH2Cl2,
2 ml of 20% piperidine in DMF was added to the solid
support. The beads were shaken for 15 min at room
temperature and washed with 5×2 ml DMF, 5×2 ml
CH2Cl2. A solution of Fmoc amino acid (86.4 mmol),
HOAt (86.4 mmol), DIC (86.4 mmol), and DIEA
(172.8 mmol) in 1 ml of DMF was added onto the beads
without preactivation. The beads were shaken for 6 h, and
afterwards washed with 5×1 ml DMF, 5×1 ml CH2Cl2. A
solution of pyridine/acetic anhydride/N-methylimidazole
(4:1:0.5) was added to the beads. After 10 min of shaking,
the beads were washed with 5×1 ml of CH2Cl2, and 5×1 ml
of DMF. The beads were treated with 1 ml of 20% piper-
idine for 15 min to remove the Fmoc protecting group,
washed with 5×1 ml of DMF and 5×1 ml of CH2Cl2.
After each complete coupling cycle, 5–6 mg of the
beads were isolated, suspended in 25 ml of 20% piperidine
in DMF over 10 min and subjected to a UV absorption
measurement at 301 nm in order to determine the
coupling yield. For all the amino acids, except arginine
analogues for which a second coupling cycle was required,
a single coupling cycle was enough to obtain a yield ca.
95%.
Na-9-Fluorenylmethyloxycarbonyl-b-(2,2,5,7,8-penta-
methylchroman-6-sulfonyl)-guanidino-l-a-alanine (4a).
Nitrogen was bubbled for 10 min through a solution of
4.8 g (8.7 mmol) of 14 in 15 ml of methanol, 15 ml of
dioxane and 1ml of acetic acid. To this solution was
added 1 g of palladium on charcoal (10%). The suspension
was hydrogenated overnight at 45 psi. After filtration, the
obtained solution was evaporated to dryness. The residue
was dissolved in 50 ml of 10% aqueous Na2CO3. To the
solution was added 30 ml of 1,4-dioxane and 2.5 g
(9.6 mmol) of 9-fluorenylmethyloxycarbonyl chloride at
0ЊC. The reaction mixture was stirred overnight and was
poured into 60 ml of H2O and the solution was extracted
three times with diethyl ether. The aqueous layer was acidi-
fied with a 2N HCl and the white suspension was extracted
three times with ethyl acetate. The combined organic layer
was dried on anhydrous MgSO4, filtered and evaporated to
dryness. The crude product was crystallized from diethyl
ether to afford compound 4a (4.5 g, 7.1 mmol, 81.5%).
Mp 192ЊC; nmax 3435, 3340 2967 (NH, OH), 1702
B. Removal of the protecting groups and cleavage of the
heptapeptide from the solid support. After completion of
the coupling of the last amino acid, the beads were treated
with 1 ml of 20% piperidine in DMF over 15 min, washed
with 5×1 ml of DMF, 5×1 ml of CH2Cl2, and then capped
with 1 ml solution of pyridine/acetic anhydride/N-methyl-
imidazole (4:1:0.5) over 10 min. After washing with 5×1 ml
of CH2Cl2, and 5×1 ml of DMF, the resin was treated with
1 ml of a mixture of TFA/H2O/thioanisole (10:0.5:0.5) for
90 min. The deprotection cocktail was removed and the
beads were washed with 5×1 ml cold diisopropyl ether.
The collected suspension was centrifuged for 10 min and
the precipitated pellet was dried in vacuo, dissolved in
(CvO), 1619, 1542 (NH), 1296 (SvO), 1106, 735 cmϪ1
.
1H NMR [(CD3)2CO] d 1.25 (s, 6H, 2×Pmc CH3‘s) 1.75(t,
2H, Pmc 30-H), 2.00 (obscured by d5-acetone, 3H, 1×CH3 m
to –SO2Ϫ), 2.58–2.66 (m, 8H, 2×CH3‘s o to –SO2Ϫ, Pmc 40-
H), 3.61, 3.77 (br, 2H, b-H), 4.18–4.42 (m, 4H, a-H, Fmoc
CH, CH2), 6.40–7.00 (m, 3H, guanidino NH’s), 7.28–7.88
(m, 8H, aromatics) ppm. 13C NMR [(CD3)2CO] d 172.19
(COOH), 157.60 (Gua-C), 157.20 (OCONH), 153.91 (Pmc-
90), 145.06–118.75 (aromatic C’s) 74.25 (Pmc-20) 67.47
(Fmoc-CH2), 55.59 (C-a), 47.88 (Fmoc-CH), 42.73 (C-b),
33.32 (Pmc-40), 26.92 (2×CH3 on Pmc-20), 21.89 (Pmc-30),
18.83, 17.71, 12.21 (2×CH3‘s o to –SO2Ϫ, 1×CH3 m to
–SO2Ϫ) ppm. Exact mass (LSIMS, thioglycerol:NaOAc)
calcd for C33H38N4O7SNa [MϩNa]ϩ 657.23590; found
657.23603; elem. anal. for C33H38N4O7S calcd C 62.44, H
6.03, N 8.83; found C 62.18, H 6.31, N 8.67; [a]2D0ˆϪ22.86
(cˆ0.29 in MeOH). Compound 4b with [a]2D0ˆϩ23.70
(cˆ0.31 in MeOH)was prepared from d-asparagine in
exactly the same way as described for 4a.
H2O, and purified on a Nova-Pak᭨ C18 column (60 A,
˚
4 mm), 150/3.9 (Waters, US). The peptides were eluted off
the column by using a linear gradient from 0 to 100% B in
30 min, with a flow rate of 1 ml/min [eluent A: 0.1% TFA in
CH3CN–H2O (5/95); eluent B: 0.1% TFA in CH3CN–H2O
(80/20)], and detected by UV at 220 nm. The major
compound was isolated and analyzed by high resolution
mass spectrometry (the peak which comes out with 76%
B corresponds to thioanisole). Peptide 1: Exact mass (ESI)
for C39H55N10O9 [MϩH]ϩ calcd 807.41532, found
807.4132. Peptide 2: Exact mass (ESI) for C41H59N14O9
[MϩH]ϩ calcd 891.45891, found 891.4626.
Determination of the enantiomeric purity of the alanine
derivatives (4a, 4b). The enantiomeric purity of the newly
synthesized amino acids was measured on a Hewlett
Packard-1100 Liquid Chromatograph (Waldbronn,
Germany). A mixture of the two isomers was injected
onto a Chirobiotic T-5 mm column 250×4.6 mm (ASTEC,
US). A complete separation was achieved with a mobile
phase of 95% MeOH (0.01 M ammonium acetate) and 5%
water, at a flow rate of 1.0 ml/min at 30ЊC. The peaks were
detected by a diode array detector set at 220.4 nm and
254.4 nm. Subsequently the two isomers were injected
one by one, with both compounds showing a single
peak. The compound with the l configuration displayed a
shorter retention time than the compound with the d
configuration.
Acknowledgements
The work was supported by a grant from K. U. Leuven
(GOA 97/11). We are grateful to Dr Volker Gnau (PE
SCIEX) for recording the mass spectrum for peptide 1 and
peptide 2. We thank Jaak Hoebus for optical rotations, Ivo
Quintens for infrared resonance analyses, and Chantal
Biernaux for editorial help.

2522
Z. Zhang et al. / Tetrahedron 56 (2000) 2513–2522
¨
¨
Nukleinsauren-bindende Oligomere mit N-Verzweigung fur
Therapie und Diagnostik (EP 0 646 595 A1).
14. Arnold, L. D.; Kalantar, T. H.; Vederas, J. C. J. Am. Chem.
Soc. 1985, 107, 7105–7109.
References
1. Declercq, E. Collect. Czech. Chem. Commun. 1998, 63, 449–
479.
15. Waki, M.; Kitajima, Y.; Izumiya, N. Synthesis 1981, 266–268.
16. Yong, Y. F.; Kowalski, J. A.; Lipton, M. A. J. Org. Chem.
1997, 62, 1540–1542.
17. Shibanuma, T.; Shiono, M.; Mukaiyama, T. Chem. Lett. 1977,
575–576.
18. Kim, K. S.; Qian, L. Tetrahedron Lett. 1993, 34, 7677–7680.
19. Feichtinger, K.; Zapf, C.; Sings, H. L.; Goodman, M. J. Org.
Chem. 1998, 63, 3804–3805.
20. Uno, T.; Beausoleil, E.; Goldsmith, R. A.; Levin, B. H.;
Zuckermann, R. N. Tetrahedron Lett. 1999, 40, 1475–1478.
21. Bernatowicz, M. S.; Wu, Y.; Matsueda, G. R. J. Org. Chem.
1992, 57, 2497–2502.
22. Rzeszotarska, B.; Masiukiewicz, E. Org. Prep. Proced. Int.
1988, 20, 427–464.
23. Rink, H.; Sieber, P.; Raschdorf, F. Tetrahedron Lett. 1984, 25,
621–624.
24. Atherton, E.; Sheppard, R. C.; Wade, J. D. J. Chem. Soc.
Chem. Commun. 1983, 1060–1062.
2. Declercq, E. Trends Drug Res. II 1998, 91–104.
3. Declercq, E. Int. J. Antimicrob. Agents 1997, 9, 21–36.
4. Kingsman, S. M.; Kingsman, A. J. Eur. J. Biochem. 1996, 240,
491–507.
5. Frankel, A. D. Curr. Opin. Genet. Dev. 1992, 2, 293–298.
6. Calnan, B. J.; Tidor, B.; Biancalana, S.; Hudson, D.; Frankel, A.
D. Science 1991, 252, 1167–1171.
7. Puglisi, J. D.; Tan, R.; Canlan, B. J.; Frankel, A. D.;
Williamson, J. R. Science 1992, 257, 76–80.
8. Aboul-ela, F.; Karn, J.; Varani, G. J. Mol. Biol. 1995, 253,
313–332.
9. Terrett, N. K.; Gardner, M.; Gordon, D. W.; Kobylecki, R. J.;
Steele, J. Tetrahedron 1995, 51, 8135–8173.
10. Medynski, D. Biotechnology 1994, 12, 709–710.
11. Houghten, R. A.; Pinilla, C.; Blondelle, S. E.; Appel, J. R.;
Dooley, C. T.; Cuervo, J. H. Nature 1991, 354, 84–86.
12. Youngquist, R. S.; Fuentes, G. R.; Lacey, M. P.; Keough, T.
J. Am. Chem. Soc. 1995, 117, 3900–3906.
¨
13. Lobberding, A., Mielke, B., Schwemler, C., Schwenner, E.,
25. Ramage, R.; Green, J.; Blake, A. J. Tetrahedron 1991, 47,
6353–6370.
¨
Stropp, U., Springer, W., Kretschmer, A., Potter, T. In