Solid-phase synthesis of dihydrovirginiamycin S1, a streptogramin B antibiotic

Article abstract of DOI:10.1002/chem.200400267

We describe the first solid-phase synthesis of dihydrovirginiamycin S ₁, a member of the streptogramin B family of antibiotics, which are nonribosomal-peptide natural products produced by Streptomyces. These compounds, along with the synergisti

Full text of DOI:10.1002/chem.200400267

FULL PAPER
Solid-Phase Synthesis of Dihydrovirginiamycin S1, a Streptogramin B
Antibiotic
Alex Shaginian,[a] Marissa C. Rosen,[a] Brock F. Binkowski,[b] and Peter J. Belshaw*[a, b]
Abstract: We describe the first solid-
phase synthesis of dihydrovirginiamy-
cin S1, a member of the streptogra-
min B family of antibiotics, which are
nonribosomal-peptide natural products
produced by Streptomyces.These com-
pounds, along with the synergistic
group A components, are ™last line of
defense∫ antimicrobial agents for the
treatment of life-threatening infections
such as vancomycin-resistant entero-
cocci.The synthesis features an on-
resin cyclization and is designed to
allow production of streptogramin B
analogues with diversification at posi-
tions 1’, 1, 2, 3, 4, and 6.Several syn-
thetic challenges known to hinder the
synthesis of this class of compounds
were solved, including sensitivity to
acids and bases, and epimerization and
rearrangements, through the judicious
choice of deprotection conditions, cou-
pling conditions, and synthetic strategy.
This work should enable a better un-
derstanding of structure activity rela-
tionships in the streptogramin B com-
pounds, possible identification of ana-
logues that bypass known resistance
mechanisms, and perhaps the identifi-
cation of analogues with novel biologi-
cal activities.
Keywords: antibiotics ¥ cyclic pep-
tides ¥ depsipeptides ¥ solid-phase
synthesis ¥ virginiamycins
Introduction
The streptogramin (or synergi-
mycin) family of antibiotics
consist of two components, A
and B, that synergistically inhib-
it protein synthesis by binding
to distinct sites on the 50S sub-
unit of the prokaryotic ribo-
some.The group A components
are polyunsaturated macrolac-
tones and the group B compo-
a
nents are N1 -acylated cyclic
hexadepsipeptides
(Figure 1).
Although the virginiamycins
have been used for many years
as a growth-promoting additive
in livestock feed, limited water
Figure 1.Structures of streptogramin antibiotics.Dihydrovirginiamycin S has a trans-hydroxyl group replacing
the carbonyl of residue 5.
1
solubility has hampered the clinical utility of streptogramin
antibiotics.Recently, the semisynthetic water-soluble deriva-
[a] A.Shaginian, M.C.Rosen, Prof.P.J.Belshaw
Department of Chemistry
tives quinupristin and dalfopristin (marketed as Synercid
TM) were approved for the treatment of Gram-positive
coccal infections including vancomycin-resistant enterococci
(Figure 1).[1] Here we describe the first solid-phase synthesis
of a group B streptogramin.Our synthesis is designed to
enable the synthesis of streptogramin B analogues at posi-
tions 1’, 1, 2, 3, 4, and 6.
University of Wisconsin-Madison
Madison, WI 53706 (USA)
Fax : (+1)608-265-4534
E-mail: belshaw@chem.wisc.edu
[b] B.F.Binkowski, Prof.P.J.Belshaw
Department of Biochemistry
University of Wisconsin-Madison
Madison, WI 53706 (USA)
Fax : (+1)608-265-4534
The genes responsible for the biosynthesis of pristinamy-
cin I in Streptomyces pristinaespiralis and virginiamycin S1 in
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DOI: 10.1002/chem.200400267
Chem. Eur. J. 2004, 10, 4334 4340

4334 4340
Streptomyces virginiae have
been sequenced, and the ho-
mologous nonribosomal peptide
synthetases (NRPS) that assem-
ble these natural products in a
template-directed fashion have
been identified.[2] Recent work
has shown that it is possible to
re-engineer the substrate speci-
ficity of NRPSs, thus enabling
the possibility of cost-effective
Figure 2.Retro-synthetic disconnections for dhVS 1.All =allyl, Alloc=allyloxycarbonyl, Boc=tert-butyloxycar-
bonyl.
production
of
analogues
through engineered biosynthe-
sis in microorganisms.[3] Although significant progress has
been made in the combined chemo-enzymatic synthesis of
libraries,[4] the exclusive biosynthetic production of nonribo-
somal peptide libraries remains a challenge.
epimerization during cyclization.This strategy also allows
the Phg ester to be constructed in solution and introduced
late in the synthesis to minimize complications with this sen-
sitive functionality.
We have developed a solid-phase synthesis of dihydrovir-
giniamycin S1 (dhVS1)[5] to enable the synthesis of strepto-
gramin B analogues for investigation of structure activity
relationships in these antibiotics.The hexapeptide macro-
cycle of the streptogramin B antibiotics has many features
that make it a favorable scaffold for the identification of an-
alogues with new biological activities: the parent compounds
are cell permeable; the macrocycle is rigidified by a trans-
annular hydrogen bond between the carbonyl oxygen atom
Results and Discussion
The synthesis of Fmoc-trans-4-hydroxypipecolic acid tert-
butyl ester 3 is outlined in Scheme 1.The commercially
available benzylamine salt of Boc-trans-4-hydroxypipecolic
acid was silylated and converted to the tert-butyl ester (1).
ꢀ
of Pro and the amide N H group of Phg; the conformation
of the macrocycle directs the side chains (potential diversity
elements) in a radial manner and could provide a diverse
set of three-dimensional structures;[6] and the side chains are
projected over a relatively large surface area, a property
that may be beneficial for binding to protein protein inter-
action surfaces.Protein protein interactions are of great in-
terest as new pharmacological targets, yet difficult to modu-
late with small molecules.[7]
Previous syntheses of streptogramin B natural products[8]
and analogues[9] have been conducted in solution with clo-
sure of the macrocycle through formation of an amide bond
from a linear depsipeptide precursor.Along with these total
syntheses, efforts toward the production of semi/hemisyn-
thetic analogues revealed several challenges:[10] compounds
containing homochiral N-alkyl amino acid triads are sensi-
tive to strong acids, the Phg ester is sensitive to both epimer-
ization and elimination, cyclization is nearly always accom-
panied by epimerization, and the des-N-hydroxypicolinic
acid derivatives are susceptible to rearrangements giving in-
active macrolactam and oxazoline byproducts.
We targeted the synthesis of dhVS1, since this analogue is
known to be active, the hydroxyl group on the hydroxypipe-
colic acid residue provides a handle for attachment to the
resin, and this residue has been extensively modified with
many analogues retaining activity.[10c] Our retrosynthesis
(Figure 2) disconnects the macrocycle into three subfrag-
ments: a linear tetrapeptide anchored to the resin through
the hydroxypipecolic acid, a fragment containing Phg with
an ester bond to the side chain of Thr, and the exocyclic 3-
hydroxypicolinic acid.These disconnections were chosen to
allow cyclization at the Thr1-d-Abu2 peptide bond by
means of activation of a carbamate-protected Thr to prevent
Scheme 1.Synthesis of Fmoc-hyPip-O tBu.Reagents and conditions:
a) 0.5m HCl(aq) in NaCl(sat), EtOAc, 08C, 20 min; b) TBS-Cl, imidazole,
2,6-lutidine, THF, 48 h, 98% for two steps; c) tBuOH, DIC, DMAP(cat)
,
CH2Cl2, 94%; d) TMS-OTf (1 equiv), CH2Cl2/toluene, 4 h; e) Fmoc-Cl,
pyridine, CH2Cl2, 15 h, 77% for two steps; f) HF/pyridine, THF, 2 h,
96%.hyPip =(2S,4S)-4-hydroxypiperidine-2-carboxylic acid, Bn=benzyl,
tBu=tert-butyl, TBS=tert-butyldimethylsilyl, Fmoc=9-fluorenylmethyl-
oxycarbonyl, THF=tetrahydrofuran, DIC=1,3-diisopropylcarbodiimide,
DMAP=4-(dimethylamino)pyridine, TMS=trimethylsilyl, Tf=trifluoro-
methanesulfonyl.
Selective Boc cleavage with one equivalent of TMS-OTf,[11]
followed by Fmoc protection afforded 2.Selective removal
of the TBS group in the presence of the tert-butyl ester with
HF/pyridine afforded 3.[12]
The synthesis of the dipeptide amine 5 is shown in
[13]
Scheme 2.Boc-Thr-OAll
was condensed with Troc-Phg-
OH[14] at ꢀ258C in the presence of 1,3-diisopropylcarbodi-
imide (DIC) and a catalytic amount of DMAP to afford
ester 4 without epimerization.Troc deprotection with zinc in
acetic acid afforded amine 5.
Our synthesis of dhVS1 is outlined in Scheme 3.We chose
Danishefsky×s bis(silyl ether) linker[15] for attachment of
trans-4-hydroxypipecolic (hyPip) to the resin as it is stable
to mild bases and palladium cleavage conditions, yet readily
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P.J.Belshaw et al.
FULL PAPER
resin-bound intermediate 6.Removal of the Alloc protec-
ting group with palladium under mild acidic conditions, and
subsequent coupling with Fmoc-Pro-OH using DIC/HOAt/
2,6-lutidine yielded the tripeptide resin.Fmoc deprotection
and coupling to Alloc-d-Abu-OH yielded resin-bound tetra-
peptide 7.Subsequently, the tert-butyl ester was deprotected
under neutral conditions (TMS-OTf/lutidine),[17] and then
coupled with amine 5 to yield linear hexapeptide resin 8.Si-
multaneous deprotection of the allyl and Alloc groups with
palladium, followed by PyAOP-mediated cyclization afford-
ed the Boc-protected macrocycle 9.Boc removal was ac-
complished under neutral conditions with TMS-OTf/lutidine,
and PyAOP/lutidine-mediated coupling with 3-hydroxy-
picolinic acid afforded 10.HF/pyridine-mediated cleavage
from resin yielded dhVS1 in 15% overall isolated yield after
purification by silica gel chromatography.LC/MS analyses
of synthetic intermediates cleaved from the resin indicated
that the on-resin cyclization largely determined the final
yield of dhVS1.
Scheme 2.Synthesis of dipeptide ester 5.a) Troc-Phg-OH, DIC,
DMAP(cat), CH2Cl2, 2 h, ꢀ258C, 95%; b) Zn, AcOH, 2 h, 88%.Troc =
2,2,2-trichloroethoxycarbonyl.
Several issues encountered during the solid-phase synthe-
sis of dhVS1 are worth noting.In our initial experiments we
attempted to employ the allyl ester of hyPip in place of tert-
butyl ester 3.However, upon deprotection of Fmoc-MePhe-
hyPip(O-resin)-OAll with piperidine, the free amine rapidly
cyclized to form the 2,5-diketopiperazine (DKP).The com-
bination of the bulky tert-butyl ester on pipecolic acid, Alloc
deprotection under acidic conditions, and DIC/HOAt/luti-
dine coupling under neutral conditions were required to
allow successful peptide extension without any DKP forma-
tion.
We also investigated approaches to dhVS1 involving cycli-
zation with 3-hydroxypicolinic acid installed on the exocyclic
amine of Thr1.However, solution-phase experiments with
model compound 11 revealed a rapid elimination immedi-
ately upon activation of Thr, resulting in the cleavage of the
ester bond (Scheme 4).This possibly occurs via formation of
Scheme 3.Solid-phase synthesis of dhVS 1.a) diisopropyldichlorosilane,
imidazole, 30 min; b) hydroxymethyl-polystyrene resin, 48 h; c) 30% pi-
peridine, CH2Cl2, 30 min; d) Alloc-MePhe-OH, HOAt, DIC, 2,6-lutidine,
DMF, 48 h; e) Pd(PPh3)4(cat), Bu3SnH, 5% AcOH, CH2Cl2, 3 h, f) Fmoc-
Pro-OH, HOAt, DIC, 2,6-lutidine, DMF, 48 h (î3); g) 30% piperidine,
CH2Cl2, 30 min; h) Alloc-d-Abu-OH, HOAt, DIC, 2,6-lutidine, DMF,
24 h; i) TMS-OTf, 2,6-lutidine, CH2Cl2, 5 h; j) MeOH, 20 min; k) 5,
HOAt, DIC, 2,6-lutidine, DMF, 6 h; l) [Pd(PPh3)4](cat), PhSiH3, CH2Cl2,
5 h; m) PyAOP, 2,6-lutidine, CH2Cl2, 48 h; n) TMS-OTf, 2,6-lutidine,
CH2Cl2, 2 h, 238C, then 20 min, 48C; o) 3-hydroxypicolinic acid, PyAOP,
2,6-lutidine, THF, 4 h, 48C, then 10 h, 238C; p) HF/pyridine, THF, 1 h,
15% overall.HOAt =1-hydroxy-7-azabenzotriazole, DMF=N,N-dimeth-
ylformamide, Abu=2-aminobutyric acid, PyAOP=7-aza-benzotriazole-
1-yloxytris(pyrrolidinophosphonium) hexafluorophosphate.
Scheme 4.Elimination of N a-protected Phg during activation of side-
chain acylated Thr.
removed with HF/pyridine.The synthesis commences with
loading of Fmoc-hyPip-OtBu (3) onto the hydroxymethyl
polystyrene resin.Fmoc removal followed by DIC/1-hy-
a 5(4H)-oxazolone 12 that eliminates to give the unstable
intermediate 13, which was trapped with 4-methoxybenzyl
amine to yield 14 (data not shown).Protection of the Thr
exocyclic amine as a urethane during cyclization avoided
this elimination pathway.
droxy-7-azabenzotriazole
(HOAt)/2,6-lutidine-mediated
coupling[16] of Alloc-MePhe-OH resulted in the formation of
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Dihydrovirginiamycin S1
4334 4340
Another potential complication was the instability of 1-
(des-3-hydroxypicolinoyl) pristinamycin IA (15), as reported
by Barriere and co-workers,[10e] which can rearrange to
lactam 16 and oxazoline 17 (Scheme 5).Expecting that basic
conditions would promote these rearrangements in the relat-
avoided in THF or can be suppressed with an excess of pyri-
dine.
To confirm the identity of our synthetic dhVS1, we pre-
pared semisynthetic dhVS1 from the natural product virgin-
iamycin S1 isolated from Stafac,[18] by reduction with sodium
1
borohydride.[5] Synthetic dhVS1 displayed identical H NMR
spectral data to the compound prepared from the natural
product.To confirm the biological activity of dhVS , we as-
1
sayed our synthetic and semi-synthetic dhVS1 for activity
against B. subtilis, both alone and in combination with vir-
giniamycin M (Table 1).Both samples displayed equal activ-
Table 1.Growth inhibition assays.The growth of B. subtilis strain BR151
(ATCC 33677) in liquid culture was monitored in the presence of varying
concentrations of compound(s).
Compound(s)[a]
MIC[b] [mgmLꢀ1
]
synthetic dhVS1
natural dhVS1
VS1
50
50
2
VM
3
synthetic dhVS1/VM
natural dhVS1/VM
VS1/VM
1/1.5
1/1.5
0.4/0.6
[a] VS1/VM compound mixtures were present in
a 3:7 molar ratio.
dhVS1 =dihydrovirginiamycin S1, VS1 =virginiamycin S1, VM=virginia-
mycin M.[b] MIC =minimal concentration required to completely in-
hibit growth after
a 10 h incubation from a starting inoculum of
~2000 cfumLꢀ1
.
Scheme 5.Rearrangements of 1-( des-3-hydroxypicolinoyl) pristinamy-
cin IA.
ity and a synergistic increase in activity with virginiamy-
cin M, albeit with a slight decrease in potency compared to
the parent natural product, consistent with previous re-
ports.[5]
ed compound in our synthesis, we chose to protect this
amine with a Boc group (intermediate 9, Scheme 3), as it
can be cleaved under neutral conditions with TMS-OTf/luti-
dine.[17] Moreover, in our initial studies we noticed that
TMS-OTf could promote epimerization of the sensitive Phg
residue.To minimize competing epimerization, O !N acyl
shift, and oxazoline formation, the Boc deprotection was
carried out in CH2Cl2 for 2 h at room temperature.Shorter
reaction times at room temperature and longer reaction
times (5 6 h) at 48C resulted in incomplete deprotection or
epimerization of Phg, respectively.Acylation with 3-hydroxy-
picolinic acid was then achieved in THF with PyAOP/luti-
Conclusion
We have developed a rapid and efficient solid-phase synthe-
sis of dihydrovirginiamycin S1 that should enable the prepa-
ration of novel streptogramin B analogues.These com-
pounds could be used to identify structure activity relation-
ships in this important class of antibiotics, possibly identify-
ing new compounds with increased potency, the ability to
bypass resistance mechanisms,[19] or completely new biologi-
cal activities.Having determined conditions for the success-
ful acylation of MePhe4 and the exocyclic amine of Thr1 in
solid-phase synthesis, our synthetic route should allow the
facile construction of analogues at positions 1’, 2, 3, and 4,
and variations at Thr1 and Phg6, which could be readily ac-
complished through the incorporation of appropriate ester-
containing fragments.
dine.Other solvents such as CH Cl2 and DMF and coupling
2
agents such as DIC/HOAt gave less satisfactory results.This
strategy of utilizing neutral deprotection and coupling condi-
tions with HOAt-active esters minimizes exposure of the
free amine and may prove generally useful for difficult
amide bond formations in which undesired side reactions of
the free amine compete.These strategies enabled successful
acylation of MePhe4 and the exocyclic amine of Thr1, there-
by allowing both of these positions to be sites for diversifica-
tion in our synthetic route.
The conditions for cleavage from resin were also critical
to avoid acidic endopeptolysis of the Pro3-MePhe4-hyPip5
triad of N-alkyl amino acids.[10b] Cleavage with HF/pyridine
in dichloromethane led exclusively to endopeptolysis from
rupture of the MePhe4-hyPip5 peptide bond.Interestingly,
the endopeptolysis was solvent-dependent and is completely
Experimental Section
General procedures: 1-Hydroxy-7-azabenzotriazole (HOAt) and (7-aza-
benzotriazole-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate
(PyAOP) were obtained from Applied Biosystems, (Foster City, CA).
Amino acids were obtained from Advanced ChemTech (Louisville, KY)
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¹ 2004 Wiley-VCH Verlag GmbH & Co.KGaA, Weinheim
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P.J.Belshaw et al.
FULL PAPER
and EMD Biosciences (San Diego, CA).Diisopropyldichlorosilane was
obtained from Fluka (Milwaukee, WI).The benzylamine salt of ( 2S,4S)-
N-Boc-4-hydroxypiperidine-2-carboxylic acid was obtained from Chiro-
tech Technology Ltd (Cambridge, UK).Hydroxymethyl polystyrene res-
in SS, 100 200 mesh, 1% DVB, 1.0 mmolgꢀ1 was obtained from Ad-
vanced ChemTech (Louisville, KY).All other reagents were obtained
from Aldrich Chemical (Milwaukee, WI) and used without further purifi-
cation.Solid-phase reactions were conducted inside 50 mL round-bot-
tomed flasks or 10 mL polypropylene poly-prep chromatography columns
obtained from Bio-Rad Laboratories (Hercules, CA) and agitated on a
Labquake shaker.Tetrahydrofuran (THF) and toluene were distilled
from sodium benzophenone ketyl; dichloromethane (CH2Cl2) was distil-
led from phosphorous pentoxide.Analytical thin-layer chromatography
(TLC) was carried out on EM Science TLC plates precoated with silica
gel 60 F254 (250 mm layer thickness).TLC visualization was accomplished
by using a UV lamp and/or charring solutions of either ninhydrin or
phosphomolybdic acid (PMA).Flash column chromatography (FCC) was
performed on Silicycle silica gel 60 (230 400 mesh). 1H NMR spectra
were recorded in deuterated solvents on a Bruker AC-250 (250 MHz),
Bruker AC-300 (300 MHz) or a Varian UNITY-500 (500 Mhz) spectrom-
eter.Chemical shifts are reported in parts per million (ppm, d) relative
to tetramethylsilane (TMS, d 0.00) or relative to residual solvent signals
(CDCl3 7.27 (1) or CD3CN 1.94 (5)). Coupling constants (J values) are
given in Hz, and peak multiplicities are denoted by s (singlet), d (dou-
blet), dd (doublet of doublets), ddd (doublet of doublet of doublets), dq
(doublet of quartets), dt (doublet of triplets), m (multiplet), q (quartet),
and t (triplet). 13C NMR spectra were recorded in deuterated solvents on
a Bruker AC-250 (62.5 MHz), Bruker AC-300 (75 MHz) or a Varian
UNITY-500 (125 MHz) spectrometer.Chemical shifts are reported in
parts per million (ppm, d) relative to residual solvent signals (CDCl3
77.23 (3) or CD3CN 1.39 (7)). For compounds with multiple rotamers all
observed signals are reported.Optical rotations were obtained on a
Perkin Elmer 241 digital polarimeter at room temperature with an Na
lamp.Concentrations ( c) are reported in g per 100 mL.Fourier transform
infrared (FT-IR) spectra were obtained on a Mattson Polaris instrument.
High-resolution electrospray ionization mass spectra (HRESI-MS) were
obtained on a Micromass LCT.
mogeneous by TLC analysis. Rf =0.66 (hexanes/Et2O 7:3); [a]=ꢀ17.2
1
(c=0.087 in CHCl3); H NMR (300 MHz, CDCl3, 258C): d=0.065 (d, J=
0.7 Hz, 6H), 0.88 (s, 9H), 1.44 (s, 9H), 1.47 (s, 10H), 1.60 (m, 1H), 1.79
(m, 1H), 2.30 (m, 1H), 2.99 (m, 1H), 3.58 (m, 1H), 3.99 (m, 1H),
4.74 ppm (m, 1H); 13C NMR (75 MHz, CDCl3, 258C): d=ꢀ4.6, 18.2,
25.9, 28.1, 28.4, 34.5, 34.8, 36.1, 40.2, 40.7, 54.5, 55.6, 67.0, 79.9, 81.4,
155.5, 155.6, 170.6, 170.8 ppm; FT-IR (thin film): n˜ =2976, 2930, 2857,
1739, 1704, 1473 cmꢀ1; MS (HRESI-MS) calcd for [C21H41NO5Si+Na]+:
438.2652; found: 438.2656.
Fmoc-(2S,4S)-hyPip(OTBS)-OtBu 2: Boc-(2S,4S)-hyPip(OTBS)-OtBu 1
(30 mg, 0.072 mmol) was dissolved in CH2Cl2 (0.7 mL) and treated with a
solution of TMS-OTf in toluene (0.292m, 0.25 mL, 0.073 mmol) at room
temperature.The reaction mixture was stirred at room temperature
under an atmosphere of N2 for 4 h and was quenched with MeOH
(1 mL), concentrated and purified by chromatography on silica gel (50:1
Et2O/MeOH) to give the a-amino ester as a liquid that was homogene-
ous by TLC analysis.The product was dissolved in CH 2Cl2 (2 mL), pyri-
dine (50 mL, 0.62 mmol) was added, and the system was cooled to 08C.
Fmoc-Cl (26 mg, 0.1 mmol) was added and the reaction mixture was stir-
red at room temperature under an atmosphere of N2 for 15 h.The mix-
ture was concentrated and purified by chromatography on silica gel (9:1
hexanes/Et2O) to give 2 as a solid (30 mg, 77%) that was homogeneous
by TLC analysis. Rf =0.40 (hexanes/Et2O 7:3); [a]=ꢀ11.4 (c=0.70 in
CHCl3); 1H NMR (300 MHz, CDCl3, 258C): d=0.12 (s, 6H), 0.93 (s,
9H), 1.46 (m, 1H), 1.50 (s, 9H), 1.66 (m, 1H), 1.87 (m, 1H), 2.40 (m,
1H), 3.17 (m, 1H), 3.67 (m, 1H), 4.14 (m, 1H), 4.35 (m, 3H), 4.89 (m,
1H), 7.36 (m, 4H), 7.60 (m, 2H), 7.78 ppm (d, J=7.5 Hz, 2H); 13C NMR
(75 MHz, CDCl3, 258C): d=ꢀ4.5, 18.3, 26.0, 28.2, 34.7, 34.8, 36.3, 40.6,
40.7, 47.4, 47.5, 55.2, 55.3, 66.9, 68.0, 82.0, 82.2, 120.1, 125.3, 127.3, 127.8,
141.5, 144.1, 144.3, 155.9, 156.3, 170.4 ppm; FT-IR (thin film): n˜ =3067,
2953, 2929, 1736, 1708, 1451 cmꢀ1
; MS (HRESI-MS) calcd for
[C31H43NO5Si+Na]+: 560.2808; found: 560.2815.
Fmoc-(2S,4S)-hyPip-OtBu 3: Fmoc-(2S,4S)-hyPip(OTBS)-OtBu
2
(90 mg, 0.167 mL) was dissolved in THF (5 mL) and HF/pyridine
(3.0 mL) was injected at 08C.The reaction mixture was stirred at room
temperature for 2 h and diluted with Et2O.The mixture was extracted
with saturated aq NaHCO3 (î1), aq HCl (0.1m, î2), and H2O (î1).
The organic layer was collected, concentrated, and purified by chroma-
tography on silica gel (1:4 hexanes/Et2O) to give 3 as a solid (68 mg,
96%) that was homogeneous by TLC analysis. Rf =0.45 (Et2O); [a]=
ꢀ23.2 (c=1.10 in CHCl3); 1H NMR (300 MHz, CDCl3, 258C): d=1.26
(m, 1H), 1.47 (s, 9H), 1.60 (m, 1H), 1.97 (m, 1H), 2.08 (s, 1H), 2.50 (m,
1H), 3.14 (m, 1H), 3.70 (m, 1H), 4.12 (m, 1H), 4.36 (m, 3H), 4.90 (m,
1H), 7.35 (m, 4H), 7.58 (m, 2H), 7.78 ppm (d, J=7.6 Hz, 2H) ; 13C NMR
(75 MHz, CDCl3, 258C): d=28.2, 34.2, 35.7, 40.4, 40.6, 47.4, 47.5, 55.0,
55.1, 66.0, 66.1, 68.0, 82.2, 82.4, 120.2, 125.2, 127.2, 127.9, 141.4, 144.0,
144.2, 155.9, 156.3, 170.2 ppm; FT-IR (thin film): n˜ =3435, 3066, 2975,
Boc-(2S,4S)-hyPip(OTBS)-OH: Aqueous HCl (0.5m) saturated with
sodium chloride (10 mL) was slowly added to the suspension of the ben-
zylamine salt of (2S,4S)-N-Boc-4-hydroxypiperidine-2-carboxylic acid
(1.0 g, 2.84 mmol) in EtOAc (10 mL) at 08C.The mixture was stirred at
08C for 20 min, diluted with EtOAc, and extracted with saturated aq
NaCl.The organic layer was collected, dried (Na SO4), and concentrated,
2
affording acid as a solid.The obtained acid was dissolved in THF
(30 mL) and imidazole (1.2 g, 17.04 mmol), 2,6-lutidine (1.3 mL,
11.36 mmol), and TBS-Cl (1.3 g, 8.52 mmol) were added at 08C.The re-
action mixture was stirred at room temperature under an atmosphere of
N2 for 48 h.The mixture was diluted with EtOAc and extracted with aq
HCl (0.1m, î2).The organic layer was collected and concentrated.The
resultant residue was suspended in MeOH (20 mL), stirred for 5 h, after
which the mixture was concentrated and purified by chromatography on
silica gel (50:50:1 hexanes/Et2O/AcOH) to give the side-chain TBS-pro-
tected acid as a solid (1.0 g, 98%) that was homogeneous by TLC analy-
sis. Rf =0.32 (hexanes/Et2O/HOAc 25:25:1); [a]=ꢀ23.3 (c=0.18 in
2931, 1733, 1704, 1451 cmꢀ1
[C25H29NO5+Na]+: 446.1943; found: 446.1921.
Alloc-l-MePhe-OH: solution of allyl chloroformate (1.5 mL,
;
MS (HRESI-MS) calcd for
A
13.9 mmol) in dioxane (20 mL) and aq NaOH (1m, 20 mL) were simulta-
neously added dropwise at 08C to a solution of H-l-MePhe-OH¥HCl
(2.0 g, 9.27 mmol), H2O (30 mL), aq NaOH (1m, 20 mL), and Et2O
(20 mL).The ice-bath was removed, and the reaction mixture was stirred
for 12 h at room temperature.The mixture was diluted with EtOAc and
extracted with saturated aq NaHCO3.The aqueous layer was collected,
acidified with concentrated HCl, and extracted with EtOAc.The organic
layer was collected, dried (Na2SO4), and concentrated affording a solid
(1.95 g, 80%) that was homogeneous by TLC analysis. Rf =0.35 (hex-
anes/Et2O/HOAc 30:20:1); [a]=ꢀ65.4 (c=2.09 in MeOH); 1H NMR
(250 MHz, CD3CN, 678C): d=2.81 (s, 3H), 3.10 (dd, J=10.7, 14.5 Hz,
1H), 3.33 (dd, J=5.2, 14.4 Hz, 1H), 4.51 (d, J=5.2 Hz, 2H), 4.88 (dd, J=
5.2, 10.5 Hz, 1H), 5.20 (m, 2H), 5.89 (m, 1H), 7.28 (m, 5H), 8.24 ppm (s,
1H); 13C NMR (62.5 MHz, CD3CN, 678C): d=33.0, 36.1, 61.9, 67.2,
117.8, 127.9, 129.8, 130.3, 134.7, 139.3, 157.5, 172.8 ppm; FT-IR (thin
film): n˜ =3078, 3029 2938, 1743, 1700, 1653, 1401 cmꢀ1; MS (HRESI-MS)
calcd for [C14H16NO4]ꢀ: 262.1079; found: 262.1069.
1
CHCl3); H NMR (300 MHz, CDCl3, 258C): d=0.060 (d, J=1.3 Hz, 6H),
0.87 (s, 9H), 1.45 (s, 9H), 1.72 (m, 3H), 2.36 (m, 1H), 2.98 (m, 1H), 3.65
(m, 1H), 4.01 (m, 1H), 4.92 (m, 1H), 11.35 ppm (s, 1H); 13C NMR
(75 MHz, CDCl3, 258C): d=ꢀ4.8, 17.9, 25.7, 28.2, 34.3, 35.4, 39.8, 40.5,
53.6, 54.5, 66.7, 80.6, 155.2, 155.7, 176.4, 176.6 ppm; FT-IR (KBr pellet):
n˜ =2954, 2856, 1750, 1630, 1439 cmꢀ1
[C17H32NO5Si]ꢀ: 358.2050; found: 358.2045.
Boc-(2S,4S)-hyPip(OTBS)-OtBu 1:
;
MS (HRESI-MS) calcd for
Boc-(2S,4S)-hyPip(OTBS)-OH
(0.92 g, 2.56 mmol) was dissolved in CH2Cl2 (5 mL) and anhydrous 2-
methyl-2-propanol (0.72 mL, 7.68 mmol) was added, followed by DMAP
(94 mg, 0.77 mmol). The solution was cooled to 08C, and DIC (0.80 mL,
5.12 mmol) in CH2Cl2 (5 mL) was injected dropwise over 15 min.The re-
action mixture was stirred at 08C under an atmosphere of N2 for 15 min
and then at room temperature for 5 h.The mixture was diluted with
Et2O and extracted with aq HCl (0.1m, î2) and H2O (î1).The organic
layer was collected, concentrated, and purified by chromatography on
silica gel (9:1 hexanes/Et2O) to give 1 as a solid (1.0 g, 94%) that was ho-
Alloc-d-Abu-OH: (d)-Aminobutyric acid (1.0 g, 9.7 mmol) was dissolved
in a mixture of H2O (15 mL), aq NaOH (1m, 10 mL), and Et2O (10 mL)
and the system cooled to 08C.A solution of allyl chloroformate (14. mL,
12.9 mmol) in dioxane (13 mL) was added slowly and simultaneously
4338
¹ 2004 Wiley-VCH Verlag GmbH & Co.KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2004, 10, 4334 4340

Dihydrovirginiamycin S1
4334 4340
with aq NaOH (1m, 13 mL).The ice-bath was removed, and the reaction
mixture was stirred for 16 h at room temperature.The mixture was dilut-
ed with EtOAc and extracted with saturated aq NaHCO3.The aqueous
layer was collected, acidified with concentrated HCl, and extracted with
tin hydride (1.43 mL, 5.31 mmol) and tetrakis(triphenylphosphine)palla-
dium(o) (100 mg, 0.087 mmol). The mixture was agitated at room temper-
ature in the dark for 3 h, drained, and washed with CH2Cl2 (20 mL),
MeCN (20 mL), and again with CH2Cl2 (20 mL).The resin was dried
under vacuum and treated with Fmoc-l-Pro-OH (0.90 g, 2.66 mmol), a
solution of HOAt in DMF (0.5m, 10 mL, 5.0 mmol), DIC (0.78 mL,
5.0 mmol), and 2,6-lutidine (1.16 mL, 10.0 mmol). The mixture was agitat-
ed for 48 h at room temperature, drained, and washed with CH2Cl2
(20 mL), MeOH (20 mL), MeCN (20 mL), and again with CH2Cl2
(20 mL).The same coupling protocol was repeated again twice, after
which the resin was washed and dried under vacuum.The resin was then
treated with a solution of 30% piperidine in CH2Cl2 (20 mL) for 30 min,
drained, and washed with CH2Cl2 (20 mL), MeCN (20 mL), and again
with CH2Cl2 (20 mL).The resin was dried under vacuum, suspended in
CH2Cl2 (5.0 mL), and treated with a solution of Alloc-d-Abu-OH in
CH2Cl2 (0.48m, 5.5 mL, 2.66 mmol), PyAOP (2.6 g, 5.0 mmol), and 2,6-lu-
tidine (1.16 mL, 10.0 mmol). The mixture was agitated for 24 h at room
temperature, drained, and washed with CH2Cl2 (20 mL), MeOH (20 mL),
MeCN (20 mL), and again with CH2Cl2 (20 mL).The resin was dried
under vacuum.
EtOAc.The organic layer was collected, dried (Na SO4), and concentrat-
2
ed, affording a liquid (1.85 g, 100%) that was homogeneous by TLC
analysis. Rf =0.50 (EtOAc/HOAc 50:1); [a]=ꢀ10.0 (c=0.70 in CHCl3);
1H NMR (300 MHz, CD3CN, 258C): d=0.94 (t, J=7.4 Hz, 3H), 1.78 (m,
2H), 4.10 (m, 1H), 4.53 (d, J=5.0 Hz, 2H), 5.18 (dd, J=1.4, 10.5 Hz,
1H), 5.29 (d, J=17.3 Hz, 1H), 5.90 (m, 1H), 6.02 (d, J=7.0 Hz, 1H),
9.61 ppm (s, 1H); 13C NMR (75 MHz, CD3CN, 258C): d=10.5, 25.7, 56.1,
66.3, 118.3, 134.2, 157.4, 174.9 ppm; FT-IR (thin film): n˜ =3323, 3085,
2973, 2882, 1718, 1533, 1235 cmꢀ1
[C8H12NO4]ꢀ: 186.0764; found: 186.0771.
; MS (HRESI-MS) calcd for
Ester 4: Boc-l-Thr-OAll (0.95 g, 3.66 mmol) was dissolved in CH2Cl2
(15 mL), and Troc-l-Phg-OH (1.32 g, 4.03 mmol) was added. The system
was cooled to ꢀ308C, and DIC (0.63 mL, 4.03 mmol) was injected, fol-
lowed by DMAP (4.5 mg, 0.0366 mmol). The reaction mixture was main-
tained between ꢀ30 and ꢀ208C under an atmosphere of N2 for 2 h, dilut-
ed with Et2O, and quenched with aq HCl (0.1m).The mixture was ex-
tracted with aq HCl (0.1m, î2), saturated aq NaHCO3 (î2), and H2O (î
1).The organic layer was collected and concentrated.The crude residue
was purified by chromatography on silica gel (2.3:1 hexanes/Et2O) to
give 4 as a solid (1.98 g, 95%) that was homogeneous by TLC analysis.
Rf =0.58 (hexanes/Et2O 1:1); [a]=+43.9 (c=0.62 in CHCl3); 1H NMR
(300 MHz, CDCl3, 258C): d=1.16 (d, J=6.5 Hz, 3H), 1.45 (s, 9H), 4.48
(dd, J=2.5, 9.5 Hz, 1H), 4.61 (d, J=6.0 Hz, 2H), 4.68 (d, J=12.0 Hz,
1H), 4.76 (d, J=12.2 Hz, 1H), 5.25 (m, 4H), 5.48 (m, 1H), 5.85 (m, 1H),
5.98 (d, J=7.1 Hz, 1H), 7.36 ppm (m, 5H); 13C NMR (75 MHz, CDCl3,
258C): d=16.4, 28.4, 57.2, 58.3, 66.7, 72.8, 74.9, 80.6, 95.4, 119.5, 127.3,
129.1, 129.3, 131.5, 135.7, 153.7, 155.9, 169.4, 169.7 ppm; FT-IR (thin
film): n˜ =3336, 2980, 1746, 1720, 1508 cmꢀ1; MS (HRESI-MS) calcd for
[C23H29Cl3N2O8+Na]+: 589.0887; found: 589.0867.
Resin-bound linear hexapeptide 8: The resin-bound tetrapeptide 7 was
suspended in CH2Cl2 (4.5 mL) and treated with 2,6-lutidine (4.0 mL,
34.5 mmol) and TMS-OTf (3.0 mL, 16.6 mmol). The mixture was agitated
at room temperature for 5 h, drained, and washed with CH2Cl2 (20 mL),
MeOH (20 mL), MeCN (20 mL), and again with CH2Cl2 (20 mL).The
resin was suspended in MeOH (10 mL) and agitated at room tempera-
ture for 20 min, after which it was drained and washed with CH2Cl2
(20 mL), MeOH (20 mL), MeCN (20 mL), and again with CH2Cl2
(20 mL).The resin was dried under vacuum, suspended in a solution of
HOAt in DMF (0.5m, 10 mL, 5.0 mmol), and treated with amine 5
(0.69 g 1.77 mmol), DIC (0.78 mL, 5.0 mmol), and 2,6-lutidine (1.16 mL,
10.0 mmol). The mixture was agitated for 6 h at room temperature,
drained, and washed with CH2Cl2 (20 mL), MeOH (20 mL), MeCN
(20 mL), and again with CH2Cl2 (20 mL).The resin was dried under
vacuum.
Amine 5: Ester 4 (1.8 g, 3.17 mmol) was dissolved in AcOH (5 mL) and
zinc dust (2 g) was added.The reaction mixture was stirred at room tem-
perature for 1 h, after which additional AcOH (1.5 mL) and zinc dust
(2 g) were added.The mixture was stirred at room temperature for an-
other hour and purified by chromatography on silica gel (10:1 Et2O/
EtOAc) affording 5 as a solid (1.1 g, 88%) that was homogeneous by
TLC analysis. Rf =0.52 (Et2O/MeOH 20:1); [a]=+66.7 (c=0.75 in
Resin-bound cyclic hexapeptide 9: The resin-bound linear hexapeptide 8
(690 mg, 0.226 mmol, based on 0.5 mmolgꢀ1 initial loading and subse-
quent weight increase of the resin) was placed into a 10 mL polypro-
pylene poly-prep chromatography column.The resin was suspended in
CH2Cl2 (6.0 mL), treated with phenylsilane (2.0 mL, 16.2 mmol), H2O
1
CHCl3); H NMR (300 MHz, CD3CN, 258C): d=1.11 (d, J=6.4 Hz, 3H),
(100 mL),
and
tetrakis(triphenylphosphine)palladium(o)
(100 mg,
1.42 (s, 9H), 1.95 (s, 2H), 4.42 (dd, J=2.3, 9.5 Hz, 1H), 4.54 (m, 2H),
4.75 (s, 1H), 5.20 (dd, J=1.1,10.4 Hz, 1H), 5.30 (ddd, J=1.5, 3.1,
17.4 Hz, 1H), 5.40 (m, 1H), 5.86 (m, 1H), 5.89 (d, J=10.5 Hz, 1H),
7.38 ppm (m, 5H); 13C NMR (75 MHz, CDCl3, 258C): d=17.0, 28.4, 57.1,
58.4, 66.8, 73.1, 80.4, 119.1, 127.3, 129.3, 129.4, 131.8, 136.9, 156.3, 170.9,
0.087 mmol). The mixture was agitated at room temperature in the dark
for 5 h, drained, and washed with CH2Cl2 (20 mL), MeOH (20 mL),
MeCN (20 mL), and again with CH2Cl2 (20 mL).The resin was dried
under vacuum, resuspended in CH2Cl2 (8.0 mL), and treated with
PyAOP (0.50 g, 0.96 mmol), and 2,6-lutidine (1.0 mL, 8.61 mmol). The
mixture was agitated for 48 h at room temperature, drained, and washed
with CH2Cl2 (20 mL), MeOH (20 mL), MeCN (20 mL), and again with
CH2Cl2 (20 mL).The resin was dried under vacuum.All the subsequent
reactions through the completion of the synthesis were conducted in the
same poly-prep chromatography column.
171.0 ppm; FT-IR (thin film): n˜ =3313, 2981, 2936, 1745, 1716, 1506 cmꢀ1
;
MS (HRESI-MS) calcd for [C20H28N2O6+Na]+: 415.1845; found:
415.1837.
Resin-bound dipeptide 6: Imidazole (0.30 g, 4.43 mmol) was dissolved in
CH2Cl2 (4.0 mL) and diisopropyldichlorosilane (0.16 mL, 0.89 mmol) was
injected dropwise.The mixture was stirred at room temperature under an
Resin-bound acylated macrocycle 10: The resin-bound cyclic hexapeptide
atmosphere of N2 for 5 min, and
a
solution of alcohol
3
(0.375 g,
9 was suspended in CH2Cl2 (4.0 mL) and treated with 2,6-lutidine
0.885 mmol) in CH2Cl2 (3.0 mL) was added slowly dropwise over 15 min
with a canula.The mixture was stirred at room temperature under an at-
mosphere of N2 for 30 min, followed by addition of the hydroxymethyl
polystyrene resin (0.65 g, 0.65 mmol). The mixture was agitated at room
temperature for 48 h, drained, and washed with MeOH (20 mL), CH2Cl2
(20 mL), and Et2O (20 mL).The resin was dried under vacuum and sub-
jected to Fmoc quantitation.The loading level was determined to be
0.50 mmolgꢀ1.The resin was then treated with a solution of 30% piperi-
dine in CH2Cl2 (20 mL) for 30 min, drained, and washed with CH2Cl2
(20 mL), MeCN (20 mL), and again with CH2Cl2 (20 mL).The resin was
dried under vacuum and treated with Alloc-l-MePhe-OH (0.70 g,
2.66 mmol), a solution of HOAt in DMF (0.5m, 10 mL, 5.0 mmol), DIC
(0.78 mL, 5.0 mmol), and 2,6-lutidine (1.16 mL, 10.0 mmol). The mixture
was agitated for 48 h at room temperature, drained, and washed with
CH2Cl2 (20 mL), MeOH (20 mL), MeCN (20 mL), and again with
CH2Cl2 (20 mL).The resin was dried under vacuum.
(3.0 mL, 25.8 mmol) and TMS-OTf (2.0 mL, 11.1 mmol). The mixture
was agitated at room temperature for 2 h and then for 25 min at 48C.
The resin was washed with precooled-to-48C THF (20 mL), immediately
suspended in precooled-to-48C THF (6.0 mL), and treated with 3-hy-
droxypicolinic acid (0.20 g, 1.44 mmol), PyAOP (0.50 g, 0.96 mmol), and
2,6-lutidine (0.60 mL, 5.17 mmol). The mixture was agitated at 48C for
4 h and then for 10 h at room temperature.The resin was drained and
washed with CH2Cl2 (20 mL), MeOH (20 mL), THF (20 mL), DMF
(20 mL), MeCN (20 mL), and again with CH2Cl2 (20 mL).The resin was
dried under vacuum.
Dihydrovirginiamycin S1: The resin-bound macrocycle 10 was suspended
in THF (7.0 mL) and treated with HF/pyridine (1.0 mL). The resin was
agitated at room temperature for 1 h.The resin was drained and washed
with THF (20 mL), methoxytrimethylsilane (20.0 mL, 145.64 mmol),
CH2Cl2 (20 mL), MeOH (20 mL), MeCN (20 mL), and again with
CH2Cl2 (20 mL).All the washes were combined with the original filtrate,
stirred at room temperature for 1 h and, concentrated.The residual
amount of pyridine was removed under vacuum.The product was puri-
Resin-bound tetrapeptide 7: The resin-bound dipeptide 6 was suspended
in a 5% solution of AcOH in CH2Cl2 (10.0 mL) and treated with tributyl-
Chem. Eur. J. 2004, 10, 4334 4340
www.chemeurj.org
¹ 2004 Wiley-VCH Verlag GmbH & Co.KGaA, Weinheim
4339

P.J.Belshaw et al.
FULL PAPER
fied by chromatography on silica gel (25:1 CH2Cl2/MeOH) to give dihy-
drovirginiamycin S1 (28 mg, 15%) as a solid that was homogeneous by
TLC analysis. Rf =0.32 (CH2Cl2/MeOH 20:1); [a]=ꢀ4.4 (c=0.54 in
CHCl3); 1H NMR (500 MHz, CDCl3, 258C): d=0.33 (dt, J=11.6, 5.1 Hz,
1H; 5b2), 0.92 (t, J=7.3 Hz, 3H; 2g), 1.25 (m, 1H; 5d2), 1.14 (m, 1H;
3b1), 1.33 (d, J=6.5 Hz, 3H; 1g), 1.31 (m, 1H; 3g2), 1.33 (d, J=6.5 Hz;
3H; 1g), 1.58 (m, 1H; 3g1), 1.67 (m, 1H; 2b2), 1.73 (m, 1H; 2b1), 1.90 (m,
1H; 5d1), 1.97 (m, 1H; 3b2), 2.38 (dd, J=12.3, 4.8 Hz, 1H; 5b1), 2.45 (dt,
J=13.4, 2.1 Hz, 1H; 5e2), 3.11 (s, 3H; 4N-Me), 3.13 (m, 1H; 4b2), 3.25
(dd, J=14.6, 8.6 Hz, 1H; 4b1), 3.33 (q, J=7.5 Hz, 1H; 3d2), 3.51 (m, 1H;
3d1), 4.27 (m, 1H; 5g), 4.51 (t, J=6.7 Hz, 1H; 3a), 4.60 (d, J=13.7 Hz,
1H; 5e1), 4.78 (q, J=7.4 Hz, 1H; 2a), 4.85 (dd, J=9.6, 1.2 Hz, 1H; 1a),
5.12 (m, 1H; 5a), 5.43 (t, J=8.1 Hz, 1H; 4a), 5.61 (d, J=7.8 Hz, 1H;
6a), 5.89 (dq, J=6.7, 1.2 Hz, 1H; 1b), 6.60 (d, J=9.3 Hz, 1H; 2NH), 7.10
(m, 2H; aromatic), 7.17 (dd, J=8.5, 4.3 Hz, 1H; 1’H5), 7.29 (m, 9H; aro-
matic), 7.70 (dd, J=4.4, 1.1 Hz, 1H; 1’H6), 8.38 (d, J=9.5 Hz, 1H;
1’NH), 8.58 (d, J=7.9 Hz, 1H; 6NH), 11.67 ppm (s, 1H; 1’OH);
13C NMR (125 MHz, CDCl3, 258C): d=10.3, 16.7, 25.0, 25.3, 27.9, 31.0,
34.0, 35.5, 35.6, 38.1, 47.9, 51.8, 52.1, 56.3, 56.9, 57.7, 57.8, 65.0, 71.5,
127.0, 127.6, 128.2, 128.4, 129.0, 129.1, 129.3, 130.5, 135.7, 136.2, 139.9,
158.6, 167.6, 168.8, 169.7, 170.3, 170.5, 171.4, 173.0 ppm; FT-IR (thin
[3] a) P.J.Belshaw, C.T.Walsh, T.Stachelhaus,
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[9] a) M.A. Ondetti, P.L. Thomas, J. Am. Chem. Soc. 1965, 87, 4373;
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Int.
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;
[10] a) M.J.O.Anteunis, N.K.Sharma,
Bull. Soc. Chim. Belg. 1988, 97,
Int. J. Pept. Protein
MS (HRESI-MS) calcd for [C43H51N7O10+Na]+: 848.3595; found:
281; b) M.J.O.Anteunis, C.Van der Auwera,
848.3575.
Res. 1988, 31, 301; c) J.M.Paris, J.C.Barriere, C.Smith, P.E.Bost,
Recent Prog. Chem. Synth. Antibiot. 1990, 183; d) M.C. Moerman,
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Acknowledgements
[11] D.Y. Maeda, J.E. Ishmael, T.F. Murray, J.V. Aldrich,
Chem. 2000, 43, 3941.
J. Med.
We thank Prof.B. Weisblum for advice and bacterial strains and R.C.
Nelson for assistance with spectral characterization.We gratefully ac-
knowledge the NIH (GM065406), the W.M. Keck Foundation and the
Burroughs Wellcome Fund for financial support of this work, and NSF
(CHE-9208463) and NIH (RR08389) for support of NMR facilities.
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Received: March 19, 2004
Published online: July 19, 2004
4340
¹ 2004 Wiley-VCH Verlag GmbH & Co.KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2004, 10, 4334 4340